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ORIGINAL RESEARCH ARTICLE

Determination of 1,4-dichlorobenzene, naphthalene

and thymol residues in honey using static headspacecoupled with GC-MS

Ren Badertscher*, Verena Kilchenmann , Agathe Liniger and Peter Gallmann

Swiss Bee Research Centre, Agroscope Liebefeld-Posieux Research Station ALP, Schwarzenburgstrasse 161CH-3003 Bern, Switzerland.

Received 7 October 2009, accepted subject to revision 24 March 2010, accepted for publication 6 May 2010.

*Corresponding author: Email: rene.badertscher@alp.admin.ch

Summary

A simple, fast, sensitive and robust analytical method was developed for the simultaneous determination of 1,4-dichlorobenzene (p-DCB),

naphthalene and thymol. The method is based on gas chromatography-mass spectrometry (GC/MS) coupled with a direct, static headspace

(HS) injection. With 1-bromo-4-chlorobenzene (p-BCB) used as internal standard, quantification achieved a high sensitivity with a linear range

in honey from 0.2 to 100 gkg1for p-DCB, 0.5 to 100 gkg1for naphthalene, and 3 to 1000 gkg1for thymol with a correlation coefficient

(r2) ranging from 0.9549 to 0.9884, respectively. The trueness was checked by repeated measurements of spiked honey samples on 3

concentration levels. There was no significant difference between the theoretical value and the obtained value. The mean Horwitz-Ratio

(HORRATrwith Thompson-correction) shows very good precision for p-DCB and naphthalene (0.5; 0.68) and good precision for thymol (1.23).

The detection limits for p-DCB, naphthalene and thymol in honey were 0.04, 0.09 and 1.4 gkg1, respectively.

Keywords: 1,4-dichlorobenzene, naphthalene, thymol; honey, residues

Journal of ApiProduct and ApiMedical Science 2 (3): 87 - 92 (2010) IBRA 2010DOI 10.3896/IBRA.4.02.3.01

Introduction

Historically, the use of common household moth poisons based on

1,4-dichlorobenzene (p-dichlorobenzene,p-DCB) have been illegally

applied by many beekeepers against wax moths (Galleria

mellonella). p-DCB enters the cycle of beeswax production and

contaminates commercial beeswax and honey (Wallner, 1992;

Bogdanovet al., 2004). In Switzerland, Germany, Greece and some

other countries up to 30% of randomly selected honey samples

containedp-DCB and a substantial part of these samples exceeded

the Swiss tolerance level of 10 gkg1(Bogdanovet al., 2004).

Alternative moth control products based on naphthalene are even

more toxic thanp-DCB and naphthalene residues have recently been

found in Swiss honey (Bogdanovet al., 2004). These unacceptable

levels of p-DCB and naphthalene residues have been the main

reason driving the need to develop a rapid sensitive and accurate

analytical method to monitor potentially harmful compounds in

honey. Although a number of papers have been published for the

determination of pesticide residues such as acaricides and

organophosphorus pesticides in honey that are used against Varroa

destructorand bee diseases (Formica and Schnabel, 1979; Formica,

1984 ;Taccheoet al., 1988; Paoli and Barbina, 1992; Cabraset al.,

1993; Fernandez-Muinoet al., 1995; Jimenezet al., 1995; Korta et

al., 2001; Martel and Zeggane, 2002; Bogdanov, 2003; Cobanoglu

and Tuze, 2008; Karazafiriset al., 2008; Ryooet al., 2008), there has

been only limited research related to the determination of p-DCB and

naphthalene (Wallner, 1992; Bogdanovet al., 2004; Tananakiet al.,

2005; Tananakiet al., 2006; Beyoglu and Omurtag, 2007; Harizanis

et al., 2008; Tsimeli et al., 2008). Also there has been limited

research related to the determination of thymol, an acaricide for the

control of V. destructor, which is a new potential residue compound

in honey. At higher residue concentrations thymol can also influence

the taste of honey.

In this study, the simultaneous detection of p-DCB,

naphthalene and thymol residues in honey, using HS-GC/MS with an

external calibration on real honey matrix with 1-bromo-4-

chlorobenzene (p-BCB) as the internal standard is presented for the

first time.

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Materials and methods

Materials

p-DCB (Pestanal analytical standard), naphthalene (puriss. p.a. >99.7%)

and thymol (purum >99%) were purchased from Fluka (Buchs,

Switzerland). The internal standard 1-bromo-4-chlorobenzene (for

Synthesis > 98%) was supplied from Merck (Darmstadt, Germany).

Stock solutions of all standards at the concentration of 100 mgL1

forp-DCB, naphthalene, thymol and the internal standard solutionat

the concentration of 10 mgL1were prepared in ethanol and were

kept at 4C.

Gas chromatography

An Agilent model 6890 gas chromatograph with a pulsed splitless

injector, equipped with an Agilent 5793 mass spectrometer (Agilent

Technologies, Basel, Switzerland) and a CombiPAL headspace

autosampler (CTC-Analytics AG, Zwingen, Switzerland) was used.

Headspace conditions: incubation at 95C, (90 min), syringe

temperature 120C, sample volume 750 L, fill speed 100 Ls-1and

inject speed 1 mLs-1.

The capillary column was DB5 fused silica, 0.25 m film

thickness (30 m 0.25 mm I.D.) (Agilent Technologies, Basel,

Switzerland). Sample introduction was via the pulsed pressure mode

(150 kPa until 1.00 min) using an injection volume of 750 L and a

helium flow rate at 0.8 mLmin

1. The injector temperature was set

to 200C. The column temperature was programmed as follows:

initial temperature 50C for 1 min, followed by a 15C min1

increase to 170C, then an increase at 20C min1to 290C, which

was held for 5 min. The MS interface temperature was set to 280C,

the ion source temperature to 230C, the quadrupole temperature

to 150C and the ionization energy to +70 eV.

Measurement procedure

Residue free honey samples containing concentrations of 1, 10, and

100 gkg1 of p-DCB, naphthalene and thymol, respectively were

prepared by spiking 500 g residue free honey (a mixture of polyfloral

and honeydew honey) with the appropriate amounts of stock

solutions.A well mixed honey sample of 4 g were filled into 20 mL

headspace vials. After adding 30 L of internal standard solution, the

vials were sealed with screw top caps with PTFE/silicon septa and

placed in the headspace sampler. The external calibration for the

quantitative detection of all three target analytes was done with

standard honey samples containing concentrations 1, 10, and 100

gkg1ofp-DCB, naphthalene and thymol respectively with the same

procedure.

ResultsSelected ion monitoring (SIM)

Qualitative and quantitative analyses were carried out by selectively

monitoring the detector response of characteristic ions at m/z 146

and 148 forp-DCB, m/z 190 and 192 for internal standardp-BCB, m/

z 127 and 128 for naphthalene and m/z 135 and 150 for thymol. The

dwell time for monitoring each m/z was 150 ms. Quantification was

performed by calculating for each analyte the ratio of peak area of

the quantifier ion between the unknown sample and the internal

standard. The quantifier ions were m/z 146, 192, 128, and 135 for p-

DCB, p-BCB, naphthalene, and thymol, respectively. The

chromatogram of a standard honey sample containing 100 gkg1of

p-DCB, naphthalene, and thymol and 300 gkg1ofp-BCB as internal

standard after analysis with the described method is presented in Fig. 1.

The retention times were 6.13 for p-DCB, 7.11 for p-BCB, 8.02 for

naphthalene, and 8.90 min for thymol.

The mass spectra for all three substances and the internal

standard are shown inFig. 2.

88 Badertscher, Kilchenmann, Liniger, Gallmann

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

5 6 7 8 9 10Time (min)

Abundance

p-DCB

Int. Std. Naphthalene

Thymol

Fig.1. Characteristic ion chromatograms of a standard honey sample, containing 100 gkg1each ofp-DCB, naphthalene and thymol and

300 gkg1ofp-BCB as the internal standard.

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Determination of DCB, naphthalene and thymol in honey 89

0

10

20

30

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100 110 120 130 140 150 160

m/z

Abundance

111

75

50

85

146

148

0

10

20

30

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

m/z

Abundance

111

75

50

192

85

96

190

155

0

10

20

30

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100 110 120 130 140

m/z

Abundance

77

51

39

64

128

87

102

0

10

20

30

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100 110 120 130 140 150 160

m/z

Abundance

1077739

91

135

150

5165

115

Fig. 2. Ion mass spectra of 1,4-dichlorobenzene, 1-bromo-4-chlorobenzene, naphthalene and thymol.

Cl

Cl

1,4-dichlorobenzene

Cl

Br

1-bromo-4-chlorobenzene

naphthalene

OH

2-isopropyl-5-methylphenol(thymol)

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Method validation

Specificity

No interfering peaks for each compound were observed at the

retention time of the characteristic ions by analyzing blank honey

samples.

Trueness

The trueness was checked by repeated measurements of spiked

honey samples at 3 levels of concentration. As shown in Table 1,

there were no significant differences between the theoretical values

and the obtained values for each analyte calculated at each level

with a two sided t-test.

Linearity

A linear calibration curve was observed over a range between 0.2 to

100 gkg1 for p-DCB, 0.5 to 100 gkg1 for naphthalene and 3 to

1000 gkg1 for thymol (Fig. 3.). The coefficients of determination

(r2) ranged from 0.9549 to 0.9884 for all analytes.

Precision

Precision was expressed as CV values, calculated from different

experiments under repeatability or reproducibility conditions. The

mean CVs were 7.2% for p-DCB, 9.9% for naphthalene and 17.9%

for thymol.

90 Badertscher, Kilchenmann, Liniger, Gallmann

Table 1.Validation data

p-DCB Naphthalene

Thymol

Level (mgkg-1) 0.00100 0.01000 0.10000 0.00100 0.01000 0.10000 0.00100 0.01000 0.10000

Mean (mgkg-1) 0.00104 0.01017 0.10286 0.00092 0.00991 0.10428 0.00115 0.00985 0.08965

n 10 9 9 10 9 9 10 9 9

Bias (mgkg-1) 3.97E-05 1.75E-04 2.86E-03 -7.71E-05 -9.22E-05 4.28E-03 1.55E-04 -1.55E-04 -1.04E-02

CV (% )

7.12 7.67 8.57 11.68 9.68 11.91 30.82 16.69 25.63

t-value 1.70 0.67 0.97 2.26 0.29 1.03 1.37 0.28 1.35

H=H0(p=0.95) yes yes yes yes yes yes yes yes yes

p-DCB: r2= 0.9884

thymol: r2= 0.9549

naphthalene: r2= 0.9883

0

50

100

150

200

250

300

350

0 0.02 0.04 0.06 0.08 0.1 0.12Residue concentration in honey (mg/kg)

Signal (-)

Fig.3. Calibration curve for 1,4-dichlorobenzene, naphthalene and thymol in honey

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The mean Horwitz-Ratio (HORRATr with Thompson-correction)

showed better precision for p-DCB and naphthalene (0.5; 0.68) than

for thymol (1.23), but the results showed excellent applicability of

the proposed method for the determination of the target compounds

in honey samples.

Limits of detection and quantification

The limits of detection and quantification were estimated from the

analysis of spiked honey samples at low concentration levels.

The detection limits were 0.04, 0.09 and 1.4 gkg1for

p-DCB, naphthalene and thymol respectively. The LOQs were

0.2 gkg1forp-DCB, 0.5 gkg1for naphthalene and 30 gkg1for

thymol.

Discussions

This paper describes a method for the simultaneous detection of

p-DCB, naphthalene and thymol residues in honey, based on

Headspace-GC/MS.

The proposed method is robust and sensitive using an

internal standard (1-bromo-4-chlorobenzene) and an external

calibration on real honey matrix. The proposed method was

optimized and validated over a wide range of concentrations for the

three target analytes. The method has been used in our institute

since 2006 for the monitoring of Swiss honey and has been applied

to more than 2000 samples. Its successful application to real

samples indicates that it is simple, fast, cost effective and suitable

for the analysis of large numbers of samples.

Acknowledgements

We thank Mark Kerry Greco from our institute for critical reading of

the manuscript.

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