Makedonski farmacevtski zbornik_57-f.indb

волумен 57 (1, 2) 2011 / volume 57 (1, 2) 2011

Македонско фармацевтско друштво, ул. Маршал Тито 13б/8, Скопје, Македонија

Macedonian Pharmaceutical Society, Marshal Tito 13b/8, Skopje Macedonia

MRAMFD MRA

Macedonian pharmaceutical bulletin, 57 (1, 2) 3 - 16 (2011)ISSN 1409 - 8695

UDC: 615.212 Review

Introduction Background

Synapses are specialized contact site of two neurons and are important for transfer of a signal from one neuron to another. Synaptic transmission is chemical and is medi-ated by neurotransmitter compounds. As it is presented in the Fig. 1, neurotransmitters are released by presynaptic cell into synaptic cleft and bound by the receptor on the postsynaptic cell.In the resting position, when the agonist is not bound to the ligand binding domain (LBD), the ion-channel is closed. When the agonist is bound to the LBD, the ion channel is opening. The third form of the receptor is desensitized form when the agonist is still present in the LBD, but the ion-channel is closed by different mechanism than deactivation as described latter in this review.

(S)-Glutamic acid (Glu) is one of the most abounded excitatory neurotransmitter in the central nervous system (CNS) and has been implicated in a number of neurological

Ionotropic Glutamate Receptors (iGluRs): Overview of iGluR2

ligand binding domain in complex with agonists and antagonists

Zorica Serafi moska1*, Tommy N. Johansen2, Karla Frydenvang2, Ljubica Suturkova1

1 Institute for Pharmaceutical chemistry, Faculty of Pharmacy, “St Cyril and Methodius” University Skopje, Macedonia2Department of Madical chemistry, Faculty of Pharmaceutical Sciences, University Copenhagen, Denmark

Received: October 2011; Accepted: November 2011

Abstract

Ionotropic glutamate receptors (iGluRs) constitute a family of ligand gated ion channels subdivided in three classes, NMDA, AMPA (iGluA1-4) and KA (1-5) according to the agonists that selectively activate them. iGluRs are tetrameric assemblies of highly homologous receptor subunits. They are critically important for normal brain function and are considered to be involved on neurological disorders and degenerative diseases such as schizophrenia, Alzheimer’s disease, brain damage following stroke and epilepsy. Since the fi rst publication of the structure of recombinant soluble protein of ligand binding domain of GluA2 extensive studies on this group of receptors were per-formed and many crystal structures as complexes of GluA2-LBD with agonists, partial agonists and antagonists were obtained. The struc-tural information in combination with functional data makes good platform for consecutive investigation and design of new selective drugs which will be used in treatment of neurodegerative diseases.

Key words: Ionotropic glutamate receptors (iGluRs), AMPA(iGluA1-4), neurological disorders, degenerative diseases schizophrenia, Alzheimer’s disease, epilepsy.

* [email protected]

and psychiatric diseases. Glu activates two different types of receptors: ionotropic (iGluRs) and metabotropic gluta-mate receptors (mGluRs) (Nakanishi, 1992). The iGluRs constitute a family of ligand-gated ion channels that are essential for mediating fast synaptic transmission in CNS. These receptors play an important role for the development and function of nervous system, and are key factors in learn-ing and memory (Dingledine et al., 1998; Meldrum, 2000). They are critically important for normal brain function and are considered to be involved on neurological disorders and degenerative diseases such as schizophrenia, Alzheimer’s disease, Parkinsons disease, brain damage following stroke and epilepsy as well as neuropathic pain (Medsen et al., 2005; Foster and Kamp, 2006; Bowie, 2008).

Ionotropic glutamate receptors (iGluRs) constitute a family of ligand gated ion channels subdivided into three classes distinguished by amino acid sequence, pharma-cological profi le, functional behavior and biological role (Armstrong and Gouaux, 2000). The iGluRs have been found to be homo- or heteromeric assemblies that form te-trameric ion channel complexes (Laube et al.,1998; Mano

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Maced. pharm. bull., 57 (1, 2) 3 - 16 (2011)

Zorica Serafi moska, Tommy N. Johansen, Karla Frydenvang, Ljubica Suturkova

and Teichberg, 1998; Rosenmund et al.,1998). Those com-posed of GluA1–4 are activated by AMPA (amino-3-hy-droxy-5-methylisoxazole-4-propionic acid) and are termed AMPA receptors, while Kainate has high affi nity for KARs GluK1-5 (Coligrudge et al., 2009) and the third class are re-ceptors activated byNMDA(N-methyl-D-aspartate). AMPA and glutamate evoke similar electrophysiological respons-es even though AMPA binds approximately 20-fold more tightly than glutamate, depending on the receptor subunit (Brauner-Osborne et al., 2000). Functional NMDA recep-tors are composed of assemblies of the NR1 together with NR2A-D subunits (Hollman and Heinemann, 1994). The involvement of these receptors in neurological diseases has aroused widespread interest in their structure and function.

Fig. 1. A schematic representation of postsynaptic AMPA receptors. Postsynaptic AMPA receptors in the closed form in the absence of an agonist (I), in the open, agonist bound form (II), and in the desensitized form (III)

The iGluRs are membrane-bound and form tetramers assembled as dimers-of-dimers (Jin, 2003; Naur, 2005).Occupancy of the two binding sites is necessary for chan-nel opening. There were four sites for agonist binding one in each subunit of the tetrameric assembly. From this and some other functional analysis is assumed that tetrameric

assembly is formed through dimerisation of subunit mono-mers (via N-terminal domain) followed by dimerization of two subunit dimers (Ayalon and Stern-Bach, 2001). When the non-bounded receptor in apo state is attracted by an agonist both dimers are closing, the channel is opening and ion fl ow is enabled, but when antagonist is bound the dim-ers are in more open conformation compared to apo state and the ion fl ow is stopped by channel closure.

Membrane topology

Molecular organization of glutamate receptor subunit is presented on Fig. 2 and 3.

COOH

M1 M2 M3

P

S1S2

ATD

Fig. 2. Modular organization of a glutamate receptor. Membrane topology of an ionotropic glutamate receptor with three membrane spanning domains (M1, M3, and M4) and a re-entrant loop (P) and the amino(ATD) and carboxyl(CTD) terminal do-main. The ligand binding domain is formed by the S1 and S2 regions of the protein which come to-gether to form a hinged clamshell-like structure.

COOH

M1 M3 M4P

*

*, Q/R site

S1 S2 Flip/flop

H N 2

Fig. 3. Scheme of AMPA receptor subunits. The transmembrane topology is shown along with the fl ip/fl op alternatively spliced exon, and the two ligand-binding domains (S1 and S2).

* is Q/R editing site and glycosylation sites in the N-terminal region.

Макед. фарм. билт., 57 (1, 2) 3 - 16 (2011)

5 Ionotropic Glutamate Receptors (iGluRs): Overview of iGluR2 ligand binding domain in complex with agonists and antagonists

An amino terminal domain (ATD) is the site of action for a number of molecules that modulate glutamate receptor function (Herin and Aizenman, 2004) and structure-func-tion studies have shown that this region is also involved in assembly of subunits within the receptor complex (Stern-Bach et al., 1994; Ayalon and Stern-Bach, 2001). The li-gand-binding domain consists of two regions termed S1 (N-terminal of M1 transmembrane spanning domain) and S2 (between regions M3 and M4) (Gouaux, 2004; Mayer and Armstrong, 2004; Mayer, 2005b). The hydrophobic membrane-associated region consists of three transmem-brane spanning α-helixes (M1, 3 and 4) and a reentry loop (M2; analogous to the P-loop found in voltage-gated po-tassium channels) and residues from this M2 region line the lumen of the ion channel pore and control ionselec-tivity and permeability (Wollmuth and Sobolevsky, 2004). An intracellular carboxy terminal domain (CTD) contains a number of structural motifs that allow the interaction with numerous signal transduction and scaffolding pro-teins and is important for correct regulation, traffi cking and localisation of the receptor protein (Peґrez-Otano and Ehlers, 2005). N-terminal half of AMPA receptor subunits are much less conserved than the sequence of C-terminal half. N-terminal segment has several sites appropriate for N-glycosylation (Everst et al., 1997), whereas C-terminal half of AMPA subunits has appropriate sites for phospho-rylation (Roche et al., 1996).

N-Terminal domain

In AMPA and KA receptor subunits ATD does not bind glutamate but instead forms dimers that are major assem-bly control points limiting oligomerization to the members of the same iGluRs subfamily (Ayalon and Stern-Bach, 2001; Ayalon et al., 2005; Mayer, 2005b). Amino termi-nal domain is the site of action for a number of molecules, like allosteric modulators, that modulate glutamate recep-

tor function.In NMDA, the ATD is associated with pH sensitivity,

glycine independent desensitization and regulation of re-ceptor function by compounds like polyamines, histamine, Mg 2+ and Zn 2+ (Dingladine et al., 1999; Zheng et al., 2001; Mayer 2005b).

Ligand-binding domain (S1S2)

Two separate extracellular regions of approximately 150 residues each, (S1) preceding the fi rst membrane seg-ment, and (S2) located between M3 and M4 segments, are interrupted by ion-channel pore (Stern-Bach et al., 1994; Mayer, 2005). The studies with chimeric AMPA/KA and NMDA receptor subunits have shown that this region (S1S2 ligand binding core) is important determinant of li-gand pharmacology.

S1S2 binding region also contributes to AMPA recep-tor desensitization and redox modulation. Flip/fl op seg-

ment in C-terminal part of S2 affects the receptor desen-sitization (Fig.3) (Partin et al., 1994). This variants have small differences in ligand-binding properties (Nakanishi, 1992), different desensitization kinetics and different sen-sitivity to desensitization modulators CTZ (Partin et al., 1995) and PEPA (4-[2(phenylsulfonilamino) ethy [thio]-2,6-difl uoro-phenoxyacetamide) (Sekiguchi et al., 1997). The fl ip forms of most subunits desensitize more slowly and less profoundly than the fl op forms.

Some of GluA2-4 AMPA subunits are edited at R/G site at an intronic site preceding the fl ip/fl op exons. Replacing the glycine codon (IGA) with arginine codon (AGA) in GluA3 and GluA4 subunits also contributes to the kinet-ics of the glutamate evoked responses and speed up the recovery from agonist-induced desensitization (Lomeli et al.,1994). This is observed in GluA2, 3 and 4, but not in GluA1, due to the absence of the essential element that fa-vors editing at the R/G site (Fletcher and Lodge, 1996).

S2 region have two Cys residues corresponding to Cys-744 and Cys-798 in NMDA receptor N1 which play impo-tent role in stabilization of the structure of ligand binding core of the iGluRs (Sutcliffe et al., 1996). This conserved cystein pair is responsible for pH sensitivity and binding of Zn2+ in NMDA receptors (Sullivan et al., 1994). S1S2 binding region of NR1 subunit of NMDA is bound to co-agonist glycine, whereas the same region of NR2 is bound to glutamate (Kuratov et al., 1994; Laube et al., 1997).

Transmembrane channel

Some glutamate receptor RNAs are post-transcription-ally modifi ed by RNA editing, which leads to single amino acid exchanges (Seeburg, 1996). RNA editing Q/R-site in M2 segment determinates the Ca2+ permeability of AMPA channels. All the AMPA receptor assemblies, that con-tain CluA2 subunit are Ca2+ impermeable and have linear steady-state current voltage relation, since GluA2 subunits are editing almost 100% and have Arg residue at Q/R site (Hollman et al., 1991; Hume et al., 1991; Burnashev et al., 1992) (Fig. 3). Gln residue at Q/R editing site is respon-sible for high permeability of GluA1, 3 and 4 subunits for Ca2+ and their inward rectifi cation. The rectifying I-V rela-tionship is blocked by intracellular polyamines, spermine and spermidine, which bind to the Gln at Q/R-site (Bowie and Mayer, 1995; Washburn and Dingledine, 1996).Similarly, blockage of Ca2+ permeable AMPA receptors by argiotoxin has been assigned to the Gln residue at the Q/R site (Meucci and Miller, 1998). The GluA2 Q/R site is the most vigorously edited site. Physiological role, if any, of unedited GluA2 (Q) is unclear.

The M3 segment of AMPA receptors affects gating properties of the receptors. The mutation of Ala to Thr at a highly conserved C-terminal segment of the M3 at AMPA and KA receptor subunits makes the receptors continuously active or renders them to be activated by CNQX, normally an antagonist (Taverna et al., 2000; Schwarzet al., 2001).

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Maced. pharm. bull., 57 (1, 2) 3 - 16 (2011)


C-terminal domain

The greatest diversity among iGluRs subunits is found in their citoplasmatic domain, which varies in size from less than 20 to around 500 amino acids. The GluA1 subunit has a C-terminal domain of 81 amino acids, C-terminal domain of the GluA3 subunit is only 50 residues long (Keinänen et al., 1990). The GluA-2 and -4 subunits have either 68 or 50 amino acids in their C-terminal domains due to alterna-tive splicing (Köhler et al.,1994). The C-terminal domain of KA receptors are of similar size to GluA1-4 subunits, where as the C-terminal domain of the NMDA receptor NR2 subunits are signifi cantly larger, approximately 620-640 amino acids (Sprengal and Seeburg, 1993). This is the key site of allosteric modulation by calcium/calomodulin kinase II (CAMKII), protein kinase C (PKC) protein ki-nase A (PKA) and phospatases and is potentially subjected to phosphorylation and dephosphorylation (Roche et al., 1996; Mayer, 2005b).

Functionally, this domain is involved in cytoskeletal interactions, subunit traffi cking, and modulation of chan-nel conductance and contains a variety of sites for post transcriptional/posttranslational modifi cations (McFeaters and Oswald, 2004).

Mechanism of AMPA receptor activation and

desensitization

The binding of glutamate and other agonists to all glutamate receptors induces activation and opening of

the transmembrane ion channel which is due to a confor-mational change in the receptor structure (Clements et al.,1998). Glutamate, as a natural agonist, is rapidly re-moved from synaptic cleft by uptake to glia cells or by diffusion, which leads to rapid closure of the channel by deactivation (Jonas, 2000). Deactivation caused by agonist appears after their dissociation. AMPA receptor activation occurs with the time constant of sub-milliseconds, whereas deactivation occurs in the time scale of one or two mil-liseconds. In high frequency stimulation, glutamate that is normally rapidly removed, may remain in the cleft for prolonged times. In the continuous presence of glutamate, and other agonist, the channel is closed by desensitization, which is particularly fast and strong forAMPA receptors and approximately three fold slower (1-6ms) than deacti-vation. The process of activation and desensitation of glu-tamate receptors is shown in Fig. 4. The extent of desen-sitization of the AMPA receptors depends on the type of agonist. L-glutamate and AMPA induce fast desensitisa-tion (with a time scale of 1-6milliseconds), whereas kain-ate as partial agonist does not allow full desensitization of the AMPA receptors (Armstrong, 1998). Since the kainate-bound structureis not fully closed, as AMPA- and gluta-mate-bound structures are, it seems that desensitization but not activation requires full closure of the binding domain (Armstrong and Gouaux, 2000).

In addition, the desensitized-state of the AMPA recep-tors show increased affi nity to agonists. Desensitization appers to be a general mechanism of many ligand-gated

D2

D1

RestingOpen cleft

Closed channel

BoundOpen cleft

Closed channel

OpenClosed cleft

Open channel

DesensitizedClosed cleftClosed channel

D2

D1

D2

D1

D2

D1

D2

D1

Fig. 4. A model for glutamate receptor activation and desensitization. Domain 1 and domain 2 of the ligand-binding core are labelled D1 and D2, respectively. Transmembrane segments of each subunit are indicated by a single cylinder and the N-terminal domain (ATD) has not been included in the model. Each subunit binds a single agonist (A, circle) and exists in three distinct conformations: closed (C), open (O)and desensitized (D). After the binding of agonist, closure of domain 2 towards domain 1 opens the channel gate, whereas closure of domain 1 towards domain 2 disrupts the dimer interface and desensitizes the receptor. The states are connected by using a simplifi ed model for activation and desensitization, more complex versions of which quantitatively describe AMPA receptor responses.

Макед. фарм. билт., 57 (1, 2) 3 - 16 (2011)


channels to cut ionfl ow into the cell in the presence of the ligand.

AMPA and KA receptors have conserved gating mech-anisms and to desensitize, their ligand binding cores must undergo large conformational rearrangements. AMPA re-ceptors recover from desensitization with time constants that are approximately 10-fold faster than KA receptors, which is the one of the main kinetic distinctions between AMPA and KA receptors activated by the endogenous neurotransmitter glutamate (Dingdeline et al., 1999).The differences in extend of desensitization observed for glu-tamate versus kainate (Pataeau et al., 1992) is likely the result of differences in domain closure that in turn lead to differences in intersubunit contacts. KA receptors, which are fully activated by kainate, have a valine instead of leu-cine at the position equivalent to 650 in GluA2. Leu650Val mutant of GluA2 allows the ligand binding core to adopt a glutamate-like conformation in the presence of bound kainate, and in terms of desensitization, kainate acts on the mutant receptor as full agonist (Armstrong and Gouaux, 2000).

In comparison, NMDA receptors are activated with a time scale of 10-50 milliseconds. The deactivation andde-sensitization also occurs with a much slower time course (in the time scale of tens orhundreds of milliseconds) than in AMPA receptors. The desensitization is complex, with va-riety of different processes that involved extracellular gly-cine, intracellular Ca2+ and certain intracellular proteins.

Crystal structure of iGluA2-S1S2 provides a new as-pect of desensitization mechanism. Beside the subunit composition of receptor and type of bound agonist as ex-plained previously, AMPA receptor desensitization kinetic is controlled also by alternative splicing affecting the fl ip/fl op region and the RNA editing of the R/G site (Dingledine et al., 1999).

Crystal structure of the ligand-binding domain of GluA2

Since the fi rst publication in 1998 of structure of a re-combinant soluble protein composing the ligand-binding domain of GluA2 extensive studies were performed in these families of receptors. Until recently, structural inves-tigations were limited to studies of soluble constructs of individual domains, N-terminal domain (Jin et al., 2009) and the ligand binding domain (LBD) (Armstrong and Gouaux, 2000; Ahmed et al., 2009a and Kasper et al., 2008). Sobolevsky and coworkers in 2009 have solved the structure of the full-length homotetrameric GluA2 re-ceptor. The determination was made at 3.6Å resolution in complex with the competitive antagonist ZK 200775 (pdb-code 3KG). The receptor shows an overall axis of two-fold symmetry but with the ion channel domain displaying four-fold symmetry. However, the soluble LBD protein is a good model system of the full length-receptor for study-ing binding of agonists and antagonists as well as bind-ing of allosteric modulators at the dimer interface. Studies

using radio-label binding assays (Armstrong and Gouaux, 2000; Hogner et al., 2002; Jin et al., 2003) have shown that GluA2 LBD has essentially identical properties to the cor-responding domain in intact, membrane-bound protein.

The extracellular domains, defi ned as S1and S2 are joined together by a short, fl exible peptide linker, replac-ing the membrane segments M1-M3 of the intact recep-tor. A large-scale production of histidine tagged S1S2 con-struct (HS1S2) by in vitro Escherichia coli expression was achieved by Chen and Gouaux, (1997). Gouaux and co-workers were able to confi rm the ‘clamshell’ or ‘venus fl y trap’ mechanism of the binding cleft when they obtained the fi rst crystal structures of kainate-bound iGluA2-S1S2 fusion protein (Armstrong et al., 1998). This reaction in-volves initial rapid association of a ligand and a protein fol-lowed by a slower conformational rearrangement, which results in a structure with a ligand locked deeply in the binding pocket of the protein. In addition, this structure of the iGluA2-S1S2Ј fragment defi ned the location of the dis-ulphide bond conserved in all iGluRs and the location of the conserved hydrophobic residues, predicted to form sub-unit-subunit contacts in the intact receptor. Crystallization of the ligand-bound protein allowed identifi cation of un-equivocally, key residues and water molecules, that inter-act with different ligands. iGluA2-S1S2 (denoted as S1S2I or J depending on the length of the interdomain peptide linker) crystals were found to existas dimerized subunits, implying that the tetrameric AMPA receptor complex may consist of a dimer of dimers confi guration (Armstrong and Gouaux, 2000).

Agonist binding in the GluA2-LBD

In GluA2 domain 1 is composed of the N-terminal part of S1 and a short C-terminal segment of S2, and do-main 2 is composed mostly of the S2segment and a short C-terminal segment of the S1 (Armstrong et al., 1998). Both are β-plated sheet surrounded by α−helices. As shown in the picture below domain 1 is formed by six α−helices: A, B, C and D from S1 segment, and J and K-helices, which are formed by S2 segment. Domain 2 forms α−helices: E, F, G, H and I (Armstrong and Gouaux, 2000).

Structure of fl op variant of GluA2 LBD with (S)-glutamate was fi rst solved by Armstrong and Gouaux (2000) and later by Ahmed et al., 2009a. Structure of fl ip variant of GluA2 LBD with (S)-glutamate was published by Gregor et al., 2006. Since 1980, when Krogsgaard –Larsen and co-workers, reported that AMPA is bioisoster of glutamate and a very potent excitant when applied in CNS and spinal cord, and its selectivity towards GluA1-4 over other GluRs led to defi nition of class of AMPA receptors (Foster and Fagg, 1984; Olsen et al., 1987; Waterkins et al., 1990), structures of GluA2 with AMPA and seven other isoxasol contain-ing agonists have been published (Armstrong and Gouaux, 2000; Hogner et al., 2002; Kasper et al., 2002; Lunn et al., 2003;Nielsen et al., 2005; Vogensen et al., 2007).

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There are 20 key residues in S1S2 binding pocket that are responsible for interactions and rearrangements in the binding pocket (Lunn et al., 2003). According to the ’Venus fl y trap’ mechanism, the ligand fi rst interacts with the side chains of residues Tyr450 and Glu402 of the S1. In the sec-ond step, slower one, S1S2 changes its conformations and forms a hydrogen-bond network between the ligand and both protein domains S1 and S2 (Armstrong et al., 1998; Abele et al., 2000).

A

A

C

D

E

F

G

B

T480

P478

R485

S654

T655

E705

Y450

Y405

E402

M708

T686

L650Y702

Fig.5. Schematic presentation of the agonist binding site. Forsimplicity, to represent the 3D natureof the binding cleft, Tyr450 and Glu705 are shown over and under the agonist subsites, respectively. Subsites (A, D, E,and F) are occupied by hydro-gen bond acceptors, while subsite B is occupied bya hydrogen bond donor.

The schematic diagram of amino acids of the LBD that are responsible for interacting with ligands is presented in Fig. 5. α−carboxilate and α-amino groups of the ligands can be responsible for the ligand recognition because they become largely desolvated and bind directly to the protein. These groups interact with the ligand binding core through 7 hydrogen and ion-pair bonding interactions to domains 1 and 2. Three residues from the S1 domain (Pro478, Thr480 and Arg485) and from the S2 domain (Ser654, Thr655 and Glu705) make direct hydrogen bonds with glutamate.Two hydrogen bonds are formed with guanidinium group of the Arg485 residue. Negatively charged α−carboxiyl group is bound by hydrogen bonds and electrostatic interactions to this residue. Kinetic experiments with mutants of the Arg485 residue leads to complete loss of the channel func-tion; thus the presence of this group is important for ligand binding (Spasenskiy and Kurnikova, 2005). α−carboxyl group of the ligand is also hydrogen bonded to the back-bone NH-group of Thr480 and Ser654.

Negatively charged Glu705 is the primary source of the negative potential at the position of the positive-ly charged α-amino group of the ligands. Amino acids

Glu705 and Thr480 are responsible for the formation of a three-way hydrogen-bond network with the α-amino group of the glutamate ligand. Side chain hydroxyl of Thr480 and carboxylate of Glu705 make strong hydrogen bonds with α-amino group. This triangular network connects the two domains of the protein and is important for stabiliz-ing the protein-ligand complex, e.g.,glutaric acid in which α-amino group is absent does not bind to iGluRs (Abele et al., 2000). α−amino group is also hydrogen bonded to the backbone carbonyl oxygen of Pro478 (Armstrong and Gouaux, 2000).

Thearomatic side chain of Tyr450, while not directly hydrogenbonding with glutamate, forms an electron-dense ring structure above the ligand-binding pocket and muta-tions of this tyrosine residue with smaller alanine or larger tryptophan, alter agonist potency and desensitization ki-netics in GluA2-containing receptors. In the S1S2J-(S)-glutamate complex Tyr450 (OH) forms a hydrogen bond with Glu402 (OE2), thereby stabilizing the interdomain interaction between Glu402 and Thr686. Tyr450 can only weekly stabilize Glu402 in the kainate complex. Because Tyr450Thr mutation disrupts the hydrogen bond between Tyr450 and Glu402 this might affect the potency of gluta-mate in a greater extent than for kainate.

GluA2 residue Met708 is fl exible residue that under-goes major conformational changes (induced fi t) to opti-mize van der Waals stabilization of the bound agonist. This allows the hydrophobic pocket to change in size in order to accommodate substituents of various sizes.

DD

G G

EE

F F

1

1

2

2

3

4W4

W3

5

Glutamate AMPA

Fig. 6. Modes of Agonist Binding. Subsite binding regions occupied by the γ-carboxylate of glutamate and water W3. Subsite binding regions occupied by the isoxasolering of AMPA and water W4. Water molecules are shown as spheres. The subsites include the following residues and atoms: Ser654 NH and Thr655 NH of subsite D, Thr655 OH of subsite E, Glu705 NH of subsite F, side chains of Glu402, Tyr450, Pro478, Glu705, Met708 and Tyr732 of subsite G.

The “γ” group binding pocket is composed of four subsites (D, E, F, and G in Fig. 6). Subsites D and E are

Макед. фарм. билт., 57 (1, 2) 3 - 16 (2011)


located at the base of helix F. The distal anionic group of the iGluA2 agonists is hydrogen bounded to the base of α−helix (helix F). The hydrogen bonds are formed by the backbone NHgroups of Ser654 and Thr655, the hydrox-yl of Thr655, a water molecule bound to the base of he-lix F (water 3), and a water molecule (2) tethered to the NH group of Leu650 and the carbonyl oxygen of Leu703. While the γ-carboxyl groups of glutamate and kainate in-teract with the same subsites (hydroxyl and backbone-NH-groups of Ser654 and Thr655 and two water molecules), theisoxazole group of AMPA interacts differently, binds in subsites E, F and G and subsite D is empty (normally oc-cupied by the γ-carboxyl group of glutamate or kainate). In the case of AMPA, a water molecule (4) is recruited to this unoccupied subsite D thus enabling AMPA tobehave like a bioisosteric mimic of glutamate (Armstrong and Gouaux, 2000). This water molecule makes close interaction with isoxazole hydroxyl (2.45 Å), an α-carboxyl oxygen, and residues at the base of helix F.

The four water molecules surrounding the aria around distal anionic group form a network that establishes indi-rect hydrogen bonds between the ligand and side-chains of amino acids within the partially hydrophobic pocket and are essential for the ligand binding and for determination of receptor subtype specifi city.

The agonists adopt the two different binding modes, glutamate-like or AMPA-like.The size of the substituent in the 5-position of the isoxazolol ring is a major determinant of the ligand-binding mode for AMPA-type agonists. A large substituent results in a glutamate-like binding mode, whereas small or no substituents leads to AMPA-like bind-ing.The isoxazolol moiety is not able to control thebinding mode (Kasper et al., 2002).

All of 20 amino acid residues in the biding pocket ex-cept Tyr702 are conserved among AMPA receptors. Banke et al. 2001 identifi ed this nonconserved Tyr residue in AMPA preferring subunits as being the main contributor to the selectivity of (S)-Br-HIBOfor GluA1 over GluA3. In GluA1, this residue is conserved (Tyr698), whereas it is a phenylalanine (Phe706) in GluA3. There is a previ-ously reported structure of a single-pointmutant of the iGluA2-S1S2J construct, (Tyr702Phe) iGluA2-S1S2J, in complex with (S)-Br-HIBO (Hogner et al., 2002). This mu-tation accounts for an 18-fold decrease in affi nity for Br-HIBO (Banke et al. 2001). The AMPA analogue Br-HIBO has been identifi ed as a subtype-selective agonist among AMPA receptors, differentiating the homomeric receptors GluA1o from GluA3o by a ca70-fold difference in affi nity of the racemate (Coquelle et al., 2000).

Domain closure

In the resting state (apo structure; pdb-code1FTO), the GluA2 LBD is in an open state (Armstrong and Gouaux, 2000). When the ligand is interacting with the iGluR2-S1S2J protein an increase in the angle of domain closure

compared to the unliganded (apo) conformation is observed between the twov binding lobes.This domain closure re-sults in the opening of ion channel in full-length receptor (activation). The electrophysiology experiments obtained an excellent correlation between agonist-induced domain closure of the GluA2 LBD, linker-linker (Ile633-Ile633) distance and compound effi cacy.

Great increase in domain closure of the clamshell was observed for full agonists (20-22°) and this correlates with agonist effi cacy, while partial agonists inducing a degree of domain closure inbetween those induced by full ago-nists or antagonists, (13-19°). (Armstrong and Gouaux, 2000; Hogner et al., 2002; Jin et al., 2002; Armstrong et al., 2003). This is best exemplifi edby a series of 5-substituted willardiines that differ in the size of a halide substituent, for which an increase in substituent size sterically hindered domain closure in a graded fashion (Jin et al., 2003). In agreement with the evidence that partial agonists induce less domain closure than full agonists, molecular dynamics simulations have shown that partial agonists such as kain-ate are held less rigidly within the iGluR2 pocket than full agonists (glutamate) and, as a consequence of this, water mobility at specifi c subsites within the pocket is agonist dependent (Arinaminpathy et al., 2006).

In general terms, the rotation describing the domain closure motion occurs about an axis that runs through the two inter-domain β-strands and along helix I from domain 2. Helix I from domain 2 reorients with domain 1, while remainder of domain 2 moves as a separate rigid body, because helix I is linked via a disulfi de bond to domain 1.The center of rotation deffers in the apo to kainate tran-sition compared to the apo to AMPA/glutamate transition. The peptide segment Asp651–Gly653 has been shown to undergo a rearrangement of the backbone upon the transi-tion from the open state (apo) to the fully closed state (e.g. AMPA) (Armstrong and Geuoux, 2000). The trans peptide bond connecting Asp651 and Ser652 undergoes a ~180° fl ip relative to its orientation in the kainate, DNQX and apo structure. Neither of the residues Asp651 and Ser652 nor Glu653 are in direct contact with the ligands, but the surrounding residues Leu650 and Ser654 make hydrogen bonds / van der Waals interactions with the agonists.The peptide bond rearrangement results in two additional hy-drogen bonds between domain 1 and 2 in the AMPA-bound state; the backbone carbonyl of Ser652 hydrogen bonds to the backbone amide of Gly451, and a water-mediated hy-drogen bond connects the backbone carbonyl of Asp651 to the backbone amide of Tyr450 (Hogner et al., 2002). The isopropenyl group of kainate acts as a ‘foot in a door’ col-liding with Tyr450 from domain 1 and with van der Waals contact with Leu650 in domain 2, thus preventing domain 2 from moving to a glutamate-like position (Armstrong and Gouaux, 2003). A correlation between peptide fl ip and relative domain closure has been suggested, as a large de-gree of closure (e.g. 20° for (S)-AMPA relative to the apo structure) has been observed in conjunction with a fl ipped

10

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peptide bond, in contrast to less closed structures (e.g. 12° in kainate).

Agonists (Fig. 7) can be divided into two groups with respect to the peptide conformation, except for glutamate, which induces multiple conformations. The fi rst group con-sists of the three high-affi nity agonists 2-Me-Tet-AMPA, ACPA, and AMPA which all induce full domain closure relative to the apo structure.The second group consists of the lower-affi nity agonists Br-HIBO and KA, as well as the antagonist DNQX, which all stabilise the unfl ipped confor-mation and induce less domain closure. The peptide seg-ment is located just before the two key residues Ser654 and Thr655, which are involved in ligand interactions; how-ever, none of the residues involved in the peptide fl ip is in direct contact with the ligands (Hogner et al., 2002).

Binding antagonists and comparison of ATPO, DNQX

and NS1209 with GluA2-S1S2LBD binding mode

The development of antagonists for glutamate receptor subtypes is a great interest because of their neurortective action of variety of neurodegenerative disorders. Today, structures of eight antagonists presented on Fig. 8 in com-plex with the GluA2 LBD are published. The relative do-main closure made by these antagonists in comparison to non-bounded apo structure is presented in Table 1. It is important to stress that all antagonists prevent the forma-tion of an important D1-D2 interdomain contact between Glu402 and Thr686 (Armstrong and Gouaux, 2000).

The antagonists FQX, CNQX, DNQX and ATPO make small domain closures from 2.5 to 8.1°, whereas (S)-NS1209, UBP282 and ZK200775 lead to 4.3-9.6° of hyper-

AMPA 2-Me-Tet-AMPA ACPA Br-HIBO

Fig. 7. GluA2 agonists. Compound names: AMPA- 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid; 2-Me-Tet-AMPA 2-methyl-tetrazolyl-[2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid]; ACPA-(S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid; Br-HIBO-(S)-2-amino-3-(4-bromo-3-hydroxy-isoxazol-5-yl)propionic acid

Fig. 8. GluA2 antagonists. Compound names: ATPO: 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazlyl]propi-onic acid; CNQX; 6-cyano-7-nitroquinoxaline-2,3-dione; DNQX: 6.7-dinitroquinoxaline-2,3-dione; FQX:[1,2,5]oxadiazolo[3,4-g]quinoxaline-6,7(5H,8H)-dione 1-oxide; NS1209: 8-methyl-5-(4-(N,N-dimethylsulfamoil)phenyl)-6,7,8,9-tetrahydro-1H-pyrrolo[3,2,h]-isoquinoline-2,3-dione-3-O-(4-hydroxybutyrate-2-yl)oxime; UBP277; 3-(2-carboxyethyl)willardine; UBP282: 3-(4-carboxybenzyl) willardine; ZK200775: [1,2,3,4-tetrahydro-7-morpholinyl-2,3-dioxo-6-(trifl uoromethyl)quinoxalin-1-yl]methylphosphonate

Макед. фарм. билт., 57 (1, 2) 3 - 16 (2011)


extension of the LBD in comparison to the apo unbounded structure due to steric hindrance(PǾhlsgaard et al., 2010).

(RS)-NS1209 is a glutamate antagonist which differs in binding and structure from two previously character-ized antagonists ATPO and DNQX. One interesting fi nd-ing for this ligandis that the two protein molecules were observed in the asymmetric unit of the crystal structure of (S)-NS1209. In one molecule, the antagonist (S)-NS1209 is bound within the ligand-binding cleft, whereas the ago-nist (S)-glutamate is bound to the other protomer (Kasper et al., 2006).A similar mixed dimer was published for an-tagonist ZK200775 which is bound in one of the protomers (Sobolevski et al., 2009).

The antagonists UBP277 and UBP282 are derivate of naturally occurring willardine. These two antagonists bind to domain D1 in a similar manner to the agonists, but dif-ferences in binding to D2 residues lead to differences in the position of the uracil ring. UBP277 introduces a domain closure similar to that of (S)-NS1209, whereas the binding of UBP282 produces the largest hyperextension of domains D1 and D2 yet reported for an AMPA receptor (Ahmed et al., 2009b). The carboxyethyl moiety of UBP277 and the carboxybenzyl moiety of UBP282 keep the domains sepa-rated by a “foot-in-the-door”mechanism(Pøhlsgaard et al., 2010).

Quinoxalinedione compounds, CNQX and DNQX are the most commonly used AMPA receptor antagonists. They induce a small domain closure in GluA2 LBD of 6.0-8.1 and 3.4-6.5°, respectively.

In the crystal structure of ATPO and DNQX the li-gands are bonded in the both protomers although some dif-ferences are observed in the bounding conformations. In the Fig. 9 (a and b) and 10 are presented the binding modes of these three antagonists.

Conserved group of residues Pro478, Thr480 and Arg485 from domein 1 and Glu705 from domein 2 make similar interactions with all three antagonists. α-carboxylate and the α-ammonium groups of ATPO overlay well with the two carbonyl groups and one of the amide nitrogens

Table 1. Relative domain closure made by antagonists binding for iGluA2

Antagonist Agonist Domain closure (deg.)a PDB codesb References

ATPO 2.5-5.1 1NOT Hogneret al. (2003)CNQX 6.0-8.1 3B7D Menuz et al. (2007)DNQX 3.4-6.5 1FTL Armstrong and Gouaux (2000)FQX 3.4-5.5 3BKI Cruz et al. (2008)NS1209 Glutamate -5.4 (22.4) 2CMO Kasper et al. (2006)UBP277 -4.3-(-6.4) (B.Gc) 3H03 Ahmed et al.(2009b)UBP282 -5.6- (-9.6) (JLNPd) 3H06 Ahmed et al. (2009b)ZK200775 Glutamate (22.7)e 3KGC Sobolevsky et al. (2009)

a Domain closure was calculated relative to the apo structure od GluA2 LBD (pdb-code 1FTO, molA) using DynDom (Hayward and Berendsen, 1998).b Structures deposited in the Protein Data Bank.c Domain closure could only be calculated for molecules B and G using DynDom. e Domain closure could only be calculated for molecules J, I, N and P using DynDom.f Domain closure could not be calculated for molecule containing the antagonist using DynDom.

(a)

(b) Fig. 9. (b) DNQX -GluA2-S1S2J binding mode. DNQX-

GluA2-S1S2J binding mode

12

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of the quinoxalinedione ring of DNQX. The two carbonyl groups mimic α−carboxyl group common to ATPO and agonists, forming hydrogen bonds to Arg485 and the hy-droxyl and backbone amide of Thr480. A DNQX amide nitrogen and α-ammonium group of ATPO makes a hydro-gen bond to the backbone carbonyl of Pro478.

Fig. 10. (S)-NS1209-GluA2-S1S2J binding mode

As it is shown on Fig.10 the oxime nitrogen as well as the carbonyl oxygen atoms and the nitrogen atom of the pyrole ring of (S)-NS1209 mimic the α-carboxylateand

α−ammonium groups and they make comparable inter-actions with the conserved residues of domain 1 (Kasper et al., 2006).The interaction with domain1 is further sta-bilisedby two additional hydrogen bonds formed be-tween (S)-NS1209 and the amino acid residues Tyr450 and Gly451. These two bonds have not been observed in any other antagonist or agonist complexes solved so far. Water molecules located at positions corresponding to W1 in the (S)-NS1209 structure are observed also in the (S)-ATPOand DNQXstructures. In the latter two cases, this water molecule forms a hydrogen bond to the main-chain nitrogen atomof Tyr450, whereas in the (S)-NS1209 struc-ture, thewater molecule is directly hydrogen bonded to the antagonist (Kasper et al., 2006).

The side-chain conformations of Tyr450, Glu705 and Met708 are different in NS1209 from those that found in the (S)-ATPO and DNQX structures. DNQX is binded near the top of the cleft. The quinoxalinedione ring of DNQX lays~3.6 Å directly below and parallel to the aromatic ring of Tyr450, thus maximizing π–stacking interactions.As a due to the presence of the bulky phenyl sulfonamide sub-stituent in (S)-NS1209, this antagonist is positioned clos-er towards D1 and makes even closer π−π overlap with

Tyr450 than DNQX or (S)-ATPO (Fig.10).The side-chain of Glu705 normally interacts with

the α-amino group of ligands derivedfrom amino ac-ids (Armstrong et al. 1998; Hogner et al. 2003; Jin et al. 2003;). Glu705 adopts different positions in the protomer A and protomer B of DNQX-GluA2 complex. It adopts an extended conformation in protomer B with the γ-carboxyl group directly under the quinoxalinedione rings, while in the protomer A the γ−carboxylof Glu705 is bent slightly away from DNQX. In (S)-NS1209 γ-carboxylate group of Glu705 adopts a different conformation compared to cor-responding atoms in the (S)-ATPO and DNQX structures, and forms an ion pair interaction with N3 of (S)-NS1209. In addition, the Glu705 side-chain is hydrogen bonded to a novel water molecule, W2, whose position corresponds to the OE2 atom of Glu705 in the (S)-ATPO and DNQX structures. The side-chain OE2 atom of Glu705 forms hy-drogen bonds to both the α-ammonium group of ATPO and to the oxygen O4 of the phosphonate group, implying that O4 is protonated.

In GluR2 complexes, Met708 adopts a wide variety of conformations to optimise its van der Waals interactions with the bound ligand. Theside-chain conformation of Met708 is the same in the DNQX and (S)-ATPO structures, pointing away from the ligands, where as it leans towards the ligand in the (S)-NS1209 structure. Met708 is also in van der Waals contact with Thr686. As with (S)-ATPO and DNQX, (S)-NS1209 acts by competitively occupying the binding site, while hindering D1–D2 domain closure and by stabilisingan open form of the ligand-binding core. The phenyl sulfonamide moiety of (S)-NS1209 functions in two ways. Firstly, it takes effect by sterically hindering domain closure and pushing D1 and D2 apart. Secondly, it acts via hydrogen bonding to a residue of D2. O7 of the sulfonamide moiety of (S)-NS1209 is hydrogen bonded to the backbone nitrogen and potentially also to the side-chain oxygen atom of Thr686. (S)-NS1209 (like DNQX, molecule A) interacts directly with a residue of the lock through hydrogen bonding and stabilises the open form by preventing an interdomain interaction between Glu402 in D1 and Thr686 in D2 essential residues for stabilising the activated domain-closed form of the receptor (Armstrong et al., 2000; Jin et al., 2003; Nilsen et al., 2005). The phe-nyl group probably acts as a spacer and serves to increase affi nity by displacing loosely bound water molecules. It is also within van der Waals interaction range of residues of both D1and D2.

The isoxazole ring of ATPO forms only a single, in-direct interaction with the protein,from the nitrogen atom via a water molecule W2 to Glu705 and it plays a role of a spacer in the binding pocket (Hogner at al., 2003).

The bulky tert-butyl group at the 5-position of the isoxazole ring in ATPO is partly buried in a pocket formed by residues Glu402, Tyr405 and Tyr450 of domain 1 and Glu705, Thr707, Met708 and Tyr732 of domain 2. The 6-nitrogroup of DNQX interacts with Tyr-732 and a pro-

Макед. фарм. билт., 57 (1, 2) 3 - 16 (2011)


tein-bound water molecule, and the 7-nitro moiety is hy-drogen bondedto the hydroxyl of Thr-686(2.95 Å) (Fig. 9 a and b).

The phosphonate group of ATPO is involved in an-extensive hydrogen bonding network, interacting directly with Ser654, Thr655, and Glu705, and forming indirect contacts with the protein through six water molecules The side-chain of Glu705 forms hydrogen bond to the oxygen O4 of the phosphonate group. (Hogner et al., 2003). The phosphonate group of ATPO appears approximately at the same position as the sulfate ion in the S1S2J:DNQX-B (Armstrongand Gouaux, 2000) and GluR2-S1S2J:(S)-NS1209 (Kasper et al., 2006) complexes. This indicates that the protein environment in this region is favorable for accommodating a large negatively charged group. Also a water molecule is located between DNQXand the bound sulfate in the crystal structure. The sulfate interacts with helix F via 3 hydrogen bonds to the protein and 3 hydrogen bonds to solvent molecules, mimicking the interactions that the anionic “R” groups of agonists make with the base of helix F. The hydroxy group of (S)-NS1209 makes a hy-drogen bond to this sulphate ion, which again forms hydro-gen bonds to Ser654 and Thr655. Unlike (S)-ATPO, (S)-NS1209 makes no direct interactions with Thr655 (Kasper et al., 2006).

Interestingly, three water molecules in the (S)-ATPO structure correspond to hydrogen-bonding atoms (O1, O3, O4) of (S)-NS1209 (although O4 is only a weak acceptor).These oxygen atoms are all from the 4-hydroxybutyratemoiety of (S)-NS1209, which reaches out into the opening of the ligand-binding cleft. This indicates that these are favoured positions for a hydrogen-bonding donor or acceptor, and that (S)-NS1209 can replace water molecules at thesepositions.

(S)-NS1209 occupies a new site IX where the phenyl sulfonamide moiety is embedded, and this site is bounded by residues Arg684, Thr685 and Thr686. This part of the molecule is positioned in an area of the cleft that has been unoccupied in all previously solved complex structures (agonists and antagonists).

Antagonists prevent further domain closure by differ-ent mechanisms. Even though ATPO and DNQX occupy different volumes of the binding site, both ligands fulfi ll the criteria for an antagonist. ATPO stabilizes the open cleft via direct interactions between the phosphonate group and residues in the base of the helix F much like the sulfate anion in the GluR2-DNQX complex in domain 2. At the same time, the tert-butyl group is accommodated within the pocket formed by residues in domain 1 and domain 2. Thus, further domain closure is prevented by steric inter-ference between the receptor and the tert-butyl group on-one hand, and between the phosphonate moiety and do-main 2 on the other.

By contrast, the 6-nitro groupof DNQX prevents an interdomain interaction between Glu402 in domain 1 and Thr686 in domain 2 and forms a long hydrogen bond (3.5

Å) to Thr686. In the closed agonist induced conformation-of S1S2J, these two residues interact strongly (Hogner et al., 2002). By occupying the ligand binding cleft near the axis of rotation that describes the relative movement of do-mains 1 and 2 in the apo to AMPA transition, DNQX stabi-lized the open cleft conformation via antagonist receptor-interactions.

(S)-NS1209 is the only competitive antagonist that stabilized even more open conformation of D1 and D2 domains of the ligand binding core than that of the apo structure due to a steric hindrance. Sulfonamide moiety of (S)-NS1209 is hydrogen bonded to the backbone nitro-gen and potentially also to theside-chain oxigen atom of Thr686. (S)-NS1209, like DNQX, interacts directly with a residue of the lock through hydrogen bonding and stabilis-es the open form by preventingan inter domain interaction between Glu402 in D1and Thr686.

Conclusion

iGluRs are ligand-gated ion channels that play sub-stantial role in development and function of the nervous system. Their involvement in numerous neudegenerative diseases, as well as in the process of learning and memory, has aroused widespread interest in their function and struc-ture. Since the fi rst publication of the crystal structure of a soluble construct of the GluA2-LBD, extensive studies were performed on GluA2 and the other iGluR subunits. Recently, a crystal structure of full-length GluA2 was de-termined, and it was proved that the soluble GluA2 LBD is a model system of the full-length receptor, as the dimeric unit is very similar. Structural studies on wild type and mu-tant GluA LBD construct in complex with agonists, par-tial agonists, antagonists, combined with functional data provide detailed understanding of these group of receptors, their activation, desensitation, inhibition and subunit selec-tivity. In this review we give one global picture of nowa-days fi ndings made on these receptors which are suitable base for future research as well as achievement in design of new selective drugs used for treatment of numerous neu-rodegenerative diseases.

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Резиме

Јонотропно глутамински (iGluRs): Преглед на iGluR2

лиганд врзувачкиот домен во комплекс со агонисти и

антагонисти

Зорица Серафимоска1*, Томи Н. Јохансен2, Карла Фриденванг2,

Љубица Шутуркова1

1 Институт за фармацевтска хемија, Фармацевтски факултет, Универзитет “Св. Кирил и Методиј” Скопје,

Македонија2Институт за медицинска хемија, Факултет за фармацевтски науки, Универзитет Копенхаген, Данска

Клучни зборови: Јонотропни глутамински рецептори (iGluRs), AMPA(iGluA1-4), невролошки пореметувања, невродегенеративни

заболувања, шизофренија, Алцхајмерова болест, епилепсија.

Јонотропните глутамински рецептори (iGluRs) претставуваат фамилија на лиганд врзувачки јонски канали поделени во три

класи, NMDA, AMPA(iGluA1-4) и KA (1-5). Оваа поделба е направена во зависност од агонистот кој врши нивна селективна акти-

вација. iGluRs се тетрамерни групации од високо хомологни рецепторни подединици. Овие рецептори се од круцијално значење

за нормалната функција на мозокот и се смета дека тие се вклучени во бројни невролошки пореметувања и невродегенеративни

заболувања како шизофренија, Алцхајмерова болест, епилепсија и оштетувања на мозокот кои настануваат после мозочен удар.

По објавувањето на структурата на првиот рекомбинантен растворлив протеин на лиганд врзувачкиот домен од GluA2 рецеп-

торот спроведени се бројни студии врз оваа група на рецептори и добиени се многу кристални структури од комплексите на

GluA2-LBD со агонисти, парцијални агонисти и антагонисти. Податоците добиени од структурните анализи во комбинација со

функционалните податоци даваат добра основа за понатамошно испитување и дизајнирање на нови селективни лекови кои ќе

бидат користени во третманот на невродегенеративните заболувања.


UDC: 615.216.5.074:543.544.5 Original scientifi c paper

1. Introduction

Rocuronium bromide (Roc) is widely used as amin-osteroidal non-depolarizing neuromuscular blocking agent with rapid onset of action and intermediate duration of the blocking effect. Since 1994 it is avaible as a solution dos-age form intended for intravenous or intramuscular injec-tion (Blazewich et al., 2007; Gao et al., 2001). Rocuronium bromide is a polar drug with octanol/water partition co-effi cient of 0.5 (Moffat et al., 2004). This compound has eight identifi ed impurities, labeled from A to H. Impurity A (N-desallylrocuronium) and impurity C (17-desacetylro-curonium) (Fig. 1) are not only main metabolite products,

Determination of Rocuronium bromide by hydrophilic

interaction liquid chromatography (HILIC)

Natalija Nakov1*, Rumenka Petkovska1, Liljana Ugrinova2, Suzana Trajkovic-Jolevska1, Aneta Dimitrovska1

1Institute of Applied Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy, University “Ss Cyril and Methodius”,

Vodnjanska 17, Skopje, Macedonia2Center of drug quality control, Faculty of Pharmacy, University “Ss Cyril and Methodius”, Vodnjanska 17, Skopje,

Macedonia

Received: November 2011; Accepted: January 2012

Abstract

A new method involving hydrophilic interaction liquid chromatography (HILIC) has been developed for determination of rocuronium bromide in presents of its main impurities (impurity A and impurity C), which are also its main degradation products, in solution for injec-tion. The infl uence of the critical chromatographic parameters such as content of acetonitrile in the mobile phase, ionic strength and pH val-ue of the buffer used in the mobile phase were investigated using the Design of experiments approach (DoE). The mechanism of retention of rocuronium bromide on bare silica column was also investigated. Optimal chromatographic conditions were obtained using mixture of acetonitrile and ammonium formate (107.5mM, pH 7.0) in ratio 90:10 as a mobile phase. The validation results have shown that the meth-od is suitable for determination of rocuronium bromid in solution for injection.

Keywords: rocuronium bromide, HILIC, Design of experiments, retention mechanism

* tel: +389 23126032 ext.123; fax: +389 [email protected]

but they are also main degradation products of rocuronium (Blazewich et al., 2007).

The analytical techniques for determination of rocuro-nium bromide and its main metabolites in biological sam-ples presented in the literature included liquid chromatog-raphy-mass spectrometry (Cirimele et al., 2003; Gutteck-Amstel and Rentsch, 2000; Farenc et al., 2001; Fuchs-Buder et al., 2004; Sayer et al., 2004) and gas chromatog-raphy with mass spectrometry (Gao et al., 2001). There is only one published method for determination of rocuro-nium and its impurities in pharmaceutical preparation, which includes liquid chromatography with electrochem-ical detection (Blazewich et al., 2007). Ph.Eur 7.0 recom-mends determination of assay of rocuronium bromide us-ing titration and its related substances by ion-pair reverse-phase liquid chromatography (European Pharmacopeia 7.0, 2010). USP-32 recommends determination of rocuro-nium bromide substance and its related compounds by re-

18

Maced. pharm. bull., 57 (1, 2) 17 - 24 (2011)

Natalija Nakov, Rumenka Petkovska, Liljana Ugrinova, Suzana Trajkovic-Jolevska, Aneta Dimitrovska

verse-phase liquid chromatography with ion-pair mobile phase (United States Pharmacopeia 32, 2009). Generally, ion-pair chromatography methods have number of disad-vantages such as long equilibration time (in this case 4 hours), disturbance of equilibration by injection and ire-versed change of the stationary phase caused by the use of ion-pair reagent (Dejaegher and Vander Heyden, 2010; Hemstrom and Irgum, 2006; Snyder et al., 2010).

Hydrophilic Interaction Liquid Chromatography (HILIC) provides an alternative approach for effectively separation of polar compounds on polar stationary phases. This technique employs polar stationary phase (most often bare silica) with mobile phase containing small amount of water phase (at least 3%) and large amount (60-97%) of po-lar organic solvent (Dejaegher and Vander Heyden, 2010; Hemstrom and Irgum, 2006; McCalley, 2007, 2010a).

The aim of this work was to develop a HILIC method for quantitative determination of rocuronium bromide in the presents of its main impurities (impurity A and impu-rity C) using the Design of Experiments approach (DoE). Generally it is accepted that the use of DoE affords the most convenient way to deal with optimization of methods since the traditional step-by-step approach involves a large number of independent runs.

The retention mechanism in HILIC is complex and partition mechanism may occur together with adsorption, ion exchange and even hydrophobic interaction under ap-propriate experimental conditions. In order to gain more insight into the HILIC mechanism, assessment was made to investigate the relative contribution of adsorption, par-tition and ion-exchange mechanism to the retention of ro-curonium bromide on bare silica column.

2. Experimental

2.1. Chromatographic conditions

Separation was performed on Agilent Rapid Resolution HPLC System 1200 Series, using Purospher STAR Si (150 x 4,6 mm, 5 µm partical size) with a mixture of ammoni-um formate (107.5 mM, pH 7.0) and acetonitrile in ratio 90:10 (v/v, %), with fl ow rate 2.0 ml/min at 30 °C. The in-

jection volume was 10 µl and detection was carried out at

210 nm.

2.2. Design of Experiments

Central Composite Face Centered (CCF) Design was

used for method optimization. The MODE 8.0 software

was used for generation and evaluation of the experimen-

tal design.

2.3 Reference substances, reagents and chemicals

Rocuronium bromide working standard was obtained

from Hameln rds. Modra, Slovakia. Ammonium formate

was supplied from Fluka, Switzerland; formic acid and

acetonitrile (HPLC grade) by Merck, Germany, ammoni-

um hydroxide solution (c.c. 25% NH3) by Sigma-Aldrich,

Germany. The reagents were analytical grade and water of

HPLC grade was used.

2.4 Standard and sample solution preparation

Rocuronium bromide standard solution in concentra-

tion of 1mg/ml was prepared. Test solution was prepared

from Rocuronium bromide Kabi 10 mg/ml solution for

injections in concentration of 1 mg/ml. Mixture of ace-

tonitrile and water in ratio 90:10 (v/v, %) was used as a

solvent for standard and test solution. Standard solution of

Roc in concentration 0.5 mg/ml was degradated for 1 hour

in oven at 105 °C in order to obtain impurity A and impu-

rity C.

Five standard solutions were prepared for the calibra-

tion curve in the range of 0.5 to1.5 mg/ml (from 50-150%

of the working concentration). Another fi ve standard so-

lutions were prepared in concentration from 0.015 to 0.05

mg/ml (from 1% to 5% of the working concentration) for

determination of the limit of detection and limit of quan-

tifi cation of the method. The determination of accuracy of

the method was done by method of standard additions, by

adding known amounts of rocuronium bromide standard

to the sample placebo at three concentrations (80%, 100%

and 120%).

a)

b)

c)

Fig. 1. Structure of: a) Rocuronium bromide, b) Impurity

A (R=CO-CH3), c) Impurity C (R=R’=H)

Макед. фарм. билт., 57 (1, 2) 17 - 24 (2011)

19 Determination of Rocuronium bromide by hydrophilic interaction liquid chromatography (HILIC)

3. Results and discussion

3.1 Optimization of chromatographic conditions

The physico-chemical properties of rocuronium bro-mide and the previous knowledge about the critical chro-matographic parameters in HILIC, were taking into con-sideration during the method development. The exper-imental design approach was chosen over the systematic approach, because it allowed simultaneous study of mul-tiple variables (e.g. acetonitrile content, salt concentration etc.) which led to signifi cant decrease in the number of ex-periments and also allowed to evaluate the factor interac-tions (Deming and Morgan, 1993; Dejaegher et al., 2008; Guo et al., 2007; Lewis et al., 1999).

The results obtained during the preliminary investiga-tion, had shown that Roc and impurity C are co eluting, probably due to the similarity of their structures. Impurity A was well separated from the rocuronium and it eluted af-ter the dead volume of the column. In order to obtain opti-mal resolution (Rs > 1.5) between rocuronium and impu-rity C for short retention time, CCF design was chosen for the method development. The method development includ-ed investigation of the effect of three chromatographic pa-rameters, at three different levels (Table 1).

The system response was estimated by the retention time of Roc and resolution between Roc and impurity C. Multiple linear regressions were used to estimate the coef-fi cients of the model, representing the relationship between the response variables measured and the chromatograph-ic factors studied. The regression coeffi cient plots (Fig. 2a and 2b) consist of bars that correspond to the regression co-effi cients with the magnitude of the effects proportional to the regression coeffi cients.

The plot obtained for retention time (Fig. 2a), indi-cated that the content of acetonitrile had the largest infl u-ence. Increasing the percentage of acetonitrile in the mo-bile phase has increased the retention time of rocuronium. This behavior was expected, because increasing the con-tent of acetonitrile reduces the polarity of the mobile phase and therefore resulting in longer retention time. The regres-sion coeffi cients have shown that higher concentration of the buffer and higher values for pH value of the buffer, re-duced the retention time of Roc. Larger concentration of counter ion (ammonium ion) in the buffer reduces the elec-trostatic interaction between the analyte and the surface si-lanol groups on silica column by competing with the an-alyte for these sites, resulting in reduced retention time. In order to defi ne the optimum range of the investigated factors for retention time as chromatographic system re-sponse, a contour diagram of the retention time of Roc as a function of the content of acetonitrile in the mobile phase and ionic strength of the buffer was constructed (Fig. 3).

The contour diagram of retention time of Roc showed that increasing the content of acetonitrile in the mobile phase and reducing the pH value of the buffer, leaded to longer retention of rocuronium. The shortest retention time of rocuronium bromide was obtained at pH 7.0 of the am-monium formate.

The infl uence of the content of acetonitrile in the mo-bile phase and pH value of the buffer on the resolution (Rs) between Roc and impurity C, as a second chromatographic response (Fig. 2b), was the same as for the retention time. The ionic strength of the buffer on this chromatographic re-sponse had opposite infl uence that on the retention time, so bigger values for resolution between the two peaks may be obtained by increasing the concentration of the buffer.

Table 1. Chromatographic parameters and its experimental range for method development

ParametersFactor levels

(-) (0) (+)

x1 content of acetonitrile in the mobile phase (%)

x2 ionic strength of the buffer (mM)

x3 pH of the buffer

70954.0

80107.55.5

901207.0

a b

Fig. 2. Regression coeffi cient plot: a) retention time of Roc, b) resolution between Roc and impurity C, (%ACN: ace-

tonitrile content in the mobile phase, pH: pH value of the buffer, ionic st.: ionic strength of the buffer)

20

Maced. pharm. bull., 57 (1, 2) 17 - 24 (2011)


The results obtained using Response Surface Methodology (Fig. 4) shown that resolution between Roc and impurity C was better at pH 4.0 than at pH 7.0. The op-timal resolution at pH 4.0 may be obtained if the content of acetonitrile in the mobile phase is larger than 87 %. The optimal resolution at pH 7.0 may be obtained if the con-tent of acetonitrile in the mobile phase is larger than 89 %, pointing that optimal value for resolution may be ob-tained for close values for the content of acetonitrile. The

pH value 7.0 was chosen as optimal considering the fact that the retention time of Roc at pH 4.0 was signifi cantly longer than at pH 7.0 (15.9 min compared to 7.9 min, re-spectively).

The optimal chromatographic condition, which corre-sponds to high values of Rs and short retention time, were obtained using mobile phase composition of acetonitrile and ammonium formate (107.5mM, pH 7.0) in ratio 90:10 v/v (Fig. 5).

a b c Fig. 3. A contour diagram of retention time of Roc at: a) pH = 4.0, b) pH = 5.5, c) pH = 7.0

a b c Fig. 4. A contour diagram for resolution between Roc and impurity C at: a) pH=4.0, b) pH=5.5, c) pH=7.0

Fig. 5. Chromatogram of standard solution of Roc degradated for 1 hour at 105 °C

Макед. фарм. билт., 57 (1, 2) 17 - 24 (2011)


a)

b)

Fig. 6 Plot of log k’ versus: a) volume fraction of water (partition plot), b) log mole fraction of water (ad-sorption plot)

Table 2. Values for the intercept, slope and correlation coef-fi cient obtained from partition and adsorption plot

Partition plot Adsorption plot

abR2

1.3683-0.03360.9994

3.5682-1.81270.9558

Table 3. Contribution of ion exchange (%) to retention of

rocuronium bromide

[NH4

+] (mM)Contribution of ion

exchange (%)

10 9120 7750 71100 56

3.2 Determination of contribution of partition, adsorption

and ion exchange mechanism to the retention of rocuroni-

um bromide on bare silica column

In HILIC, the retention of the analyte is mediated by

partitioning of analyte between a surface water layer and

the bulk mobile phase, adsorption via processes such as

hydrogen bonding and other dipole/dipole interactions and(or) ion-exchange interactions between ionized si-lanol groups and ionized analyte. Plotting the logarithm of the retention factor (k’) versus volume fraction of the wa-ter phase (partition plot) or logarithm of mole fraction of the water phase (adsorption plot), should give an indica-tion whether partitioning or adsorption is the dominant re-tention mechanism (Hemstrom and Irgum, 2006; Jandera, 2011; Karatapanis et al., 2011; Liu et al., 2008; Mccalley, 2010b). In this study, the percentage of the water phase (ammonium formate 20 mM, pH 7.0) varied in the range from 10 to 30 %, and the obtained adsorption and par-

tition plot were compared (Fig. 6a and 6b). The correla-

tion coeffi cient obtained for the partition plot was higher

than the correlation coeffi cient obtained for the adsorption

plot (Table 2), pointing that partition is dominant retention

mechanism for rocuronium.

Further information regarding the degree of electro-

static interaction involved in the retention can be obtained

through estimation of the contribution of ion-exchange

mechanism to the retention (Cox and Stout, 1987; Yang,

2003; Liu, 2009; McCalley, 2010b). In order to determi-

nate the contribution of ion-exchange mechanism to re-

tention, different concentrations (10 mM, 20 mM, 50 mM

and 100 mM) of ammonium formate (pH 7.0) in mixture

with acetonitrile (20:80, respectively) were used as mobile

phase. Data obtained from regression analysis of the plot

k’ vs. reciprocal values of concentration of ammonium for-

mate in the buffer (1 / [NH4

+]) were used for calculation of the percent contribution of the ion exchange to retention of the rocuronium (Table 3).

The obtained results have shown that the ion-exchange mechanism has infl uence on the retention on rocuronium bromide and its contribution was bigger at lower counter-ion concentration (91% contribution of ion exchange at 10

mM concentration of the buffer, compared to 56% at 100

mM).

3.3 Validation of the method

The method was validated according to the require-

ments of ICH (International Conference on Harmonization,

2005).

No interferences were observed due to the presents of

the main impurities (impurity A and impurity C) and the

placebo components (sodium chloride and sodium ace-

tate), so the method selectivity was confi rmed (Fig. 5, Fig.

7 and Fig. 8). The validation results (Table 4 and Table 5)

indicated that the method is linear, accurate, sensitive and

precise.

The CCF experimental design was used for robustness

testing of the method. Results obtained during the meth-

od optimization, indicated that optimal resolution may be

obtain with in investigated range of ionic strength (95-

120 mM) and pH value of the buffer, if the content of ac-

etonitrile in the mobile phase is above 88%. Therefore the

infl uence of content of acetonitrile in the mobile phase and

ionic strength of the buffer on the resolution between Roc

and impurity C was investigated (Table 6).

22

Maced. pharm. bull., 57 (1, 2) 17 - 24 (2011)


Table 4. Results obtained from testing different parameters during validation of the analytical method

Validation parameter Rocuronium bromide

Linearity Concentration range (mg/ml) Correlation coeffi cient Intercept Slope

0.5 - 1.5 0.015 - 0.0500.9998 0.999571.209 17.5861030.3 2032.7

LODConcentration (µg/ml) 1LOQ Concentration (µg/ml) 3

Precision (RSD %, n=6) 1.05

Table 5. Results obtained from testing of the accuracy of the method

Working concentration (%)

Added (mg/ml)

Determinated (mg/ml)

RSD (%)

Accuracy(P=95%)

80 0.79 0.783 0.58 99.10 ± 1.4100 1.03 1.024 0.52 99.47 ± 1.27120 1.18 1.198 0.26 101.57 ± 0.66

Table 6. Chromatographic parameters (factors) and its experimental range during robustness testing

ParametersFactor levels

(-) (0) (+)

x1x2

Content of acetonitrile (%)Ionic strength of the buffer (mM)

8895

90107.5

92120

Fig. 7. Chromatogram of placebo solution Fig. 8. Chromatogram of rocuronium bromide 10 mg/ml solution for injection

The results had shown that small but deliberate changes in the chromatographic conditions (89-91% ac-etonitrile and ionic strength of the buffer in the range of

105 - 108 mM) did not affect the resolution between ro-curonium bromid and impurity C, so the method can be applied in routine work (Fig. 9).

Макед. фарм. билт., 57 (1, 2) 17 - 24 (2011)


4. Conclusion

The proposed HILIC method enables a simple, rapid, accurate and selective quantitative determination of rocuro-nium bromide in solution for injection. The approach for optimization of the method using DoE represents an easily accomplishable approach for determination of the optimal HILIC condititions. The results obtained using the CCF de-sign, confi rmed that the content of the organic solvent and the ionic strength of the buffer are signifi cant factors that affect the retention and separation in HILIC. Investigation of the retention mechanism has shown that the partition prevails over the adsorption as retention mechanism and the ion-exchange mechanism also has considerable contri-bution to the retention of rocuronium bromide.

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Резиме

Определување на рокурониум бромид со примена на течна

хроматографија со хидрофилна интеракција (HILIC)

Наталија Наков1*, Руменка Петковска1, Лилјана Угринова2,

Сузана Трајковиќ-Јолевска1, Анета Димитровска1

1 Институт за примената хемија и фармацевтски анализи, Фармацевтски Факултет, Универзитет “Св.Кирил и

Методиј”, Водњанска 17, Скопје, Македонија2 Центар за контрола на лекови, Фармацевтски Факултет, Универзитет “Св.Кирил и Методиј”, Водњанска 17,

Скопје, Македонија

Клучни зборови: рокурониум бромид, HILIC, дизајнирање на експерименти, механизам на ретенција

Развиен е нов метод за определување на рокурониум бромид во присуство на неговите главни онечистувања (онечистување

А и онечистување С) кои воедно се и негови главни деградациони продукти во раствор за инјектирање со примена на течна

хроматографија со хидрофилна интеракција (HILIC). Испитано е влијанието на клучните хроматографски параметри како што

се удел на ацетонитрил во мобилната фаза, јонска јачина и pH вредност на пуферот кој влегува во состав на мобилната фаза

со примена на дизајнирање на експерименти. Исто така направено е испитување на механизмот на ретенција на рокурониум

бромид на необработена силика како стационарна фаза. Оптимални хроматографски услови се постигнати со примена на смеса на

ацетонитрил и амониум формат (107,5 mM, pH 7.0) во однос 90:10 како мобилна фаза. Резултатите од валидацијата покажаа дека

методот е соодветен за определување на рокурониум бромид во раствор за инјектирање.


UDC: 582.457–113.55:615.281(497.7)Original scientifi c paper

Introduction

Representatives of genus Pinus in fl ora of R. Mace-

donia include fi ve different species: Pinus nigra Arnold,

Pinus sylvestris L., Pinus mugo Turra, Pinus heldreichii

Christ. subsp. leucodermis (Antoine) Blecic and Pinus

peuce Grisebach (Micevski, 1985). Most of the Pinus spe-

cies are trees or shrubs with specifi c morphological char-

acteristics of leaves (needles) rich in strong and terpene-

aromatic essential oil. Pine needle essential oils are main-

ly used in folk medicine for the treatment of respiratory in-

Antimicrobial activity of needle essential oil of Pinus peuce

Griseb. (Pinaceae) from Macedonian fl ora

Marija Karapandzova1*, Gjose Stefkov1, Elena Trajkovska-Dokic2, Ana Kaftandzieva2, Svetlana Kulevanova1

1Institute of Pharmacognosy, Faculty of Pharmacy, University “Ss. Cyril and Methodius”, Skopje, Macedonia2Institute of Microbiology, Faculty of Medicine, University “Ss. Cyril and Methodius”, Skopje, Macedonia

Received: January 2012; Accepted: March 2012

Abstract

Chemical composition and antimicrobial activity of needle essential oil, obtained by hydrodistillation from wild Pinus peuce Griseb. (Pinaceae), growing on three different locations in R. Macedonia were investigated in period 2008/2009. Carried out GC/FID/MS analy-sis, one hundred and three constituents were identifi ed belonging to the six different classes of components: monoterpene hydrocarbons,

oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, diterpenes and other non-terpene components, rep-

resenting 88.61/94.04% of the entire oil. The most abundant constituents were α-pinene (12.89/27.34%), β-pinene (6.16/13.13%), li-

monene + β-phellandrene (2.09/6.64%) and bornyl acetate (2.92/11.67%) as well as trans-(E)-caryophyllene (4.63/7.13%) and germac-rene D (8.75/20.14%).

Antimicrobial screening of Pinus peuce needle essential oil was made by hole-plate diffusion and broth dilution method against 13 bacterial isolates of Gram positive and Gram negative bacteria and one strain of Candida albicans. The most sensitive bacteria against test-ed Pinus peuce essential oils were Streptococcus pneumonia encompassing Staphylococcus aureus, Staphylococcus epidermidis, Strepto-

coccus agalactiae, Acinetobacter spp. and Streptococcus pyogenes. Minimal inhibitory concentrations (MICs) of the oils ranged from 7.5 - 62.5 µl/ml.

Key words: Pinus peuce, Macedonian pine, essential oil composition, GC/FID/MS, antimicrobial activity.

* [email protected]; [email protected]

fections accompanied by cough, bronchitis, bronchial asth-ma, emphysema, tracheitis, sinusitis, laryngitis, pharyngi-tis, tonsillitis and infl uenza (Dervendzi, 1992). Up to now,

there has been an increased interest in studying chemical

composition as well as biological activity of the essential

oils isolated from different pine species. Generally, monot-

erpenes and sesquiterpenes like α-pinene and β-pinene,

camphene, ∆3-carene, β-myrcene, limonene, phellan-

drene, β-ocymene, β-caryophyllene, bornyl acetate, ger-

macrene D, cadinene and muurolene are mentioned as

dominant components of pine needle essential oils (Rous-

sis, 1994; Ucar, 2004; Tognolini, 2006; Naydenov, 2006,

Idzojtic, 2005; Grassmann, 2003; Grassmann, 2005; Ste-

vanovic, 2005; Ustun, 2006; Maciag, 2007; Judzentiene,

2006; Kupcinskiene, 2008; Semiz, 2007; Kainulainen,

26

Maced. pharm. bull., 57 (1, 2) 25 - 36 (2011)

Marija Karapandzova, Gjose Stefkov, Elena Trajkovska-Dokic, Ana Kaftandzieva, Svetlana Kulevanova

2002, Holzke, 2006; Dob, 2007; Menkovic, 1993; Nikol-ic, 2007; Dob, 2006; Pagula, 2006; Dormont, 1998; Yong-Suk, 2005; Oluwadayo, 2008; Sacchetti, 2005; Barnola, 2000). Some of these oils like essential oil isolated from Pinus caribaea Morelet, Pinus densifl ora S. and Z., and Pinus radiate D. Don have showed different antimicrobi-al activity against different microorganisms (Oluwadayo, 2008; Yong-Suk, 2005; Sacchetti, 2005). The antimicrobi-al activity of pine essential oils is of multipurpose thus they are used in manufacture of medicinal products and cosmet-ics as antimicrobial additives, etc. Nowadays, there are few registered pharmaceuticals such as Pinimenthol®, an oint-ment which contains pine needle essential oil and is partic-ularly suitable for the treatment for upper respiratory tract infections both in adolescents and adults (Kamin, 2007).

Pinus peuce, known as “Molica” or “Macedonian pine” is an endemic species of Balkan Peninsula mostly spread in southern and western parts of R. Macedonia (Micevski, 1985), northern part of Greece and south-eastern parts of Bulgaria, as well as in parts of Albania, Serbia and Mon-tenegro. Up to present, there are a lack of data that are re-lated to the essential oil composition of Pinus peuce pop-ulation from Macedonian fl ora and its antimicrobial activ-

ity, thus the aim to this study was to investigate the chem-

ical composition of the needle essential oil of Pinus peuce

from Macedonian fl ora and to assess its antimicrobial ac-

tivity against certain types of microorganisms that affect

respiratory, gastrointestinal and urogenital system and pro-

voke pathological conditions on skin.

Material and methods

Plant material

Plant material was collected from four different local-

ities in R. Macedonia: Baba Mtn. (Pelister), Nidze Mtn.,

Shara Mtn. and Karadzica Mtn. in July, 2008 and 2009, and

was dried at room temperature and on draft for two weeks.

Just before hydrodistillation, the needles were separated

from the branches and were properly minced.

Determination of water content

Determination of water content was made by distilla-

tion according to Ph.Eur.7 regulations (2.2.13.) from 20 g

dried plant material.

Essential oil isolation

Essential oil isolation was made by hydrodestillation

in all-glass Clevenger apparatus (Ph.Eur 7, 2.8.12.). For

that purpose, 20 g of minced needles were distilled for 4

hours. For purifi cation purpose, anhydrous sodium sulfate

was added to the isolated essential oil to remove residual

water. For GC/FID/MS analysis, the essential oil was dis-

solved in xylene (Alkaloid, R. Macedonia) to obtain 1 µl/

ml oil solution.

Gas chromatography

Essential oil samples were analyzed on Agilent 7890А Gas Chromatography system equipped with FID detec-tor and Agilent 5975C Mass Quadrupole detector as well as capillary fl ow technology which enables simultaneous

analysis of the samples on both detectors. For that purpose,

HP-5ms capillary column (30 m x 0.25 mm, fi lm thickness

0.25 µm) was used. Operating conditions were as follows:

oven temperature at 60 °C (5 min), 1 °C/min to 80 °C (2

min) and 5 °C/min to 280 °C (5 min); helium as carrier gas

at a fl ow rate of 1ml/min; injector temperature 260 °C and

that of the FID detector 270 °C. 1µl of each sample was in-

jected at split ratio 1:1.

The mass spectrometry conditions were: ionization

voltage 70 eV, ion source temperature 230 °C, transfer line

temperature 280 °C and mass range from 50 - 500 Da. The

MS was operated in scan mode.

Identifi cation of the components present in essential

oils was made by comparing mass spectra of components

in essential oils with those from Nist, Wiley and Adams

mass spectra libraries, by AMDIS (Automated Mass Spec-

tral Deconvolution and Identifi cation System) and by com-

paring literature and estimated Kovat′s (retention) indices

that were determined using mixture of homologous series

of normal alkanes from C9 to C

25 in hexane, under the same

above mentioned conditions.

The percentage ratio of essential oils components was

computed by the normalization method of the GC/FID

peak areas and average values were taken into further con-

sideration (n=3).

Antimicrobial screening

Antimicrobial activity of essential oils was studied

against 14 different microorganisms, including 13 bacterial

isolates representing both Gram-positive (Staphylococcus

aureus ATCC 29213, Stahylococcus epidermidis, Strepto-

coccus pneumoniae, Streptococcus agalactiae, Streptococ-

cus pyogenes and Enterococcus) and Gram-negative bacte-

ria (Acinetobacter spp, Escherichia coli ATCC 25927, Sal-

monella enteritidis, Klebsiella pneumoniae ATCC 700603,

Pseudomonas aeruginosa ATCC 27853, Haemophylus in-

fl uenzae and Proteus mirabilis) and one strain of Candida

albicans ATCC 10231.

Hole-plate diffusion method was used for screening

the antimicrobial activity of all essential oils (determina-

tion of growth inhibition zones of studied microorganisms

that occur around certain essential oil). This investigation

was followed by broth dilution method (determination of

minimal inhibitory concentration MIC of the particular oil

that had revealed good antimicrobial activity by hole-plate

diffusion method).

Макед. фарм. билт., 57 (1, 2) 25 - 36 (2011)

27 Antimicrobial activity of needle essential oil of Pinus peuce Griseb. (Pinaceae) from Macedonian fl ora

Hole-plate diffusion method

A nutrient agar (Mueller Hinton) was prepared by dis-solving agar (28 g) in distilled water (1000 ml). The mix-ture was heated to dissolve and autoclaved at 121 °C for 15 minutes. The nutrient agar was poured into sterile Petri dishes at uniform depth of 3 mm and allowed to solidify. Blood agar (Oxoid) was used for testing the antimicrobi-al activity against Streptococcus pyogenes, Streptococcus

agalactiae, Streptococcus pneumoniae and Enterococcus, while Sabouraund agar (bioMerieux) was used for testing the antimicrobial activity against Candida albicans. Mi-croorganisms were suspended in sterile broth with turbidi-ty corresponding to 0.5 and 1 Mc Farland (approximate by 107-108 CFU/ml) for all bacteria and for Candida albicans, respectively. The microbial suspensions were streaked over the surface of the agar media using a sterile cotton swabs to ensure uniform inoculation. After inoculation of micro-organisms, holes of 6 mm in diameter were made at well-spaced intervals. They were fi lled with 85 µl of 50% solu-tions of essential oils in dimethylsulfoxide (DMSO, Sig-ma-Aldrich, Germany) and one hole was fi lled only with DMSO as a control. The plates were incubated at 37 °C, aerobically for 24 hours. The growth inhibition zones were measured after incubation of the isolates under their opti-mal growth conditions and were ranged between 6 mm and 30 mm in diameter. The antimicrobial activity was deter-mined according to the diameters of the inhibition zones. If the diameter of the growth inhibition zones was in the range between 0 and 14 mm, those species were accepted like resistant; if the diameter was between 14 and 19 mm, the species were accepted like moderate susceptible and in the cases where the diameter was above 19 mm, species were accepted like susceptible.

Broth dilution method

This method was used for some particular essential oil (50% solution in DMSO) that had revealed good antimi-crobial activity by hole-plate diffusion method. For that purposes, 25 µl of those essential oils were diluted in equal quantities of 0.9% sodium chloride solution, to make them with the concentration of 25%. This concentration was de-creased fi ve times, subsequently, by adding 25 µl of each bacterial or fungal suspension, thus the fi nal concentrations were: 12.5%, 6.25%, 3.125%, 1.562% and 0.75% or 125 µl/ml, 62.5 µl/ml, 31.25 µl/ml, 15.26 µl/ml and 7.5 µl/ml, respectively.15 µl of each bacterial or fungal suspensions with these particular concentrations were inoculated on solid media (Miller-Hinton agar, blood agar, Sabouraund agar), depending on the microorganism. The growth of any microorganism was evaluated after its incubation under the optimal growth conditions. The lowest concentration of es-sential oil which was able to inhibit the growth of the par-ticular microorganism was considered as its minimal inhib-itory concentration (MIC).

Results and discussion

Essential oil yield, calculated on anhydrous needles of Pinus peuce is given in Table 1. The obtained essential oils were transparent, agile, light yellowish liquids with specif-ic and very strong turpentine odor.

Table 1. The yield* of Pinus peuce needle essential oils

calculated on anhydrous plant material.

SampleWater content

(ml/kg)

Yield* of essen-

tial oil (ml/kg)

± SD

Pelister Mtn. 78.21 5.04 ± 1.49%

Nidze Mtn. 68.33 9.93 ± 0.27%

Shara Mtn. 93.75 9.24 ± 2.86%

Karadzica Mtn. 80.00 2.86 ± 1.73%

*(n=3)

Table 2 shows components that were identifi ed in the Pinus peuce needle essential oils with their percentage amount and Kovat′s retention indices. Given percentage values are averages of percentages of the components that are present in the essential oils obtained from plant materi-al collected from various altitudes on four different locali-ties: Pelister, Nidze, Shara and Karadzica Mtn.

Total of one hundred and three components were iden-tifi ed in the investigated samples of Pinus peuce needle essential oils. Analysis of the essential oils composition shows seasonal variations in the content of components that are present, probably due to the infl uence of weather conditions throughout the year. The most abundant compo-nents in all samples of isolated essential oils were monot-erpenes: α-pinene, β-pinene, limonene + β-phellandrene and bornyl acetate and sesquiterpenes: trans-(E)-caryo-phyllene and germacrene D. The content of each of these 6 components varies in relatively wide range but generally they are present in amounts from 2% to 28% and are likely to determine the physico-chemical characteristics as well as chemical profi le of the oils. Large amount of monoter-penes camphene and α-terpenil acetate and sesquiterpenes δ-cadinene and α-cadinol was observed in some essential oils samples (Table 2).

Needle essential oil isolated from plant material col-lected in 2008/2009 on Baba Mtn. (Pelister) contains 12.89/19.72% α-pinene, 6.16/7.95% β-pinene, 3.08/3.97% limonene + β-phellandrene, 9.29/10.56% bornyl acetate, 4.88/7.13% trans (E)-caryophyllene and 8.75/19.90% germacrene D. These dominant components represent 54.56/59.72% of the whole needle essential oil. Essen-tial oil obtained from Pinus peuce collected in 2008 from Nidze Mtn. contains larger amount of α-pinene (27.34%), β-pinene (10.73%) and limonene + β-phellandrene (6.64%), but smaller amount of bornyl acetate (7.71%). Among dom-

28

Maced. pharm. bull., 57 (1, 2) 25 - 36 (2011)

Marija Karapandzova, Gjose Stefkov, Elena Trajkovska-Dokic, Ana Kaftandzieva, Svetlana Kulevanova T

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1162

1170.1

Pin

oca

rvone

0.2

00.0

20.0

8-

--

-

35

1165

1172.5

Born

eol

0.6

40.7

60.4

80.1

72.8

0-

-

Макед. фарм. билт., 57 (1, 2) 25 - 36 (2011)


No.

KIL

KIE

Com

pone

ntP.

Mtn

. 20

08

P. M

tn.

2009

N

. Mtn

. 20

08

Sh.

Mtn

. 20

08

Sh.

Mtn

. 20

09

K. M

tn.

2008

K

. Mtn

.20

09

3611

7711

81.9

Ter

pine

ne-4

-ol

0.08

0.02

0.03

0.06

0.09

-0.

0237

1183

1189

.4p-

Cym

ene-

8-ol

0.

080.

02-

0.08

0.11

-0.

0238

1189

1191

.6α

-Ter

pin

eol

0.3

00.2

90.1

70.4

30.2

80.1

00.2

8

39

1193

1195.2

Myrt

enal

0.2

90.1

60.2

00.1

10.1

80.1

30.3

5

40

1198

1199.2

Dodec

anet

0.2

50.2

30.3

40.3

50.3

20.1

70.1

3

41

1205

1205.6

trans-

Pip

erit

ol

0.1

90.2

00.0

4-

0.0

8-

-

42

-1207.1

Ver

ben

one

0.0

40.0

60.1

10.3

50.1

1-

-

43

1212

1218.0

trans-

Car

veo

l0.0

80.0

20.0

20.0

7-

-0.0

2

44

1220

1219.9

endo-F

ench

yl

acet

ate

0.0

30.0

20.0

30.0

20.1

1-

-

45

-1254.6

Pip

erit

one

0.0

2-

0.0

2-

0.1

0-

-

46

1256

1257.6

Lin

alool

acet

ate

0.1

20.0

20.0

20.0

80.0

5-

-

47

1261

1263.1

2-D

ecen

alt

0.0

20.1

2-

--

--

48

1285

1285.6

Born

yl

acet

ate

10.5

69.2

97.7

12.9

211.6

70.2

00.1

7

49

1294

1296.2

Tri

dec

anet

0.0

2-

0.0

30.0

70.0

8-

-

50

1314

1312.5

2E

,4E

-Dec

adie

nal

t0.0

20.0

20.0

2-

--

-

51

1339

1336.1

δ-E

lem

ene

0.2

10.1

10.1

90.1

80.1

70.9

10.2

2

52

1350

1347.5

α-T

erp

enyl

acet

ate

2.9

71.2

01.1

01.6

31.8

00.1

7-

53

1373

1371.1

α-Y

langen

e0.0

2-

0.0

20.0

2-

-0.0

2

54

1376

1374.0

α-C

op

aene

0.1

00.0

20.0

30.0

90.0

70.2

20.0

3

55

1383

1380.9

Ger

anyl

acet

ate

0.0

4-

--

--

-

56

1384

1383.3

β-B

ourb

onen

e 0.1

10.1

20.0

90.2

30.1

20.3

10.1

4

57

1391

1389.2

β-E

lem

ene

0.4

70.3

60.5

10.3

50.1

11.3

00.5

0

58

1401

1400.7

Met

hyl

eugen

ol

--

--

--

0.0

2

59

1402

1403.4

Longif

ole

ne

0.0

2-

0.0

5-

--

-

60

1418

1418.0

trans

(E)-

Car

yop

hyll

ene

7.1

34.8

84.6

35.5

56.3

55.1

53.9

2

61

-1430.1

β-C

op

aene

0.7

60.4

00.3

90.4

90.7

30.8

60.2

2

62

1439

1444.3

Aro

mad

endre

ne

0.0

4-

--

-0.1

3-

63

-1446.8

γ-A

morp

hen

e 0.1

90.1

50.1

9-

0.5

20.3

8-

64

1454

1453.6

α-H

um

mule

ne

1.5

90.7

90.6

21.2

70.6

31.2

90.5

4

65

-1469.1

Ep

i-b

icycl

ose

squip

hel

landre

ne

0.0

60.5

20.5

40.1

40.8

40.2

40.3

0

66

1473

1474.5

Dodec

anol

0.1

50.0

70.0

30.1

1-

-0.0

2

67

1477

1475.8

γ-M

uuro

lene

0.1

50.2

20.3

00.5

00.3

01.1

80.7

9

68

1480

1481.7

Ger

mac

rene

D19.9

08.7

510.1

020.1

419.3

69.9

07.1

3

69

1485

1493.9

β-S

elin

ene

0.0

3-

--

-0.3

50.0

2

70

1494

1496.3

Bic

ycl

oger

mac

rene

0.9

91.4

30.6

12.0

01.1

61.8

91.2

9

71

1495

1497.9

α-M

uuro

lene

0.9

20.2

51.5

30.0

2-

1.4

11.8

8

72

-1507.5

trans-

Cad

ina-

1(6

)4-d

iene

0.0

2-

0.0

30.1

40.0

9-

-

30

Maced. pharm. bull., 57 (1, 2) 25 - 36 (2011)

Marija Karapandzova, Gjose Stefkov, Elena Trajkovska-Dokic, Ana Kaftandzieva, Svetlana Kulevanova N

o.K

ILK

IEC

ompo

nent

P. M

tn.

2008

P.

Mtn

. 20

09

N. M

tn.

2008

S

h. M

tn.

2008

S

h. M

tn.

2009

K

. Mtn

.20

08

K. M

tn.

2009

7315

1315

13.7

γ-C

adin

ene

0.45

0.39

0.49

0.48

0.50

2.68

0.96

7415

2415

25.3

δ-

Cad

inen

e2.

711.

521.

821.

791.

708.

295.

7275

1532

1532

.0tr

ans-

Cad

ina-

1,4-

dien

e 0.

060.

390.

080.

050.

230.

230.

3076

1538

1540

.6α

- C

adin

ene

0.1

30.3

10.3

30.2

00.2

50.3

10.3

2

77

1547

1546.4

α-C

alac

ore

ne

0.0

20.1

40.0

20.0

20.0

20.0

20.0

2

78

1564

1561.4

Ner

oli

dol-

(E)

0.1

80.0

50.1

00.2

00.0

23.1

20.5

4

79

1568

1564.8

Dodec

anoic

aci

dt

0.5

00.0

60.0

30.0

80.0

90.0

2-

80

-1571.3

β-C

alac

ore

ne

0.0

2-

--

0.0

20.0

2-

81

1570

1575.2

Hex

enyl

bez

oat

e (3

-Z-)

0.0

50.0

20.0

20.0

2-

0.2

20.2

6

82

1574

1577.0

Ger

mac

rene-

4-o

l0.1

0-

0.0

20.2

90.2

33.7

10.4

2

83

1576

1583.0

Sp

athule

nol

0.3

20.0

80.2

30.1

50.1

50.0

20.1

8

84

1581

1586.4

Car

yop

hyll

ene

oxid

e0.5

70.0

70.1

90.4

1-

0.6

80.3

3

85

1590

1598.0

Vir

idifl

oro

l0.0

3-

--

--

-

86

1606

1613.0

Hum

ule

ne

epoxid

e II

0.1

2-

0.1

00.1

20.2

50.4

3-

87

1627

1637.8

1-e

pi-

Cub

enol

0.0

70.0

20.0

30.0

60.0

20.3

10.0

2

88

1640

1641

1644.3

1647.4

τ-C

adin

ol

(ep

i-α

-Cad

inol)

+

τ-M

uuro

lol

(ep

i-α

-Muuro

lol)

1.5

80.5

20.7

51.1

81.1

34.4

62.3

0

89

1645

1650.1

α-

Muuro

lol

(Torr

eyol)

0.2

40.2

30.0

20.1

30.7

00.7

60.1

6

90

1653

1657.0

α-C

adin

ol

1.9

90.4

60.3

21.5

21.5

17.0

42.8

5

91

-1693.1

Am

orp

ha-

4,9

-die

n-2

-ol

0.1

70.0

30.0

30.1

3-

0.3

40.1

0

92

1733

1746.3

Op

lop

anone

--

0.0

2-

0.0

6-

-

93

1762

1769.9

Ben

zyl

ben

zoat

e0.0

50.0

20.0

20.1

00.1

00.0

7-

94

-1884.5

Hex

enyl

cinnam

ate

(3-Z

-)0.1

51.2

30.0

40.1

30.0

40.3

60.4

8

95

1929

1946.7

Cem

bre

ne

0.0

4-

0.0

3-

--

-

96

-2009.4

Man

ool

oxid

e 0.0

9-

0.0

30.0

80.0

80.3

3-

97

1967

2013.0

Scl

aren

e0.0

20.0

60.0

80.0

20.1

1-

0.1

5

98

2054

2073.7

Ab

ieta

trie

ne

0.0

20.0

20.0

2-

0.0

4-

0.0

2

99

2080

2101.4

Ab

ieta

die

ne

0.0

2-

0.0

4-

0.0

40.1

10.1

3

100

-2166.4

Ab

ienol

--

--

-0.2

0-

101

2220

2253.2

Scl

areo

l-

-0.1

3-

--

-

102

2263

2296.5

Deh

ydro

abie

tal

0.0

2-

0.0

2-

0.0

50.4

10.0

2

103

2302

2332.1

Ab

ieta

l-

0.0

80.0

8-

--

3.7

4

TO

TA

L89.7

288.6

194.0

492.9

792.5

288.7

281.3

1

n=

3;

KIL

- K

ov

at’s

in

dex

lit

erat

ure

; K

IE –

Ko

vat

’s i

nd

ex e

stim

ated

; P.

Mtn

., N

.Mtn

., S

h.M

tn. an

d K

.Mtn

. -

Pel

iste

r, N

idze

, S

har

a an

d K

arad

zica

Mounta

in, re

spec

tivel

y;

(-)

- not

found, t

– t

enta

tive

iden

tifi

cati

on,

No

- o

rdin

al n

um

ber

of

the

com

po

nen

t ac

cord

ing

to

its

ret

enti

on t

ime.

Макед. фарм. билт., 57 (1, 2) 25 - 36 (2011)


inant components in the essential oil obtained from plant material collected in 2008/2009 on Shara Mtn., α-pinene

(12.95/23.91%), β-pinene (8.40/13.13%) and germac-

rene D (19.36/20.14%) were the most abundant while li-

monene + β-phellandrene (2.09/3.06%) and bornyl acetate

(2.92/11.67%) were present in the smaller amount. These

components represent 67.15% and 61.79/67.74% of whole

needle essential oil isolated from Pinus peuce from Nidze

and Shara Mtn., respectively. Essential oil composition of

cultivated Pinus peuce from Karadzica Mtn. (samples col-

lected in 2008/2009) is signifi cantly different from the oth-

ers essential oils mentioned above. This essential oil con-

tains smaller amount of α-pinene (9.21/12.22%), β-pinene

(4.88/6.03%), bornyl acetate (0.17/0.20%) and germac-

rene D (7.13/9.90%), but it contains larger amount for two

more components, δ-cadinene (5.72/8.29%) and α-cadinol

(2.85/7.04%). Large amount of diterpene abietal (3.74%)

was measured in the essential oil isolated from plant mate-

rial collected in 2009.

Data analysis of the chemical composition of Pinus

peuce needle essential oils revealed six different class-

es of components: monoterpene hydrocarbons, oxygenat-

ed monoterpenes, sesquiterpene hydrocarbons, oxygenat-

ed sesquiterpenes, diterpenes and other non-terpene com-

ponents. Among different classes of components that are

present in the essential oil isolated from plant material

from Baba (Pelister), Nidze, Shara and Karadzica Mtn, the

major constituents were terpene hydrocarbons (62.44%,

76.59%, 69.86% and 54.46%, respectively) while oxygen-

ated terpenes were present in smaller amount (Fig. 1.). The

most abundant fraction among the total terpene amount

were monoterpenes (47.2% in essential oil samples from

Pelister, 62.16% in essential oil samples from Nidze Mtn.

and 49.35% in samples from Shara Mtn.) (Fig.2.). Domi-

nant sesquiterpene fraction (45.23%) despite monoterpenes

(23.96%) is present only in the needle essential oils isolat-

ed from cultivated Pinus peuces from Karadzica Mtn. This

is probably due to the environmental conditions that affect

terpene biosynthesis of Pinus peuce in this region.

Up to now, there are only few data related to the chem-

ical composition of Pinus peuce needle essential oils (Hen-

ning, 1994; Papadopoulou, 1996; Koukos, 2000; Petrakis,

2001; Nikolic, 2008; Karapandzova, 2010a; Karapand-

zova, 2010b; Karapandzova, 2011). Generally, there are

some differences in chemical composition of essential oils

isolated from Pinus peuce growing in Greece, Serbia and

Montenegro in terms of presence of the dominant compo-

nents in the oil. Compared with essential oils isolated from

Pinus peuce from Macedonia, the needle essential oil iso-

lated from Pinus peuce from Greece contain larger amount

of β-pinene (22.00%) and citronellol (13.42%), smaller

amount of trans (E)-caryophyllene (3.05%), but it doesn’t

contain germacrene D (Koukos, 2000). On the other hand,

the needle essential oil isolated from Pinus peuce from

Montenegro and Serbia contain larger amount of α-pinene

(36.5%) and camphene (8.5%) (Nikolic, 2008).

The most sensitive bacteria against tested needle es-

sential oils were Streptococcus pneumoniae. Streptococ-

cus agalactiae showed similar sensitivity as previous bac-

teria and was susceptible to the effects of needle essen-

tial oils isolated from Pinus peuce from Shara and Nidze

Mtn. as well as moderate susceptible to the effects of the

needle essential oil from Pelister. Streptococcus pyogenes

was susceptible to the effects of essential oils isolated from

Fig. 1. Abundance of terpene hydrocarbons (THC) and oxigenated terpenes (OT) regarding diterpenes (DT) and other com-

ponents (OC) in Pinus peuce needle essential oils.

32

Maced. pharm. bull., 57 (1, 2) 25 - 36 (2011)


Pinus peuce from Shara Mtn. and moderate susceptible to the essential oils from Pelister and Nidze Mtn. Bacte-ria such as Staphylococcus aureus, Staphylococcus epider-

midis and Acinetobacter spp. showed sensitivity to the ef-fects of essential oils isolated from plant material collect-ed from Pelister and Nidze Mtn. Other investigated bacte-ria and Candida albicans were resistant to the antimicrobi-al effects of the Pinus peuce needle essential oils.

Summarizing the obtained results it is evident that the essential oil isolated from Pinus peuce from Pelister showed antimicrobial effect toward Streptococcus pneu-

moniae, Staphylococcus aureus, Staphylococcus epider-

midis and Acinetobacter spp. Essential oil isolated from plant material from Shara Mtn. showed antimicrobial ac-tivity against Streptococcus pneumoniae, Streptococcus

agalactiae and Streptococcus pyogenes, while the needle essential oil from Pinus peuce from Nidze Mtn. showed effects against Streptococcus pneumoniae, Staphylococcus

aureus, Staphylococcus epidermidis, Streptococcus aga-

lactiae and Acinetobacter spp. (Fig. 3.). This essential oil showed the best antimicrobial activity among all tested Pi-

nus peuce needle essential oils. Minimal inhibitory concentration (MIC) values of Pi-

nus peuce needle essential oils for tested microorganisms determined by broth dilution method are given in Table 3. The lowest MIC (less than 7.5 µl/ml) is determined in essential oil isolated from Pinus peuce from Shara Mtn. against Streptococcus pyogenes, while the highest MIC value (62.5 µl/ml) is determined in essential oil obtained from plant material collected on Baba Mtn. (Pelister) to-ward Staphylococcus aureus.

Table 3. Minimal inhibitory concentrations (MIC) of Pi-

nus peuce needle essential oils determined by broth dilution method (µl/ml).

MIC (µl/ml)

MicroorganismPelister

Mtn.Shara Mtn.

Nidze Mtn.

Streptococcus

pneumoniae31.25 15.62 15.62

Staphylococcus

aureus62.5 n.a. 31.25

Staphylococcus

epidermidis31.25 n.a. 15.62

Streptococcus

agalactiaen.a. 31.25 15.62

Streptococcus

pyogenesn.a. < 7.5 n.a.

Enterococcus n.a. n.a. n.a.

Haemophilus

infl uenzae n.a. n.a. n.a.

Acinetobacter

spp. 31.25 n.a. 31.25

Escherichia

colin.a. n.a. n.a.

Salmonella

enteritidis n.a. n.a. n.a.

Candida

albicans n.a. n.a. n.a.

n.a. - no activity found

Fig. 2. Abundance of monoterpenes (MT), sesquiterpenes (ST), diterpenes (DT) and other components (OC) in Pinus

peuce needle essential oils.

Макед. фарм. билт., 57 (1, 2) 25 - 36 (2011)


Although the biological activity of essential oils is

well known, the mode of their action is not completely un-

derstood. Generally, the activity of essential oils is the re-

sult of the combined effect both of their active and inac-

tive components. Inactive components may affect resorp-

tion, speed of reaction and bioavailability of active com-

ponents (Colgate, 1993; Svoboda, 1995), while the active

components can have a synergistic effect. Essential oils are familiar with antibacterial, antifungal and antiviral activity. Many studies have explained antibacterial activity of es-sential oils against Gram-positive and Gram-negative bac-teria (Reichling, 1999a; Weseler, 2005; Reichling, 1999b; Youesf, 1980; Dorman, 2000; Takarada, 2004) and have confi rmed their antifungal activity (Hammer, 1998). There are differences in the biological activity of essential oils, especially when it comes to different strains of Gram-posi-tive and Gram-negative bacteria. The different susceptibil-ity of bacteria to the action of essential oils are not only due to a different chemical composition of the oils, but the dif-ferences in the composition and structure of the cell wall, lipid and protein composition of cytoplasmic membrane and specifi c physiological processes in different bacterial species. The way and mechanism of antibacterial activity of essential oils are not suffi ciently explained yet. Nowa-days, it is accepted that some essential oils and their com-ponents mainly act on bacterial cytoplasmic membrane (Ulrich, 2008). According to the literature data, there are only three references that are related to the antimicrobi-al screening of pine needle essential oils. Oluwadayo et al. (2008) reported that the major constituents of essential oils isolated from the needles of Pinus caribaea Morelet are β-phellandrene (67.9%), β-caryophyllene (10.2%) and

α-pinene (5.4%). This essential oil exhibited moderate ac-

tivity against Pseudomonas aeruginosa at minimum inhib-

itory concentration of 1000 µl/ml and no activity against

other investigated microorganisms as Candida albicans,

Bacillus subtilis, Staphylococcus typhi, Bacillus aureus

and Proteus mirabilis. According to Sacchetti et al. (2005),

the most abundant components of Pinus radiata D. Don

are β-pinene (35.21%), α-pinene (21.9%), β-phellandrene

(12.6%), iso-silvestrene (8.42%), α-terpineol (3.01%),

terpinolene (2.21%), estragole (2.07%) and citronellol

(1.87%). This essential oil showed a moderate inhibiting

activity against tested yeasts as Candida albicans, Rhodo-

torula glutinis, Schizosaccharomyces pombe, Saccharo-

mycea cerevisiae and Yarrowia lypolitica at minimum in-

hibitory concentrations of 0.14, 0.09, 0.02, 0.06 and 0.29

mg/ml, respectively. The antimicrobial effects of vola-

tile components extracted from needles of Pinus densi-

fl ora S. and Z. was examined by Youg-Suk et al. (2005).

α-Ocimene (29.3%), sabinene (10.9%), β-myrcene (9.6%),

β-caryophyllene (8.0%), β-cadinene (7.3%), α-terpinolene

(4.9%), 2-hexanal (4.5%) and β-pinene (4.3%) were con-

sidered as dominant components. Antimicrobial activity

was screening against six different microorganisms: Ba-

cillus cereus, Salmonella typhimurium, Vibrio parahae-

molyticus, Listeria monocytogenes, Staphylococcus aureus and Escherichia coli. Among tested microorganisms, the growth of Bacillus cereus, Salmonella typhimurium and Staphylococcus aureus was completely inhibited.

Compared to our results, composition of Pinus peuce needle essential oil differs a lot from above tested pine oils. Antimicrobial screening was performed on different bacte-rial and fungi strains. All mentioned results indicate that the

Fig. 3. Antimicrobial activity (growth inhibition zones in mm) of Pinus peuce needle essential oils deter-mined by hole-plate diffusion method: 0-14 mm resistant, 14-19 mm moderate susceptible and 19-30 mm susceptible microorganisms.

34

Maced. pharm. bull., 57 (1, 2) 25 - 36 (2011)


antimicrobial activity of pine needle essential oils depend on the type of microorganism as well as chemical compo-sition of the oil. For example, antimicrobial activity of Pi-

nus radiata essential oil against Candida albicans is prob-ably due to the higher amount of α-pinene (21.9%) and β-pinene (35.21%) in comparison with Pinus peuce nee-

dle essential oil which contains lower amounts of these two

components and hasn’t showed antimicrobial effect against

tested fungi. On the other hand, the mechanism of antimi-

crobial activity of needle essential oil from different pine

species has not been clarifi ed yet thus the action of essen-

tial oils on bacterial cytoplasmic membrane may be con-

sidered as a superior mechanism of antimicrobial activity

of Pinus peuce needle essential oil.

Conclusion

The yield of needle essential oils obtained by hydro-

distillation of Pinus peuce from Pelister, Nidze, Shara and

Karadzica Mtn. ranged from 5.04, 9.24, 9.93 and 2.86 ml/

kg, respectively. The obtained essential oils were transpar-

ent, agile, light yellowish liquids with specifi c and very

strong turpentine odor.

Using GC/FID/MS, one hundred and three compo-

nents were identifi ed in the Pinus peuce needle essen-

tial oils. The most abundant components in all samples of

isolated oils were monoterpenes: α-pinene, β-pinene, li-

monene + β-phellandrene and bornyl acetate and sesquiter-

penes: trans-(E)-caryophyllene and germacrene D.

The most sensitive bacteria against tested Pinus peuce

needle essential oils were Streptococcus pneumoniae,

while Streptococcus agalactiae, Streptococcus pyogenes,

Staphylococcus aureus, Staphylococcus epidermidis and

Acinetobacter spp. showed different sensitivity to the anti-

microbial effects of the essential oils deepening on the area

of collection of plant material. Essential oil obtained from

plant material collected from Nidze Mtn. showed the high-

est antimicrobial activity against tested microorganism.

As the most promising activity of Pinus peuce nee-

dle essential oil was found against Streptococcus pneumo-

niae, this oil could be recommend for inhalation for infec-

tions of upper respiratory tract or for treatment of different

skin condition provoked by Staphylococcus aureus. Fur-

ther examination of safety of Pinus peuce needle essential

oil should be done before their fi nal recommendation.

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36

Maced. pharm. bull., 57 (1, 2) 25 - 36 (2011)


Резиме

Aнтимикробнa активност на етерично масло од

иглички од молика (Pinus peuce Griseb., Pinaceae) од

флората на Р. Македонија

Марија Карапанџова1*, Ѓоше Стефков1, Елена Трајковска-Докиќ2, Ана Кафтанџиева2, Светлана Кулеванова1

1Институт за Фармакогнозија, Фармацевтски факултет, Универзитет „Св. Кирил и Методиј“, Скопје,

Македонија2Институт за Микробиологија, Медицински факултет, Универзитет „Св. Кирил и Методиј“, Скопје, Македонија

Клучни зборови: Pinus peuce, Македонски бор, хемиски состав на етерично масло, GC/FID/MS, антимикробна активност.

Хемиски состав и антимикробна активност се испитувани кај етерично масло добиено со дестилација со водена пареа на иг-лички од молика (Pinus peuce Griseb., Pinaceae) која самоникнатo расте на три различни локалитети во Р. Македонија. Со GC/FID/MS анализа, идентификувани се 103 компоненти што припаѓаат на шест различни класи соединенија: монотерпенски ја-глеводороди, оксидирани монотерпени, сесквитерпенски јаглеводороди, оксидирани сесквитерпени, дитерпени и други нетере-пенски компоненти и истите сочинуваат 86,61/94,04% од севкупното масло. Најзастапени компоненти во етеричното масло се α-пинен (12,89/27,34%), β-пинен (6,16/13,13%), лимонен + β-феландрен (2,09/6,64%) и борнил ацетат (2,92/11,67%), како и trans (E)-кариофилен (4,63/7,13%) и гермакрен D (8,75/20,14%).

Антимикробната активност на етерични масла од иглички од молика е испитувана со агар дифузиона и со агар дилуциона метода на 13 бактериски изолати на Грам позитивни и Грам негативни бакетии и еден изолат на габата Candida albicans. Најголе-ма осетливост на дејството на маслата од молика покажа бактеријата Streptococcus pneumoniae, следена од Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Acinetobacter spp. и Streptococcus pyogenes. Минималните инхибиторни кон-центрации на етеричните маслата се движат од 7,5 до 62,5 µl/ml.


UDC: 547.943:543.544.5Original scientifi c paper

Introduction

The European Pharmacopoeia, in the monographs for the Opium dry extract, standardised (01/2008:1839); Opi-um, raw (01/2008:0777); and Opium tincture, standardised (01/2008:1841) provides HPLC-UV assay method for mor-phine and codeine and test method for thebaine (Ph.Eur.7). These natural products, however, can contain even more alkaloids that originate from opium poppy Papaver som-

niferum L. (Papaveraceae), including: noscapine papaver-

Determination of relative response factors of the opium

alkaloids with HPLC-DAD

Jelena Acevska1*, Gjoshe Stefkov2, Natalija Nakov1, Marija Karapandzova2, Svetlana Kulevanova2, Aneta Dimitrovska1

1Institute for Applied Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy, Vodnjanska 17, 1000 Skopje, Republic

of Macedonia 2 Institute for Pharmacognosy, Faculty of Pharmacy, Vodnjanska 17, 1000 Skopje, Republic of Macedonia

Received: December 2011; Accepted: February 2012

Abstract

In this work, a convenient method for determination of relative UV response factors (RRFs) of morphine, codeine, thebaine, oripavine, noscapine and papaverine by high performance liquid chromatography (HPLC) equipped with a diode array detector (DAD) was present-ed. Pholcodine was selected as the reference compound for calculating the relative response factors of the alkaloids.

The separation of all seven compounds was obtained with optimized gradient elution with high pH value of the mobile phase on a re-versed phase column with bidentate C18-C18 bonding technology. The RRFs of the alkaloids were determinate by three different approach-es: ‘regression analysis/mass concentration’, ‘regression analysis/molar concentration’and ‘detector sensitivity’ approaches.

The ‘regression analysis/molar concentration’ approach gave the accurate approximation of the exact amount of the substance that en-ters in the detector and the statistically relevant calculation includes several points of different concentrations (at least fi ve), which makes this approach most advantageous one.

This method is suitable for quality assessment of the standardised opium dry extract, raw opium and standardised opium tincture by quantitative analysis of not only morphine and codeine as indicated in the respective European Pharmacopoeia monographs, but as well as the major impurities that originate from opium poppy Papaver somniferum L. (Papaveraceae).

Key words: HPLC-DAD, Relative response factors (RRFs), opium alkaloids, internal standard

* [email protected]. +38923126032; Fax: +38923123054

ine and oripavine (Fig. 1). These alkaloids are expected to be identifi ed with the TLC method provided for identifi ca-tion in the cited monographs.

All of the above mentioned alkaloids are important for the quality of the cited natural products described in the monographs. The presence of some alkaloids other than those defi ned in the content can be considered as impuri-ties if their pharmacological action is different from the de-sired one.

Providing reference standard for all the analytes of in-terest in natural products is sometimes diffi cult, especial-ly for the non pharmacopoeial substances, or substances which are subject to international narcotics control, so the content determinations often include quantitation based

38

Maced. pharm. bull., 57 (1, 2) 37 - 41 (2011)

Jelena Acevska, Gjoshe Stefkov, Natalija Nakov, Marija Karapandzova, Svetlana Kulevanova, Aneta Dimitrovska

on relative HPLC peak areas obtained at a specifi c wave-length. In order for this quantitation to accurately refl ect weight percentages of the compounds, the relative UV re-sponse factors (absorptivities) at the given wavelength must be known (Sun et al., 2008; Ph.Eur.2.2.46).

The objective of this study was to determine UV rel-ative response factors (RRf) of opium alkaloids at differ-ent wavelengths and to propose a method for rapid, rou-tine analysis for the content determination of six alkaloids in standardised opium dry extract, raw opium and stan-dardised opium tincture.

Experimental

Materials: Authentic samples of morphine, codeine, oripavine, thebaine, noscapine, papaverine and pholcodine (free base each) were obtained from Alkaloid A.D., Skopje (Skopje, R.Macedonia).

Reagents: Methanol (HPLC grade), Trifl uoroacetic

acid (for synthesis) and Phosphoric acid 85% (pro analy-sis) were purchased from Merck, Damstad, Germany. Tri-

ethylamine (pro analysis) was purchased from Sigma-Al-drich, Steinheim, Germany. Water (highly purifi ed) was obtained with a TKA-LAB Reinstwasser system (Nie-derelbert, Germany).

Standard Solution of Alkaloids: Standard solutions of alkaloids were prepared from methanolic stock solutions of 2 mg/ml morphine, 1 mg/ml codeine, 1 mg/ml thebaine, 1 mg/ml oripavine, 1 mg/ml papaverina, 2 mg/ml noscap-ine and 2 mg/ml pholcodine. Standard solution of alkaloids for a HPLC method was prepared in the same manner to obtain a concentration of 200 µg/ml morphine and pholco-dine (as internal standard), 100 µg/ml codeine and noscap-ine and 50 µg/ml thebaine, papaverine and oripavine.

Chromatographic Conditions: The separation of the six alkaloids and the internal standard was performed on Agilent 1200 HPLC/DAD system and a 250 mm x 4.6 mm i.d., 5 µm particle size, Zorbax Extend C-18 column (Agi-lent Technologies). The eluents were a solution of 0.1% tri-fl uoroacetic acid (TFA) in water, pH adjusted to 9.6 with triethylamine (TEA) (= solvent A) and methanol (= solvent B), both fi ltered through 0.45 µm Millipore fi lters. Elution was performed with optimized gradient (Acevska, 2012 a). The fl ow rate was 1.5 mL/min and the injection volume 20 µl. The column temperature was maintained at 40ºC. The measurements were made with UV detection at wavelength of 280 nm, and the injection volume was 20μl. Pholcodine was selected as the reference compound for calculating the relative response factors of the alkaloids. The RRFs of the alkaloids were determined by linear calibration curves and through absolute response factors of the alkaloids.

Statistical analysis: The regression analysis was per-formed using Excel Analysis Tool Pack. The regression curves were obtained by plotting mass concentration or molar concentration of the alkaloids vs. their HPLC peak areas. The signifi cance of the difference (d) between the RRf values obtained by different approaches was calculat-ed using equitation d = (RRf

ni− RRf

nt)/RRf

nt × 100% at lev-

el of signifi cance p=0.05.


According to Ph.Eur. methods, the detection of mor-phine and codeine is performed at wavelength of 280 nm. Since the alkaloids have different absorptivities at pH 9.6 (Fig. 2), as for the proposed mobile phase (Acevska et al., 2012 a; Acevska 2012 b), the content determination must be based on RRfs for all the compounds of interest.

The conventional way to determine UV RRf is to an-

Fig.1. Chemical structure of the alkaloids from opium poppy, Papaver somniferum L. (Papaveraceae)

Макед. фарм. билт., 57 (1, 2) 37 - 41 (2011)

39 Determination of relative response factors of the opium alkaloids with HPLC-DAD

alyze two pure compounds in defi ned quantities under the

same detection conditions and calculate ratio of slopes of

the linear regression curves. Unless otherwise indicated,

the regression analysis is done with the mass concentration

of the compounds. Since the mass concentration does not

include the exact amount of the component that enters the

detector, more appropriate way for performing the regres-

sion analysis is with a series of molar concentrations. Lin-

ear calibration curves for all seven compounds were con-

structed using the peak areas and analyte’s mass concentra-

tions in the range of 1–150% (w/w) of the working concen-

tration. The linearity data using mass and molar concentra-

tion of all analytes were summarized in Table 1 and 2, re-

spectively. The UV relative response factor (RRf) was de-

termined as the ratio of slope of the regression line for each

alkaloid and pholcodine.

Another approach for determination of RRf is to deter-

mine the detector sensitivity, defi ned as absolute response

factor (signal output per unit concentration of a substance

in the mobile phase entering the detector) (Ph.Eur.2.2.46).

The relative response factor is expressed as the sensitiv-

ity of a detector for a given substance relative to a stan-

Fig. 2. Absorption maxima of the opium alkaloids at pH value 9.6

Table 1. Regression data (mass concentration) and UV relative response factors of opium alkaloids

Compound Conc. range (μg/ml) Regression equitation (a) RRf(b) Cf (c)

Morphine 2-300 y=3.56 x+1.69 1.23 0.82

Oripavine 0.5-75 y=18.85x-0.26 6.49 0.15

Codeine 1-150 y=4.00x-1.18 1.38 0.73

Pholcodine 2-300 y = 2.90x + 0.23 1.00 1.00

Papaverine 0.5-75 y=15.14x-2.28 5.21 0.19

Thebaine 0.5-75 y=17.39x-1.37 5.99 0.17

Noscapine 1-150 y=4.92x+0.22 1.70 0.59

(a)The regression curves were obtained by plotting mass concentration (x) vs. peak area (y). (b)RRf is UV relative response factor determined in reference to

pholcodine at 280 nm. (c) Cf is correction factor for the quantitation of the alkaloids at 280nm.

Table 2. Regression data (molar concentration) and UV relative response factors of opium alkaloids

Compound Conc. range (μmol/l)Molar mass (g/

mol)Regression equitation (a) RRf(b) Cf (c)

Morphine 7-1051 285.33 y = 0.0010 x + 1.6873 0.83 1.20

Oripavine 2-252 297.34 y = 0.0056x - 0.2599 4.67 0.21

Codeine 3-501 299.36 y = 0.0012x - 1.184 1.00 1.00

Pholcodine 3-337 398.00 y = 0.0023x + 0.2296 1.00 1.00

Papaverine 1-221 339.29 y = 0.0051x - 2.2833 4.25 0.24

Thebaine 2-241 311.37 y = 0.0054x - 1.3733 4.50 0.22

Noscapine 2-363 413.43 y = 0.0020x + 0.2240 1.67 0.60

(a)The regression curves were obtained by plotting molar concentration (x) vs. peak area (y). (b)RRf is UV relative response factor determined in reference

to pholcodine at 280 nm. (c) Cf is correction factor for the quantitation of the alkaloids at 280nm.

40

Maced. pharm. bull., 57 (1, 2) 37 - 41 (2011)

Jelena Acevska, Gjoshe Stefkov, Natalija Nakov, Marija Karapandzova, Svetlana Kulevanova, Aneta Dimitrovska

dard substance, i.e. ratio of absolute response factors of the substance and the standard (Table 3). The results for the detector sensitivity, defi ned as absolute response factor (ARf), determined for each compound are summarized in Table 3. The RRf was expressed as a ratio of ARf of each compound and pholcodine. This approach gave more ac-curate RRf values than the conventional (mass concentra-tion) regression analysis, because the calculation includes the exact amount of the substance that enters in the detec-tor. However, unlike the ‘regression analysis/molar con-centration’ approach, the calculation for ‘detector sensitiv-ity’ approach includes only one concentration of the ana-lyte, which is the major disadvantage of this approach.

For all the compounds having RRf values outside the range 0.8-1.2, a correction factor should be applied in the quantifi cation (Ph.Eur.2.2.46). The correction factors (Cf) were calculated as a reciprocal of the relative response fac-tor and are presented in the fi nal columns of the Tables: 1, 2 and 3.

The different approaches used for determination of rel-ative UV response factors (RRFs) of morphine, codeine, thebaine, oripavine, noscapine and papaverine by HPLC-DAD showed different values for the RRf. The statistical comparison between the RRf values obtained from differ-ent approaches (Table 4) showed that the difference be-tween the ‘regression molar concentration’ and ‘detector sensitivity’ approaches can be considered as insignifi cant

and is due to the differences of the statistical calculation (fi ve vs. one concentration point). The signifi cant statisti-cal difference between the ‘regression mass concentration’ approach and the other two approaches is an indicator that misjudgment can be made if the correction factor is calcu-lated using the mass concentration of the standards. The most accurate way to determine RRf values is to calcu-late ratio of slopes of the linear calibration curves obtained from series of molar concentrations, because in the calcu-lation is included the exact amount of the substance that enters in the detector. For the accurate determination of the content of alkaloids is necessary to include a correction factor (Cf), given in the last column of Table 2.

Conclusion

Different approaches for the determination of relative UV response factors (RRf) of morphine, codeine, thebaine, oripavine, noscapine and papaverine by HPLC-DAD, us-ing pholcodine as the reference compound, were present-ed.

The ‘regression analysis/molar concentration’ approach gives the accurate approximation of the exact amount of the substance that enters in the detector and the calculation includes statistically enough concentration points (at least fi ve), which makes this approach most advantageous one. The correction factor to be included for alkaloid determi-

Table 3. Detector sensitivity and UV relative response factors of opium alkaloids

Compound n (nmol) ARf(a) RRf(b) Cf(c)

Morphine 1.402 509.8 0.88 1.14Oripavine 0.336 2803.8 4.84 0.21Codeine 0.668 596.3 1.03 0.97

Pholcodine 1.005 578.90 1.00 1.00Papaverine 0.295 2577.5 4.45 0.22Thebaine 0.321 2699.3 4.66 0.21

Noscapine 0.484 1019.2 1.76 0.57

(a)ARf is an absolute response factor determined as ratio between the signal output (mAU*s) measured at 280 nm and the amount of each compound (nmol) in the defi ned injection volume. (b)RRf is UV relative response factor determined as ratio of absolute response factors of each compound and the reference compound. (c)Cf is correction factor for the quantitation of the alkaloids at 280nm.

Table 4. Statistical comparison between the relative response factors of the target alkaloids, determined by: ‘regression anal-ysis/mass concentration’ (RRf1), ‘regression analysis/molar concentration’ (RRf2) and ‘detector sensitivity’ ap-proach (RRf3)

RRf 1 RRf 2 RRf 3 d(RRf1/RRf2)(a) d(RRf2/RRf3)(a) d(RRf1/RRf3)(a)

Morphine 1.23 0.83 0.88 47.13 5.68 39.21Oripavine 6.49 4.67 4.84 39.15 3.79 34.07Codeine 1.38 1.00 1.03 37.77 3.00 33.75Papaverine 5.21 4.25 4.45 22.67 4.76 17.09Thebaine 5.99 4.50 4.66 33.06 3.62 28.42Noscapine 1.70 1.67 1.76 1.73 5.64 -3.69

(a)d = (RRfni− RRf

nt)/RRf

nt × 100%, p=0.05, DF=1, (t

tab=6.314)

Макед. фарм. билт., 57 (1, 2) 37 - 41 (2011)

41 Determination of relative response factors of the opium alkaloids with HPLC-DAD

nation is expressed as the reciprocal value of the factor of relative detector response calculated by regression analy-sis of dependence between the detector response and molar concentration of the alkaloids.

This method is suitable for rapid, routine analysis for the content determination of six alkaloids in standardised opium dry extract, raw opium and standardised opium tinc-ture, through relative response factors of the opium alka-loids.

References

Acevska, J., Stefkov, G., Petkovska, R., Kulevanova, S. and Dimitrovska, A., 2012a. Chemometric approach for development, optimization and evaluation of different

chromatographic methods for separation of opium alkaloids, Analytical and Bioanalytical Chemistry 403, 1117-1129.

Acevska, J., Dimitrovska, A., Stefkov, G., Brezovska, K., Karapandzova, M. and Kulevanova, S., 2012b. Development and validation of RP-HPLC method for determination of alkaloids from Papaver somniferum L., Papaveraceae, Journal of AOAC International 95(2), 399-405.

European pharmacopeia 7th Edition European Directorate for the Quality of Medicines & HealthCare (EDQM), Council of

Europe, Strasbourg, France 2011, 2.2.46, pp.76-77.

Sun, P., Wang, X., Alquier,L., Maryanoff, C.A., 2008.

Determination of relative response factors of impurities in

paclitaxel with high performance liquid chromatography

equipped with ultraviolet and charged aerosol detectors.

Journal of Chromatography A 1177, 87–91.

Резиме

Определување на фактор на релативен одговор на

детекторот на опиумските алкалоиди со примена на

HPLC-DAD

Јелена Ацевска1*, Ѓоше Стефков2, Наталија Наков1, Марија Карапанџова2, Светлана Кулеванова2, Анета Димитровска1

1 Институт за применета хемија и фармацевтски анализи, Фармацевтски факултет, Водњанска 17, 1000 Скопје,

Република Македонија 2 Институт за фармакогнозија, Фармацевтски факултет, Водњанска 17, 1000 Скопје, Република Македонија

Kлучни зборови: HPLC-DAD, Фактор на релативен одговор на детекторот (RRF), опиумови алкалоиди, внатрешен стандард

Во рамки на овој труд, претставен е соодветен метод за определување на фактор на релативен одговор на UV детектор (RRF) на морфин, кодеин, тебаин, орипавин, папаверин и носкапин со примена на високо-перформансна течна хроматографија (HPLC) и детектор со диодна решетка (DAD). Како референтна компоненета за определување на RRF вредностите на алкалоидите беше избран фолкодин, стандардна супстанција.

Разделувањето на сите седум соединенија е постигнато со оптимизирано градентно елуирање со висока pH вредност на мо-билнaтa фаза, на реверзно-фазна колона со специфично обработена стационарна фаза (технологија на двојно C18-C18 сврзување). RRF вредностите на алкалоидите се утврдени со три различни пристапи: „регресиона анализа / масена концентрација“, „регреси-она анализа / молска концентрација“ и „осетливост на детекторот“.

Пристапот „регресиона анализа / молска концентрација“ е најсоодветен за определување на RRF вредностите на алкалоиди-те, затоа што на овој начин се добива точна апроксимација за концентрација на аналитот што влегува во детекторот, а пресметка-та е статистички релевантна со оглед дека вклучува неколку различни концентрации (најмалку пет) на аналитот.

Предложениот метод е соодветен за проценка на квалитетот на стандардизиран сув екстракт од опиум, сиров опиум и стан-дардизирана тунктура од опиум, преку квантитативна анализа не само на морфин и кодеин, како што е наведено во соодветните монографии на Европската фармакопеја, туку и определување на главните онечистувања што потекнуваат од афион Papaver som-

niferum L. (Papaveraceae).


UDC: 582.477–113.55(497.7) Original scientifi c paper

Introduction

Common juniper, Juniperus communis L. (Cupressaceae) is widely spread throughout the territory of Republic of Macedonia (Micevski, 1998). The berries of this plant are extensively utilized in production of blended teas and other herbal medicinal products, in food industry, as a spice, in production of alcoholic beverages, etc. The cone-berries (Juniperi pseudofructus) are used traditional-ly to cure cystitis, digestive disorders, in therapy of chron-icle arthritis and other indications. They contain essential oil with characteristic conifer-like aroma and bitterly taste. The pharmaceutical and the medicinal use of the juniper

Chemical composition of berry essential oils from Juniperus

communis L. (Cupressaceae) growing wild in Republic of

Macedonia and assessment of the chemical composition in

accordance to European Pharmacopoeia

Floresha Sela, Marija Karapandzova, Gjoshe Stefkov, Svetlana Kulevanova

Institute of Pharmacognosy, Faculty of Pharmacy, University “Ss Cyril and Methodius”, Skopje, Republic of Macedonia

Received: December 2011; Accepted: February 2012

Abstract

Chemical composition of fi fteen samples of juniper essential oil was analyzed using GC/FID/MS method. Thirteen samples of berries were collected on different locations in south-western part , two of them in central-north region of Republic of Macedonia. The essential oils were obtained by hydrodistillation in a Clevenger type apparatus using offi cial method of European Pharmacopoeia. GC/MS analysis revealed 74 identifi ed components. The predominant fractions of the oils were monoterpene hydrocarbons representing 39.11-73.38%. Great variability in the chemical composition and content of some components was observed. The most variable components were α-pinene (15.59-43.19%), β-pinene (1.65%-5.35%), β-myrcene (2.89%-26.50%), sabinene (2.80-11.77%), and limonene (2.90-4.46%). In the fraction of oxidized monoterpenes the most abundant was terpene-4-ol (trace - 6.32%) followed by α-ter pineol (0.18-1.63%). In the sesquiterpene fraction predominant components were: germacrene D (2.76-10.22%), β-ele mene (1.13-3.40%) and trans-(E)-caryophyllene (1.8%- 4.05%). Twelve samples of Macedonian juniper oils comply with European Pharmacopoeia chemical composition requirements for juniper oil and three samples did not, due to lower amount of α-pinene.

Key words: Juniperus communis, Juniper oil, common juniper, Republic of Macedonia

* [email protected]

oil are of multi-purpose: diuretic, antiseptic, digestive, sto-machic, antireuma thic, etc. (Yarnell, 2002). Antimicrobial (antibacterial and antifungal) activity of the oil was studied in vitro and many data on this issue point out on a strong antimicrobial activity that could be of interest in medicine and other fi elds (Filipowitz et al., 2003; Stassi et al., 1995; Emami et al., 2007; Pepeljnjak et al., 2005; Asili et al., 2008; Kumar et al., 2009; Miceli et al., 2009; Glisic et al., 2007; Öztürk et al., 2010; Consentino et al., 2003). Juniper oils are used in production of medicinal products with di-uretic and antiseptic activity than in food industry, produc-tion of alcohol beverages, cosmetic and perfume produc-tion, etc.

The yield and the composition of the essential oils of the juniper berries depends on the geographical origin of

44

Maced. pharm. bull., 57 (1, 2) 43 - 51 (2011)


the plant, the maturity of the berries, the age of the plant, the meteorological condition (temperature, insolation, etc.) and other environmental factors (Orav, 2010; Chatzapoulou and Katsiotis 1993, 1995). Therefore the essential oil com-position can vary signifi cantly and that is the reason for very wide-ranging the values in European Pharmacopoeia monograph’s requirements for characteristic constituents of the juniper oil (Ph. Eur. 7).

The juniper berries and the juniper essential oil are nat-ural drugs that are exported from Republic of Macedonia, for years behind. Until now, there was no detailed study on the chemical composition of Macedonian juniper oil, which led us to the goal of the present research - determi-nation of the chemical composition and possible chemi-cal variability of the juniper essential oils from wild grow-ing plants in the Republic of Macedonia and assessment of the essential oil chemical composition according to the European Pharmaco poeia.

Materials and methods

Plant material

The samples of the ripe juniper berries were collected in a late autumn in 2010 on different locations in Republic of Macedonia. The samples were labeled with marks JC1-JC15, relating to the name of the nearest city or name of mountain where the collections were made: JC1 – Mavrovo (1); JC2 – Skopje; JC3 – Resen; JC4 – Demir Hisar; JC5 – Prilep, JC6 – Karaorman Mtn.; JC7 – Kicevo (1); JC8 – Kicevo (2); JC9 – Debar; JC10- Mavrovo (2); JC11 – Kicevo (3); JC12 – Jakupica Mtn., JC13 – Pelister Mtn., JC14 – Bistra Mtn.; JC15 – Makedonski Brod. Thirteen samples originated from south-western part of the coun-try and two of them were collected from north-central part (Skopje and Jakupica Mtn.). All samples were left to air dry and then put in a paper bags and stored at cool, dry and dark place, until analysis.

Distillation of the essential oils

The essential oils were isolated by steam-distilla-tion in a Clevenger-type apparatus using the method from European Pharmacopoeia (Ph. Eur. 7). The oils were dried over anhydrous sodium sulfate and stored in vials under re-frigeration prior to analysis.

GC/FID/MS analyses

Essential oil samples were analyzed on Agilent 7890А

Gas Chromatography system with fl ame ionization detec-

tor (FID), and Agilent 5975C mass spectrometer (MS) also

equipped with capillary fl ow technology which enables si-

multaneous analysis of the sample on both detectors. HP-

5ms (30 m x 0.25 mm, fi lm thickness 0.25 µm) capillary

column was used. Operating conditions were as follows:

oven temperature 60 °C (5 min), 1 °C/min to 80 °C (2

min); 5 °C/min 280 °C (5 min); fl ow rate of 1ml/min (He);

injector T=260 °C; FID T= 270 °C; 1 µl injection volume

at split ratio 1:1.

The mass spectrometry conditions were: ionization

voltage 70 eV, ion source temperature 230 °C, transfer

line temperature 280 °C and mass range from 50-500 Da.

The MS was operated in scan mode. Identifi cation of the

components present in essential oils was made by com-

paring mass spectra of components in essential oils with

those from Nist, Wiley and Adams mass spectra libraries,

by AMDIS (Automated Mass Spectral Deconvolution and

Identifi cation System) and by comparing literature and es-

timated Kovat′s (retention) indices that were determined

using mixture of homologous series of normal alkanes

from C9 to C

25 in hexane, under the same above mentioned

conditions.

The percentage ratio of essential oils components was

computed by the normalization method of the GC/FID

peak areas and average values were taken into further con-

sideration (n=3).


Chemical composition of the essential oils

By the means of GC/FID/MS method, all 15 juniper

oil samples were analyzed and 74 different components

were indentifi ed, representing 91.08 – 99.83% of the en-

tire oils (Table 1). Monoterpene components were present-

ed in larger amounts in all samples of the oils than sesqui-

terpenes (Fig. 1.). Beside mono and sesquiterpenes, small

amounts of few non-terpene components were identifi ed,

such as undecanone-2 and tricycle ne (Table 1).

Terpene hydrocarbons represented the most signifi cant

part of the oil rather than oxygen containing terpenes (Fig.

2.). Predominate fraction was monoterpene hydrocarbons,

representing the most abundant fraction in all oil samples,

ranging from 39.11% (JC13) to 78.38% (JC14) (Fig. 3).

In the monoterpene hydrocarbons fractions dominated

components were: α-pinene (15.59 JC9 – 43.19% JC14),

β-pinene (1.65 JC9 – 5.35 % JC14), β-myrcene (2.89 JC5

– 26.50% JC6), sabinene (2.80 JC5 – 11.77% JC6) and

limonene (2.90 JC4 – 4.46% JC2). The oxygen contain-

ing monoterpenes were present only from 2.24% (JC3) –

8.00% (JC9) in all samples. The most abundant was ter-

pene-4-ol, in traces and up to 6.32 % (JC9) followed by

α-ter pineol (0.18 JC3-1.63% JC3).

The sesquiterpene hydrocarbons were present from

17.45% (JC14) to 42.25% (JC14) in all samples (Fig. 3.).

The predominant components were: germacrene D (2.76

JC14-10.22% JC7); β-ele mene (1.13 JC14-3.40% JC7)

and trans-(E)-caryophyllene (1.8 JC10-4.05% JC5). The

oxygen containing sesquiterpenes have appeared with

0.59% (JC3) to 5.76% (JC13). The component α-cadinol

varied from 0.03 % (JC4) to 1.13 % (JC13). The mixture

τ-murolol+τ-cadi nol were presen ted from 0.07 % (JC15) to

Макед. фарм. билт., 57 (1, 2) 43 - 51 (2011)

45 Chemical composition of berry essential oils from Juniperus communis L. (Cupressaceae) growing wild in.... T

able

1.

Che

mic

al c

ompo

siti

on o

f th

e es

sent

ial

oils

obt

aine

d by

hyd

rodi

stil

lati

on f

rom

ber

ries

of

Mac

edon

ian

Junip

erus

com

munis

a

No.

dK

IbK

I expc

Com

pone

nts

JC1

JC2

JC3

JC4

JC5

JC6

JC7

JC8

JC9

JC10

JC11

JC12

JC13

JC14

JC15

Mon

oter

pene

hyd

roca

rbon

s

2.93

194

6.5

α−T

huje

ne1.

171.

202.

340.

770.

701.

181.

031.

491.

051.

210.

830.

970.

800.

740.

71

3.93

995

1.8

α−

Pin

ene

22.1

27.1

528

.86

22.0

127

.65

18.6

218

.32

22.2

015

.59

27.0

632

.02

26.4

122

.01

43.1

939

.14

4.95

396

0.5

Cam

phen

e0.

190.

240.

240.

190.

230.

140.

620.

170.

150.

18-

0.26

0.10

0.14

0.35

5.97

597

8.7

Sab

inen

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165.

8010

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3.47

2.80

11.7

76.

107.

654.

446.

565.

14

4.75

3.89

4.68

4.77

6.98

098

0.6

β−

Pin

ene

2.50

2.81

3.61

2.24

2.69

2.17

3.75

2.59

1.65

2.78

4.10

2.79

2.64

5.35

3.29

7.99

199

3.8

Myr

cene

(β

)16

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.82

20.1

17.0

114

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26.5

010

.47

12.7

88.

5321

.519

.75

11.9

92.

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0310

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α−

Phe

llan

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e0.

140.

190.

230.

130.

160.

100.

060.

190.

220.

090.

210.

180.

220.

180.

82

9.10

0710

07.7

∆3 -

Car

ene

0.09

0.16

0.15

0.04

tr

0.11

0.08

0.15

0.17

tr0.

150.

170.

140.

160.

05

10.

1018

1013

.6α

−T

erpi

nene

0.

650.

731.

280.

370.

600.

420.

871.

121.

221.

041.

100.

650.

620.

830.

42

11.

1025

1020

.6p-C

ymen

e0.

120.

160.

220.

130.

240.

090.

210.

340.

410.

630.

410.

520.

500.

230.

24

12.

1031

1024

.6L

imon

ene

3.40

4.46

4.33

2.90

3.07

3.62

3.28

3.81

3.39

4.03

4.41

3.67

3.27

3.47

2.90

13.

1040

1044

.5β

−ci

s-O

cym

ene

0.07

trtr

0.08

1.

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-tr

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14.

1062

1053

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Ter

pine

ne1.

191.

302.

270.

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672.

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101.

791.

831.

121.

051.

310.

84

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1088

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) T

erpi

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221.

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740.

881.

091.

142.

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40

1.30

1.

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350.

930.

871.

030.

92

To

tal

(%)

49.3

356.2

75.5

751.0

455.4

866.7

649.3

555.9

540.2

268.2

171.4

754.6

539.1

178.3

869.4

6

Ox

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11

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90.0

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70.1

0

21

.11

66

11

73

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a-1

,5-d

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--

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60.0

70.0

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50.3

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22

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77

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82

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61.5

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02.8

02.7

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261

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64

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40.0

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0-

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46

Maced. pharm. bull., 57 (1, 2) 43 - 51 (2011)

Floresha Sela, Marija Karapandzova, Gjoshe Stefkov, Svetlana KulevanovaN

o.d

KIb

KI ex

pcC

ompo

nent

sJC

1JC

2JC

3JC

4JC

5JC

6JC

7JC

8JC

9JC

10JC

11JC

12JC

13JC

14JC

15

37.

1383

1384

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yl a

ceta

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060.

03-

0.08

0.08

0.13

tr0.

040.

06tr

0.19

0.22

0.24

0.11

0.13

Tot

al (

%)

2.26

2.45

2.24

2.44

4.06

2.32

3.56

5.59

8.0

7.51

4.07

4.58

6.68

3.27

4.25

Ses

quit

erpe

ne h

ydro

carb

ons

32.

1339

1339

.0δ−

Ele

men

e0.

310.

300.

190.

330.

280.

260.

470.

300.

320.

140.

280.

230.

290.

170.

18

33.

1351

1351

.8α

-Cub

eben

e0.9

00.7

00.6

01.0

61.3

10.7

00.7

60.7

10.6

00.5

30.5

80.6

00.6

70.5

60.7

6

35

.1

373

13

73

.8α

−Y

lan

gen

e0.1

20.1

40.0

80.1

40.1

20.0

30.0

90.0

70.0

80.0

70.0

90.0

70.0

80.0

70.0

3

36

.1

376

13

78

.2α

−C

op

aene

0.9

00.8

30.5

51.1

81.4

50.8

50.0

50.4

30.4

50.4

70.5

10.7

20.7

40.5

20.7

0

38

.1

391

13

93

.6β

−E

lem

ene

2.8

03.4

81.7

73.2

72.7

9 2

.11

3.4

02.4

43.1

71.3

31.7

22.6

02.8

11.1

31.8

3

39

.-

14

04

.4S

ibir

ene

0.3

40.7

10.3

20.4

20.3

40.0

3tr

0.4

40.3

90.4

00.2

40.4

30.4

60.2

80.2

0

40

.1

402

14

08

.4L

on

gif

ole

ne

0.0

60.0

60.0

30.0

60.1

0tr

0.0

40.0

4T

r0.0

40.0

30.0

40.0

50.0

40.0

6

41

.1

418

14

23

.8tr

ans-

Car

yop

hy

llen

e2.7

32.4

01.9

53.7

04.0

52.3

93.7

03.1

03.8

31.8

12.8

42.7

32.8

72.2

32.5

0

42

.1

420

14

33

.4β

−C

op

aene

0.4

1T

r0.2

30.4

60.4

10.3

0-

-0.3

90.3

20.5

70.0

80.5

80.5

50.2

8

43

.1

433

14

37

.4γ−

Ele

men

e2.6

52.2

21.4

22. 36

2.5

21.9

16.4

03.4

03.1

70.9

82.7

71.8

82.4

31.4

32.0

2

44

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439

14

43

.6A

rom

aden

dre

ne

0.0

70.1

00.0

70.1

40.1

20.0

30.0

80.0

70.1

00.0

50.0

60.0

70.0

70.0

30.0

3

45

.-

α−

Am

orp

hen

e0.2

70.1

40.3

00.2

40.1

50.3

80.2

10.2

40.3

30.2

40.3

30.2

90.1

2

47

.-

14

55

.4M

uu

rola

-3,5

-die

n

0.1

4-

0.0

90.1

80.1

60.0

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40.0

9tr

trtr

-0.0

5

48

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454

14

58

.9α

-Hu

mu

len

e3.5

83.1

12.0

94.6

0 4

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3.0

64.0

83.2

63.3

82.1

5 2

.90

3.3

43.5

9 2.0

2.4

6

49

.-

14

68

.3(+

)-E

pi-

bicy

clo

sesq

uiph

ella

n-dr

ene

0.38

0.66

0.28

0.43

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0.69

0.33

0.40

0.2

80.

10

50.

1477

1482

.7γ−

Muu

role

ne1.

201.

220.

691.

32tr

0.45

1.00

0.86

0.98

0.44

-1.

041.

120.

400.

33

51.

1480

1486

.9G

erm

acre

ne D

9.11

9.61

3.87

10.2

27.

208.

636.

847.

208.

554.

973.

078.

028.

582.

766.

65

52.

1485

1492

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−S

elin

ene

0.44

0.42

0.73

0.48

0.49

0.30

0.82

0.43

0.56

0.25

0.30

0.70

0.72

0.17

0.32

53.

1494

1500

.2α

−S

elin

ene

0.78

--

0.85

--

-tr

1.90

-tr

1.58

-0.

330.

86

54.

1494

1501

.7B

icyc

loge

rmac

rene

1.05

0.95

0.54

0.85

1.07

1.64

-2.

08-

0.98

-1.

58-

--

55.

1495

1503

.9α

−M

uuro

lene

0.93

1.01

0.55

1.12

0.10

0.12

2.63

tr1.

020.

521.

610.

842.

650.

870.

42

56.

1513

1511

.5δ−

Cad

inen

e0.

32

0.34

0.17

0.33

4.17

tr0.

260.

210.

290.

160.

600.

220.

250.

090.

07

57.

1524

1520

.3γ−

Cad

inen

e1.

431.

470.

661.

321.

040.

121.

070.

941.

402.

430.

631.

351.

660.

580.

46

58.

-15

32.3

Cad

inen

e (β

)3.

705.

402.

084.

634.

172.

154.

312.

814.

94-

2.10

3.82

4.24

1.57

2.02

59.

1532

1538

.8tr

ans-

Cad

ina-

1.4-

dien

e 0.

320.

330.

180.

330.

290.

130.

250.

21tr

0.14

0.13

0.21

0.26

0.11

0.10

60.

1538

1543

.9α

−C

adin

ene

0.67

0.63

0.35

0.62

0.55

0.13

0.52

0.43

0.68

0.24

0.30

0.42

0.50

0.18

0.14

61.

1542

1549

.8S

elin

a-3,

7(11

)-di

ene

0.27

0.22

0.17

0.27

0.29

0.07

0.21

0.20

0.29

0.12

0.14

0.19

0.22

0.63

0.07

63.

1556

1566

.4G

erm

acre

ne B

1.47

1.21

0.68

1.28

1.20

1.19

1.51

1.60

2.15

0.83

0.34

0.93

0.94

0.15

0.73

73.

1674

1683

.9C

adal

ene

--

--

--

--

--

-0.

11

0.06

--

Макед. фарм. билт., 57 (1, 2) 43 - 51 (2011)

47 Chemical composition of berry essential oils from Juniperus communis L. (Cupressaceae) growing wild in.... N

o.d

KIb

KI ex

pcC

ompo

nent

sJC

1JC

2JC

3JC

4JC

5JC

6JC

7JC

8JC

9JC

10JC

11JC

12JC

13JC

14JC

15

To

tal

(%)

37.4

34.1

520

.48

42.2

536

.20

26.9

636

.94

31.7

138

.50

19.5

922

.38

35.0

37.5

117

.45

23.4

9

Oxi

gen

cont

aini

ng s

esqu

iter

pene

s

62.

1549

1556

.0E

lem

ol0.

180.

130.

060.

110.

100.

120.

300.

180.

260.

070.

09

0.12

0.15

0.03

0.07

64.

1574

1584

.3G

erm

acre

ne D

-4-o

l0.

160.

12-

tr-

0.60

tr0.

160.

070.

12

0.05

trtr

0.09

0.07

65.

1576

1587

.0S

path

ulen

ol0.

240.

11tr

0.17

0.18

0.10

0.56

0.23

0.51

1.19

0.10

0.53

0.85

-0.

15

66.

1581

1592

.0C

aryo

phyl

lene

oxi

de0.

210.

12-

tr-

-0.

360.

230.

400.

33T

r0.

530.

640.

080.

21

67.

1590

1592

.1V

irid

ifl o

rol

--

--

0.16

0.04

--

--

0.10

--

-0.

02

68.

1627

1636

.61-

epi-

Cub

enol

0.14

0.20

trtr

0.12

0.07

0.36

0.30

tr0.

210.

080.

310.

420.

070.

03

69.

1630

1641

.9E

udes

mol

(γ)

0.04

--

--

-0.

080.

060.

550.

04tr

0.09

0.12

tr0.

36

70.

1641

1650

.6τ-

Mur

olol

+ τ

-Cad

inol

0.

510.

480.

210.

480.

440.

261.

410.

800.

130.

520.

341.

121.

740.

230.

07

71.

1645

1655

.2α

-Muu

rolo

l (T

orre

yol)

0.11

0.09

tr0.

090.

080.

041.

720.

150.

360.

110.

340.

22tr

0.22

0.44

72.

1653

1664

.4α

-Cad

inol

0.

720.

610.

320.

030.

610.

300.

041.

042.

730.

660.

351.

441.

89-

-

74.

-17

07.6

Eud

esm

-7(1

1)-e

n-4-

ol-

--

--

--

0.03

0.07

-0.

010.

040.

04-

-

To

tal

(%)

2.3

11.

860.

590.

881.

691.

534.

833.

125.

083.

251.

463.

474.

760.

721.

42

Non

terp

ene

com

pone

nts

1.92

694

3.6

Tri

cycl

ene

0.03

0.05

0.05

-0.

05tr

tr0.

03tr

0.05

0.03

trtr

0.03

0.07

30.

1291

1293

.8U

ndec

anon

e-2

0.06

0.11

0.05

0.07

0.10

0.05

0.08

0.08

0.09

0.05

0.04

0.07

0.07

0.04

0.06

To

tal

(%)

0.0

90.1

60.1

00.0

70.1

50.0

70.1

00.1

10.1

10.1

00.0

70.0

90.0

90.0

70.1

3

TO

TA

L

of

iden

tifi

ed

com

po

nen

ts (

%)

91.3

94.6

698

.88

96.6

197

.43

97.5

794

.68

96.3

791

.08

98.5

699

.83

97.7

088

.06

99.8

298

.62

a -

esse

ntia

l oi

ls o

f pl

ants

col

lect

ed f

rom

the

JC

1-JC

15 a

reas

; b

-

Kov

ats

inde

x: r

eten

tion

ind

ices

rel

ativ

e to

hom

olog

ous

seri

es o

f C

9 -

C24

n-a

lkan

es o

n D

B-5

MS

col

umn,

c -

Kov

ats

inde

x de

term

ined

exp

erim

enta

lly;

d -

nu

mb

er o

f co

mp

on

ents

in o

rder

to t

hei

r el

uti

on f

rom

HP

-5M

S c

olu

mn;

(-)

– n

ot

found,

tr =

tra

ce (

< 0

.02%

);

[are

as o

f co

llec

tion

: JC

1 –

Mav

rovo

(1);

JC

2 –

Sko

pje;

JC

3 –

Res

en; J

C4

– D

emir

His

ar; J

C5

– P

rile

p, J

C6

– K

arao

rman

Mtn

.; J

C7

– K

icev

o(1)

; JC

8 –

Kic

evo(

2); J

C9

– D

ebar

; JC

10-

Mav

rovo

(2);

JC

11 –

Kic

evo(

3); J

C12

– J

akup

ica

Mtn

., JC

13 –

Pel

iste

r M

tn.,

JC14

– B

istr

a M

tn.;

JC

15 –

Mak

edon

ski

Bro

d];

n=3.

48

Maced. pharm. bull., 57 (1, 2) 43 - 51 (2011)


1.74% (JC13) (Table 1).Analysis of the obtained essential oils from juniper

berries from R. Macedonia revealed a great variability in the chemical composition. Considering the components which are required for the quality assessment of juniper oil by European Pharmacopeia monograph, variability in the content of α-pinene, myrcene, sabinene, limonene and β-pinene occur in huge range (Table 1 and 2).

Evaluation of the chemical composition and the con-tent of selected components in the investigated essential oils showed that twelve Macedonian samples comply with Ph. Eur. 7 requirements (α-pinene 20-50%; myrcene 1-35.5%, sabinene < 20%, limonene 2-12%; β-pinene 1-12%; trans

(E) caryophyllene < 7%; terpinen-4-ol 0.5-10%; bornyl ac-

etate < 2% and α-phellandrene < 1%). However, three oil samples obtained from the berries from Karaorman (JC6), Kicevo (1) (JC7) and Debar (JC9) contained insuffi cient

amount of α-pinene (18.62%, 18.32% and 15.59%, respec-tively) nevertheless the amounts of other components were

satisfactory. Eventually prepared mixture of equal parts of all fi fteen investigated juniper oils, mix oil, should have

average percentages of the components that comply with

the Ph. Eur. 7 requirements for chemical composition qual-

ity (Table 2). This could be a solution for solving problems

that appeared on chemical composition of the juniper es-

sential oil from berries originated from different areas of

collection. However, for complete assessment of the quali-

ty of the Macedonian juniper oils requires further analysis

on several physical and chemical characteristics of the oils,

set under the section tests as well as other features of the oil

mentioned in the Juniper oil monograph.

Differences and the variability in the oil composition

of juniper essential oils were reported for the oils originat-

ed from different regions in Europe and in America. The

main constituents of the oils are pinenes, mostly α-pinene

which could be present in a wide range, from 27% in the

Greek samples (Chatzopoulou and Katsiotis, 1993), over

28.6-38.2% in Montenegro (Damjanovic et al., 2006),

Fig. 1. Main fractions in the essential oils from Macedonian samples of juniper berry (JC1 –JC15 oil samples).

Fig. 2. Terpene hydrocarbons/oxygen containing terpenes ratio in the essential oils of Macedonian juniper berry (JC1–JC15 oil samples).

Макед. фарм. билт., 57 (1, 2) 43 - 51 (2011)

49 Chemical composition of berry essential oils from Juniperus communis L. (Cupressaceae) growing wild in....

and up to 46,63% in the Iranian samples (Rezvani, 2010).

Other important components such as sabinene, germacrene

D, myrcene, β-pinene and limonene were found in higher

amounts in juniper oil (Chatzopoulou and Katsiotis, 1993;

Orav et al., 2010; Pepeljnjak et al., 2005). Terpine-4-ol

is an important constituent of juniper oil, usually consid-

ered to be responsible for the diuretic effect of the oil. Its

content in juniper oil can be variable and it was attained

in small amount of 1.37% in Greek samples and 2.86% in samples from Iran (Chatzopoulou and Katsiotis, 1993; Rezvani, 2010).

Conclusion

GC/FID/MS analysis of the chemical composition of fi fteen samples of juniper essential oils from berries of wild growing juniper in Republic of Macedonia revealed 74 identifi ed components that represent 91.08–99.83% of

all of the samples. The predominant fraction of the oils was

monoterpene hydrocarbons representing 39.11-73.38%.

Great variability in the chemical composition and especial-

ly in the content of some components was observed. The

most variable were α-pinene (15.59-43.19%), β-pinene

(1.65%-5.35%), β-myrcene (2.89%-26.50%), sabinene

(2.80-11.77%), and limonene (2.90-4.46%). In the frac-

tion of oxygen containing monoterpenes the most abun-

dant was terpene-4-ol, in traces and up to 6.32% followed

by α-ter pineol (0.18-1.63%). In the fraction of sesquiter-

pene the predominant components were: germacrene D

(2.76-10.22%), β-ele mene (1.13-6.40%) and trans-(E)-

caryophyllene (1.8%-4.05%). Assessment of the chemi-

cal composition quality of the oils in accordance with the

European Pharmacopoeia monograph of juniper oil showed

that twelve Macedonian samples comply with Ph. Eur. 7

requirements and three samples possess lower amounts of

α-pinene, under the requested level of min 20%.

Table 2. Variations in the main components composition of the essential oils obtained from berries of Macedonian Juniperus

communis

ComponentsJuniper oils

(%)

Ph.Eur. 7

(%)Score 1

1. α-Pinene

Min

15.59

Max

39.14 20 - 50 (−)2. Myrcene (β) 2.89 26.50 1-35,5 (+)

3. Sabinene 2.80 11.77 < 20 (+)

4. Limonene 2.89 4.46 2 - 12 (+)

5. β-Pinene 1.65 4.10 1 - 12 (+)

6. trans (E)-Caryophyllene 1.81 4.05 < 7 (+)

7. Terpinen-4-ol tr 6.32 0.5 - 10 (−)

8. Bornyl acetate 0.17 0.37 < 2 (+)

9. α-Phellandrene 0.09 0.82 < 1 (+)

(+) - fulfi lled monograph’s requirements, (−) – not fulfi lled monograph’s requirements

Fig. 3. The abundance of MHC - monoterpene hydrocarbons, SHC - sesquiterpene hydrocarbons, OCM – oxygen contain-

ing monoterpenes and OCS – oxygen containing sesquiterpenes in essential oils from Macedonian juniper ber-

ry (JC1 –JC15 oil samples).

50

Maced. pharm. bull., 57 (1, 2) 43 - 51 (2011)


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Stassi, V., Verykokidou, E., Loukis, A., Harvala, C., Philianos, S., 1995. The antimicrobial activity of the essential oils of four Juniperus species growing wild in Greece, Flav. Fragr. J., 11 (1), 71-74. DOI 10.1002/(SICI)1099-1026(199601)11:1<71::AID-FFJ536>3.0.CO;2-9.

Yarnell, E., 2002. Botanical medicines for the urinary tract, World J. Urol. 20, 285-293. DOI 10.1007/s00345-002-0293-0.

Резиме

Хемиски состав на етеричнo маслo изолирано од бобинките

од Juniperus communis L. (Cupressaceae) кој самоникнато

расте во Република Македонија и проценка на хемискиот

состав на маслото во согласност со Европската фармакопеја

Флореша Села, Марија Карапанџова, Ѓоше Стефков, Светлана Кулеванова*

Институт за Фармакогнозија, Фармацевтски факултет, Универзитет ”Св. Кирил и Методиј”, Скопје,

Македонија

Клучни зборови: Juniperus communis, етерично масло од смрека, смрека, Република Македонија.

Анализата на хемиски состав на петнаесет примероци на етерично масло изолирано од смрека е направена со помош GC/FID/MS метод. Тринаесет примероци на бобинки беа собрани на различни локации во југо-западниот дел на Р. Македонија, а останатите два во централно-северенот регион. Изолацијата на етерични масла е направе-на со официјалниот метод на Европската фармакопеја со дестилација со водена пареа со користење на апаратурата

Макед. фарм. билт., 57 (1, 2) 43 - 51 (2011)

51 Chemical composition of berry essential oils from Juniperus communis L. (Cupressaceae) growing wild in....

по Клевенџер. Со GC/MS анализа идентификувани се вкупно 74 компоненти. Доминантната фракција на маслата се

монотерпенските јаглеводороди и истите сочинуваат 39,11-73,38% од вкупното етерично масло. Утврдена е голема

варијабилност во хемискиот состав и во содржината на определени компоненти. Најголеми варијации се забележа-

ни кај компонените како што се α-пинен (15,59-43,19%), β-пинен (1,65% -5,35%), β-мирцен (2,89% -26,50%), саби-

нен (2,80-11,77%) и лимонен (2,90-4,46%). Во фракцијата на монотерпените со кислород, најзастапен е терпен-4-ол

(6,32%) заедно со α-терпинеол (0,18-1,63%). Во сесквитерпенската фракција, најдоминантни компоненти се: гермак-

рен D (2,76-10,22%), β-елемен (1,13-3,40%) и trans-(Е)-кариофилен (1,8% - 4,05%). Дванаесет примероци на етерич-

но масло изолирано од плодови од македонска смрека одговараат на барањата на Европската фармакопеја за хемис-

киот состав на етерично масло од смрека, додека три примероци не одговараат на овие барања поради помалата со-

држина на α-пиненот.


UDC: 615.213.074:543.544.5 Original scientifi c paper

Introduction

Carbamazepine (CBZ) (5-H-dibenze [b,f] azepine-5-carboxamide), a tricyclic lipophilic compound is a fi rst line antiepileptic drug used in the treatment of partial and gen-

Optimization and validation of bioanalytical SPE – HPLC

method for the simultaneous determination of carbamazepine

and its main metabolite, carbamazepine-10, 11-epoxide, in plasma

Jasmina Tonic – Ribarska1*, Zoran Sterjev2, Emilija Cvetkovska3, Igor Kuzmanovski3, Gordana Kiteva3, Ljubica Suturkova2, Suzana Trajkovic - Jolevska1

1 Institute of Applied Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy, University “Ss Cyril and Methodius”,

Skopje, Macedonia2Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, University “Ss Cyril and Methodius”, Skopje, Macedonia

3Clinic of Neurology, Faculty of Medicine, University “Ss Cyril and Methodius”, Skopje, Macedonia

Received: December 2011; Accepted: March 2012

Abstract

Carbamazepine is widely used as an antiepileptic drug in the treatment of partial and generalized tonic-clonic seizures. Carbamazepine 10,11-epoxide is the most important metabolite of carbamazepine, because it is a pharmacologically active compound with anticonvulsant properties. According to that, the routine analysis of carbamazepine 10,11-epoxide along with carbamazepine may provide optimal thera-peutic monitoring of carbamazepine treatment.

The aim of this study was to optimize and validate a simple and reliable solid - phase extraction method followed by RP-HPLC for the simultaneous determination of plasma levels of carbamazepine and carbamazepine-10,11-epoxide, in order to assure the implementation of the method for therapeutic monitoring.

The extraction of the analytes from the plasma samples was performed by means of a solid-phase extraction procedure. The separation was carried out on a reversed-phase column using isocratic elution with acetonitrile and water (35:65, v/v) as a mobile phase. The temper-ature was 30°C and UV detection was set at 220 nm.

The extraction yield values were more than 98% for all analytes, measured at four concentration levels of the linear concentration range. The method displayed excellent selectivity, sensitivity, linearity, precision and accuracy. Stability studies indicate that stock solu-tions and plasma samples were stabile under different storage conditions at least during the observed period.

The method was successfully applied to determine the carbamazepine and carbamazepine-10,11-epoxide in plasma of epileptic pa-tients treated with carbamazepine as monotherapy and in polytherapy.

In conclusion, the proposed method is suitable for application in therapeutic drug monitoring of epileptic patients undergoing treat-ment with carbamazepine.

Key words: carbamazepine, carbamazepine 10, 11-epoxide, plasma, solid-phase extraction, high-performance liquid chromatography, validation

* [email protected] ; [email protected]

eralized tonic-clonic seizures. The main metabolic path-way of CBZ is epoxidation, which is catalyzed primarily by the cytochrome P450 enzyme CYP3A4 and results in the formation of carbamazepine-10,11-epoxide (CBZ-EP). From a clinical standpoint, CBZ-EP is the most important of the 33 metabolites of CBZ that have been isolated, be-cause it is a pharmacologically active compound with an-ticonvulsant properties, as its parent compound (Sillanpaa

54

Maced. pharm. bull., 57 (1, 2) 53 - 61 (2011)

Jasmina Tonic-Ribarska, Zoran Sterjev, Emilija Cvetkovska, Igor Kuzmanovski, Gordana Kiteva, Ljubica Suturkova, Suzana Trajkovic - Jolevska

et al., 2009).Carbamazepine is highly bound (75% to 80%) to plas-

ma proteins, including albumin and α1–acid glycopro-

tein, while the epoxide binding is less strong (50% to 60%

bound). Since, the epoxide has a greater percentage in the

free form (pharmacologically active fraction) and is equi-

potent to the parent drug, epoxide is assumed that contrib-

utes signifi cantly to both therapeutic and adverse effects

(Sillanpaa et al., 2009; Potter and Donnelly, 1998).

CBZ is usually administered at oral daily doses rang-

ing from 400 to 1600 mg, which result in CBZ plasma con-

centrations in therapeutic range of 4-12 µg/ml. With mono-

therapy of CBZ, the steady-state of CBZ-EP plasma con-

centration is equivalent to 20%-25% of the parent drug

(Sillanpaa et al., 2009; Shen et al., 2001; Liu and Delga-

do, 1999).

Carbamazepine has a narrow therapeutic index, and

the relationship between dose and plasma concentrations

of carbamazepine may be unpredictable because of differ-

ences in genetics, age, gender, absorption, autoinduction

and disease state between individuals. Also, the presence

of numerous clinically signifi cant drug interactions sup-

ports the need of using therapeutic monitoring of carbam-

azepine as an essential tool in designing a safe and effec-

tive therapeutic regimen for patients with epilepsy (Sillan-

paa et al., 2009; Patsalos et al., 2008).

CBZ-EP was not routinely measured, though there

were patients for whom high concentrations of this metab-

olite could be responsible for otherwise unexplained toxic-

ity (Patsalos et al., 2008).

Therefore, the routine analysis of CBZ-EP along with

CBZ may provide optimal therapeutic monitoring of CBZ

treatment (Potter and Donnelly, 1998).

Most commonly applied methods for the routine mon-

itoring of CBZ alone in plasma were commercial reagent-

based techniques like as fl uorescence polarization immu-

noassay (FPIA) and enzyme multiplied immunoassay tech-

nique (EMIT), especially in clinical settings (Kang et al.,

2011). However, the antibodies used in these procedures,

may sometimes cross-react with CBZ metabolites, lead-

ing to an overestimation of CBZ plasma levels, and to the

lower specifi city of the immunoassays (Kang et al., 2011;

Eadie, 1998; Wilson et al., 1992).

A number of HPLC methods for simultaneous deter-

mination of CBZ and its metabolites in plasma have been

published, using pretreatment techniques such as liquid-

liquid extraction (Oh et al., 2006; Moreno et al., 2003; Ma-

tar et al., 1999; Pienimaki et al., 1995; Miller and Vrander-

ick, 1993), solid-phase extraction (Vermeij and Edelbroek,

2007; Mandrioli et al., 2001), deproteinization (Leite et al.,

2009; Yoshida et al., 2006) and stir bar-sorptive extraction

(Querioz et al., 2008). However, with some of these tech-

niques the obtained extraction yields were not satisfactory,

most of them were time-consuming, some required expen-

sive instrument, rendering them not appropriate for rou-

tine drug monitoring. Deproteinization is simple, fast and

costless technique, but offers minimal selectivity and sam-

ple cleanup as it only removes gross levels of protein from

a sample prior to analysis. Oh et al. (2006) described a liq-

uid – liquid extraction with methyl tert– butyl ether as sam-

ple pretreatment for simultaneous determination of CBZ

and CBZ-EP in plasma. Even though the method showed

excellent sensitivity, the extraction recoveries for CBZ-EP

were less than 90%, the analysis was time-consuming, and

also, the chromatographic run was 30 min. Another meth-

od for simultaneous determination of CBZ and CBZ-EP

in plasma (along with phenytoin and phenobarbital) was

described by Queiroz et al. (2008). They developed a stir

bar-sorptive extraction, but this procedure required a very

long analysis time (100 min), expensive instruments, and

the analysis was cumbersome.

LC-MS methods (Sener et al., 2007; Zhu et al., 2005;

Van Rooyen et al., 2002) have also been reported for the

determination of CBZ and its metabolites in biological fl u-

ids, but although they provide improved sensitivity and

specifi city compare with other analytical methods, MS

procedures were more sophisticated and more expensive

than HPLC-UV.

Therefore, HPLC with UV detection was the meth-

od of choice for the simultaneous monitoring of CBZ and

CBZ-EP in biological fl uids, after suitable pretreatment.

The aim of this study was to optimize and validate a

simple and reliable solid - phase extraction method fol-

lowed by RP-HPLC with UV detection for the simultane-

ous determination of plasma levels of carbamazepine and

its main metabolite, carbamazepine 10, 11-epoxide, in or-

der to obtain results of adequate quality and reliability to

assure the implementation of the proposed bioanalytical

method for the therapeutic monitoring of carbamazepine

and its active metabolite.


Chemicals and solutions

Carbamazepine (CBZ), its metabolite carbamazepine-

10,11-epoxide (CBZ-EP) and nitrazepam used as the inter-

nal standard,(IS), were purchased from Sigma-Aldrich (St.

Luis, MO, USA). Methanol and acetonitrile, HPLC grade,

were obtained from Merck (Darmstadt, Germany). For all

analysis HPLC grade water purifi ed with a TKA_LAB Re-

instwasser system (Niederelbert, Germany) was used. OA-

SIS® HLB cartridges (30mg/1mL) used for the solid-phase

extraction (SPE) procedure were supplied by Waters (Mil-

ford, MA, USA).

Stock solutions of CBZ (500 µg/ml), CBZ-EP (200

µg/ml) and the IS (500 µg/ml) were prepared by dissolv-

ing each compound in methanol. The stock solutions were

stable for at least 3 months stored at 2-8 ºC. Working solu-

tions were prepared daily from stock solutions by dilution

with purifi ed water.

Макед. фарм. билт., 57 (1, 2) 53 - 61 (2011)

55 Optimization and validation of bioanalytical SPE – HPLC method for the simultaneous determination of carbamazepine and...

Apparatus and chromatographic conditions

The assay was carried out on Agilent 1100 HPLC sys-

tem equipped with a vacuum degasser (G1322A Degas-

ser), quaternary pump (G1311A QuatPump), autosam-

pler (G1313A ALS), column compartment (G1316A COL-

COM), diode array detector (G1315B DAD), and Chem-

Station for LC 3D software for data handling (Wilming-

ton, DE).

Separation was performed on a reversed-phase column

Zorbax Extend C18 (150 x 4,6 mm, 5µm) using isocrat-ic elution with acetonitrile and water (35:65, v/v) as a mo-bile phase, at a fl ow rate of 1 ml/min. The temperature was 30°C, volume of injection 20 µl and UV detection was set at 220 nm.

Human plasma sampling

Plasma samples from both, healthy volunteers (drug – free plasma for the method validation) and from the epi-leptic patients undergoing chronic CBZ therapy, were ob-tained from Clinic of Neurology, Faculty of Medicine, Uni-versity “Ss Cyril and Methodius”, Skopje. The participa-tion of each subject was voluntary and could be cancelled by any individual at any time during this study (according to the Helsinki II declaration). The Ethics Committee at the Faculty of Pharmacy and the Faculty of Medicine, Ss. Cyril and Methodius University – Skopje, approved the re-search protocol for this study and all volunteers signed the Study Informed Consent form.

Since CBZ has a relatively short half –life, sampling time in relation to dose ingestion was important for the in-terpretation of the drug concentration. Ideally, blood sam-ples for TDM of CBZ should be drawn before the morn-ing dose (Sillanpaa et al., 2009). Thus, blood samples were collected at 08.00 h in the morning, just before the fi rst daily drug administration, into heparinised tubes (8-10 IU heparin/ml blood), and centrifuged at 3000 rpm for 10 min. The supernatant plasma was transferred into test tubes and frozen at - 20 °C until analysis.

Solid-phase extraction procedure (SPE)

Prior to analysis, plasma samples were thawed and al-lowed to equilibrate at room temperature. To 250 µl of ep-ileptic patient plasma, 200 µl ultrapure water and 50 µl of IS solution (5µg/ml) were added and the mixture was vor-tex-mixed for 30s.

The extraction of the analytes from the plasma sam-ples was performed by means of a solid-phase extrac-tion (SPE). For this purpose, OASIS® HLB cartridges (30mg/1ml) were used. The SPE procedure was carried out according to the following steps: a) conditioning with 1ml methanol; b) equilibration with 1 ml water; c) loading the plasma sample (of patients as described above and for val-idation purpose as described under the section Calibration

curves); d) washing with 1ml 5% methanol. Cartridge dry-

ing for 30 s at - 20 kPa; e) elution with 500 µl methanol. 20 µl of the eluate was injected into the HPLC system.

Bioanalytical method validation

Method validation was conducted following the rec-ommendations for validation of bioanalytical methods of EMA guideline (EMA, 2009).

According to the guideline, a complete method valida-tion should be performed for any analytical method, new or based upon literature.

Selectivity

Selectivity was assessed by comparing the chromato-grams of drug-free plasma (blank plasma) from six sources and those obtained from plasma samples spiked with ana-lytes and internal standard.

Calibration curves

The calibration standards consist of drug free plasma samples spiking with known concentration of the analyte. Aliquots of 50 µl of analytes working solutions (CBZ and CBZ-EP) at 7 different concentrations, 50 µl of IS work-ing solution, and 100 µl ultrapure water were added to 250 µl of blank plasma. The resulting plasma concentrations were: 0.25 µg/ml, 0.5 µg/ml, 1.0 µg/ml, 5.0 µg/ml, 10.0 µg/ml, 20.0 µg/ml and 25.0 µg/ml for CBZ and 0.1 µg/ml, 0.25 µg/ml, 0.5 µg/ml, 1.0 µg/ml, 2.5 µg/ml, 5 µg/ml and 10 µg/ml for CBZ-EP, containing the IS at constant con-centration of 5 µg/ml. Calibration standards were subject-ed to the SPE procedure and injected into the HPLC. The obtained analyte - IS peak area ratios were plotted versus the respective analyte concentrations and the calibration curves for CBZ and CBZ-EP were constructed by means of the least-squares method.

Accuracy and precision

The quality control samples (QC samples) were used to assess the accuracy and precision of the method. Four levels of QC samples were prepared at the concentrations of 0.25 µg/ml (lower limit of quantitation), 1.0 µg/ml (low QC sample), 5.0 µg/ml (medium QC sample) and 20.0 µg/ml (high QC sample) for CBZ and at the concentrations of 0.1 µg/ml (lower limit of quantitation), 0.5 µg/ml (low QC sample), 2.5 µg/ml (medium QC sample) and 5.0 µg/ml (high QC sample) for CBZ-EP, in same way as described above for the calibration standards. Accuracy and preci-sion were evaluated for the values of the QC samples ob-tained within a single run (the within run) and in different runs (the between-run). Within-run accuracy and precision were determined for fi ve samples per concentration level at LLOQ, low, medium and high QC samples in a single run. Between-run accuracy and precision were assessed by

56

Maced. pharm. bull., 57 (1, 2) 53 - 61 (2011)


fi ve determination per concentration per run at LLOQ, low, medium and high QC samples from three runs analyzed on two different days.

Recovery

Extraction yields were assessed at four concentration levels corresponding to the lower limit, low, medium and high point of each calibration curve (i.e. plasma concentra-tion of 0.25 µg/ml, 1.0 µg/ml, 5.0 µg/ml and 20 µg/ml for CBZ, and 0.1 µg/ml, 0.5 µg/ml, 2.5 µg/ml and 5.0 µg/ml for CBZ-EP).

Stability

Stability tests were performed on three replicates of low and high QC samples after 24 h at room temperature (short term stability), after three freeze-thaw cycles, au-tosampler stability for 12 h, and after 90 days on samples stored at - 20 °C (long term stability). Stability tests were also performed on stock solutions of analytes after 24 h at room temperature and after 3 months at 2-8 °C.


Optimization of chromatographic condition

Many studies were conducted with the aim of develop-ing new bioanalytical methods or improve existing meth-ods for TDM of antiepileptic drugs.

In order to establish the chromatographic conditions for the determination of CBZ and CBZ-EP in plasma, num-ber of literature data were reviewed and many experiments were made. During the optimization of the chromatograph-ic conditions different mobile phases (different types of or-ganic solvents and different organic to aqueous phase ra-tio) were evaluated, in order to ascertain whether a faster separation is possible, which is an important fact for rou-tine control. The experiments were made using the mixture of methanol/water, the mixture of methanol/acetonitrile/water, and the mixture of acetonitrile/water, in different ra-tio compared the organic and water phase.

The best results of good separation of all analytes were obtained with the mixture of acetonitrile–water (35 : 65, v/v), using a Zorbax Extend C18 (150 x 4,6 mm, 5µm) col-umn at temperature of 30 ºC and fl ow rate of 1ml/min.

Under these chromatographic conditions, CBZ, its ac-tive metabolite CBZ-EP and the internal standard were baseline separated in less than 7 min, in contrast to 30 min reported by Oh et al. (2006) and 20 min reported by Leite et al. (2009).

Solid-phase extraction (SPE) procedure

Application of a SPE technique was chosen for the sample pre-treatment, because it makes sample preparation more feasible, simple and accurate, less polluting than the

liquid-liquid extraction, and allows high extraction yields with good selectivity.

Mandrioli et al. (2001) performed a SPE procedure for extraction of CBZ and its fi ve metabolites from human plasma using tetrahydrofuran for elution. However, the us-age of this strong solvent caused peak broadening and in-terference, so its completely elimination from injection mixture was necessary.

In order to simplify the SPE procedure and consider-ing the good solubility of CBZ and CBZ-EP in methanol, elution of the analytes from the cartridge was performed with methanol (instead of tetrahydrofuran), thus making the method less prone to interference. Furthermore, the last step of procedure, the dryness of the eluate, was avoided.

The optimized SPE procedure included loading the conditioned cartridge with 250 µl of plasma, washing with 1 ml of 5% methanol and eluting the analytes with 500 µl of methanol. The extraction yield values were higher than 98% for CBZ and CBZ-EP, which were better than those reported in papers using liquid-liquid extraction (Oh et al., 2006) or SPE procedure (Mandrioli et al., 2001). The chro-matograms of a blank plasma sample and of a blank plasma spiked with standard solutions of analytes and IS, subject-ed to the described SPE procedure, were presented in Fig. 1. No interference from endogenous plasma components was present. Moreover, the SPE procedure gave excellent results when was applied to plasma samples of patients un-dergoing chronic treatment with CBZ, both in monothera-py and polytherapy. No interfering peaks due to the co-ad-ministered drugs were eluted at the retention times of an-alytes (Fig. 3.).

Bioanalytical method validation

Validation of the bioanalytical method is a compulso-ry step to estimate the ability of the developed method to provide accurate results for its routine application (Rozet et al., 2011).

Selectivity

The selectivity of the method was assessed by analyz-ing six different drug free plasma samples and drug free plasma spiked with CBZ, CBZ-EP and nitrazepam as in-ternal standard. The chromatograms of blank plasma and blank plasma spiked with analytes were presented in Fig.1. The retention times for CBZ, CBZ-EP and IS were 2.8 min, 4.7 min and 6.6 min, respectively. No interfering peaks for endogenous compounds were observed in the retention times of CBZ, CBZ-EP and IS.

Calibration curves

Prior to carrying out the validation of the bioanalytical method the concentration range should be justifi ed based on scientifi c information. The range should be covered by

Макед. фарм. билт., 57 (1, 2) 53 - 61 (2011)


the calibration curve range, defi ned by the lower limit of

quantitation (LLOQ) and the upper limit of quantitation

(ULOQ). LLOQ is the lowest amount of analyte in a sam-

ple (plasma) which can be quantifi ed reliably and should

be adapted to expected concentrations and to the aim of

the study.

The acceptable criterion for each standard concentra-

tion was ± 15% of the nominal value, except for the LLOQ

which was ± 20%.

Seven-point calibration curves were set up for CBZ

and CBZ-EP within the concentration range of 0.25 - 20.0

µg/ml for CBZ and 0.1 - 10.0 µg/ml for CBZ-EP. The cal-

ibration curves were linear over the defi ned concentration

ranges according to the levels of each compound expected

in patients’ plasma samples, including LLOQ.

The regression equations were y = 154.87x -19.828

with a coeffi cient of determination r2 = 0.9989 for CBZ

and y = 117.96x +1.219 with a coeffi cient of determination r2 = 0.9991 for CBZ-EP.

Accuracy and precision

The within – run and between – run accuracy and pre-cision were shown in Table 1. All the results of the tested samples were within recommended limits. Within-run as-say precision ranged from 0.6% to 1.2% for CBZ and from

0.7% to 2.0% for CBZ-EP, while within-run assay accura-

cy ranged from 98.4% to 101.3% and 97.0% to 100.6%,

for CBZ and CBZ-EP, respectively. The between-run pre-

cision and accuracy, ranged from 0.9% to 2.2% and 97.9%

to 102.1% for CBZ, while for CBZ-EP the ranges were

0.9% to 3.1% and 96.7% to 102.7%, respectively.

Recovery (Extraction yield)

Recovery describes the extraction effi ciency of the an-

alytical process. The recovery was calculated by compar-

ison of the peak areas of analytes obtained from the SPE

processed blank plasma sample previously spiked with an-

alytes versus peak areas obtained from a post – extracted

spiked blank plasma sample, which represent 100% recov-

ery (Matuszewski et al., 2003).

a)

b)

Fig. 1. Chromatograms of: a) blank plasma, and b) blank plasma spiked with 5.0 µg/ml CBZ, 2.5 µg/ml CBZ-EP and

5.0 µg/ml nitrazepam (IS), after the SPE procedure.

58

Maced. pharm. bull., 57 (1, 2) 53 - 61 (2011)


The extraction yields were evaluated at four concen-tration levels (LLOQ, low QC, medium QC and high QC sample) of the linear concentration range for both, CBZ and CBZ-EP. The obtained values were in the range of 98.5% - 99.6% for CBZ and 99.1% - 99.7% for CBZ-EP (Table 2), while the recovery value for IS was 98.9%. These results indicate excellent recoveries for all analytes which means that the proposed SPE procedure is appropriate to be ap-

plied to the determination of CBZ and its active metabolite in plasma taken from epileptic patients.

Stability

Stability experiments were performed through evalu-ation the stability of CBZ and CBZ-EP, and also IS stock

Table 1. Accuracy and precision of carbamazepine and carbamazepine-10,11-epoxide in human plasma

AnalyteNominal conc.

(µg/ml)

Within-run assay (n=5) Between-run assays (n=30)

accuracy (%) precision (CV%) accuracy (%) precision (CV%)

CBZ 20.0 99.2 1.2 98.6 2.25.0 99.5 0.8 98.5 1.11.0 101.3 1.0 102.1 0.90.25 98.4 0.6 97.9 1.8

CBZ-EP 5.0 100.6 1.1 100.9 1.02.5 99.1 2.0 97.6 3.10.5 97.3 0.8 96.7 1.70.1 97.0 0.7 102.7 0.9

Table 2. Recoveries of carbamazepine and carbamazepine-10,11-epoxide from plasma (n=5)

AnalyteNominal conc.

(µg/ml)Found conc.

(µg/ml)Recovery

(%)CBZ 20.0 19.70 98.5

5.00 4.98 99.61.00 0.99 99.00.25 0.247 98.8

CBZ-EP 5.00 4.98 99.62.50 2.48 99.20.50 0.497 99.40.10 0.099 99.1

Table 3. Stability of carbamazepine and carbamazepine-10,11-epoxide in human plasma under various conditions (n=3)

Nominal concentration (µg/ml)CBZ / CBZ-EP

Initial concentrationt = 0 min (µg/ml)CBZ / CBZ-EP

Accuracy(%)

CBZ / CBZ-EPShort term stability (24h at room temperature)

1.0 / 0.5 1.02 / 0.51 99.0 / 98.620.0 / 5.0 19.98 / 5.03 101.2 / 99.3

Autosampler stability (after 12h)1.0 / 0.5 1.01 / 0.51 97.1 / 96.020.0 / 5.0 20.04 / 4.99 98.8 / 97.4

Three freeze-thaw cycles1.0 / 0.5 1.02 / 0.49 94.8 / 93.220.0 / 5.0 20.02 / 4.97 97.7 / 94.1

Long term stability(90 days at -20 °C) 1.0 / 0.5 0.99 / 0.50 98.8 / 99.1 20.0 / 5.0 19.89 / 5.10 100.6 / 99.3

Макед. фарм. билт., 57 (1, 2) 53 - 61 (2011)


solutions and plasma samples under different conditions.

Stock solutions of analytes were stable at room temper-

ature for 24 h (accuracy: 99.6%, 99.4% and 100.3% for

CBZ, CBZ-EP and IS, respectively) and at 2 - 8 °C for

3 months (accuracy: 101.7%, 98.3% and 98.1% for CBZ,

CBZ-EP and IS, respectively). It was shown that the hu-

man plasma samples spiked with CBZ and CBZ-EP were

stable after three freeze-thaw cycles, after 12 h in autosam-

pler and at room temperature for 24 h. The study indicated

that the plasma samples could be stored at – 20 °C for 90

days. The results of stability were presented in Table 3.

The obtained results indicate that the analytes were

stable under all storage conditions described above and

that no stability related problems would be expected dur-

ing the routine plasma sample analysis.

Application to patient plasma

The proposed method was applied to the simultane-

ous determination of CBZ and its active metabolite, CBZ-

EP, in plasma samples taken from epileptic patients un-

der chronic CBZ therapy (400 – 1200 mg/day). The chro-

matogram of a plasma sample from a patient, who received

400 mg/day of CBZ, taken 12 h after the last drug intake

was reported in Fig. 2. By interpolation on the respective

calibration curves, the following concentrations were ob-

Fig. 2. Chromatogram of plasma sample from a patient treated with 400 mg/day of CBZ, subjected to the developed SPE

procedure.

Table 4. Results of analysis on patient plasma samples

Determined concentration (µg/ml)

Patient no.Dosage

(mg/day)Co-administered drugs

FPIA method

CBZ

HPLC method

CBZ

HPLC method

CBZ-EP

1 400 - 4.96 4.69 0.922 400 - 8.98 5.71 0.883 600 - 6.83 6.54 1.114 400 - 8.04 7.32 0.74

5 400 - 4.26 4.02 0.40

6 400 - 4.73 4.71 0.557 800 - 7.05 6.49 1.008 400 - 4.73 4.02 0.819 800 Levetiracetame 8.09 7.55 1.3310 800 - 6.93 6.34 0.8311 800 Phenobarbital 3.31 2.89 0.6712 600 - 8.27 7.52 1.9213 1200 Lamotrigine 8.51 8.06 1.7814 800 Valproate 10.40 7.90 2.9015 1200 Levetiracetame, Lamotrigine 7.73 6.37 1.4516 800 - 6.86 6.14 1.0917 400 - 4.66 4.65 0.9218 1200 Levetiracetame, Lamotrigine, Topiramate 8.27 5.59 0.9519 400 - 4.73 4.05 1.1420 800 - 7.80 7.01 1.68

60

Maced. pharm. bull., 57 (1, 2) 53 - 61 (2011)


a)

b)

c)

Fig. 3. Chromatograms of plasma samples from patients treated with: a) carbamazepine and phenobarbital; b) carbam-azepine and valproate; c) carbamazepine, lamotrigine, topiramate and levetiracetam, subjected to the developed SPE procedure.

tained: 4.69 µg/ml for CBZ and 0.92 µg/ml for CBZ-EP. The results obtained from the assays on 20 patient plasma samples were reported in Table 4.

In this table the CBZ plasma concentrations obtained with the proposed HPLC method were also compared with those obtained from the same samples by FPIA method pre-

Макед. фарм. билт., 57 (1, 2) 53 - 61 (2011)


formed in the laboratory at Clinic of Neurology, Faculty of

Medicine, Skopje. Statistical analysis was performed using

GraphPad Prism software (version 5.0). A signifi cant linear

correlation was obtained (r = 0.8898, p < 0.0001). Com-

parison of the values by paired t - test indicated that the

FPIA concentrations were signifi cantly greater (p<0.001)

than those obtained by HPLC method. The probable rea-

son for this was interference by CBZ metabolites, mainly

by CBZ-EP, with FPIA technique (Kang et al., 2011; Wil-

son et al., 1992).

Given that epileptic patients are often treated with

polytherapy, it was important to assess probable chromato-

graphic interferences from potentially co-administered an-

tiepileptic drugs. To evaluate the selectivity of proposed

method, plasma samples taken from patients simultane-

ously treated with valproate, lamotrigine, topiramate, phe-

nobarbital and levetiracetam (commonly prescribed drugs

along with CBZ), were analysed. The chromatograms of

plasma samples of patients treated with polytherapy (pa-

tient 11: phenobarbital along with CBZ; patient 14: val-

proate along with CBZ, and patient 18: lamotrigine, topi-

ramate and levetiracetam along with CBZ) were present-

ed in Fig.3.

It was evident that no chromatographic interference

from co-administered drugs were observed in the retention

times of CBZ, CBZ-EP and IS, hence the method could be

applied for TDM of CBZ and CBZ-EP in patients undergo-

ing chronic treatment with CBZ, either as monotherapy or

in polytherapy with other antiepileptic drugs.

Conclusion

A simple and reliable bioanalytical HPLC method has

been optimizated and validated for the simultaneous deter-

mination of carbamazepine and its active metabolite, car-

bamazepine-10, 11-epoxide in human plasma after solid-

phase extraction.

The SPE procedure applied gave excellent recovery val-

ues for carbamazepine and carbamazepine-10, 11-epoxide.

All analytes were baseline separated in less than 7 min.

The validation data demonstrate that the proposed bio-

analytical method is selective, sensitive, linear, precise and

accurate.

The method was successfully applied to separate and

determine the CBZ and CBZ-EP in plasma obtained from

epileptic patients treated with CBZ as monotherapy and in

polytherapy.

According to all obtained results, it can be conclud-

ed that the proposed method is suitable for a reliable thera-

peutic drug monitoring of patients undergoing therapy with

carbamazepine, both in monotherapy and in polytherapy.

Since, it requires only 250 µl of plasma for one complete

analysis, the proposed method could be particularly advan-

tageous when multiple blood sampling were needed, as in

pharmacokinetic studies.

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Queiroz, R.H.C., Bertucci, C., Malfara, W.R., Dreossi, S.A.C., Chaves, A.R., Valerio, D.A.R., Queiroz, M.E.C., 2008. Quantifi cation of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and Phenobarbital in plasma samples by stir bar-sorptive extraction and liquid chromatography. J. Pharm. Biomed. Anal. 48, 428-434.

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Резиме

Оптимизација и валидација на биоаналитички SPE-HPLC

метод за истовремено определување на карбамазепин и

неговиот главен метаболит, карбамазепин-10,11-епоксид, во

плазма

Јасмина Тониќ - Рибарска1*, Зоран Стерјев2, Емилија Цветковска3,

Игор Кузмановски3, Гордана Китева3, Љубица Шутуркова2,

Сузана Трајковиќ - Јолевска1

1Институт за применета хемија и фармацевтски анализи, Фармацевтски факултет, Универзитет “Св Кирил и

Методиј”, Скопје, Македонија2Институт за фармацевтска хемија, Фармацевтски факултет, Универзитет “Св Кирил и Методиј”, Скопје,

Македонија3Клиника за неврологија, Медицински факултет, Универзитет “Св Кирил и Методиј”, Скопје, Македонија

Клучни зборови: карбамазепин, карбамазепин-10,11-епоксид, плазма, цврсто-фазна екстракција (SPE), HPLC, валидација

Карбамазепинот е најчесто употребуван антиепилептичен лек во третман на парцијални и генерализирани тонично-клонични

напади. Kарбамазепин-10,11-епоксидот е најзначаен метаболит на карбамазепинот, бидејќи е фармаколошки активна супстанција

со антиконвулзивни особини. Според тоа, рутинската анализа на карбамазепим-10,11-епоксид заедно со карбамазепин, може да

даде оптимални резултати во терапевтското следење на третманот со карбамазепин.

carbamazepine and carbamazepine 10,11-epoxide in human

plasma by tandem liquid chromatography-mass spectrometry

with electrospray ionisation. J. Chromatogr. B Analyt.

Technol. Biomed. Life. Sci. 25, 1-7.

Vermeij, T.A., Edelbroek, P.M., 2007. Robust isocratic

HPLC method for simultaneous determination of seven

antiepileptic drugs including lamotrigine, oxcarbamazepine

and zonisamide in serum after SPE. J. Chromatogr. B. 857,

40-46.

Wilson, J. F., Tsanaclis, L. M., Perrett, J. E., Williams, J., Wicks,

J. F. C., Richens, A., 1992. Performance of techniques for

measurement of therapeutic drugs in serum. A comparison

based on external quality assessment data. Ther. Drug Monit.

14, 98-106.

Yoshida, T., Imai, K., Motohashi, S., Hamano, S., Sato, M., 2006.

Simultaneous determination of zonisamide, carbamazepine

and carbamazepine-10,11-epoxide in infant serum by HPLC.

J. Pharm. Biomed. Anal. 16, 1386-1390.

Zhu, Y., Chiang, H., Wulster-Radcliffe, M., Hilt, R., Wong,

P., Kissinger, C.B., Kissinger, P.T., 2005. Liquid

chromatography/tandem mass spectrometry for the

determination of carbamazepine and its main metabolite in

rat plasma utilizing an automated blood sampling system. J.

Pharm. Biomed. Anal. 38, 119-125.

Макед. фарм. билт., 57 (1, 2) 53 - 61 (2011)


Целта на овој труд е да се оптимизира и валидира едноставен и сигурен SPE-HPLC метод за истовремено определување на

карбамазепин и неговиот главен метаболит, карбамазепин-10,11-епоксид во плазма, со цел да се добијат резултати со соодветен

квалитет и сигурност со што ќе се обезбеди примена на предложениот биоаналитички метод во следење на терапевтските

концентрации на карбамазепин и неговиот активен метаболит.

Екстракцијата на аналитите од примероците на плазма е изведена со помош на цврсто-фазна екстрактивна постапка.

Разделувањето на екстрахираните аналити е спроведено на реверзно фазна колона со изократско елуирање со мобилна фаза

составена од ацетонитрил и вода (35:65, v/v), на температура од 30 °С. Детекцијата е изведена на бранова должина од 220 nm.

Добиените вредности за приносот на екстракција се повисоки од 98% за сите аналити, определени во четири концентрациски

точки од линеарниот концентрациски опсег. Воспоставениот метод покажува одлична селективност, осетливост, линеарност,

прецизност и точност. Студиите за стабилност укажуваат дека работните раствори и примероците од плазма, чувани под различни

услови, се стабилни во тек на целиот период потребен за изведување на анализите. Методот е успешно применет за определување

на карбамазепин и карбамазепин-10,11-епоксид во плазма добиена од пациенти третирани со карбамазепин како монотерапија

или во политерапија.

Како заклучок, воспоставениот биоаналитички метод е соодветен за примена во следење на терапевтските концентрации

на карбамазепин и на неговиот активен метаболит, карбамазепин-10,11-епоксид, кај пациенти болни од епилепсија кои се под

терапија со карбамазепин.


UDC: 615.849.2 Original scientifi c paper

Introduction

The development of Nuclear Medicine towards molec-ular diagnostic imaging modality in the past two decades was followed by intensive research and applicative work resulting with introduction of wide variety of new genera-tion radiopharmaceuticals. Labeled peptides have proven their potential as targeting agents for receptor scintigraphy, especially the synthetic analogs with preferable biological half-life and in vivo stability. Octreotide, a somatostatin analog, was used to compound 111In-DTPA-octreotide (Oc-treoScan), the fi rst commercial radiolabeled peptide, reg-ulatory approved in Europe and USA for clinical applica-tion. It became the imaging agent of choice for detection of

“In-house” preparation of 99mTc-EDDA/HYNIC-TOC, a specifi c

targeting agent for somatostatin receptor scintigraphy

Sonja Kuzmanovska*, Olivija Vaskova, Marina Zdraveska Kocovska

Institute of Pathophysiology and Nuclear Medicine, Medical Faculty, University “Sts Cyril and Methodius”, Vodnjanska

17, Skopje, Republic of Macedonia,

Received: November 2011; Accepted: January 2012

Abstract

The use of radiolabeled peptide ligands as diagnostics and therapeutics in nuclear oncology has increased recently. One of the most frequently used radiopharmaceutical is 99mTc-EDDA/HYNIC-TOC, a somatostatin analog with affi nity for certain types of somatostatin re-ceptors, overexpressed in tumors of neuroendocrine origin. The radiopharmaceutical is not readily available; therefore we introduced its “in house” preparation within project activities supported by the International Atomic Energy Agency (IAEA). We optimized the radiolabeling protocol, prepared a small batch of frozen kits, performed ITLC quality control and animal biodistribution during the preclinical evaluation procedures. The co-ligand exchange labeling procedure was carried out at 100°C during 10 min, resulting in radiochemical purity >90%. The biodistribution scintigrams in normal Wistar rats showed rapid blood clearance after 15 min and predominant kidney accumulation af-ter 4 h, in accordance with the data reported by other authors. Storage stability of the formulated small batch frozen kit (-20°C) was evalu-ated within 6 months, with radiolabeling yield ranging between 94,3% and 96,9%. We conclude that frozen kit can be a safe alternative to the freeze-dried for small batch in house production, and after the satisfactory preclinical evaluation, the “in house” prepared 99mTc-EDDA/HYNIC-TOC can be introduced in clinical practice as specifi c targeting agent for somatostatin receptor scintigraphy.

Key words: radiopharmaceutical, technetium-99m, tyr3-octreotide, receptor scintigraphy, in house production, quality control

* [email protected],+389 2 3112831, fax: +389 2 3147203

somatostatin (SST) receptors, overexpressed in neuroen-docrine tumors (NET), with evidence of higher sensitiv-ity in comparison with CT and MR scanning (Shi et al., 1998). Besides the specifi c somatostatin receptor imaging properties, somatostatin receptor scintigraphy can be used as tumor staging agent (Lebtahi et al., 1997), as well as a predictor of the effectiveness of therapy with somatosta-tin analogues (Krenning et al., 1993). Despite of the fa-vorable features, 111In-DTPA-octreotide has limited avail-ability, suboptimal imaging gamma energy and high cost. Therefore, efforts were made by different research groups worldwide to replace the radiometal 111In with 99mTc, the most preferable imaging radionuclide for single photon emission computed scintigraphy (SPECT) up to date (Mai-na et al., 1995; Vallabhajosula et al.,1996; Decristoforo et al.,2000).

Peptide labeling with 99mTc-pertechnetate is however a complex task and considering the small size of peptide

66

Maced. pharm. bull., 57 (1, 2) 65 - 70 (2011)

Sonja Kuzmanovska, Olivija Vaskova, Marina Zdraveska Kocovska

molecules and limited number of donor atoms, the indi-rect labeling approach is shown to be the most preferable technique (Liu et al., 1997). Thus, a bifunctional chelator is used to conjugate the peptide molecule and simultane-ously form a coordination bound with the reduced 99mTc-pertechnetate. One of the earliest effi cient labeling meth-ods was reported by Maecke (Maecke and Béhé, 1996) and later developed by Decristoforo (Decristoforo and Mather, 1999). They used Tyr3octreotide (TOC) conjugated with HYNIC (hydrazinonicotic acid) as a bifunctional chelator. When using HYNIC as a bifunctional chelator, the com-plex has to be stabilized through the addition of a co-ligand or a mixture of co- ligands. To stabilize the 99mTc - HYNIC-TOC complex, EDDA (ethylendiamine N, N’ diacetic acid) was used as co-ligand, resulting with favorable biodistribu-tion, low blood levels and high renal excretion of the radio-pharmaceutical.

Since 99mTc-EDDA/HYNIC-TOC was evidenced to be an effi cient tool for detecting SST2 and SST5 recep-tor subtypes, the main targets for the somatostatin receptor scintigraphy are carcinoids, insulinomas, pituitary adeno-ma, pheochromocytoma, neuroblastoma, medulary thyroid carcinoma. The radiopharmaceutical is not readily avail-able, therefore our goal was to introduce it as “in house” product within the project activities supported by the Inter-national Atomic Energy Agency (IAEA), for updating and expanding the range of nuclear oncology services provid-ed by our institute.


- Chemicals with high purity grade were purchased by Merck, Germany (SnCl

2 ·

2H

2O), or granted by IAEA

(EDDA from Fluka Chemie Gmbh, Tricine from Sigma-Aldrich Chemie Gmbh).

- The peptide conjugate (HYNIC-TOC) was provided by Radioisotope Centre POLATOM, Poland

- 99mTc-pertechetate was obtained as fresh eluate from commercial 99Mo/99mTc generator (ELUMATIC-III) sup-plied by Cis biointernational, France

- For the product purifi cation SepPak C-18 mini car-tridges (Waters, Milford, USA) were used, and for the fi nal sterilization of the radiopharmaceutical 0,20 μ sterile Mil-

lipore fi lters

- For the quality control TLC-SG (Merck, 5553 Silica

gel 60) aluminum sheets were used

- Biodistribution studies were performed on normal fe-

male Wistar rats in accordance with EC Directive 86/609/

EEC for animal experiments

Preparation of the radiopharmaceutical

In order to optimize the radiopharmaceutical produc-

tion protocol concerning own facilities two approaches

were implemented:1. Production of a single dose wet labeled 99mTc-ED-

DA/HYNIC-TOC2. Production of a small-batch frozen kits for labeling

with 99mTc-pertechnetateThe optimized labeling conditions (pH, temperature,

reaction time, specifi c activity) applied during the wet la-beling procedure were identical during the labeling of fro-zen kits.

Wet labeling protocol was conducted according the publication of Decristoforo (Decristoforo and Mather, 1999), via co-ligand exchange as follows: in a sterile glass vial 20μg of HYNIC-TOC were mixed with 1 mL of so-

lution mixture (1:1) consisting of 20 mg EDDA and 10 mg tricine, 15 μg of SnCl

2·2H

2O dissolved in 0,1 M NCl

and incubated with addition of 1000 MBq 99mTc-pertech-

netate (up to fi nal volume of 2 mL) in water bath at 70

°C for 30 minutes (protocol 1). The solutions were fresh-

ly prepared and purged with sterile nitrogen. To optimize

the reaction time we followed the von Guggenberg (von

Guggenberg et al., 2003) modifi cation of the previous la-

beling protocol and heated the reaction mixture at 100 °C

for 10 minutes (protocol 2). The pH of the labeled prod-

uct was 7 ( measured semi-quantitative by Merck pH- indi-

cator strips). Finally, the radiopharmaceutical was SepPak

purifi ed and sterile fi ltered.

Kit formulation was prepared for a mini batch of 12

vials. We modifi ed the protocol published for freeze-dried

kit formulation by von Guggenberg (von Guggenberg et

al.,2004). Initially, 150 mg of EDDA and 300 mg of tricine

were dissolved in 14 mL sterile H2O (in a vial) by heating

in water bath. 12 mL of the co-ligand mixture were trans-

ferred into 50 ml sterile evacuated closed vial trough 0,20

μ Millipore fi lter. 250 μL (1 mg/mL) of HYNIC-TOC so-

lution was added with syringe in the vial and mixed. Final-

ly, 150 μL of instant dilution of SnCl2x2H

2O in 0,1 M HCl

consisting 240 μg was injected and mixed properly. The

pH of this solution was 4. Finally, the solution was dis-

pensed in 12 sterile evacuated vials trough Millipore GV

(low protein binding) fi lter in 1 mL volume. The vials were

fi lled with sterile nitrogen and stored frozen at -20 °C.

The labeling procedure of the frozen kit was per-

formed after thawing at room temperature, adding 0,5 mL

of 0,2N Phosphate buffer and 0,5 mL of 99mTc-pertechne-

tate. The mixture was incubated for 10 minutes in boiling

water bath. The fi nal pH was 7. The radiopharmaceutical

was SepPak purifi ed and sterile fi ltered.

SepPak purifi cation procedure

A SepPak C-18 mini cartridge was initially activated

with 5 mL of 100% ethanol, washed with 5 mL of 0.9% sa-

line and dried with 5 mL of air. The radiolabeled solution

was passed through the cartridge which was washed after-

wards with 5 mL of saline. The labeled peptide was elut-

ed from the cartridge with 0.6 mL of 80% ethanol through

a low-protein binding sterile fi lter, and fi nally diluted with

5 mL of saline. The content of the ethanol in the fi nal solu-

tion was not likely to induce side effects.

Макед. фарм. билт., 57 (1, 2) 65 - 70 (2011)

67 “In-house” preparation of 99mTc-EDDA/HYNIC-TOC, a specifi c targeting agent for somatostatin receptor scintigraphy

Quality Control

Radiochemical purity

Instant thin –layer chromatography (ITLC) was per-

formed on Silica gel strips with different mobile phases:

Methylethylketone (MEK) to determine the percentage of

the free 99mTcO4

- fraction (Rf=1), acetonitryle (ACN) 50%

for the 99mTc-colloid fraction (Rf=0). For the non-peptide

bound 99mTc-co- ligand 0.1M Sodium citrate buffer (pH=5)

(Rf=1) and optionally 0.9% NaCl (Rf=1) was used. Ra-

diochemical purity was calculated by supstraction the sum

of the impurities from 100%. Radiochromatograms were

obtained by Veenstra Radiochromatogram scanner VCS

101, with a software package.

Sterility

The small-batch of frozen kits was tested on sterility,

in compliance with the internal GMP Production Protokol.

Biodistribution

For biodistribution studies 7,4 MBq of the radiophar-

maceutical (consisting 1-2 μg of peptide) in a volume of

300 μL was injected into the tail vein of the rats. Animal

scintigrams were obtained after 15, 60 min, 2h and 4h p.i.

in a supine position, with a single-head Simens (e.cam)

gamma camera, using a pinhole collimator. Biodistribution

within the organs was performed 4h p.i.; blood sample was

collected with heparinized syringe by heart puncture, the

animals were sacrifi ced afterwards and other organs of in-

terest (liver, kidneys, spleen, pancreas, adrenals, gut and

muscle) extracted. The organs were weighed and radioac-

tivity measured respectively in well-type gamma scintilla-

tion counter. The results were expressed as percentage of

the injected dose per gram of the organ (% ID/g).

Results and Discussion

For the optimization of the labeling protocol, the ra-

diochemical impurities obtained by ITLC-SG for the dif-

ferent wet labeling protocols were displayed (Table 1) and

compared.

The proportion of free pertechnetate was low (0,53%

for protocol 1 and 0,36% for protocol 2) as well as the 99mTc-coloid impurities (3.4% and 2.46% respectively). Al-

though the results for the radiochemical impurities were

similar in both protocols, the 10 min. boiling water (proto-

col 2) was chosen as more convenient, as suggested by von

Table 1. Labeling conditions and radiochemical impurity of the wet labeled 99mTc-EDDA/HYNIC-TOC

Labeling conditions

Radiochemical impurity (mean% ± SD) n=3

99mTcO4

(MEK)

99mTc-RH

(ACN 50%)

99mTc-non peptide bound

(0.1N Citrate buff.)

70˚C, 30 min. 0.53 ± 0.15 3.4 ± 0.72 5.4 ± 0.82

100˚C, 10 min. 0.36 ± 0.11 2.46 ± 0.76 5.63 ± 1.5

Fig. 1. Gamma camera images of 99mTc-EDDA/HYNIC-TOC a) 15 min and b) 4h post injection into normal Wistar rats

68

Maced. pharm. bull., 57 (1, 2) 65 - 70 (2011)


Guggenberg et al (2003). The percentage of the overall ra-diolabeled impurities was less than 10%, accepted as up-per limit and reported by other authors (Plachcinska et al., 2003; von Guggenberg et al., 2004; Melero et al., 2009). For the assessment of non-peptide bound impurities 0.9% NaCl was used elsewhere (Bangard et al. 2000; Plachcins-ka et al.2004). We obtained lower percentage for the above mentioned impurities with 0.9% NaCl (2.8% ±1.06) com-pared to 0.1 M Citrate buffer mobile phase. For more pre-cise detection of peptide related side products, HPLC anal-ysis with radiation detector would be preferable method, but it was not available in our laboratory during this study.

Animal biodistribution studies, integrated in our qual-ity control protocol, were carried out with wet labeled kits. The results from the imaging study are presented in Fig. 1.

The radiopharmaceutical was cleared rapidly from the blood, as shown on the 15 min static image, and the most intense radioactivity was observed in the kidneys and blad-der. The late, 4 h image confi rmed the predominant renal uptake, as reported by Decristoforo et al. (1999). The pro-portion of the injected dose/g in the kidneys was 2, 95% (see Fig. 2), and with lesser extent in the gut (1,16%) and the liver (1,15%). No activity was observed in the thyroid, which was in accordance with the low percentage of free 99mTc-petrechnetate impurity found by ITLC-SG quality control.

Fig. 2. Biodistribution of 99mTc-EDDA/HYNIC-TOC in normal Wistar rats 4h p.i (n=2)

Although the wet labeling protocol was confi rmed as effi cient and reliable procedure for 99mTc labeling of /HYNIC-TOC, it was more time and labor consuming in comparison with frozen kit labeling. Therefore, prior in-troducing the radiopharmaceutical in the clinical routine, we decided to produce a small kit batch. After the prepa-ration of frozen kits, which were found sterile, their stor-age stability on -20 °C was tested within the period of 6 months. Protocol 2 was used for the radiolabeling, after adding 0,2N phosphate buffer to the mixture. The labeling yield during period observed (expressed as mean of dupli-cate assessment) is displayed on Fig. 3. The range of the ra-

diolabeling yield was between 94,3% and 96,9%. After 6 months, no decrease of radiolabeling yield was observed.

Fig. 3. Storage stability of the frozen kit formulation up to 6 months post production (p.p.= immediately post production)

Fig. 4. Radiochromatograms (ITLC) of 99mTc-EDDA/HYNIC-TOC kit after 6 months storage at -20 °C: a) MEK (Rf=1) free 99mTc-pertechnetate; b) 50% ACN (Rf=0) 99mTc-coloid; c) 0.9% NaCl (Rf=1) 99mTc-non peptide bound coligand + free 99mTc-pertechnetate

Макед. фарм. билт., 57 (1, 2) 65 - 70 (2011)

69 “In-house” preparation of 99mTc-EDDA/HYNIC-TOC, a specifi c targeting agent for somatostatin receptor scintigraphy

The radiochromatograms obtained from the ITLC

strips for the three main radiochemical impurities (Fig.

4.) illustrate the good quality of our radiopharmaceutical

and indicate that frozen kit can be a safe alternative to the

freeze-dried for small batch in house production.

We could not compare our stability fi ndings with those

published in the literature available, because they are main-

ly related to the in house wet labeling procedures (Mel-

ero et al., 2009; Pusuwan et al., 2010) or in house freeze

dried kits (von Guggenberg et al., 2004; Gandomkar et al.,

2006; Tasdelen, 2011). In house production of 99mTc-ED-

DA/HYNIC-TOC is usually carried out within Coordinat-

ed Research Projects or Technical Cooperation Projects

of IAEA in labs worldwide due to its restricted availabili-

ty. The only commercially released kit so far is Tektrotyd

from Radioisotope Centre POLATOM, Poland.

Conclusion

In house kit production, performed in compliance

with GMP and GRP regulations within hospital premis-

es is sometimes the only way to introduce novel radiop-

harmaceuticas, especially in developing countries. Fol-

lowing the proscribed and optimized procedures, we pro-

duced 99mTc-EDDA/HYNIC-TOC, a specifi c SST recep-

tor targeting agent which fulfi lled the quality control cri-

teria for radiochemical purity, sterility, normal biodistribu-

tion and stability. Our future activities will be focused on

the clinical application and evaluation on selected group

of patients.

Acknowledgements

This work was supported by IAEA Technical Co-op-

eration Project MAK3/004 ”Developing of 99mTc labeled

peptides and monoclonal antibodies”.

We wish to thank C. Decristophoro and E. von Guggen-

berg from the University Clinic for Nuclear Medicine,

Innsbruck, Austria for sharing their experience in EDDA/

HYNIC-TOC labeling, and preclinical and clinical evalua-

tion of the radiopharmaceutical.

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Melero, L.T.U.H., Araujo, E.B., Mengatti, J., 2009.

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Резиме

“In house” производство на 99mTc-EDDA/HYNIC-

TOC, специфичен насочувачки агенс за соматостатин -

рецепторна сцинтиграфија

Соња Кузмановска, Оливија Васкова, Марина Здравеска Кочовска

Институт за патофизиологија и нуклеарна медицина, Медицински факултет, Универзитет ,,Св. Кирил и Мето-

диј”, Водњанска 17, Скопје, Република Македонија

Клучни зборови: радиофармацевтик, технециум-99m, тир3-октреотид, рецепторна сцинтиграфија, домашно производство, конт-

рола на квалитет.

Употребата на радиообележени пептидни лиганди како дијагностици и терапевтици во нуклеарната онкологија е зголемена во

последно време. Еден од најчесто користените радиофармацевтици е 99mTc-EDDA/HYNIC-TOC, соматостатински аналог кој спе-

цифично се врзува за одредени типови соматостатински рецептори со висок дензитет кај тумори од невроендокрино потекло. Ра-

диофармацевтикот не е широко комерцијално достапен и поради тоа го воведовме како „in house“ производ, како дел од проект

поддржан од Меѓународната агенција за атомска енергија (МААЕ). Во рамките на процедурите за предклиничка проценка, оп-

тимизиран е протоколот за радиообележување, произведена e мала серија китови, извршена e контрола на квалитет со инстант

тенкослојна хроматографија и се направени биодистрибуциони студии. Процесот на радиообележување на пептидот, изведен со

измена на ко-лиганди на температура од 100 °C за време од 10 мин., резултираше со радиохемиска чистота > 90%. Биодистрибу-

ционите сцинтиграми кај нормални Wistar стаорци покажаа брз клиренс од циркулацијата по 15 мин. и доминантна акумулација во

бубрезите по 4 часа, што е во согласност со податоците објавени од други автори. Беше проценета стабилноста на препаратот, про-

изведен во мала серија, на -20 °C при складирање за време од 6 месеци, при што ефикасноста на радиообележување се движеше

од 94,3% до 96,9%. Нашите резултати покажаа дека смрзнатите китови можат да бидат сигурна алтернатива на лиофилизираните,

во услови на домашно производство на мали серии, а по задоволителната предклиничка проценка 99mTc-EDDA/HYNIC-TOC може

да биде воведен во клиничката практика како специфичен насочувачки агенс за соматостатин - рецепторна сцинтиграфија.

Macedonian pharmaceutical bulletin, 57 (1, 2) 71 - 76 (2011)

ISSN 1409 - 8695

UDC: 582.929.4–113.55(497.7) Original scientifi c paper

Introduction

Most of the species from genus Salvia have me-dicinal and horticultural importance as they produce many useful natural constituents including terpenes and fl a-

vonoids (Kelen and Tepe, 2008). They are counted as one

of the largest members of the Lamiaceae family that in-

cludes around 900 species and has an almost cosmopolitan

distribution (Mediterranean, Asia Minor, Central Europe

and America etc).

“Sage”, the dialect name of the genus Salvia is attrib-

uted to different species that are widely used in the food,

drug and fragrance industry. The high diversity in second-

ary metabolites (essential oils and the phenolic derivatives)

isolated from sage plants, possess excellent antimicrobial

Essential oil composition of wild growing Sage from

R. Macedonia

Gjoshe Stefkov, Ivana Cvetkovikj*, Marija Karapandzova, Svetlana Kulevanova

Institute of Pharmacognosy, Faculty of Pharmacy, University “Ss. Cyril and Methodius” – Skopje, R. Macedonia

Received: February 2012; Accepted: March 2012

Abstract

The main objective of this study was to analyze and identify the essential oil composition of S. offi cinalis populations growing in Republic of Macedonia and to evaluate these data according to different standards’ requirements for, commercially most utilized, Dalmatian sage. The essential oil yield, obtained after hydrodestilation from leaves, of three different populations of Salvia offi cinalis L. from Republic of Macedonia was determined, varying from 1.40 to 3.46%. The GC/FID/MS analysis of the composition of the essential oils revealed 63, 57 and 51 components in Galicica Mtn., Jablanica Mtn. and Karaorman Mtn. sage populations, respectively. The main components of the oil, in all three samples, were the terpene hydrocarbons, encompassing the monoterpenes: camphor (13.15 - 25.91%), α-thujone (19.25 - 26.33%), β-thujone (2.03 - 5.28%), 1,8-cineole (6.51 – 13.60%), α-pinene (0.93 – 1.47%), borneol (1.07 – 4.67%), then sesquiterpenes: trans (E)-caryophyllene (1.72 – 5.33%), α-humulene (2.89 – 7.99%), viridifl orol (4.27 – 7.99%), and the diterpene manool

(2.13 - 3.79%).

Thus, our results for the essential oil composition of sage complied with the reference values specifi ed in the DAC 86 monograph for

Salvia essential oil.

Key words: Salvia offi cinalis, essential oil composition, GC/FID/MS, R. Macedonia.

* [email protected];

[email protected]

activity as well as antioxidant capacity and some are used

as anticancer agents or have hypoglycemic effect (Kintzios,

2000; Miladinovic and Miladinovic, 2000; Khalil and Li,

2011). Of the many existing Salvia species, Salvia offi ci-

nalis also known as “Dalmatian sage” or “Garden sage”,

has economic importance and can be used for prepara-

tion of various extracts and herbal remedies with antisep-

tic and antibacterial properties which are attributed to the

rich chemical content of the essential oil and proven by

the modern medical science (Velickovic et.al., 2003; Avato

et.al., 2005; Delamare et.al., 2005; Bernotiene et.al., 2007).

Although sage is an ancient spice and remedy, its impor-

tance today is quite limited to the Medi terranean coun-

tries (starting from Italy till Greece) (Mockute et.al., 2003;

Hager, 2006). Some of the Salvia species, including S. of-

fi cinalis (S. offi cinalis folium) and S. triloba (S. triloba fo-

lium) can be found in many pharmacopeias (Flamini et.al,

2005; Eur.Ph.7.0, 2010). Pharmacopoeias monographs of

S. offi cinalis essential oil (Salviae aetheroleum - Helv VII;

72

Maced. pharm. bull., 57 (1, 2) 71 - 76 (2011)


Salviae offi cinalis aetheroleum - DAC 86) are available, as well. The essential oil can be obtained by steam distillation of the leaves of S. offi cinalis (Helv VII), or from aboveg-round parts of the sage thus obtaining essential oil rich in thujone (DAC 86).

The well studied Dalmatian sage oil show differenc-es in the total oil yield varying from 1.20 to 3.60% (with maximum in July) and gives characteristic sequence of the major constituents: α + β-thujone> camphor> cineole. The principal components in the sage essential oil are the vol-atile monoterpenes, present in the following average dis-tribution: 8.4 to 24.0% camphor, cineol (8.4 to 24.0%), α-thujone from 22.2 to 36.8%, β-thujone from 4.0 to 27.5%, borneol (2.1%), bornyl acetate (1.6%), camphene (4.4%), β-caryophyllene (3%), α-humulene (4.4%), α-pinene (3.5%), β-pinene (2.2%) and viridifl orol (6%). The content and composition of the oil is subjected to periodic daily variations and fl uctuations as well as to other ecological factors (climatic and soil conditions) (Hager, 2006). The relative amounts of major constituents in the sage essential oil are regulated with the German Drug Codex and the ISO 9909 standard (Table 3).

The Macedonian fl ora includes 37 species of genus Salvia. Up to present there are no evident data about essen-tial oil composition of S. offi cinalis populations growing in Republic of Macedonia. The importance of S. offi cinalis

drew our attention to examine and identify the active ingre-dients of the essential oils obtained by hydrodistillation of sage leaves collected from R. Macedonia.

Materials and methods

Plant material

The plant material from three different populations was harvest from Galicica Mtn., Jablanica Mtn., and Karaorman Mtn., located in the western part of Macedonia, nearby the Albanian border, during June year 2009 and 2010. The leaves were air dried and stored in a cool and dark place un-til distillation. The herb was authenticated as Salvia offi ci-

nalis L. (Lamiacecae) by Dr. Gjoshe Stefkov, and voucher specimens (No. So-MKD 13/10; So-MKD 14/10; So-MKD 15/10) were deposited at the Herbarium at the Department of Pharmaceutical Botany, Institute of Pharmacognosy, Faculty of Pharmacy, Skopje, Macedonia.

Essential oil isolation

Essential oil isolation from sage leaves was performed by hydrodistillation in all-glass Clevenger apparatus fol-lowing the procedure from European Pharmacopeia (Ph. Eur.7.0, 2010).

Analysis of essential oils’ chemical composition

Each sample of essential oil was dissolved in xylene (1:1000 v/v) and further analyzed on Agilent 7890А Gas

Chromatography system equipped with FID detector and Agilent 5975C mass spectrometer. HP-5ms 5% phenyl 95% dimethylpolysiloxane bonded phase capillary col-umn (30 m x 0.25 mm, fi lm thickness 0.25 µm) was used. Analytical conditions were: oven temperature at 60 °C for 5 min, then increased to 80 °C at rate of 1 °C/min and held 2 min and at the end increased to 280 °C at rate of 5 °C/min and held 5 min; helium as carrier gas at a fl ow rate of 1ml/

min; temperature of the injector 260 °C and that of the FID

detector 270 °C; the GC split ratio 1:1. 1µl of each sample was injected per GC run. The mass spectrometry conditions were: ionization voltage 70 eV, ion source temperature 230 °C, transfer line temperature 280 °C and mass range from 50 - 500 Da. The MS was operated in scan mode.

The compounds were identifi ed on the basis of liter-ature and estimated Kovat′s (retention) indices that were determined using mixture of homologous series of normal alkanes (C

9-C

25) analyzed under Automated Mass Spectral

Deconvolution and Identifi cation System (AMDIS)’ condi-tions. Confi rmation was done by comparing the mass spec-tra obtained from AMDIS with the reference spectra from Nist, Wiley and Adams mass spectra libraries.

Quantifi cation of the essential oils components was performed using the normalization method of the GC/FID peak areas.


The essential oil yields, obtained with hydrodistilla-tion of the upper leaves from each sage population from Macedonia were as followed: So-MKD 13/10 = 1.8%, So-MKD 14/10 = 1.4% and So-MKD 15/10 = 3.46%. The amount of oils from Galicica and Jablanica populations (1.8% and 1.4%, respectively) were in accordance with lit-erature references 1-2.5% (ESCOP Monographs) or 1-3% (Eur.Ph.7), while the isolate from Karaorman Mtn. (essen-tial oil, 3.46%) was above these values, and close to the upper limit for the Dalmatian essential oil.

By the means of GC-MS, total of sixty nine compo-nents, in all three samples of S. offi cinalis were identifi ed and presented in Table 1.

The GC-MS analysis revealed sixty three components (37 monoterpenes, 17 sesquiterpenes, 5 diterpenes and 4 other components) in the essential oil from Galicica sage population, fi fty seven (34 monoterpenes, 12 sesquiter-penes, 5 diterpenes and 6 other constituents) from Jablanica sage population and fi fty one from Karaorman sage popu-lation (27 monoterpenes, 13 sesquiterpenes, 5 diterpenes and 6 other components - aromatic and aliphatic hydro-carbons, esters and etc). The quantities of the constitutive chemical groups of the essential oils of Salvia offi cinalis populations are shown in Fig. 1.

The most abundant constituents, in all three samples, were the terpene hydrocarbons, encompassing the vola-tile monoterpenes: camphor (13.15 - 25.91%), α-thujone (19.25 - 26.33%), β-thujone (2.03 - 5.28%), 1,8-cineole

Макед. фарм. билт., 57 (1, 2) 71 - 76 (2011)

73 Essential oil composition of wild growing Sage from R. Macedonia

Table 1. Chemical composition of essential oils isolated from three different populations of S. offi cinalis from R. Macedonia

No. KIL KIE ComponentSo-MKD 13/10 %

(m/m)

So-MKD 14/10

% (m/m)

So-MKD 15/10

% (m/m)

1 / 921.0 4-heptanol / 0.26 0.42

2 926 924.7 tricyclene / 0.08 0.08

3 931 927.9 alpha-thujene 0.05 / /

4 939 935.8 alpha-pinene 1.20 0.93 1.47

5 953 951.2 camphene 2.91 1.94 1.96

6 978 976.7 1-octen-3-ol 0.07 0.07 0.05

7 980 980.0 beta-pinene 0.78 0.22 0.46

8 991 991.2 myrcene 0.80 0.50 0.81

9 994 969.1 mesitylene 0.09 0.06 0.10

10 1000 999.9 n-decane 0.05 0.04 0.06

11 1005 1007.1 alpha-phellandrene 0.09 0.06 0.03

12 1018 1018.6 alpha-terpinene 0.16 0.15 0.10

13 1026 1026.1 p-cymene 0.28 0.41 0.79

14 1031 1030.4 limonene 2.15 1.84 1.63

15 1033 1033.5 1,8-cineole 6.51 9.92 13.60

16 1062 1059.8 gamma-terpinene 0.29 0.19 0.54

17 1065 1069.0 cis-sabinene hydrate 0.05 / /

18 1088 1090.6 terpinolene 0.51 0.26 0.26

19 1098 1101.3 linalool 0.60 0.40 0.30

20 1110 1110.4 cis-thujone 19.98 19.25 26.33

21 1111 1119.4 trans-thujone 2.03 3.26 5.28

22 1125 1128.6 alpha-campholenal 0.06 0.03 0.03

23 1133 1136.9 iso-3-thujanol 0.07 0.06 /

24 1139 1143.7 trans-sabinol 0.10 0.07 0.10

25 1143 1150.1 camphor 25.91 23.79 13.15

26 1156 1160.0 isoborneol 0.07 0.06 /

27 1160 1163.9 trans-pinocamphone / 0.06 0.16

28 1165 1168.9 borneol 4.26 4.67 1.07

29 1177 1179.3 terpinen-4-ol 0.39 0.43 0.44

30 1183 1186.3 p-cymene-8-ol 0.06 0.09 0.04

31 1189 1192.6 alpha-terpineol 0.20 0.17 0.11

32 1194 1199.2 myrtenol 0.04 0.26 0.26

33 1217 1220.1 trans-carveol 0.07 0.17 0.04

34 1226 1225.0 cis-carveol 0.03 / /

35 1233 1230.0 isobornyl formate 0.03 0.05 /

36 1235 1237.2 neral 0.02 0.04 /

37 1237 1241.6 pulegone 0.03 / /

38 1255 1254.0 geraniol 0.02 / /

39 1267 1269.0 iso-3-thujanol acetate 0.03 / /

40 1273 1276.0 neo-3-thujanol acetate 0.06 0.08 /

41 1285 1288.4 bornyl acetate 3.86 3.53 0.43

42 1290 1293.8 trans-sabinyl acetate 0.31 0.33 0.16

43 1298 1301.2 carvacrol 0.05 0.12 0.10

44 / 1327.5 myrtenyl acetate / 0.05

45 1337 1338.4 trans-carvyl acetate 0.03 0.07 /

46 1342 1344.2 piperitenone / 0.06 /

47 / 1381.7 6,9-guaiadiene 0.11 / /

48 1386 1390.4 alpha-isocomene 0.18 / 0.14

74

Maced. pharm. bull., 57 (1, 2) 71 - 76 (2011)


No. KIL KIE ComponentSo-MKD 13/10 %

(m/m)So-MKD 14/10

% (m/m)So-MKD 15/10

% (m/m)

49 1409 1418.6 alpha-gurjunene 0.05 / /

50 1418 1424.2 (E)-caryophyllene 1.716 4.21 5.33

51 / 1454.8 trans-cadina-1(6),4 -diene 1.69 1.32 /

52 1453 1458.5 alpha-humulene 2.89 5.21 7.34

53 1467 1465.5 9-epi-(E)-carryophyllene 0.33 0.44 0.34

54 1478 1479.0 gamma-muurolene 0.44 / /

55 1493 1499.5 viridifl orene 2.67 2.77 2.04

56 1528 1510.9 zonarene 0.07 0.07 0.04

57 1524 1527.1 delta-cadinene 0.15 0.09 0.04

58 1581 1588.2 caryophyllene oxide 0.25 0.40 0.35

59 1590 1597.3 viridifl orol 4.27 / 7.99

60 1593 1600.0 humulene epoxide I 0.16 / /

61 1606 1614.4 humulene epoxide II 0.49 0.75 0.81

62 1630 1637.0 muurola-4,10(14)-dien-1-b-ol 0.45 1.21 1.19

63 1664 1674.8 14-hydroxy-(Z)-caryophyllene 0.17 0.77 0.46

64 / 1810.2 alpha-bisabolene / 0.16 0.11

65 1894 1901.8 rimuene 0.08 0.08 0.05

66 1950 1917.4 isopimara-9(11)-15-dien 0.19 0.21 0.16

67 19601668.8

sandaracopimara- 8(14),

15- diene 0.25 0.21 0.13

68 / 1977.7 labd-7,13-dien-15-ol 0.11 0.09 0.06

69 2056 2061.2 manool 2.13 3.79 3.06

Total: 93.13 95.73 100.00

*No. - Ordinal number according to the elution order of the components; KIL - Kovat’s Index Literature; KIE - Kovat’s Index Estimated, So-MKD 13/10

– Salvia offi cinalis population from Galicica Mtn.; So-MKD 14/10 – Salvia offi cinalis population from Jablanica Mtn.; So-MKD 15/10 – Salvia offi cinalis population from Karaorman Mtn.

Fig. 1. Presentation of different classes of terpenes and their abundance in the essential oils of Macedonian sage plants *MT - monoterpene; OCM - oxygen containing monoterpene; ST - sequiterpene; OCS - oxygen containing sesquterpene; DT - diterpene; OT - other type (aliphatic and aromatic hydrocarbons, aromatic esters etc.); So-MKD 13/10 – Salvia offi cinalis population from Galicica Mtn.; So-MKD 14/10 – Salvia offi cinalis population from Jablanica Mtn.; So-MKD 15/10 – Salvia offi cinalis population from Karaorman Mtn.

Макед. фарм. билт., 57 (1, 2) 71 - 76 (2011)

75 Essential oil composition of wild growing Sage from R. Macedonia

(6.51 – 13.60%), α-pinene (0.93 – 1.47%), borneol (1.07 –

4.67%), the sesquiterpenes: trans (E)-caryophyllene (1.72

– 5.33%), α-humulene (2.89 – 7.99%), viridifl orol (4.27 –

7.99%), and the diterpene manool (2.13 - 3.79%).

Our results for the essential oil composition of sage,

thus, comply with the reference values specifi ed in the

German Drug Codex monograph for Salvia offi cinalis es-

sential oil. The composition analysis of the volatile con-

stituents of the three Macedonian sage populations showed

that only the essential oil from Karaorman Mtn. population

belongs to the thujone-rich oils, while essential oils isolated

from Galicica Mtn. and Jablanica Mtn. populations had

camphor as major component. Typically, according to some

authors (Perry et.al., 1999; Walch et.al., 2011), three are

three Dalmatian Sage chemotypes with low (9%), medium

(22-28%), and high (39-44%) thujone contents. Concerning

the latter, essential oils from S. offi cinalis samples from

Galicica Mtn. and Jablanica Mtn. populations, belong to

the Sage group containing medium thujone amounts and

cannot meet with the requirements for the sage essential

oil chemical composition reported in other available scien-

tifi c literature where the demand of thujone content is stated

as 35-60% (e.g. Radulescu et.al., 2004; Maksimovic et.al.,

2007). Due to the camphor predominance in essential oil

isolated from Galicica Mtn. population, this essential oil

cannot meet the ISO 9909 standard (Table 2).

This inter-specifi c comparison of the essential oils

isolated from three Salvia offi cinalis populations from

Macedonia showed differences in the amount of the prin-

ciple components especially in the oils obtained from

Galicica and Jablanica populations what emphasize the

role of the environmental factors (light, soil, water, time

of harvest, drying, etc.) on the yield and chemical compo-

sition of the essential oil even when they are from same

region.

Conclusion

The essential oils’ yields obtained after hydrodesti-

lation of leaves from three different populations of sage

from Macedonia varied from 1.40 to 3.46%. The essen-

tial oil composition of all three sage populations complies

with the reference values specifi ed in the DAC 86 mono-

graph for Salvia essential oil. Yet, the requirements of the

ISO 9909 standard have not been met. Nevertheless, the

set of components identifi ed in the three samples of the

Macedonian sage oil match up with the essential oil com-

position of the Dalmatian sage, though the set differs in

relative content of the principle components.

References

Avato, P., Fortunato, I.M., Ruta, C., D’Elia, R., 2005. Grandular

hairs and essential oils in micropropagated plants of Salvia

offi cinalis L. Plant Science 169, 29-36.

Bernotiene, G., Nivinskiene, O., Butkiene, R., Mockute, D.,

2007. Essential oil composition variability in sage (Salvia

offi cinalis L.). Chemija 18 (4), 38-43.

DAC 86 – Deutsher Arzneimittel Codex (German Drug

Formulary) 86.

Delamare, L.A.P., Moschen-Pistorello, I.T., Artico L., Atti-

Serafi ni, L., Echeverrigaray, S., 2005. Antibacterial activity

of the essential oils of Salvia offi cinalis L. and Salvia triloba

L. cultivated in South Brazil. Food Chemistry 100 (2), 603-

608.

Table 2. Comparison of the dominant constituents of the essential oils obtained after hydrodestilation of the three Salvia of-

fi cinalis populations from R. Macedonia with referent literature

Constituents

% (m/m)

So-MKD

13/10

So-MKD

14/10

So-MKD

15/10DAC 861 ISO 99092 Serbia3 Romania4 Croatia5

1,8-cineole 6.51 9.92 13.61 6.00-16.00 5.50-13.00 9.79 6.72 0.90

cis-thujone 19.98 19.25 26.33 >20.00 18.00-43.00 24.88 21.85 57.00

trans-thujone 2.03 3.26 5.28 3.00-8.50 / 5.51 15.00

camphor 25.91 23.79 13.15 14.00-37.00 4.50-24.50 16.03 11.25 3.30

borneol 4.26 4.67 1.07 ≤5.00 / / 2.58 /

bornyl acetate 3.86 3.53 0.43 ≤5.00 <2.50 / 3.22 /

α-pinene 1.20 0.93 1.47 / 1.00-6.50 / / /

α-humulene 2.89 5.21 7.34 / 0-12.00 / 4.51 /

camphene 2.91 1.94 1.96 / 1.50-7.00 / 1.66 /

limonene 2.15 1.84 1.63 / 0.50-3.00 / / /

viridifl orol 4.27 / 7.99 / / / 11.71 14.20

manool 2.13 3.79 3.06 / / / 9.15 /

1Monograph for Salvia offi cinalis essential oil in DAC 86 (German Drug Formulary 86); 2ISO 9909 standard for medicinal uses regulates the amounts of

nine constituents in the sage essential oil, Ref: Mockute et.al., 2003; Bernotiene et.al., 2007; 3Salvia offi cinalis essential oil from Serbia, Ref.: Miladinovic

and Miladinovic, 2000; 4Salvia offi cinalis essential oil from Romania, Ref.: Radulescu et.al., 2004; 5Salvia offi cinalis essential oil from Croatia, Ref.:

Maksimovic et.al., 2007; So-MKD 13/10 – Salvia offi cinalis population from Galicica Mtn.; So-MKD 14/10 – Salvia offi cinalis population from Jablanica

Mtn.; So-MKD 15/10 – Salvia offi cinalis population from Karaorman Mtn.

76

Maced. pharm. bull., 57 (1, 2) 71 - 76 (2011)


European Pharmacopoeia 7.0, 2010.ESCOP, 2003. Salviae offi cinalis folium (Sage leaf). ESCOP

Monographs (2nd ed), 452-455. Flamini, G., Cioni, P.L., Morelli, I., Bader, A., 2007. Essential

oils of the aerial parts of three Salvia species from Jordan: Salvia lanigera, S. spinosa and S. syriaca. Food Chemistry 100, 732-735.

Hager, 2004. Salvia. HagerROM 2004, Springer, Heidelberg. Kelen, M., Tepe, B., 2008. Chemical composition, antioxidant

and antimicrobial properties of the essential oils of three Salvia species from Turkish fl ora. Bioresource Technology 99 (10), 4096-4104.

Khalil, R. and Zheng-Guo, L., 2011. Antimicrobial activity of

essential oil of Salvia offi cinalis L. collected in Syria. African

Journal of Biotechnology 10 (42), 8397-8402.

Kintzios, S.E., 2000. Sage: the genus Salvia, Harwood academic publisher, Australia, Canada.

Maksimovic, M., Vidic, D., Milosh, M., Sholic, M.E., Abadzic, S., Siljak-Yakovlev, S., 2007. Effect of the environmental conditions on essential oil profi le in two Dinaric Salvia species: S. brachyodon Vandas and S. offi cinalis L. Biochemical systematic and ecology 35, 473-478.

Miladinovic, D., Miladinovic, Lj., 2000. Antimicrobial activity of essential oil of sage from Serbia. Facta Universitatis, Series: Physics, Chemistry and Technology 2 (2), 97-100.

Mockute, D., Nivinskiene, O., Bernotiene, G., Butkiene, R., 2003. The cis-thujone chemotype of Salvia offi cinalis L. essential oils. Chemija 14, 216-220.

Perry, N.B., Anderson, R.E., Brennan, N.J., Douglas, M.H., Heaney, A.J., McGimpsey, J.A., Smallfi eld, B.M., 1999. Essential oils from dalmatian sage (Salvia offi cinalis

L.): variations among individuals, plant parts, seasons, and sites. Journal of Agricultural Food Chemistry 47 (5), 2048-54.

Radulescu, V., Chiliment, S., Oprea, El., 2004. Capillary gas chromatography-mass spectrometry of volatile and semi-volatile compounds of Salvia offi cinalis. Journal of Chromatography A 1027, 121-126.

Velickovic, A.S, Ristic, M.S., Velickovic, D.T., Ilic, S.N., Mitic, N.D., 2003. The possibilities of the application of some species of sage (Salvia L.) as auxiliaries in the treatment of some diseases. Journal of Serbian Chemical Society 68 (6), 435-445.

Walch, S.G., Kuballa, T., Stuhlinger, W., Lachenmeier, D.W., 2011. Determination of the biologically active fl avour substances thujone and camphor in foods and medicines containing sage (Salvia offi cinalis L.). Chemistry Central Journal 5 (44).

Резиме

Состав на етерично масло од диво растечки жалфии

од Р. Македонија

Ѓоше Стефков, Ивана Цветковиќ*, Марија Карапанџова, Светлана Кулеванова

Институт за Фармакогнозија, Фармацевтски факултет, Универзитет “Св. Кирил и Методиј”, Скопје, Р.

Македонија

Клучни зборови: Salvia offi cinalis, состав на етерично масло, GC/FID/MS, Р. Македонија.

Главна цел на студијата беше анализа и идентификација на составните компоненти на етеричното масло изолирано од популациите на S. offi cinalis кои растат во Република Македонија и нивна споредба со далматинска жалфија. Приносот на етерично масло добиено со хидродестилација на листовите од трите различни популации на Salvia offi cinalis L. од Република Македонија се движи од 1.46 до 3.46%. Со GC/FID/MS анализата на етеричните масла идентификувани се 63, 57 и 51 компонента кај популациите од Галичица, Јабланица и Караорман, соодветно. Како главни компоненти на маслата во сите три примероци се терпенските јаглеводороди, вклучувајќи ги монотерпените: камфор (13.15 - 25.91%), α-тујон (19.25 - 26.33%), β-тујон (2.03 - 5.28%), 1,8-цинеол (6.51 – 13.60%), α-пинен (0.93 – 1.47%), борнеол (1.07 – 4.67%), по што следат сескитерпените: транс (Е)-кариофилен (1.72 – 5.33%), α-хумулен (2.89 – 7.99%), виридифлорол (4.27 – 7.99%) и дитерпенот манол (2.13 - 3.79%). Следствено, составот на етеричното масло од испитуваните жалфии од Р. Македонија одговара на референтните вредности утврдени во монографијата за етерично масло на жалфија во Германскиот кодекс за лекови (DAC 86).


ISSN 1409 - 8695

UDC: 615:35.072.6(497.7)Professional paper

Global Characteristics of the Pharmaceutical

Sector

The fi ndings confi rm that the so called «life cycle of

medicine» - Fig. 1 is, in essence, a strictly regulated tech-nical system with many elements of complex nature. To be-gin with, this system encompasses elements for new medi-cines research and development. The most successful can-didates for becoming medicines are subjected to clinical trials and patent procedure. Medicine production licens-ing is another very important element of this system. Put-ting the medicines in a strictly organized process of indus-

Harmonization of Inspection Supervision in the Pharmaceutical

Sector of the Republic of Macedonia in conformity with the

Recommendations of the European Legislation and WHO

Vasilka Nicha, Renata Slaveska Raichki* and Tatjana Kadifkova Panovska

Public Institution in the Health Sector, for the needs of PHI University Clinics, Institution and Urgent Centre, Skopje

Faculty of Pharmacy, University “Ss Cyril and Methodius”, Skopje

Received: November 2011; Accepted: De pan>

Abstract

The new Law on Inspection Supervision (Offi cial Gazette 50/2010, implementation as of 1.04.2011) was passed in April 2010 with the aim of improving the quality of inspection in the pharmaceutical sector, as well.

The new Law covers the spectrum of weaknesses recognized over time. The system of quality inspection encompasses a set of com-monly required quality management process, objectives, conditions, policies, formal rules and procedures. The organizational scheme ad-dresses all aspects of inspection activity. The inspection service has to assure that its personnel are not under any undue internal or exter-nal commercial, fi nancial or other kind of pressure and infl uence that may adversely affect the quality of their work. Moreover, the inspec-tion service needs a suffi cient number of employees with the necessary education, training, technical knowledge and experience to perform inspection activities according to specifi ed requirements and standards. An inspector’s engagements have to be within the scope of the re-sponsibilities arising from his/her activities. Continued training in inspection activities should be established, including advanced training programs that offer various levels of inspectors’ qualifi cation. In regard to their specifi c activity, new rule books governing inspection su-pervision in the pharmaceutical sector should be developed and adopted in the near future.

This permanent upgrading process of the existing national policies in compliance with the EU legal policy has also become character-istic of the Macedonian pharmaceutical sector.

Key words: pharmaceutical inspection supervision

* [email protected]

trial production is burdened by numerous challenges, re-sulting in diffi culties in establishing the conditions, crite-ria and procedures. The medicine becomes available to the end user once the marketing authorization is obtained.

The price of the medicine is determined using select-ed national methodology (IDPI, 2009). In keeping with the priorities in population health care, some of the med-icines with marketing authorizations are selected and in-cluded in the national list of essential medicines, as per the World Health Organization recommendations (WHO, 2004; WHO, 2010).

The components of the pharmaceutical system known as medicines supply and distribution provide smooth ac-cess and availability of medicines (of domestic origin or imported) for the end users (OGRM, 2007a; WHO, 1999).

78

Maced. pharm. bull., 57 (1, 2) 77 - 83 (2011)

Vasilka Nicha, Renata Slaveska Raichki and Tatjana Kadifkova Panovska

The element of providing ethical promotion of medicines

to health professionals and end-users (patients) is no less

important.

In the light of the existence of globally harmonized

legislation and technical guidelines on the separate func-

tions in the pharmaceutical sector: GMP (Good Manufac-turing Practice), GCP (Good Clinical Practice in Clinical Trials), GQCLP (Good Quality Control Laboratory Prac-tice), GLP (Good Laboratory Practice), GSP (Good Stor-age Practice), GDP (Good Distribution Practice), GPhP (Good Pharmacy Practice), guidelines for ethical promo-tion of medicines and the like, it is recognized that the in-tegration of all these elements in the national pharmaceu-tical sector and their successful application and evaluation in practice is of vital importance. Bearing in mind that the issue here is the essential regulation of the pharmaceutical sector, the inspection supervision in the spectrum of func-tions is recognized as one of the paramount activities of the competent bodies in this sector.

The aim of the inspection supervision in the pharma-ceutical sector, i.e. inspection of medicines and medical devices production, import, export, distribution, advertis-ing and the like, is to ensure that these functions are per-formed in compliance with the national legislation and the approved standards and guidelines. To that end, the prima-ry objective is to ensure that medicines intended for the population in the country are safe, effi cient and of con-trolled quality. In addition to the responsibilities in the of-fi cial market of medicines, inspectorates should guaran-tee that there are no illegal activities such as smuggling of medicines, medicines sale in uncontrolled open markets, as well as no counterfeited medicines and medicines that do not meet the required quality standards.

The fi rst step to be taken by the national authorities in order to institute an effi cient inspection supervision, is to

establish a legal basis. In other words, a consistent imple-mentation of inspection supervision in the area of medi-cines and medical devices requires development and on-going improvement of the laws and regulations within the national legislation. The regulations in the area of inspec-tion supervision should, at the same time, prevent any pos-sibility of inspectors’ illegal activities and abuse of offi cial powers, and ensure appropriate sanctions in cases of such activities. In line with these principles and the global expe-riences of many years, it is realized that it is imperative to implement a system of control of the inspection activities that will combine a set of basic requirements with regard to quality assurance and management with the inspectorates in their accomplishment of the set goals, tasks and condi-tions. That is to say, the system should be founded on re-spect for the existing legislation and other elements stem-ming from and integrated into this sphere. The organiza-tional structure of inspection services should cover all as-pects of their activities.

Development of a National Inspection Supervi-

sion in the Pharmaceutical Sector

At present, the primary task and one of extreme im-portance is the harmonization of the provisions of the Law on Inspection Supervision (OGRM, 2010) with the Law on Medicines and Medical Devices (OGRM, 2007b) and the passing of by-laws foreseen with the Law on Inspec-tion Supervision. These two laws should be the realistic ba-sis for the building of a quality system for the pharmaceu-tical inspection services, embodied in a concrete quality manual (Inspectorate Quality Manual) (PIC/S, 2007). The implementation of the quality system for the pharmaceuti-cal inspection services and the compliance with the EU di-

Fig. 1. Life cycle of medicine

Макед. фарм. билт., 57 (1, 2) 77 - 83 (2011)

79 Harmonization of Inspection Supervision in the Pharmaceutical Sector of the Republic of Macedonia in conformity with...

rectives should ensure and guarantee a future sustainable

system as a minimum criterion for mutual recognition and

acceptance of the national pharmaceutical inspectorate by

the European Economic Area (EEA) member countries as

well as with and by other pharmaceutical inspectorates out-

side the national framework. Recognizing, primarily, the

need for a number of activities to be undertaken with the

aim of implementing the policy on inspectorates quality

systems and of achieving the objectives of the function,

WHO, PIC/S (The Pharmaceutical Inspection Conven-tion and Pharmaceutical Inspection Co-operation Scheme) and EMA (European Medicines Agency) have published a framework for the essential requirements (conditions) with regard to a quality system that will help the pharmaceuti-cal inspection services to develop their own quality sys-tems and to be recognized by other national pharmaceuti-cal authorities.

Elements in the Building of a Quality System for

the Pharmaceutical Inspectorate (PhI)

Administrative Structure, Organization and Management

of Pharmaceutical Inspectorates

The policy on the quality of inspection services should be rooted in the basic principles set out in the Law on In-spection Supervision: legality, public interest, equality, in-dependence, impartiality, transparency, objectivity, princi-ples of material truth, prevention, proportionality and sub-sidiarity (OGRM, 2010). All these principles shall be re-fl ected in concrete quality requirements established for the given inspectorate. The Pharmaceutical Inspectorate man-agement structure should be offi cially committed to sup-port the recommended quality system requirements by en-suring that the quality policy of the inspectorate is docu-mented, that it is relevant to its objectives and that it is im-plemented. Furthermore, the Pharmaceutical Inspectorate management structure should have suffi cient resources and competent staff at all levels, to ensure effective and suc-cessful functioning, and it should nominate a person to car-ry out the quality assurance function (quality system im-plementation and maintenance) and to perform periodical revisions of the quality systems.

The primary need with regard to the administrative structure and organization is to have clearly defi ned and documented procedures and criteria for the selection of persons, advisory committees, agencies or organizations to perform certain delegated activities as set out in a contract (this applies to persons who are part of the inspectorate, a regulatory body in the area of medicines or sub-contracted experts). The criteria to be met in the selection or employ-ment of the PhI personnel should be:

available as a written document and available to - the public, and stating at least the required profes-sional qualifi cations (e.g. pharmacist, physician, chemist and the like);stating precise data on the minimum years of past -

experience;supported by recommendations from professional - associations, previous positions, and employment of the candidate for inspector; supported by testimonials to successful comple-- tion of inspectors trainings.

Inspectors’ job descriptions, responsibilities, and au-thorities, as well as the reporting structure, should be clearly defi ned and documented in rulebooks. The struc-ture should be defi ned in organization charts and support-ed by job descriptions for each member of the PhI staff. The rules for deontology, ethics and confl ict of interests (OGRM, 2007c) should be clearly defi ned for both the PhI staff and the persons performing (or sub-contracted to per-form) certain assignments for the inspectorate. The sys-tem for obtaining fees must not infl uence the quality of in-spection. All cases requiring joint supervision shall follow a documented procedure for the cooperation between the inspectorate and other agencies and/or sub-contracted ex-

perts. The inspectorate shall implement a policy of distin-

guishing between inspection supervision and providing of advisory services to the clients.

Human Resources - Inspectors

Financial stability and sources of fi nances and tech-nical assistance are clearly essential for the unhampered functioning of the inspection services. In distributing the resources and in determining the priorities, the Pharmaceu-tical Inspectorates should apply the principle of risk assess-ment and risk management. However, inspectors are an equally important element in the successful functioning of the supervision. Inspectorates need to have suffi cient avail-able personnel with adequate qualifi cations, competences, technical skills, specialized training, knowledge based on experience in performing inspection activities, and such that meet the specifi c requirements and standards (EMEA, 2007). Inspection services should guarantee that the per-sonnel engaged is free from any “external” or “internal” infl uences of commercial, fi nancial or any other nature that might affect the quality of their performance (ORGM, 2001 and OGRM, 2006). Personal integrity, maturity, transpar-ency, understanding of the complexity of the subject, the ability to make a sound and complete judgment, as well as confi dence, consistency, the ability to see given situation in a realistic way and practice their analytical skills, are just some of the principal traits inspectors have to possess. As regards inspector’s personal qualities, it should be not-ed that during the inspection supervision inspectors should create an atmosphere of openness and transparency, give objective answers to all questions and offer explanations when needed in the course of the inspection, while being mindful not to assume the role of consultants.

Inspection supervision is performed by trained inspec-tors with experience in, for instance, medicines produc-

80

Maced. pharm. bull., 57 (1, 2) 77 - 83 (2011)


tion, medicines quality control, pharmaceutical work etc. Inspectors shall have acquired their skills and been trained in accordance with the internationally adopted guidelines and programs of the agencies such as: The Pharmaceutical Inspection Convention/ Pharmaceutical Inspection Co-op-

eration Scheme, and the European Compliance Academy –

ECA. They shall perform their activities in a highly profes-

sional manner demonstrating integrity and honesty.

Inspectors’ Qualifi cations and Training

Inspectors’ qualifi cation is on an equal level with the qualifi cation for any other qualifi ed person (QP- Qualifi ed person) which means that, in essence, they need to possess real knowledge of national and EU legislation. Their basic training for inspectors should cover a number of areas and enable them to acquire knowledge in:

national and EU medicines-related legislation;- technical standards such as GMP, GCP, GQCLP, - GSP, GDP, GPP, guides relating to ethical adver-tising of medicines, etc;principles of quality assurance and quality man-- agement systems (EN, ISO standards);technical aspects of medicines production (exper-- tise in pharmaceutical technology, processes and HVAC-systems, validation, computerized sys-tems, analytical instruments, microbiology etc); organization and quality systems for competent - authorities/inspectorates and training in compli-

ance with the relevant national and internation-al standards, operational procedures (SOPs) and procedures related to inspection supervision;marketing systems and production authorization - and the relationship between the two;regulation of the cooperation relations in the - sphere of licensing, inspection, sampling proce-dure and analyses;knowledge of the agreements with the Medicines - Regulatory Agencies and EU agreements;inspection techniques acquired through participa-- tion in relevant trainings and/or in the presence

and/or run by qualifi ed inspectors;administrative procedures: management of plan-- ning, organization, communications or providing answers to the entity subjected to inspection su-pervision with regard to the process of evaluation of the existing situation and notifi cation thereof;medicines development: QRM (Quality Risk - Management), PQS (Pharmaceutical Quality Sys-tem) (ICH Q8,Q9, Q10); international organiza-tions and their activities – EDQM (European Di-rectorate for the Quality of Medicines and Health Care), ICH (International Conference on Harmo-nization), PIC/S, WHO;knowledge of other methodologies applicable in - the achievement of quality assurance principles.

The national inspectorates may, when necessary, orga-nize training in inspection supervision techniques, commu-nication skills and notifi cation skills, technical terms used in communication, legal aspects and management aspects.

Inspectors’ continued training and maintenance of their competences are achieved through a variety of forms including: participation in training courses, seminars, pro-fessional committees and conferences organized by na-tional and international scholarly and professional organi-zations (on subjects such as new production technologies, computerized systems); use of relevant sources of informa-tion, methods of joint inspections, or visits for training in other countries members of the EU etc.

Further continued training of inspectors can be run by senior inspectors, who will instruct them on the theory of inspection supervision using practical, concrete and real-life case studies and will discuss with them the importance and purposes of the inspection supervision.

In maintaining their competence inspectors shall be subjected to periodical knowledge assessments consistent with the requirements of the national legislation and the in-spection services quality system.

Documentation in Inspectorates

Pharmaceutical inspectorates should establish and maintain a system for control of all documentation, re-cording of relevant documents and handling of documents, timeframe for the keeping of superseded and/or replaced documents, current versions previously approved by an au-thorized person, available versions kept by nominated in-spectors. The existence of mechanisms for identifi cation of any changes to documents, the controlled manner in which changes are made for the purposes of documents faster up-dating and approval by the authorized person, and for with-drawal and fi ling of the changed documents – such mech-anisms are necessary in good documents management. Pharmaceutical inspectorates should prepare and regularly upgrade Inspectorate Quality Manual, incorporating all the elements of the quality system, and serving as a source of all referential working materials to be used as guides in in-spectors’ individual performances of their inspection activ-ities. These Guides shall guarantee consistency and trans-parency of the inspection supervision process, as well as prevention of subjectivity in the performance of the super-vision activities. On the other hand, written guides for in-spection services shall be of great help in the assessment of inspectors’ performance of their activities in compliance with the guides and the procedures. The pharmaceutical in-spection service Quality Manual may contain samples of forms used in inspection supervision: check list used in the performance of inspection supervision, certifi cates, reports and examples of their processing, keeping, fi ling and dis-posal. In the area of good document keeping it is also nec-essary to set up a system for preparation of reports (as re-

Макед. фарм. билт., 57 (1, 2) 77 - 83 (2011)


quired under the national legislation) related to the activi-

ties of the inspection service. Where relevant, documents

from applicants for or holders of licenses shall also be in-

cluded into the system. The reports refer to: detailed infor-

mation on inspection supervision planning, description of

the manner in which each inspection supervision was per-

formed, follow-up activities after inspection supervision,

sanctions imposed, and recommendations addressed to the

body in charge of issuing licenses. All reports shall be con-

sidered confi dential, unless otherwise regulated under the

laws on free access to information (OGRM, 2006).

Internal audit and quality improvement by means of cor-

rective/preventive actions and handling of complaints

The improvement of the quality of inspection supervi-

sion, or of the Pharmaceutical Inspectorate, is achieved by

establishment of quality indicators related to both the in-

spectorate activities and the procedures for the analysis of

the deviation from the quality system ascertained by exter-

nal or internal audits carried out by the inspectorate. Pe-

riodic audits of the Pharmaceutical Inspectorate activities

are necessary to assess compliance with the requirements

of the quality system. The results of the audits and the cor-

rective measures taken are reviewed by the management as

part of the management review process. The processes and

the documents relating to the audit processes, as well as the

auditors qualifi cations, should be clearly defi ned. The re-

ports should be kept for a defi ned period of time.

The procedure for corrective/preventive actions should

include prescribing, implementation and verifi cation of the

corrective actions resulting from reports, investigation of

complaints and other observations relating to inspectors’

activities. The Pharmaceutical Inspectorate should estab-

lish a procedure for the investigation of complaints re-

garding the activities of the inspection services, its person-

nel, persons or organizations sub-contracted by the inspec-

torate, as well as written procedure on fi ling complaints

against fi rst instance decisions and a guide for complaints

management.

Other elements related to the requirements for a quality

system for Pharmaceutical Inspectorate

A sustainable quality system requires that the follow-

ing requirements be established, documented and imple-

mented:

requirement to establish a system for the issue -

or withdrawal of licenses or GMP certifi cate or,

where appropriate, for advising on the issue or

withdrawal of licenses or GMP certifi cate (in ac-

cordance with the national and EU legislations);

requirement to defi ne the time limits within which -

the assessment shall be carried out;

requirement to establish a system under which ap--

propriate actions will be taken in cases of an ad-

verse inspection report, with description of the ac-

tions to be taken;

requirement to defi ne the liaisons in the licensing -

system when it is not part of the pharmaceutical

sector functions;

requirement to establish a system for handling of -

reports on suspected defects in medicinal prod-

ucts, based on QRM;

requirement to establish a Rapid Alert System – -

RAS;

requirement to establish, maintain and update a -

list of recalls;

requirement to legally defi ne the liaison with an -

offi cial lab for the purposes of medicines control;

requirement to exchange information concerning -

medicines or procedures of taking samples from

starting materials for the production or for medic-

inal products, as per validated SOPs.

Inspection Supervision – Methods, Strategies and Docu-

menting

At a global level, inspectors perform two types of in-

spection supervision: (I) in the period before licensing, and

(II) in the period after licensing. The role of inspection su-

pervision in the period before licensing is as follows:

to evaluate the compliance with the requirements -

specifi ed in the guides with regard to business ca-

pacities, spatial capacities, personnel, equipment,

processes etc.;

to evaluate the implemented procedures and meth--

ods of control used in the production, import, ex-

port, distribution etc.;

to evaluate the completeness and accuracy of the -

information provided by the applicant.

The information gathered in the process of inspection

supervision in the period before licensing is a very impor-

tant segment in the evaluation of the application for regis-

tration and approval. The purpose of inspection supervi-

sion following licensing is to check the continuity in ensur-

ing compliance with the approved standards, guidelines,

procedures and the national regulations on medicines. De-

pending on the purpose of the inspection supervision, the

inspectors apply a number of methods for supervision of

pharmaceutical capacities, including:

comprehensive or routine inspections;-

concise inspections; -

follow-up inspections; -

special inspections; and-

investigative inspections. -

The strategies used in the process of inspection, with

the aim of achieving a higher level of objectivity and re-

ducing the possibilities for unethical practices in supervi-

sion to a minimum, include:

82

Maced. pharm. bull., 57 (1, 2) 77 - 83 (2011)


inspection supervision by a team of inspectors -

with a selected chief inspector leading the team;rotation of inspectors, by means of a system for - development of inspection supervision schedule; rotation of inspectors in different geographic ar-- eas; inspection supervision carried out in the presence - of a peer inspector that will write a report on the inspector’s work; andexternal assessment, i.e. independent evaluation - of the inspection supervision.

The inspector is required to submit his report with rec-ommendations to the inspection authorities that will evalu-ate its compliance with the guidelines and procedures. The recommendations should be subjected to substantial evalua-tion with regard to possible contradictions in the fi ndings as-certained during the inspection process and/or possible am-

biguity and unclear formulations. In addition, it is necessary

to confi rm that the inspector has discussed his/her fi ndings with the responsible representatives of the management of the applicant subjected to inspection supervision, and thus met the requirement for transparency of inspection. Inspec-tion supervision should represent a well documented pro-cedure based on relevant laws and regulations the aim of which it is to enable supervision (as in the cases of produc-tion and distribution operations), in conformity with the na-tional legislation, the offi cial guidelines and the formal in-spection plan. The set of documents should include: written instructions, written standards and procedures, work sheets, references etc. The documents shall be regularly updated and made easily available to the personnel.

It is necessary to distinguish between whether the in-spection supervision was performed by a team of inspec-tors or by one inspector, and to see if the report (in a de-fi ned format) was adequately prepared by a person autho-rized to prepare the report, in the case of a team. Further-more, the subject of inspection supervision should autho-rize a person that will receive the report, which is a quali-fi ed person (QP).

Guidelines for management of confl icts of interests and

for declaration of confl ict of interests

An inspector must not use his/her offi cial powers to obtain privileges of any nature, including those associat-ed with fi nancial, commercial and other interests incom-patible with his/her job description and duties. These strict requirements apply also to the members of his/her family, friends and associates. The minimum criteria in the written guides on confl ict of interest management and for the dec-laration of a confl ict of interests should include the follow-ing elements:

defi nition of all situations that represent confl icts - of interests in the inspector’s job description;rules on the acceptance of gifts; -

rules on declaring interests;- a mechanism for protection of the persons inform-- ing on the existence of a confl ict of interests; actions to be undertaken in cases of ascertained - omissions and breaches in the adherence to the principle and in confl icts of interests manage-ment; evidence of the continuous and systematic updat-- ing of information by inspectors and civil servants involved in the inspection supervision.

All these elements of the pharmaceutical inspection supervision represent obligations arising from the Law on Inspection Supervision of the Republic of Macedonia (OGRM, 2010) and the adequate bylaws. The permanent process of developing and establishment of concrete re-quirements and recommendations ensuing from this Law includes reducing any possibilities for their ineffi cient im-plementation in practice to the minimum.

References

EMEA, 2007. Guideline on Training and Qualifi cations of GMP Inspectors (http://www.gmpcompliance.org/eca_news_1336_5865,5592,5816,5587,5946.html)

IDPI, 2009. International Drug Price Indicator Guide (http://erc.msh.org/dmpguide/pdf/DrugPriceGuide_2010_en.pdf).

ORGM, 2001. Civil Servants Code of Ethics (Offi cial Gazette of RM No бр.96/2001; Code to supplement the Civil Servants

Code of Ethics, 16/2004; 48/2007).

OGRM, 2006. Law on Free Access to Information of Public

Interest (Offi cial Gazette of RM No. 13/2006; latest

amendment 14 July 2008).

OGRM, 2007a. Law on Public Procurements (Offi cial Gazette of

RM No. 136/2007 and amendments: 130/08; 97/10;53/11).

OGRM, 2007b. Law on Medicines and Medicinal Devices

(Offi cial Gazette of RM No.106, of 5 September 2007;

amendments 15 and 16 July 2010.

OGRM, 2007c. Law on Prevention of Confl icts of Interests

(Offi cial Gazette of RM No.70/2007;114/2009- statement of

interests.

OGRM, 2010. Law on Inspection Supervision (Offi cial Gazette

of RM No. 50 of 13 April 2010; implementation as of 1 April

2011).

PIC/S, 2007. Recommendation on Quality Systems Requirements

for Pharmaceutical Inspectorates (http://www.ccd.org.cn/

ccd/fs/web_edit_fi le/20110510141908.pdf).

WHO, 1999. Operational Principles for Good Pharmaceutical

Procurement, Essential Drugs and Medicines Policy,

Interagency Pharmaceutical Coordination Group,WHO/

EDM/PAR/99(http://www.who.int/3by5/en/who-edm-par-

99-5.pdf).

WHO, 2004. Priority medicines for Europe and the World,

Essential Drugs and Medicines Policy, Ed.WHO, pp23-28

WHO, 2010. Model List of Essential Medicines List, 16th (updated)

March (http://whqlibdoc.who.int/hq/2010/a95060_eng.pdf).

Макед. фарм. билт., 57 (1, 2) 77 - 83 (2011)


Резиме

Хармонизација на Инспекцискиот надзор во

фармацевтскиот сектор во Република Македонија согласно

препораките на европската регулатива и СЗО

Василка Нича, Рената Славеска Раички и Татјана Кадифкова Пановска

Јавна установа од областа на здравството за потребите ЈЗУ Универзитетски Клиники, Завод и

ургенетен центар, Скопје

Фармацевтски факултет, УКИМ, Скопје

Клучни зборови: фармацевтски инспекциски надзор

Новиот Закон за инспекциски надзор (Службен весник 50/2010, имплементација од 2011/04/01) беше донесен во април 2010

година со цел да се унапреди квалитетот на инспекцискиот надзор и во фармацевтскиот сектор. Новиот Закон покрива спектар

на тековно идентификувани слабости.

Системот за квалитет на инспекцискиот надзор обединува збир од основни барања за квалитет во управување со процесот,

цели, услови, политики, официјални закони и процедури. Организациската шема се однесува на сите аспекти на инспекциски-

те активности. Инспекциската служба мора да гарантира дека нејзиниот персонал не е под било какви внатрешни или надвореш-

ни трговски, финансиски или друг вид на притисоци и несоодветни влијание кои што може негативно да влијае на квалитетот на

нивната работа.

За извршување на инспекцискииот надзор во согласност со спецофичните барања и стандарди, инспекциската служба тре-

ба да располага со доволен број на вработен персонал кој ги поседува потребните степени на образование, обука, техничко поз-

навање и искуство. Ангажираноста на инспекторот треба да е во рамките на одговорностите кои што произлегуваат од неговите/

нејзините активности. Потребно е да се востанови континуирана обука за инспекциските активности вклучувајќи ги и напредни-

те програми за обука кои нудат различни нивоа на квалификација на инспекторите.

Во блиска иднина, се наметнува потреба од развивање и усвојување на правилници со кои ќе се регулира инспекцискиот над-

зор во фармацевтскиот сектор во однос на нивната специфична активност.

Овој процес на перманентно надградување на постоечката национална релулатива согласно легалните политики на Европс-

ката Унија е една од карактеристиките на фармацевтскиот сектор во Р. Македонија.


ISSN 1409 - 8695

UDC: 615.11:34(4)Professional paper

Introduction

Satisfying the expectations of its’ fi nal users - the pa-tients by obtaining safe, effi cient, easily available, high quality affordable medicines is a major goal of the generic pharmaceutical industry.

This paper presents an overview of the ICH Q8, Q9,

Analysis and critical review of ICH Q8, Q9 and Q10 from

a generic pharmaceutical industry view point

Ljuba Karanakov1*, Jasmina Tonic-Ribarska2, Marija Glavas-Dodov3, Suzana Trajkovic-Jolevska2

1Replek Farm Ltd, Kozle 188, Skopje, Macedonia 2Institute of Applied Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy, Ss Cyril and Methodius University,

Vodnjanska 17, Skopje, Macedonia3Institute of Pharmaceutical Technology, Faculty of Pharmacy, Ss Cyril and Methodius University,Vodnjanska 17, Skopje,

Macedonia

Received: December 2011; Accepted: January 2012

Abstract

Generic industry aims to produce safe, effi cient, built-in quality medicines that will satisfy patients’ requirements and will be competitive on the market. In this paper, assessment of the need for quality by design (QbD) and process analytical technology (PAT) implementation by generic industry was made, by analysis of the ICH Q8, Q9 and Q10 guidelines and their implementation in European regulation. The review of the guidelines indicates differences in the life cycle of a generic medicine, leading to a fi nal conclusion in terms of generic industry.

PAT provides statistical analysis and real time quality monitoring, as the basis for proactive quality management. Using QbD/PAT, quality is proved and improved throughout the entire life cycle.

Better understanding of the product and processes within a defi ned design space leads to easier proof of built-in quality throughout the life cycle of the medicine, faster and easier regulatory evaluation, faster time to market, as well as post marketing savings regarding costs and time.

Implementation of QbD/PAT as a systematic approach together with risk assessment as part of quality management system is a useful challenge to the generic industry and gives an opportunity for technological, temporal, fi nancial and quality improvement.

It was concluded that having in mind its’ own manufacturing capabilities the applicant should optimize the implementation of QbD in accordance with current good manufacturing practice guidelines. Implementation of QbD/PAT is an innovative challenge for the generic industry. Managing pharmaceutical quality system allows the top management to make right decisions at the right time.

Key words: ICH Guidelines, Pharmaceutical development, Quality Risk Management, Pharmaceutical Quality System, Quality by Design, Process Analytical Technology

* [email protected]

Q10 guidelines for generic medicines for human use. The goal is to recognize the benefi ts, challenges and

opportunities deriving from implementation of the guide-lines, including Quality by design (QbD) and Process ana-lytical technology (PAT), from a critical viewpoint of the generic pharmaceutical industry related to the regulation and authorities evaluation of the application, concerning medicine quality.

Description and analyses of the ICH Q8, Q9, and Q10 focused on determination of QbD and PAT, as well

86

Maced. pharm. bull., 57 (1, 2) 85 - 96 (2011)

Ljuba Karanakov, Jasmina Tonic-Ribarska, Marija Glavas-Dodov, Suzana Trajkovic-Jolevska

as European quality regulations and guidelines, will point out the contrast of the generic medicine life cycle, consid-ering their implementation, concluded from aspect of the generic industry.

ICH Q8, Q9, Q10

Implemented as scientifi c guideline in European reg-ulation (Volume 3), ICH Q8 - Pharmaceutical develop-

ment gives guidance for: optimization of medicine quality through its life cycle, presentation of science based data in CTD Module 3 and meaning of transfer from fi nished product inspection to build in quality and real time release testing. This guideline gives possibility and guidance for development studies where risk management, as part of ef-fi cient pharmaceutical quality system, is implemented. The guidelines ICH Q9 Quality Risk Management and ICH

Q10 Pharmaceutical Quality System, differing from Q8, are not defi ning the content of the CTD Module 3. They only provide guidance, for basic, expectations for presen-tation of the development and certifi cation of the med-icine quality. Adopted ICH Q9 and ICH Q10 are imple-mented as sections of PART III of European Union Good Manufacturing Practice (ЕМА, 2011). The aim of PART

III is to explain the regulatory expectations and should be

considered as source of information for current best prac-

tice (ECA, 2011).

QbD/ PAT Implementation

The empirical approach is based on predefi ned

specifi cation that available data should comply with.

Determination of one variable at a time may end up with

Out of Specifi cation results, leading to quarantine or with-

drawal of the product. Controls of materials and processes

are repeated after each cycle, production process is fi xed,

without possibility of unauthorized changes, and its vali-

dation is based on minimum three pilot batches manufac-

tured at industrial facilities. The control strategy is out of

site testing and inspection, focused on repeatability and

optimization of processes, controlled in terms of specifi -

cation approved with the Marketing Authorization (MA).

Retrospective quality assurance and corrective measures,

based on in-process and fi nished product analysis are used,

where opportunities for statistical and basic problem cause

analysis are limited (ICH Q8, 2008).

Differing from the empirical approach, QbD/PAT im-

plementation represents a systematic-multidimensional de-

termination of Critucal Quality Atributes (CQAs) within

design space. Design space is defi ned depending on Quality

Target Product Profi le (QTPP) and available resources, us-

ing prior scientifi c knowledge, risk assessment and de-

sign of multivariable experiments, to better understand

the qualitative attributes of input materials and processes.

Product quality is refl ected in the approved design space.

Data on safety and effi cacy in this phase are obtained by

performing in vitro studies and in vitro/in vivo correlations

(EMA Inspection Assessment, 2007). Specifi cation in ac-

cordance with regulatory requirements and risk assessment

of CQA, for materials and product quality, results in deter-

mination of CQAs of the performance for testing and PAT

tools should be used. Production processes are fl exible and

scale up of manufacturing to industrial facilitates is easier

and faster. QbD product almost never fails the bioequiva-

lence study and transfer - scale up to commercial manufac-

turing is predictable. Ongoing quality management leads

to processes validation through the life cycle of the medi-

cine. Control strategy is based on scientifi c knowledge and

risk assessment. PAT enables statistical analysis and qual-

ity monitoring at real time, as a base for proactive quality

management (EMA Refl ection paper, 2006). Using QbD/

PAT, the quality performances are improved through the

entire life cycle.

Selection of components, formulation, manufactur-

ing process and control strategy development for easi-

er and faster commercialization of a product with a con-

sistent quality is at major focus of the generic industry.

There are no strict requirements for QbD/PAT application.

Depending on his needs and possibilities of proving built

in quality, the applicant should optimize QbD implementa-

tion in compliance with the Current Good Manufacturing

Practice (cGMP) guidelines. Implementation of QbD/PAT

is innovative challenge for the generic industry with op-

portunities for robust processes, cost reduction, lowered

rate of batch failures and faster science based regulatory

assessment (Varu, 2010). QbD enables real time release

improvement of the product and proceses after marketing

within the approved design space with no need to apply

for post marketing variations (ICH Q8, 2008). This leads

to reduced postmarketing costs. Variations may apper dur-

ing the development as well. For example variable disso-

lution rate at the end of the development will prolonge the

time to market. Investing in DoE at the beginning of de-

velopment helps to realze where the variations can come

from and this will shorten the time to market (Snee, 2008).

Implementation of QbD/PAT gives an opportunity for pre-

dictable fi nal outcome, easier and faster transfer of manu-

facturing proces from development to industrial capacities

wich leads to faster time to market (IMB, 2005). Managing

quality pharmaceutical system enables the top manage-

ment to bring right decisions at right time.

Generic industry critical point of view for imple-

mentation of ICH Q8, Q9, Q10 guidelines and

PAT

The guidelines ICH Q8, Q9 и Q10 are complemen-

tary to each other. ICH Q8 is tightly connected with ICH

Q9. Quality Risk Management (QRM) is based on the ex-

periments performed during the pharmaceutical develop-

ment. Risk assessment enables designing a high quality

product during implementation of QbD, defi nition of the

Макед. фарм. билт., 57 (1, 2) 85 - 96 (2011)

87 Analysis and critical review of ICH Q8, Q9 and Q10 from a generic pharmaceutical industry view point

CQAs and quality specifi cation. Risk assessment helps to

determine a suitable and acceptable design space. ICH Q8

refers to better understanding of product and process attri-

butes, based on accumulated knowledge and risk assess-

ment, due to constant improvement of product quality. On

the other hand ICH Q10 gives guidance for implementa-

tion and management of modern quality system. While it

doesn’t talk for a specifi c product but for the system as a

whole, however, the focus is on specifi c measurements of

product and processes quality that demonstrate continuous

improvement of the realized product, that should satisfy

consumers’ quality requirements. ICH Q10 provides man-

agement of changes within the design space. Any success-

fully designed pharmaceutical quality system contains el-

ements of risk management (ICH Q9, 2005). From this it

can be concluded that to enjoy the benefi ts of implement-

ing ICH Q8 and ICH Q9 implementation of ICH Q10 can-

not be avoided. For successful implementation of QbD and

PAT, the industry should follow the directions given in the

guidelines ICH Q8, Q9, Q10.

Because of the rigorous requirements set for the manu-

facturing processes, the pharmaceutical industry is far be-

hind other industries in terms of improving the techniques

and processes for on line control. On the other hand, regu-

lators require following of new technology achievements,

and the PAT represents a chance for the pharmaceutical in-

dustry to bring innovation in the development and control

of manufacturing processes.

PAT is the basis for successful implementation of ICH

Q8, Q9 and Q10 in the development of a medicine wheth-

er it is innovative or generic. Implementation of ICH Q8,

Q9, Q10 and PAT will allow distribution of products with

proven quality, safety and effi ciency to end users - patients.

Modern approach to development through improvement of

technology brings a lot of benefi ts.

Production time, number of defective products, life cy-

cle of improved quality medicine, documentation, manufac-

turing costs and manual error are reduced. Manufacturing

capacities, use of high technology and safety of the operator

are increased. This approach enables marketing the prod-

uct under the principle of real time release, use of state-of-

the-art technologies and guaranteed quality level (unit to

unit) in production (Varu, 2010). Establishment of continu-

ous processing and improvement of the effectiveness and

management of variables, gives an opportunity for more

pragmatic approach of medicines agencies and continu-

ous improvement during evaluation, which will enable the

manufacturer to be more competitive on the market.

For all this to be achieved manufacturers and the

competent authorities will have to exert a lot of effort.

Reciprocal communication will have to rise to a higher lev-

el and all issues that will arise will need to be considered

interactive.

Besides the chance for quality improvement, safety

and effi cacy of the product and processes, the challenge for

the industry to transit from traditional to more modern and

improved access to development, without costs increase,

must be taken into account. But this challenge without the

investment is almost impossible.

Successful implementation of the new paradigm for

most of the generic manufacturers will represent a change

of the company policy, and even change of the organiza-

tional structure. The guidelines are accepted by European

Medicines Agency (EMA) but they are not mandatory, ex-

cept for elements prescribed in other guides such as: ICH

M4 (R1), International organization for standardization

(ISO), cGMP national guidelines. It’s up to the manufac-

turer to decide which elements in what extent should be

implemented. Whether for a generic industry is worthwhile

to introduce new elements and principles to existing prod-

ucts or only to new products a good analysis should be

done. Introduction of the new approach to existing prod-

ucts will entail reporting variations among authorities, and

therefore additional costs.

Deciding upon QbD/PAT implementation

Since the implementation of the new improved ap-

proach is at an early stage, there is still no accurate data on

the possible cost benefi t. However, based on the available

data on manufacturing costs of a generic medicine assump-

tion can be made.

Development of medicine using a traditional approach

takes approximately 4.5 million dollars. If costs are shown

for each stage of the process separately, an assumption can

be made in which phase of the process the costs will be in-

creased or reduced and what the implementation of a mod-

ern approach would mean for a generic industry. Table 1

refers to the development costs of generic prescription

medicine in Canada (Canadian generics, 2010)

Table 1. Structure of costs for development of a generic

medicine using traditional approach, given by

stages expressed in dollars

Generic medicine

development stages

Structure of costs

expressed in dollars ($)

API providing 250 000

Formulation 250 000

Testing, manufacturing process,

scale - up, manufacturing1 000 000

Bioequivalence 1 000 000

Regulatory affairs until and

after granting of the marketing

authorization

2 000 000

From the foregoing data on the benefi ts of the imple-

mentation of QbD/PAT an assumption can be made that

the costs associated with regulatory activities would be re-

88

Maced. pharm. bull., 57 (1, 2) 85 - 96 (2011)


duced by 10-15%, the cost of transfer from laboratory to industrial production would be reduced by 10-20% and the cost of providing active substance would be reduced by 4%, but the initial costs for formulation will need to be in-creased. After the time for return on investment, the costs for this part of the process should be reduced as well.

In what extent the costs for research and formulation development will increase depends on the manufacturer’s decision, to what extent, how and weather, to improve its performance by introducing innovation.

Taking into account the structure of manufacturing costs of a generic medicine, that the generic industry should implement ICH Q8, Q9, Q10 and PAT can be concluded.

The generic industry doesn’t have any costs for devel-opment of new molecules and therefore selling price of a generic medicine is much lower than the selling price of the innovative medicine, and profi t margins are higher than for innovative companies (Malhotra, 2010).

Also selling price of the generic medicine is many times higher than the costs of active substance and formu-lation that gives the opportunity for generic manufacturers to invest in the implementation of QbD/PAT and again to be profi table (Malhotra, 2010).

To support the concept, simpler and faster access to quality safety and effi cacy medicines for all, generic man-ufacturers should be reviewed, at what level of the road to success is and where he wants to be, and depending on the above he will decide whether the new paradigm is the way to success.

If the manufacturer makes a positive conclusion, the whole process of implementation should be accepted as an investment, which depending on the extent will sooner or later return, and products processes will be constantly improved.

If the generic manufacturer decides to begin the in-novation by implementing the ICH Q8, Q9, Q10 and PAT, consultations with the competent authorities, for almost every part of the process, will be necessary.

For positive decision of a generic company on imple-mentation of the new paradigm QbD/PAT, primarily the top management has to realize that the implementation is necessary and it’s a useful challenge and opportunity for the company.

Need of management awareness is clearly shown by Woelbeling and co-workers (Woelbeling et al., 2008). Assessment by interviews of 11 PAT case studies, where PAT projects were executed was performed. The following categories have been defi ned and considered for this as-sessment: quality, process, risk, cost, personnel, tools, time validation, organization, regulatory. It was concluded that when PAT is professionally implemented the quality, risk validation and regulatory aspects can be summarized as a positive experience. Even the companies with less expe-rience in PAT implementation have positive expectations. PAT enables increasement of process understanding and the utilization of different technologies accompanied with

management of a lot more data which improves the com-munication between departments, as well as advanced and comprehensive data analysis and assessment. Considering cost benefi ts it’s still early for precise calculations, but experience shows decreased indirect costs. Benefi ts are claimed in terms of higher yields, reduced cycle times and fewer rejections/reworks rather than in terms of fewer personnel.

Once management has accepted the need for imple-mentation of QbD/PAT, the next step is to decide whether the implementation will be conducted for existing or new products planned, keeping in mind that changes will need to reported as variations to the authorities. Whatever has been decided, a plan and framework for implementation should be made. The framework will present the entire life cycle of the generic medicine, and defi ne in which phase of the life cycle, and in what extent should be undertaken for suc-cessful implementation of the new paradigm. Structuring of the data allows assessment of resources, technologies, personnel and time for development, which in turn is a ma-jor challenge for the generic manufacturer for faster plac-ing on market of high quality product.

Framework for QbD/PAT implementation

Proposed framework for the implementation of QbD/PAT is given in the study of Chatterjee (Chatterjee, 2010). The framework consist of fi ve phases concerning the core principles of QbD defi ned as product and process design and understanding, process predictability and process performance.

Phase 1 addresses the fi rst steps in generic development such as product selection and Site Capability Analysis. This is an opportunity for the manufacturer to realize its’ capa-bilities and recognize the increased basic costs and poten-tial standard costs of pursuing a new product.

In this phase a Product Design Specifi cation summa-rizing the product’s key attributes, risk analysis from a clinical perspective for the process predictability and vali-dation, and project timeline should be developed as criteria for project performance. As regulatory required some de-sign elements, such as dosage form, strength, route of ad-ministration, identity, assay and content uniformity are ex-pected to be constant during development.

The second phase describes the elements of develop-ment and characterization. For better process understand-ing, a platform approach should be adopted for one type of product development, at a time (Chatterjee, 2010). The for-mulation knowledge gained from key excipient, binder and other functional excipient elements can be used for all de-velopment activity. In vitro/in vivo correlation if provided by the innovator and risk analysis from phase 1 will steer the product design and characterization. For better under-standing of the component variability active pharmaceuti-cal ingredient (API) as well as excipients should be well characterized and presented in the development dossier.

Макед. фарм. билт., 57 (1, 2) 85 - 96 (2011)


Measurement Sensitivity Analysis (MSA) should be used on all in-process and release tests used to steer and mea-sure the process stability and product performance. This will ensure that the measurement tools being used to evalu-ate the process have the resolution to tell good from bad.

Phase 3 addresses the: location, selection, process de-sign and technology transfer. Technology transfer depends on the level of process understanding during the develop-ment. A risk analysis before process design and MSA after the method transfer should be repeated. These elements en-able simple process design with maximum possibility for success.

The fourth phase considers the data showing in the application submitted for MA. Characterized process and product performance, batch record and control strat-egy within a robust design space should be shown in MA documentation.

Phase 5 shows that the measurement of true critical process parameters at the right point of the process, de-fi ned during structured development and process design

gives an opportunity for real time intervention if needed.

Compliance measurements and use of corrective and pre-

ventive actions, generated by each product can be used

as a driver for continuous improvement of the business

performance.

Knowledge management and knowledge gained from

the past product as structured routine evaluation is bene-

fi ciary for the developing product and processes and will

limit the risk profi le. Decisions and choices for the devel-

oping product will be simplifi ed.

Following a structured framework with clearly defi ned

metrics at each stage gate will reduce any confusion and

variability in the product development process and will fa-

cilitate a faster time to market. The structured approach al-

Fig. 1. Monitoring of tabletting process and proces control using wet granulation method when QbD/PAT is implemented.

90

Maced. pharm. bull., 57 (1, 2) 85 - 96 (2011)


lows the organization to measure and focus its continuous improvement activities upon those elements that have the largest potential to increase and sustain business perfor-mance (Chatterjee, 2010).

For better understanding of the importance for devel-opment of process controls in comparison with testing by one parameter, examples for monitoring of manufacturing processes are presented in Figs. 1, 2 and 3.

Fig. 1 shows typical tabletting process where wet gran-ulation method is used, when QbD/PAT is implemented. API and excipients are characterized. CQA defi ned in de-sign space are measured by PAT. During wet granulation CQA such as mixing time, wetting rate, temperature, mix-ing speed, granulation time and torque end point were de-termined for assessment of granule properties. Tablet prop-erties were assessed by performing suitable dissolution test. When QbD/PAT is implemented fi nal product is real time released and 100% in compliance with product qual-ity specifi cation.

Fig. 2 is showing a manufacturing process of modi-fi ed release multiple-unit dosage form, capsules containing imediate release (IR) and controlled release (CR) pellets, and its control without the implementation of QbD/PAT. Finished product is in compliance with quality criteria if it meets the requirements from quality specifi cation, and quality control is performed at another location, testing one parameter at the time. Processes and testing are performed in accordance with cGMP, but (i) API and excipients have not been characterized; instead of that they are sampled and tested if they comply with a predefi ned specifi cation; (ii) intermediate processes in the manufacturing of modi-fi ed release capsules, such as preparation of API layering solution, coating of sugar spheres (cores) with a layer of API using Wurster fl uid-bed process, sieving, preparation of the polymeric coating suspension, CR coating of pellets and re-sieving are fi xed. Therefore, they are without abil-ity to monitor and determine parameters such as API con-tent in the layering solution, content and content uniformi-ty and size of IR pellets, polymeric coating suspension vis-

Fig. 2. Monitoring of the manufacturing process and process control of modifi ed release capsule (pellets fi lled with an im-mediate release and controlled release pellets) without implementation of QbD/PAT

Макед. фарм. билт., 57 (1, 2) 85 - 96 (2011)


cosity, the thickness of CR membrane, the size of CR pel-lets and drug release from CR pellets. Instead of that, the control of API layering solution, IR pellets, suspension for CR coating and CR pellets are at the end of the processes, by sampling and testing. Filled capsules are also sampled and tested due to quality control. The fi nal outcome may be

a fi nished product that meets specifi cation requirements, or

does not, leading to product failure, retrospective analysis

of the cause of the malfunction and the time and costs for

placing the medicine on the market are increased.

Unlike the traditional approach, implementation of

QbD/PAT offers a lot of possibilities, as shown in Fig. 3.

When QbD/PAT is implemented product is in compliance

with quality criteria if it meets the specifi cation, but mea-

surements of critical process parameters are performed

during processes and not after fi nishing. Process and test-

ing are carried out in accordance with cGMP as well, but

(i) API and excipients are characterized which allows de-

fi ning optimal solubility of the API and stability of the API

layering solution and maximal achievement of content uni-

formity; (ii) intermediate processes are fl exible and critical parameters are controlled during process. Real time testing is performed during pellets production. The fi nal outcome is a fi nished product that meets specifi cation requirements and the time and costs for placing the medicine on the mar-ket are decreased.

Competent authorities are also concerned by the imple-mentation of the new paradigm and will have to change the system of evaluation and inspection. The evaluation should be based on science and risk assessment. Specialists in all areas of evaluation will need to be recruiting and continu-ously trained. Presentation of data from the traditional and new approach in the documentation is different, therefore the competent authorities should be more fl exible, and over time setting of more specifi c directions may be needed. Decisions regarding the design space and its’ extent should be made without infringement of the confi rmed medicine quality. In the future assessment of the need for variations submission should be devised and even need for change of medicines control and system reorganization may appear.

Fig. 3. Monitoring of the manufacturing process and process control of modifi ed release capsule (pellets fi lled with an im-mediate release and controlled release pellets) with implementation of QbD/PAT

92

Maced. pharm. bull., 57 (1, 2) 85 - 96 (2011)


Inspectors should be thoroughly trained in terms of risk

management tools and principles in order to perform prop-

er inspection, companies’ pharmaceutical quality systems.Alignment of industry and competent authorities will

be very long and diffi cult journey, but the goal can only be achieved by mutual cooperation and understanding.

Conclusion

Investment in the development studies, as a source of scientifi c knowledge or knowledge gained from previous experience, enables the applicant to establish a wider de-sign space and better understand the product and process-es. This leads to easier improvement of the built in qual-ity through the life cycle of the medicine, faster and easier regulatory assessment, as well as savings on post approval costs and time. Due to economic competition, wider design space leads to faster commercialization and reduced post-approval variations.

Implementation of QbD/PAT as a systemic approach using risk assessment, as a part of the Quality management system, is benefi cial, challenging and offers the generic in-dustry opportunities for technological, time consuming, fi -nancial and quality improvements.

Any way it’s up to generic industry to decide for the way and extent of QbD implementation. But any industry for own improvement should be lead be Nelsons’ saying “You cannot do today’s job with yesterdays method and be in business tomorrow” (Horatio Nelson Jackson).

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Saari, H-L., 2004. Risk management in drug development projects. [online] Available at: http://jobfunctions.bnet.com/abstract.aspx?docid=154472 [Accessed 5 December 2010]

Sabir, A., AAPS, 2010. Life Research Hope, Generics Embracing

Макед. фарм. билт., 57 (1, 2) 85 - 96 (2011)


QbD / PAT Globally. Tampa, FloridaSam, T., 2008. WHO Training Workshop on Pharmaceutical

Development with Focus on Pediatric Formulations, Quality by Design. [workshop] May. Mumbai, India

Shah, B.R., 2009. AAPS. Quality by design in Pharmaceutical manufacturing. Chicago. Available at: http://www.aapspharmaceutica.com/inside/discussiongroups/chicago/imagespdfs/ShahNov2009.pdf (Accessed 5 April 2011)

Smith, C.G.,O’Donnell, J.T., eds., 2006. The Process of New Drug Discovery and Development. 2nd Edition, New York: Informa Healthcare

Snee, D.R., Cini, P., Jason, J., Kamm, J.J., Meyers, C.A., 2008. Quality by design shortening the path to acceptance. (Tunnell Consulting) [online] Available at:http://www.tunnellconsulting.com/library/published-аrticles/synopsis.php?id=19 [Accessed 11 December 2010]

Somma, R. SommaTech. Using Quality by Design (QbD) in Designing Effi cient FDA compliant Pharmaceutical

Manufacturing Processes and Facilities. Available at: http://

www.sommatechconsulting.com/site/images/stories/news_

room/events/using_qbd.pdf (Accessed 10 May 2011)

The European Agency for the Evaluation of Medicinal Products

Human Medicines Evaluation Unit, Note for Guidance,

1996/155/CPMP/QWP, 28 January 1998 on development

pharmaceutics

Turner, J. R., 2007. New Drug Development - Design, Methodology

and Analysis, New Jersey: John Wiley & Sons

Urkin, E., Magenheim B., 2009. Challenges in Global Product

Development 1, The ICH Quality Vision & Implementation

Model Q8, Q9, Q10, March 31, April 1-2, Bar Ilan University,

Israel

Wilkinson, N., 2008. QP Symposium. ICH Q10 – Delivering a

Modern Effective Pharmaceutical Quality System. April.

London

World Health Organization., 2010. Quality by Design. [workshop]

21 to 25 June. Beijing, PR of China

Wyvratt, J., 2009. FDA Advisory Committee on Pharmaceutical

Science & Clinical Pharmacology. ICH Quality IWG

Questions & Answers: Quality by Design Aspects. 5 August

Резиме

Анализа и критички осврт на ICH Q8, Q9 и Q10 од аспект

на генеричка фармацевтска индустрија

Љуба Каранаков1*, Јасмина Тониќ-Рибарска2, Марија Главаш-Додов3,

Сузана Трајковиќ-Јолевска2

1Реплек Фарм Ltd, Козле 188, Скопје, Македонија2Институт за применета хемија и фармацевтски анализи, Фармацевтски факултет, Универзитет „Св. Кирил и

Методиј“, Скопје3Институт за фармацевтска технологија, Фармацевтски факултет, Универзитет „Св. Кирил и Методиј“,

Скопје

Клучни зборови: ICH водичи, фармацевтски развој, управување со ризикот за квалитет, фармацевтски систем за квалитет,

квалитет преку дизајн, процесна аналитичка технологија

Генеричката индустрија има за цел да произведе сигурни, ефикасни лекови со вграден квалитет, кои ќе ги задоволат

барањата на пациентите и ќе бидат конкурентни на пазарот. Во овој труд преку анализа на водичите ICH Q8, Q9 и

Q10 и нивна имплементација во Европската регулатива, направена е процена за потребата од имплеметација на

квалитет преку дизајн (QbD) и процесна аналитичка технологија (PAT) од страна на генеричка индустрија. Прегледот

на водичите укажува на разликите во животниот циклус на генерички лек, водејќи до краен заклучок од аспект на

генеричка индустрија.

PAT овозможува статистичка анализа и следење на квалитетот во реално време, како основа за проактивно

управување со квалитетот. Со употреба на QbD/PAT, квалитетот се докажува и подобрува во текот на целиот животен

циклус.

Изведен е заклучок дека апликантот треба да ја оптимизира имплементацијата на QbD во согласност со важечките

водичи за добра производна пракса, земајќи ги во предвид своите производствени можности. Имплементацијата на

QbD/PAT е иновативен предизвик за генеричката индустрија. Управувањето со фармацевтски систем за квалитет му

овозможува на највисокиот менаџмент да носи вистински одлуки во вистинско време.

96

Maced. pharm. bull., 57 (1, 2) 85 - 96 (2011)


Подоброто разбирање на производот и процесите во рамките на дефиниран простор во дизајн води кон полесно

докажување на вграден квалитет во текот на животниот циклус на лекот, побрза и полесна регулаторна евалуација,

побрза комерцијализација, како и заштеда на постмаркетиншки трошоци и време.

Имплементацијата на QbD/PAT како системски пристап кој користи процена на ризик, како дел од систем за

управување со квалитет, претставува корисен предизвик за генеричката индустрија и нуди можности за, технолошко,

временско и финансиско унапредување, како и подобрување на квалитетот.

INSTRUCTIONS FOR AUTHORS

Macedonian Pharmaceutical Bulletin is an offi cial publication f the Macedonian Pharmaceutical Association. The journal publishes original scientifi c papers, short com-munications, reviews, mini-reviews and professional pa-pers from all fi elds of pharmacy and corresponding sci-entifi c fi elds of interest for pharmacy (pharmaceutical and medicinal chemistry, immunology and imunochemistry, molecular biology, pharmaceutical analyses, drug quali-ty control, pharmaceutical technology, pharmacoinformat-ics, pharmacoeconomics, biopharmacy, pharmacology, ap-plied botany, pharmacognosy, toxicology, clinical pharma-cy, food and nutrition, physical pharmacy, organical syn-thesis, social pharmacy, history of pharmacy etc.)

The Macedonian Pharmaceutical Bulletin, also, pub-lishes and other contributions (recommendations and an-nouncements, reports of meetings, important events and dates, book reviews, various rubrics).

Types of paper

Original scientifi c papers (full length manuscripts) should contain own unpublished results of completed orig-inal scientifi c research.

Short communications also should contain completed but briefl y presented results of original scientifi c research.

The article should be prepared as described for full length

manuscripts, except for the following: the number of pag-

es should not exceed 10 (including 2 illustrations, fi gures

or tables). An Abstract should be included as well as a full

reference list.

Reviews and mini-reviews are written at the invitation

of the Editorial Board. “Mini-reviews” of a topic are espe-

cially welcome.

They should be surveys of the investigations and

knowledge of several authors in a given research area, the

competency of the authors of the reviews being assured by

their own published results.

Professional papers report on useful practical results

which are not original but help the results of the original

scientifi c research to be adopted into practical use. Profes-

sional papers might be based on the elaborating of theoret-

ical data.

Language

Original scientifi c papers, short communications, re-

views and mini-reviews should be written in good English

(American or British usage is accepted, but not a mixture

of these), while professional papers and all other contribu-

tions may be submitted in Macedonian.

Submission declaration

Submission of an article implies that the work de-

scribed has not been published previously (except in the

form of an abstract or as part of a published lecture or ac-

ademic thesis), that it is not under consideration for publi-

cation elsewhere, that its publication is approved by all au-

thors and tacitly or explicitly by the responsible authorities

where the work was carried out, and that, if accepted, it

will not be published elsewhere in the same form, in Eng-

lish or in any other language.

Policy and ethics

The work described in your article must have been

carried out in accordance with The Code of Ethics of the

World Medical Association (Declaration of Helsinki)

for experiments involving humans http://www.wma.net/

en/30publications/10policies/b3/index.html;

EC Directive 86/609/EEC for animal experiments

http://ec.europa.eu/environment/chemicals/lab_animals/

legislation_en.htm;

Uniform Requirements for manuscripts submitted to

Biomedical journals http://www.icmje.org. This must be

stated at an appropriate point in the article.

Submission

Please submit the manuscript electronically (e-mail ad-

dress: [email protected]) as a single PDF fi le, which

will be used in the peer-review process. All correspon-

dence, including notifi cation of the Editor’s decision and

requests for revision, takes place by e-mail removing the

need for a paper trail.

98

Maced. pharm. bull., 56 (1, 2) 79 - 82 (2010)

Editor

Referees

Please submit, with the manuscript, the names, ad-

dresses and e-mail addresses of 3 potential referees. Note

that the editor retains the sole right to decide whether or not

the suggested reviewers are used.

Papers received by the Editorial Board are sent to ref-

erees. The suggestions/comments of the referees and Edi-

torial Board are sent to the author(s) for further action. The

revised article should be returned to the Editorial Board as

soon as possible but in not more than 30 days.

Preparation of manuscripts

Use of wordprocessing software

It is important that the fi le be saved in the native for-mat of the wordprocessor used. The text should be typed (1 ½ spaced) on A4 paper with margins of 3.0 cm on each side in single-column format, font Times New Roman, Mac C Times, Macedonian Times and size 11, Keep the layout of the text as simple as possible. Most formatting codes will be removed and replaced on processing the article. In par-ticular, do not use the wordprocessor’s options to justify text or to hyphenate words. However, do use bold face, italics, subscripts, superscripts etc. When preparing tables, if you are using a table grid, use only one grid for each in-dividual table and not a grid for each row. If no grid is used, use tabs, not spaces, to align columns. The electronic text should be prepared in a way very similar to that of conven-tional manuscripts. To avoid unnecessary errors you are strongly advised to use the “spell-check” and “grammar-check” functions of your wordprocessor.

The pages in the article should be numbered. Finally, please create PDF fi le before sending the arti-

cle. After acceptance, you will be asked to supply the arti-cle as wordprocessing document (zip-fi le).

Appendices

If there is more than one appendix, they should be identifi ed as A, B, etc. Formulae and equations in appen-dices should be given separate numbering: Eq. (A.1), Eq. (A.2), etc.; in a subsequent appendix, Eq. (B.1) and so on. Similarly for tables and fi gures: Table A.1; Fig. A.1, etc.

Abbreviations

Defi ne abbreviations that are not standard in this fi eld in a footnote to be placed on the fi rst page of the article. Such abbreviations that are unavoidable in the abstract must be defi ned at their fi rst mention there, as well as in the footnote. Ensure consistency of abbreviations through-out the article.

Units

Follow internationally accepted rules and conventions: use the international system of units (SI). If other units are mentioned, please give their equivalent in SI.

The names of substances should be in accordance with the IUPAC recommendations and rules or Chemical Ab-

stracts practice.

Math formulae

Present simple formulae in the line of normal text where possible and use the solidus (/) instead of a horizon-

tal line for small fractional terms, e.g., X/Y. In principle, variables are to be presented in italics.

Footnotes

Footnotes should be used sparingly. Number them consecutively throughout the article, using superscript Ar-abic numbers. Many wordprocessors build footnotes into the text, and this feature may be used. Should this not be the case, indicate the position of footnotes in the text and present the footnotes themselves separately at the end of the article. Do not include footnotes in the Reference list.

Table footnotes

Indicate each footnote in a table with a superscript lowercase letter.

Figures

Figures (photographs, diagrams and sketches) and structural formulae should each be given on a separate sheet (the place to which they belong in the text should be indicated). The fi gures should be numbered in Arabic nu-merals (e.g. Fig. 1). Ensure that each illustration has a cap-tion. Supply all captions separately, not attached to the fi g-ure. A caption should comprise a brief title (not on the fi g-ure itself) and a description of the illustration. Keep text in the illustrations themselves to a minimum but explain all symbols and abbreviations used.

Please submit the pictures in a black and white version.

Tables

The tables should be numbered in Arabic numerals (e.g. Table 1) and each should be given on a separate sheet (the place to which they belong in the text should be in-dicated). Number tables consecutively in accordance with their appearance in the text. Place footnotes to tables be-low the table body and indicate them with superscript low-ercase letters. Be sparing in the use of tables and ensure that the data presented in the tables are not duplicated else-where in the article.

Макед. фарм. билт., 56 (1, 2) 79 - 82 (2010)

99 Instruction for Authors

Article structure

Manuscript should contain: title, abstract, key words,

introduction, material and methods, results and discussion,

conclusion, acknowledgment (if desired) references and

summary.

Subdivision

Divide your article into clearly defi ned sections (Ab-

stract, Introduction, Material and methods. etc.). Any sec-

tion or subsection may be given a brief heading. Each head-

ing should appear on its own separate line.

Essential title page information

Papers should be preceded by a title page comprising:

the title, the complete name(s) of the authors, and the au-

thor’s affi liations.

Title. Concise and informative. Avoid abbreviations

and formulae where possible.

Author names and affi liations. Where the family name

may be ambiguous (e.g., a double name), please indicate

this clearly. Present the authors’ affi liation addresses (where

the actual work was done) below the names. Indicate all af-

fi liations with a lower-case superscript arabic number im-

mediately after the author’s name and in front of the appro-

priate address. Provide the full postal address of each affi li-

ation, including the country name of each author.

Corresponding author. Clearly indicate (with *) who

will handle correspondence at all stages of refereeing and

publication, also post-publication. Ensure that telephone and

fax numbers (with country and area code) are provided in ad-dition to the e-mail address and the complete postal address.

Each paper must begin with an Abstract which should not exceed more than 250 (original scientifi c and profes-

sional papers) or 100 (short communications) words. The

abstract should state briefl y the purpose of the research, the

principal results and major conclusions. References should

be avoided, but if essential, then cite the author(s) and

year(s). Also, non-standard or uncommon abbreviations

should be avoided, but if essential they must be defi ned at

their fi rst mention in the abstract itself. Immediately after

the abstract, provide a list of 3 to 6 keywords arranged in

the order according to their importance.

Introduction

State the objectives of the work and provide an ade-

quate background, avoiding a detailed literature survey or

a summary of the results.


Provide suffi cient detail to allow the work to be repro-

duced. Methods already published should be indicated by a

reference: only relevant modifi cations should be described.

Manuscripts which are related to theoretical studies, in-

stead of Material and methods, should contain a sub-head-

ing and the Theoretical background where the necessary

details for verifying the results obtained should be stated.

Results

Results should be clear and concise.

Discussion

This should explore the signifi cance of the results of

the work, not repeat them. A combined Results and Discus-

sion section is often appropriate. Avoid extensive citations

and discussion of published literature.

Conclusions

The main conclusions of the study may be presented

in a short Conclusions section, which may stand alone or

form a subsection of a Discussion or Results and Discus-

sion section.

Acknowledgements

Collate acknowledgements in a separate section at the

end of the article before the references and do not, there-

fore, include them on the title page, as a footnote to the ti-

tle or otherwise. List here those individuals who provid-

ed help during the research (e.g., providing language help,

writing assistance or proof reading the article, etc.).

References

Citation in text

Please ensure that every reference cited in the text is

also present in the reference list (and vice versa). Any ref-

erences cited in the abstract must be given in full. Unpub-

lished results and personal communications are not rec-

ommended in the reference list, but may be mentioned in

the text. If these references are included in the reference

list they should follow the standard reference style of the

journal and should include a substitution of the publication

date with either “Unpublished results” or “Personal com-

munication”. Citation of a reference as “in press” implies

that the item has been accepted for publication and a copy

of the title page of the relevant article must be submitted.

Web references

As a minimum, the full URL should be given and the date

when the reference was last accessed. Any further information,

if known (DOI, author names, dates, reference to a source pub-

lication, etc.), should also be given. Web references can be list-

ed separately (e.g., after the reference list) under a different

heading if desired, or can be included in the reference list.

100

Maced. pharm. bull., 56 (1, 2) 79 - 82 (2010)

Editor

Reference style

Text: All citations in the text should refer to: 1. Single author: the author’s name (without initials,

unless there is ambiguity) and the year of publication; 2. Two authors: both authors’ names and the year of

publication; 3. Three or more authors: fi rst author’s name followed

by “et al.” and the year of publication. Citations may be made directly (or parenthetically).

Groups of references should be listed fi rst alphabetically, then chronologically.

Examples: “as demonstrated (Allan, 1996a, 1996b, 1999; Allan and Jones, 1995). Kramer et al. (2000) have recently shown....”

List: References should be arranged fi rst alphabeti-cally and then further sorted chronologically if necessary. More than one reference from the same author(s) in the same year must be identifi ed by the letters “a”, “b”, “c”, etc., placed after the year of publication.

Examples:

Reference to a journal publication: Van der Geer, J., Hanraads, J.A.J., Lupton, R.A., 2000.

The art of writing a scientifi c article. J. Sci. Commun. 163, 51–59.

Reference to a book: Strunk Jr., W., White, E.B., 1979. The Elements of

Style, third ed. Macmillan, New York. Reference to a chapter in an edited book: Mettam, G.R., Adams, L.B., 1999. How to prepare an

electronic version of your article, in: Jones, B.S., Smith, R.Z. (Eds.), Introduction to the Electronic Age. E-Publish-ing Inc., New York, pp. 281–304.

Journal abbreviations source Journal names should be abbreviated according to Index Medicus journal abbreviations: http://www.nlm.

nih.gov/tsd/serials/lji.html List of serial title word abbreviations: http://www.issn.

org/2-22661-LTWA-online.php; CAS (Chemical Abstracts Service): http://www.cas.

org/sent.html.Manuscripts written in English should contain a Sum-

mary in Macedonian at the end of the paper. The summa-ry should contain: title, author(s) full-name(s), surname(s), author’s affi liations (institution and address), key words and abstract. Professional papers written in Macedonian should contain a summary in English in which the same data should be included.

Submission checklist

It is hoped that this list will be useful during the fi nal checking of an article prior to sending it to the journal’s

Editor for review. Please consult this Guide for Authors for further details of any item.

Ensure that the following items are present: One Author designated as corresponding Author:

E-mail address - Telephone and fax numbers - All necessary fi les have been uploaded- Keywords- All fi gure captions - All tables (including title, description, footnotes)- Further considerations: Manuscript has been - “spellchecked” and “grammar-checked”References are in the correct format for this jour-- nal All references mentioned in the Reference list are - cited in the text, and vice versa Permission has been obtained for use of copy-- righted material from other sources (including the Web)

After acceptance

Proofs

One set of page proofs (as PDF fi les) will be sent by e-mail to the corresponding author. Please list the correc-tions and return them via e-mail. If, for any reason, this is not possible, then mark the corrections and any other comments on a printout of your proof and return by fax, or scan the pages and e-mail, or by post. Please use this proof only for checking the typesetting, editing, complete-ness and correctness of the text, tables and fi gures. Signif-icant changes to the article as accepted for publication will not be accepted.

We will do everything possible to get your article pub-lished quickly and accurately. Therefore, it is important to ensure that all of your corrections are sent back to us in one communication: please check carefully before replying, as inclusion of any subsequent corrections cannot be guaran-teed. Proofreading is solely your responsibility. Note that Macedonia Pharmaceutical Bulletin may proceed with the publication of your article if no response is received.

Offprints

The corresponding author, at no cost, will be provided with a PDF fi le of the article by e-mail. The PDF fi le is a watermarked version of the published article and includes a cover sheet with the journal cover image.

Additional paper offprints can also be ordered for an extra charge.

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