HPTLC for identification of

botanicals and their adulterants

Dr. Anita AnkliCAMAG – LaboratorySonnenmattstrasse 11CH-4132 Muttenz

2 The Team

3

Overview

What is HPTLC?

Reproducibility is our goal

Methodology for standardization in HPTLC

Some HPTLC methods

Identity and natural variability of botanicals,

Adulteration with other plant products or

API’s

Mixtures of plant products

Limit test

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What is HPTLC?

TLC for the 21st century

Instrumental TLC

Application

Development

Documentation

Densitometry

Truly „plug and play―

Fully cGMP compliant

A new concept

Suitable instruments

Scientific basis

Standardized methodology

Validated methods

High Performance Thin-Layer Chromatography

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Comparison TLC-HPTLC of flavonoids

TLC plate 20 x 20 cm

HPTLC plate 20 x 10 cm

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Comparison HPTLC - TLC

HPTLC TLC

Average particle size: 5-7 µm 10-15 µm

Particle size distribution: narrow wide

Separation distanze: 30 - 70 mm 100 - 150 mm

Running time: 3 - 20 min 30 -200 min

Solvent consumption: 5 - 10 ml 50 ml

Detection limit, absorb.: 10 - 100 ng 100 - 1000 ng

fluoresc.: 0.1 - 10 ng 1 - 100 ng

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Power of HPTLC

Screening

rapid , many samples, comparision on one plate

Fingerprint

for complex mixtures, disposable plates

Flexibility

kind of detection, multiple detection, mobile phase

Simple documentation

visual – electronic images

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Fields of application

Plant Drugs

ID, batch conformity, adulterations

Pharmaceutical industry

Food and cosmetic industry (lipids, colors...)

Environmental analysis (pesticides...)

Forensics

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Reproducibility is our goal

Acanthopanax

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Successful standardization

Echinacea June 30, 2005 – CSI Laboratory

Original image

published 2001

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Standardization of methodology

Plate setup and handling

Sample application (as band)

Chamber geometry and saturation

Humidity control

Developing distance

Derivatization procedure

Documentation (electronic images)

SOP

Our SOP is in full compliance with PhEur, USP, ChP

Available at: www.camag-laboratory.com

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Chamber conditioning

Twin Trough Chamber

precondition saturation unsaturation

Toluene, ethyl acetate, acetic acid = 70 : 33 : 3; HPTLC Silica gel 60 F254,

Left: Schisandra chinensis, right: S. sphenanthera

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Influence of the relative humidity

15% 30% 47% 60% RH 17% 47% 75% RH

Green tea: Polyphenoles

Toluene, acetone, formic acid = 4.5 : 4.5 : 1 Toluene

Test dye

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The basic HPTLC setupApplication Development Derivatization Documentation

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Identity of Ginger rhizome (Zingiber officinalis)

Toluene, ethyl acetate

= 9:1

Deriv.: Anisaldehyde

reagent

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 6-Gingerol2 8-Gingerol3 10-Gingerol4 6-Shogaol5 Ginger rhizome 16 Ginger rhizome 27 Ginger rhizome 38 Ginger rhizome 49 Ginger rhizome 510 Ginger rhizome 611 Ginger rhizome 712 Alpinia officinarum, rhizome13 Kaempferia galangal, rhizome14 Alpinia oxyphylla, fruit15 Alpinia katsumadai, semen

white light

(WRT)

UV 366nm

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Variability of Cimicifuga racemosa

Validated method:

Toluene, ethyl formate, formic acid = 5 : 3 : 2

Deriv.: Sulfuric acid reagent

1: Actein, 2-9: differentCimicifuga racemosa, rhizome

WRT

UV 366nm

1 2 3 4 5 6 7 8 9 10

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Adulterants of Cimicifuga racemosa

1 Isoferulic acid

2 Norcimifugin

3 Actein

4 23-epi-26-Deoxyactein

5 Cimifugin

6 Cimicifuga racemosa, rhizone 1

7 Cimicifuga racemosa, rhizome 2

8 C. foetida, rhizome

9 C. heracleifolia, rhizome

10 C. dahurica, rhizome

11 C. americana, Yellow cohosh1 2 3 4 5 6 7 8 9 10 11

white light

UV 254nm, before deriv.

5 6 7 8 9 10 11

Toluene, ethyl formate, formic acid = 5:3:2

UV 366 nm

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Collaborative trial (I)

Cimicifuga racemosa with 5 % adulteration

Acte

in

G1 G2 G3 G4

Acte

in

Appl. volume: 20 μl

Derivatization with

Boric acid/oxalic acid,

120 °C during 5 min

Fluorescent zone with

Rf = 0.24 (Rf = 0.06)

5 % C. foetida in C.

racemosa

Acte

in

Acte

in

R1 R2 R3 R4*

*

System suitability

test: Actein Rf ≈

0.37

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Collaborative trial with 5 % mixtures (II)

Derivatization with

antimony (III) chloride

120 °C for 10 min

Fluorescent zone with

Rf = 0.39 5 % C.

dahurica or C. heraclei-

folia in C. racemosa

R1 R2 R3 R4

G1 G2 G3 G4

Acte

in

Acte

in

Acte

inA

cte

in

*

*

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Collaborative trial with 5 % mixtures (III)

Evaluation: UV254 nmR1 R2 R3 R4

G1 G2 G3 G4

Fluorescence

quenching zone at

Rf=0.30 5 % of

C. americana in C.

racemosa

*

*

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PhEur 7.3 Cimicifuga racemosa

knowledge database of

PhEur (www.edqm.eu)

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ID and adulteration of Equisetum arvense

Equisetum palustre

Ethyl aceate, water, acetic acid, formic acid = 67 : 18 : 7.5 : 7.5, NP reagent,

Standards: rutin, hyperoside, caffeic acid

Equisetum arvense

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Sage oil – evidence of an adulteration

GC

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Ylang Ylang

Essential oil: toluene,

ethyl acetate = 95 : 5,

Anisaldehyde reagent

Various fatty oils:

DCM, acetic acid, acetone

= 20 : 40 : 50

Phosphomolybdic acid

reagent

Su

nflo

we

r o

il

Lin

ole

nic

acid

Arg

an

oil

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Herbal slimming supplements -

adulterated with Sibutramin (API)

Swissmedic

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Detection of sibutramin by HPTLC

UV 254nm

UV 225nm

UV spectrum

Toluene, methanol = 95 : 5

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Limit of detection of Aristolochic acid

1: Ref (a), 2: Ref (b) (2 ppm AA1), 3: Stephania, 4: Aucklandia,

5: Vladimiria, 6: Aristolochia debilis

1 2 3 4 5 6

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Screening test for Aristolochic acid PhEur 7.3

Solvent mixture: anhydrous formic acid R, water R, methanol R (1:9:40 V/V/V).

Test solution. To 1.0 g of the powdered herbal drug add 10.0 mL of the solvent mixture, sonicate for 10 min and centrifuge.

Reference solution (a). Disperse an amount of aristolochia HRS corresponding to 0.10 mg of aristolochic acid I in 20.0 mL of the solvent mixture, sonicate for 10 min and centrifuge.

Reference solution (b). Dilute 1.0 mL of reference solution (a) to 25.0 mL with methanol R.

Plate: TLC silica gel F254 plate R (2-10 µm) = HPTLC

Mobile phase: anhydrous formic acid R, water R, ethyl acetate R, toluene R (3:3:30:60 V/V/V/V); use the upper layer.

Application: 20 µL as bands of 8 mm.

Development: over a path of 6 cm.

Drying: in a current of cold air for 5 min.

Detection: spray with a 100 g/L solution of stannous chloride R in dilute hydrochloric acid R until the plate is slightly wet, heat at 100 °C for 1 min and examine in ultraviolet light at 365 nm.

System suitability:

— the chromatogram obtained with reference solution (a) shows 2 greenish-blue zones due to aristolochic acids I and II between RF = 0.35 and RF = 0.55, which may not be completely separated;

— the chromatogram obtained with reference solution (b) shows at least 1 of these zones (corresponding to 2 ppm of aristolochic acid I).

Results: in the chromatogram obtained with the test solution no zone is similar in position and fluorescence to any of the zones due to aristolochic acids in the chromatogram obtained with reference solution (a).

If the chromatogram obtained with the test solution shows any zones similar in position and fluorescence to any of the zones due to aristolochic acids I and II in the chromatogram obtained with reference solution (b), apply method B.

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Amarant - a red azo dye

- as adulterant of Bilberry extract

Image evaluated under white light

1: Bilberry dry extract

2: Bilberry dry extract spiked with

amaranth (spiking level 0.25 %)

3. Amaranth

1 2 3 1 2 3

enhanced

1-Butanol, water, formic acid

= 40 : 15 : 10

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Sources of methods

European Pharmacopoeia (EP)

New monographs feature TLC and HPTLC in parallel

British, French, German, Swiss Pharmacopoeias

Offer specific monographs not found in EP

The USP Dietary Supplement Compendium

TLC and state of the art HPTLC

Chinese Pharmacopoeia, Japanese Pharmacopoeia,

American Herbal Pharmacopoeia, Quality Standards

of Indian Medicinal Plants, Wagner, H. and Bladt, S.

„Plant Drug Analysis‖, …

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Thyme leaf plates 1 - 3

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Thyme leaf – comparison viewer

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Three channels per track

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Pattern recognition Master thesis, R. Ambühl, UNI Basel

Evaluation by principal component analysis

(PCA) and multivariate analysis of variance

(MANOVA)

Identification of probe1 () by MANOVA

Dendrogram of group means

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Thank you for your attention

anita.ankli@camag.com

www.camag-laboratory.com