multi-class pesticide analysis in human hair by gas chromatography tandem (triple quadrupole) mass...

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Analytica Chimica Acta 710 (2012) 65– 74

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Multi-class pesticide analysis in human hair by gas chromatography tandem(triple quadrupole) mass spectrometry with solid phase microextraction andliquid injection

Guillaume Salquèbrea, Claude Schummera,b, Maurice Milletb, Olivier Briandc, Brice M.R. Appenzellera,∗

a Laboratory of Analytical Human Biomonitoring – CRP-Santé, Université du Luxembourg, 162A avenue de la faïencerie, L-1511 Luxembourg, Luxembourgb Equipe de Physico-Chimie de l’Atmosphère, LMSPC (UMR 4515 CNRS-Université de Strasbourg), 1 rue Blessig, F-67084 Strasbourg Cedex, Francec French Agency for Food, Environmental and Occupational Health and Safety, Risk Assessment Department, 27-31 avenue du Général Leclerc, F-94700 Maison-Alfort, France

a r t i c l e i n f o

Article history:Received 17 July 2011Received in revised form 14 October 2011Accepted 16 October 2011Available online 21 October 2011

Keywords:PesticidesHairGC–MS/MSSPMEHuman biomonitoringExposure

a b s t r a c t

A method for the simultaneous detection and quantification of 22 pesticides from different chemicalclasses was developed using solid-phase microextraction (SPME) and gas chromatography tandem (triplequadrupole) mass spectrometry. Pesticides were extracted from 50 mg of pulverized hair with acetoni-trile. The extract was submitted to two successive steps of direct immersion-SPME at 30 ◦C and 90 ◦C orto a liquid injection without SPME in order to obtain optimized conditions for each of the 22 analytesinvestigated. Validation parameters were significantly influenced by both the injection mode (SPME vsliquid injection) and the temperature of SPME. Limits of quantification ranged from 0.05 pg mg−1 for tri-fluralin to 10 pg mg−1 for pentachlorophenol. The application of the validated method to the analysis ofsamples collected from non-occupationally exposed volunteers demonstrated the presence of pesticidesin all the samples tested. Altogether, 13 different analytes were detected at concentration above the limitof quantification.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

First thoroughly investigated in forensic contexts for the detec-tion of drugs of abuse, hair has been increasingly considered arelevant matrix for the biomonitoring of human exposure to envi-ronmental pollutants. Indeed, the possibility to reach extendedwindows of detection and to obtain information representative ofthe average level of xenobiotics concentration/entrance into thebody from a single specimen is particularly relevant in the con-text of chronic exposure biomonitoring. For this purpose, hair hasbeen tested for several environmental pollutants such as metals,PCBs, PAHs, and pesticides such as organochlorines, organophos-phates or pyrethroids [1–4]. The easy sampling of hair, whichdoes not require medical staff and generally facilitates patientcompliance, is a further advantage in the case of epidemiologicalstudies focusing on population exposure. On the other hand, thesmall amounts generally collected (limited to a few tens to a fewhundreds milligrams) and the low levels of concentration of xeno-biotics in hair, particularly in the case of environmental exposure,require the use of highly sensitive analytical methods. Analytical

∗ Corresponding author. Tel.: +352 46 66 44 67 27; fax: +352 22 13 31.E-mail address: [email protected] (B.M.R. Appenzeller).

sensitivity is also directly affected by the specificity of the method,in that sensitivity generally decreases with increasing number ofcompounds from different chemical families (with different physic-ochemical properties) that are simultaneously analyzed in one run.Nevertheless, priority lists defined by authorities are based on risksassociated with chemicals and generally include compounds fromdifferent chemical families without taking into account technicalfeasibility [5,6]. Moreover, the importance of cumulative exposureto chemical mixtures, even at low levels, is increasingly pointedout [7,8]. These considerations highlight the need to develop multi-class methods with high sensitivity for the biomonitoring of humanexposure to pesticides based on hair analysis.

Solid phase microextraction (SPME), developed by Pawliszynand co-workers in 1989 [9,10], consists in a coated metal- orglass-fiber on which analytes are adsorbed directly from an aque-ous extract (direct immersion: DI-SPME) or from the associatedheadspace (HS-SPME) that initially contains the analytes to be ana-lyzed, before their desorption into the chromatographic system.Presenting the ability to perform at the same time analyte extrac-tion from sample, concentration and purification, as well as beinga versatile injection tool, SPME represents a significant advance inanalytical chemistry for the handling of environmental and biolog-ical matrices containing low level of target analytes or/and highconcentration of impurities. Associated with gas chromatographyand microelectron-capture, mass spectrometry or high-resolution

0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2011.10.029


66 G. Salquèbre et al. / Analytica Chimica Acta 710 (2012) 65– 74

Table 1GC–MS/MS parameters.

Compound RTa (min) Quantification transition CEb (V) Confirmation transition CEb (V) Confirmation transition CEb (V)

Dichlorvos-D6 12.49 191.3 > 99.0 14Dichlorvos 12.54 109.2 > 79.0 6 109.2 > 47.0 15 185.3 > 93.0 14Trifluralin-D14 18.03 315.5 > 267.1 7Trifluralin 18.13 306.4 > 264.0 7 264.4 > 160.0 15 306.4 > 43.0 20�-HCH-D6 18.44 224.3 > 187.0 7�-HCH 18.53 219.2 > 183.0 7 109.2 > 49.0 27 181.2 > 145.0 16�-HCH 19.21 219.2 > 183.0 7 109.2 > 49.0 27 181.2 > 145.0 16�-HCH-D6 19.24 224.3 > 187.0 7�-HCH 19.34 219.2 > 183.0 7 109.2 > 49.0 27 181.2 > 145.0 16Pentachlorophenol (acetate)-13C6 19.88 314.2 > 272.0 1Pentachlorophenol (acetate) 19.98 264.2 > 165.0 26 266.2 > 167.0 26 308.2 > 266.0 1Aldrin 22.53 263.3 > 193.0 40 263.3 > 228.0 24 293.3 > 186.0 45o,p′-DDE 25.26 246.3 > 176.0 37 318.3 > 248.0 20 318.3 > 246.0 20�-Endosulfan 25.59 195.2 > 125.0 28 195.2 > 89.0 46 339.2 > 267.0 3p,p′-DDE-D8 26.67 326.4 > 254.1 18p,p′-DDE 26.74 246.3 > 176.0 35 318.3 > 248.0 25 318.3 > 246.0 18Dieldrin 26.78 263.2 > 193.0 39 263.2 > 228.0 23 277.2 > 207.0 27Oxadiazon 27.01 302.2 > 175.0 14 302.2 > 258.0 4 344.3 > 112.0 52�-Endosulfan-D4 28.26 199.3 > 92.0 46�-Endosulfan 28.35 195.2 > 159.0 8 195.2 > 125.0 28 195.2 > 89.0 46o,p′-DDT 29.04 235.4 > 165.0 28 235.4 > 199.0 18 165.0 > 115.0 17p,p′-DDT-D8 30.86 243.4 > 173.0 34p,p′-DDT 30.98 235.4 > 165.0 34 235.4 > 199.0 18 165.0 > 115.0 17Tebuconazole-D6 31.73 256.5 > 126.9 25Tebuconazole 31.77 250.4 > 125.0 25 250.4 > 70.0 7 125.3 > 89.0 21Diflufenican 32.36 394.4 > 266.0 11 266.4 > 246.0 15 266.4 > 218.0 27�-Cyhalothrin 38.20 181.3 > 152.0 29 197.3 > 141.0 12 197.3 > 161.0 5trans-Permethrin-D6 40.52 169.3 > 133.1 5trans-Permethrin 40.68 163.3 > 127.0 5 165.3 > 127.0 5 165.3 > 129.0 5Cypermethrin 42.27 163.3 > 127.0 5 165.3 > 127.0 5 165.3 > 129.0 5Fenvalerate 44.20 167.3 > 125.0 9 125.3 > 89.0 24 125.3 > 99.0 24deltamethrin 44.90 181.3 > 152.0 27 253.3 > 172.0 6 253.3 > 174.0 6

a Retention time.b Collision energy

time-of-flight mass spectrometry detection, SPME has already beenproven to be suitable for multi-class pesticide analysis in environ-mental matrices (e.g. rain, water, atmosphere) and food (e.g. bovinemilk) [11–15]. SPME has also been tested for the determination ofpesticides in human biological fluids (blood, urine, human milk),but the studies were limited to the detection of specific compoundsor families of compounds and did not aim at performing multi-class analysis [16]. Until now and despite its obvious suitability forpesticide analysis, SPME has never been used for the detection ofpesticides in hair and the use of this technique in hair analysis waslimited to the detection of medical drugs and drugs of abuse [16,17].This probably lies in the fact that hair, as a solid matrix, requirespretreatment such as hydrolysis (generally acidic or alkaline) orextraction with aqueous buffer, as it is performed for the detectionof drugs before exposing the SPME fiber to the aqueous extract.For several pesticides, acidic or alkaline hydrolysis is not recom-mended for protecting analytes from degradation. Extraction withaqueous buffer is neither adapted as it is not compatible with thehydrophobic properties of most pesticides. Until now, in most ofthe studies that investigated pesticide detection in hair, extractionwas performed with organic solvent (directly from pulverized hairor from acidic hydrolyzate) and after possible clean-up on solidphase extraction and concentration by evaporation to dryness andreconstitution in a smaller volume of solvent, the liquid extract wasinjected into the gas chromatography system coupled with massspectrometry or electron capture detection [4,18–20]. In the pub-lished literature, neither SPME nor gas chromatography coupledwith tandem (triple quadrupole) mass spectrometry (GC–MS/MS)has been tested for the detection of pesticides in human hair.

The aim of the present study has been to develop a highly sensi-tive method for the analysis of multi-class pesticides in human hairbased on SPME coupled with GC–MS/MS. Among other parameters,

the development of the full methodology included the setting upof an adapted extraction method of analytes from hair, the com-parison of DI-SPME vs HS-SPME, the optimization of the time andtemperature of fiber exposure and the validation of the method.The list of analytes investigated comprised 22 pesticides fromdifferent chemical classes including organochlorines, organophos-phates, dinitroanilin, nicotianilin, phenol, azole and pyrethroids.An optimized protocol was designed on the basis of the validationparameters and was finally applied to the analysis of field samplesthat were tested for the presence of the analytes investigated.

2. Experimental

2.1. Chemicals, reagents and standard solutions

All solvents (HPLC grade methanol, HPLC grade acetonitrile,methylene chloride, ethyl acetate) were purchased from LabscanAnalytical Sciences (Dublin, Ireland), acetic anhydride, sodiumdihydrogen phosphate and disodium hydrogen phosphate weresupplied by Merck (Darmstadt, Germany). Ultrapure water wasproduced by an AFS-8 from Millipore (Brussels, Belgium). Pesti-cides standards were purchased from Dr. Ehrenstorfer (Augsburg,Germany) (the complete list of standards and internal standards isprovided in Table 1). A stock solution at 1 g L−1 for each compoundand a mix standard solution at 10 mg L−1 were prepared in acetoni-trile. Working solutions at 100 and 10 �g L−1 in acetonitrile wereused. Supelco SPME fibers were purchased from Sigma–Aldrich(Bornem, Belgium). A working solution of all internal standardswas prepared at 0.1 mg L−1 except dichlorvos-D6, �-endosulfan-D4,tebuconazol-D6 and pentachlorophenol-13C6 of which concentra-tion was 1 mg L−1.


G. Salquèbre et al. / Analytica Chimica Acta 710 (2012) 65– 74 67

2.2. Apparatus

Experiments were carried out with an Agilent 7890A gas chro-matograph equipped with a HP-5MS capillary column (30 m,0.25 mm i.d., 0.25 �m film thickness), coupled to an Agilent 7000Atriple quadrupole mass spectrometer operating in electron impactionization mode and an Agilent CTC PAL autosampler. A ball millfrom Retsch (Haan, Germany) was used for hair pulverization.

2.3. Hair treatment and pesticides extraction

In order to remove external contamination and substances pos-sibly responsible for increase in the background noise, hair sampleswere first decontaminated with water (2 min under agitation) andwith acetonitrile (2 min under agitation) and oven-dried at 40 ◦Cbefore extraction of the target analytes. Pesticide extraction fromhair was tested with 3 different solvents (acetonitrile, methanoland acetone) on 4 different hair samples and in two different situa-tions: extraction of isotope-labeled standards added to hair sample(10 �L of 0.1 mg L−1 solution added to 50 mg of hair) and extrac-tion of analytes naturally present in hair samples. Acetonitrile wasfinally preferred for the final procedure. After pulverization in aball mill, 50 mg of pulverized hair were weighed in a 4-mL glasstube. One milliliter of acetonitrile and 5 �L of internal standardsmix (concentration presented in Section 2.2) were added and sam-ple was incubated in a sealed glass tube overnight at 40 ◦C underagitation. Sample was then centrifuged for 5 min at 5000 rpm and800 �L of supernatant was transferred into a 10-mL SPME glasstube (sealed with aluminium screw cap furnished with a PTFE-facedseptum) added with 7 milliliters of phosphate buffer at (1 M, pH 7).Finally, 20 �L of acetic anhydride was added for pentachlorophenolderivatization.

2.4. Solid phase microextraction

Five different SPME fibers were tested in both HS-SPMEand DI-SPME: two polydimethylsiloxane (PDMS) fibers withthickness of 7 and 100 �m respectively, one mixed poly-dimethylsiloxane/divinylbenzene (PDMS/DVB) fiber, onemixed carboxene/polydimethylsiloxane (CAR/PDMS) andone mixed divinylbenzene/carboxene/polydimethylsiloxane(DVB/CAR/PDMS). SPME optimization was performed with astandard solution containing all the pesticides investigated atconcentration of 100 �g L−1 each. Three protocols were tested forHS- and DI-SPME. For the first protocol, the standard mix solutionwas evaporated to dryness under nitrogen flow and the fiber wasexposed to the headspace (HS-SPME) over the dry residue. Forthe second protocol, 7 mL of phosphate buffer (1 M, pH 7) wasadded to the 800 �L acetonitrile before exposing the SPME fiberto the headspace. For the third protocol, 7 mL of phosphate buffer(1 M, pH 7) was added to the acetonitrile and the SPME fiberwas immersed into the mixture. Temperature and time of fiberexposure were optimized by applying a circumscribed centralcomposite design. Response surface was estimated using Minitab15 software (Paris, France). In the final protocol used for fieldsamples analysis, the DI-SPME was carried out using a 65 �mPDMS-DVB fiber. Two successive extractions were performed at30 ◦C during 90 min and then, at 90 ◦C during 90 min. The fiberdesorption was done at 260 ◦C during 15 min.

2.5. Gas chromatography tandem mass spectrometry analysis

GC–MS/MS measurements were performed with the pulsedsplitless injection mode with a pressure of 35 psi (1.5 min) forboth SPME desorption and liquid injection. For liquid injection,20 �L of acetonitrile extract was collected after extraction and

directly sealed into glass vials without evaporation. The volumeinjected was 1 �L. The temperature of the injector and of theGC–MS interface was set at 260 ◦C and 250 ◦C respectively. Thehelium carrier gas flow was set at 1.2 mL min−1. The initial tem-perature of the column was held at 70 ◦C for 5 min, then raisedby 10 ◦C min−1 to 200 ◦C, afterwards by 2 ◦C min−1 to 240 ◦C, andfinally by 10 ◦C min−1 to 300 ◦C. It was maintained at 300 ◦C for3 min. After each run, the temperature was maintained at 300 ◦Cfor 4 min with backflush in order to remove high boiling com-pounds through the split vent. The temperature of the MS sourcewas set at 230 ◦C. The ionization was done by electron impactand the instrument was operating in the multiple reaction mon-itoring (MRM) mode. Collision induced dissociation (CID) gas andquench gas in collision cell were respectively nitrogen and heliumand were used following supplier’s recommendations on flow andquality. Optimization of retention time, collision energy, precur-sor and fragment ions masses was performed using 0.1 mg L−1 and2.5 mg L−1 standard solutions in acetonitrile and spiked samplematrix solutions. Table 1 details the MS/MS parameters settings foreach analyte. Compounds were identified by using the retentiontime, and the relative abundance of two confirmation transitionswith respect to the target (variation had to be within 20% of theexpected values based upon standards). For quantification, stable-isotope-labeled analogues were used (Table 1). For analytes forwhich labeled analogue was not available, trans-permethrin-D6 (forpyrethroids) and p,p′-DDE-D8 (for the others) were used as internalstandard.

2.6. Hair samples collection

Fourteen hair specimens were collected from volunteers (mem-bers of the laboratory) that were not occupationally exposed topesticides. All were Caucasian adults (11 men and 3 women) agedfrom 23 to 60 years old (mean ± SD: 33 ± 11 years). In order toavoid possible contamination, none of the samples analyzed orig-inated from people involved in pesticide analysis. Three samplesdisplayed the presence of white hair shafts within pigmented onesbut the respective amount of white and pigmented shafts has notbeen evaluated since pigmented shafts were highly dominant. Haircolor varied from dark blond to black, except for one sample (brightblond) for which the volunteer declared hair bleaching. A strand ofhair was collected from the posterior vertex region of the scalp, asclose as possible to the skin, using a fine pair of scissors. All par-ticipants were fully informed about the procedure and objectivesof the study and consented to take part in the study. The studywas approved by the National Research Ethics Committee and theNational Commission for Private Data Protection.

3. Results and discussion

3.1. Extraction of analytes from hair

Solid phase microextraction is based on the affinity of targetcompounds for the SPME-fiber compared with the aqueous extract(DI-SPME) or the associated head-space (HS-SPME) that initiallycontains the analytes to be analyzed. In liquid matrices, SPME canbe performed directly without pre-analytical treatment when thematrix does not contain too many impurities, such as rain water orriver water [14]. In the case of biological fluids (e.g. blood, urine,milk), pre-analytical treatment such as dilution, addition of aque-ous buffer or alkaline or acidic treatment is generally required[16,17]. After treatment, the modified matrix still consists in anaqueous extract compatible with SPME functioning. For a solidmatrix like hair, simple extraction with aqueous buffer may be per-formed, but preliminary destructuration of the matrix with NaOH,



acid or enzymatic digestion may be applied to enhance the recoveryfrom hair. Once again, the aqueous hydrolysate finally obtained iscompatible with both SPME and the hydrophilic properties (water-soluble) of the analytes that are generally analyzed this way (e.g.medical drugs and drugs of abuse) [16].

In the case of multi-class pesticide extraction from hair, alka-line or acidic hydrolysis of the matrix is not recommended as it maydegrade some analytes [21]. Because of the hydrophobic propertiesof many pesticides, extraction from solid hair (generally pulver-ized) using aqueous buffer is not recommended either as it maylead to poor recovery. For this reason, the use of organic solventwas generally preferred in studies investigating pesticides detec-tion in hair [18,21]. Nevertheless, although this approach is suitablefor liquid injection of the extract into the chromatographic system,it is not compatible with SPME which requires an aqueous extract.

To face this limitation, the extract may be evaporated to dry-ness and reconstituted in aqueous buffer before DI-SPME, as wasperformed by Pereira de Toledo et al. [22] for the determinationof cocaine and metabolites in hair after extraction with methanol.Another approach, performed by Nadulski et al. for the deter-mination of �9-tetrahydrocannabinol after hair hydrolysis andextraction with isooctane, consisted in evaporating the solvent anddirectly performing HS-SPME above the dry extract after addinga derivatization agent [23]. Although evaporation of the organicextract was proven to be suitable for poorly volatile moleculessuch as cocaine, THC and congeners, in the case of multi-classpesticide analysis, this may induce loss of the most volatile com-pounds. For this reason, direct dilution of the organic extract withaqueous buffer without evaporation was also tested in the presentwork. This procedure requires the use of an organic solvent misci-ble with water. In the present work, three different water-miscibleorganic solvents were tested: acetonitrile, methanol and ace-tone. Although the response was sample- and analyte-dependent,the best recovery for both added isotope-labeled standards andpesticides naturally present in hair was generally obtained withacetonitrile. In details, concerning added internal standards, thebest recovery was obtained with acetonitrile for 50% of the ana-lytes, with methanol for 29% and with acetone for 21%. Concerningthe analytes naturally present in the hair samples, the best recov-ery was obtained with acetonitrile for 71% of the analytes, withmethanol for 26% and with acetone for 3%.

On the basis of the above-mentioned considerations, threeprotocols were tested for SPME after pesticide extraction fromhair with acetonitrile: (1) Headspace-SPME on dry extract, (2)Headspace-SPME on extract added to phosphate buffer pH 7, and(3) Direct immersion-SPME on extract added to phosphate bufferpH 7.

3.2. Optimization of solid phase microextraction and liquidinjection

The influence of the fiber coating was preliminarily testedusing PDMS (7 and 100 �m thickness), 65 �m PDMS/DVB, 85 �mCAR/PDMS and 50/30 �m DVB/CAR/PDMS in HS-SPME and DI-SPME. These experiments were conducted using 10 �L of standardmix solution (100 �g L−1) added to 7 mL of phosphate buffer. Forall the fibers tested, HS-SPME was performed at 70 ◦C for 30 minand DI-SPME was performed at 98 ◦C for 30 min. For most of thecompounds tested here, the best response was clearly obtainedwith PDMS/DVB for both headspace and direct immersion (datanot shown). This fiber was then adopted for the remaining experi-ments. The influence of fiber coating has been extensively studiedin previous works that investigated pesticide detection in dif-ferent matrices (e.g. milk, serum or rainwater) [12–14,24]. Thegeneral outcome was that PDMS-DVB represents the most suitable

Fig. 1. Estimated response surfaces from central composite design for pen-tachlorophenol and cyhalothrin by DI-SPME.

coating for multi-class pesticide analysis (including different chem-ical classes).

SPME was tested through the three different protocols men-tioned in Section 3.1. For protocols 2 and 3, acetic anhydride wasadded for derivatization of pentachlorophenol. For each samplingprotocol described above, time and temperature of fiber exposurewere optimized by applying a circumscribed central compositedesign. Extreme low and high values were 30 ◦C and 90 ◦C for tem-perature and 10 min and 90 min for time. The complete designconsisted in 14 experiments, including full factorial points (centerfull factorial in triplicate) and axial points (center axial in triplicate).Results clearly indicated that DI-SPME (protocol 3) was by far moreefficient than the two HS-SPME protocols (protocols 1 and 2). Onaverage, the maximal response (peak area) obtained with protocol1 and protocol 2 amounted for 6.3 and 4.6% of the response obtainedwith protocol 3 respectively. Relative response obtained for all theanalytes tested here with each protocol is presented in Table 2. Onthe basis of these results, DI-SPME was preferred to HS-SPME for thefollowing experiments. The advantage of DI-SPME over HS-SPMEwas also reported in studies investigating pesticides in milk. It wasdemonstrated that even after dilution to protect the fiber from fatand proteins during immersion, better sensitivity was achieved byusing DI-SPME in diluted milk than by using HS-SPME. Moreover,probably because of low volatility, many of the studied compoundswere not detected using HS-SPME [12,13].

Fig. 1 presents the response surface estimated for pen-tachlorophenol and �-cyhalothrin with DI-SPME according to timeand temperature exposure of the fiber. As observed for these twoanalytes, two opposite tendencies were detected among the pesti-cides investigated here: analytes with maximum response whenexposure was performed at low temperature and analytes withmaximum response when exposure was performed at high tem-perature. In both cases, response was maximal after 90 min of fiberexposure. DI-SPME at low temperature (30 ◦C) allowed the detec-tion of 17 out of the 22 pesticides investigated and DI-SPME at



Table 2Normalizeda response obtained for each SPME protocol.

Compound Protocol

1 2 3Description

HS-SPME on dry extract HS-SPME on buffer DI-SPME on buffer

Dichlorvos Not detected Not detected Not detectedTrifluralin 5.7 5.8 100�-HCH 9.4 1.4 100�-HCH 13.8 0.4 100�-HCH 15.1 1.3 100Pentachlorophenol Not detected 3.6 100Aldrin 6.4 6.7 100o,p′-DDE 1.8 2 100�-Endosulfan 13.2 4.9 100p,p′-DDE 6.2 6.6 100Dieldrin 9.6 5.6 100Oxadiazon 6.2 2.4 100�-Endosulfan 15 1.4 100o,p′-DDT 10.1 11.3 100p,p′-DDT 13.3 15.2 100Tebuconazole Not detected Not detected 100Diflufenican 0.7 0.9 100�-Cyhalothrin 0.1 8.1 100trans-Permethrin 1.8 11.7 100Cypermethrin 0.05 5.3 100Fenvalerate 0.03 4.6 100Deltamethrin 0.01 2.8 100

a The response obtained with protocol 3 was set to 100 and the response obtained with protocols 1 and 2 was expressed as percentage of this one.

Fig. 2. Chromatograms (quantification transitions) of pesticide analysis of hair supplemented at 10 pg mg−1 by GC–MS/MS with DI-SPME (A) at 30 ◦C, (B) at 90 ◦C and (C)liquid injection. Inserts represent enlarged zones for better visibility. For each of the three chromatograms, only the analytes that were quantified with the correspondinginjection mode (SPME at 30 ◦C, at 90 ◦C or liquid injection) were presented: 1, dichlorvos-D6; 2, dichlorvos; 3, trifluralin-D14; 4, trifluraline; 5, �-HCH-D6; 6, �-HCH; 7,�-HCH; 8, �-HCH-D6; 9, �-HCH; 10, pentachlorophenol-13C6; 11, pentachlorophenol; 12, aldrin; 13, o,p′-DDE; 14, �-endosulfan; 15, p,p′-DDE-D8; 16, p,p′-DDE; 17, dieldrin;18, oxadiazon; 19, �-endosulfan-D4; 20, �-endosulfan; 21, o,p′-DDT; 22, p,p′-DDT-D8; 23, p,p′-DDT; 24, tebuconazol-D6; 25, tebuconazol; 26, diflufenican; 27 and 27′ , �-cyhalothrin; 28 and 28′ , trans-permethrin; 29, trans-permethrin-D6; 30 and 30′ , �-cypermethrin; 31 and 31′ , fenvalerate; 32 and 32′ , deltamethrin. Asterisk (*) indicatesinternal standard.



Table 3Validation parameters for the detection of pesticides in hair by GC–MS/MS with each injection mode (liquid injection, DI-SPME at 30 ◦C and DI-SPME at 90 ◦C).

Compound R2 of the calibration curve LOD LOQ Accuracy (% of target) Precision (RSD %) Recovery

(pg/mg) Intradayb Interdayc Intradayb Interdayc

Added concentration (pg/mg)1 20 1 20 1 20 1 20 1 20

DI-SPME 30 ◦CTrifluralina 0.9994 0.01 0.05 111 98 100 89 16 2 15 7 87 72�-HCHa 0.9995 0.01 0.05 97 98 101 88 4 3 10 10 71 75�-HCHa 0.9995 0.02 0.1 107 118 101 97 16 1 23 22 80 82�-HCHa 1.0000 0.02 0.1 117 108 92 100 4 1 24 11 112 82Pentachlorophenola 0.9957 2 10 – 102 – 98 – 6 – 25 81 82Aldrin 0.9984 0.5 2 – 114 – 108 – 4 – 14 – 59o,p′-DDE 0.9997 0.05 0.2 122 117 112 102 2 9 16 17 75 76�-Endosulfana 0.9998 0.2 1 115 105 120 102 6 13 13 8 78 73p,p′-DDEa 0.9996 0.02 0.1 113 104 99 100 4 4 13 7 102 78Dieldrin 0.994 2 5 – 117 – 83 – 7 – 18 – 72Oxadiazona 0.9976 0.1 0.5 106 106 87 94 18 15 25 22 97 79�-Endosulfana 0.9997 0.5 2 – 107 – 98 – 3 – 12 89 86o,p′-DDTa 0.9998 0.2 1 91 101 97 102 10 10 18 11 80 74p,p′-DDT 0.9998 0.5 2 – 115 – 97 – 4 – 16 107 80Tebuconazolea 0.9997 0.2 2 – 92 – 88 – 9 – 13 – 84diflufenicana 1.0000 0.05 0.2 101 90 90 88 15 5 17 18 91 78trans-Permethrin 0.6656 ndd ndd 650 43 447 62 45 4 45 43 ndd ndd

Molecules not detected: dichlorvos, �-cyhalothrin, cypermethrin, fenvalerate, deltamethrin

DI-SPME 90 ◦CTrifluralin 0.9996 0.02 1 104 93 91 87 14 3 15 12 111 88�-HCH 0.9995 0.5 2 99 97 97 91 11 2 12 8 60 76�-HCH 0.9987 0.2 1 88 97 117 104 3 3 38 32 81 82�-HCH 0.997 0.2 1 127 122 252 155 12 14 61 57 85 82Aldrina 0.9983 0.05 0.2 111 98 106 98 7 18 18 19 74 73o,p′-DDEa 0.9998 0.02 0.1 109 91 105 92 9 2 7 9 87 84p,p′-DDE 0.9999 0.02 0.2 104 104 103 103 13 1 17 7 95 84Dieldrina 0.9972 1 5 90 99 90 106 16 9 23 15 – 84Oxadiazon 0.9995 0.5 2 – 114 – 105 – 1 – 16 – 102o,p′-DDT 0.9987 0.5 2 139 120 110 114 8 2 22 21 91 83p,p′-DDTa 0.9998 0.2 1 106 120 103 95 5 2 13 19 83 82Diflufenican 0.9986 0.05 0.2 73 106 93 110 13 3 23 25 97 88�-Cyhalothrin a 0.9984 0.2 1 111 86 100 101 11 7 14 12 92 83trans-Permethrina 0.8590 ndd ndd 1339 159 615 154 66 14 16 18 ndd ndd

Cypermethrina 0.9982 0.1 0.5 120 112 112 112 3 8 9 9 102 83Fenvaleratea 0.9995 0.2 2 – 102 – 98 – 12 – 22 92 84Deltamethrina 0.9981 1 5 – 105 – 102 – 7 – 7 – 82Molecules not detected: dichlorvos, pentachlorophenol, �-endosulfan, �-endosulfan, tebuconazole

Liquid injectionDichlorvosa 0.9944 0.5 2 – 95 – 101 – 10 – 10 – 42Trifluralin 0.9996 2 10 – 110 – 98 – 11 – 11 – 75�-HCH 0.9986 5 20 – 93 – 84 – 11 – 11 – 68�-HCH 0.9975 2 10 – 76 – 89 – 20 – 20 – 73�-HCH 0.9945 2 10 – 109 – 96 – 18 – 16 – 73Aldrin 0.9927 5 20 – 81 – 102 – 1 – 17 – 50o,p′-DDE 0.9998 0.5 2 – 95 – 97 – 22 – 18 – 71�-Endosulfan 0.9997 5 40 – – – – – – – – – –p,p′-DDE 0.9991 1 5 – 106 – 100 – 20 – 14 – 69Dieldrin 0.9976 2 10 – 93 – 95 – 18 – 16 – 49Oxadiazon 0.9983 5 20 – 107 – 101 – 17 – 14 – 81�-Endosulfan 0.9989 10 40 – – – – –o,p′-DDT 0.9978 2 10 – 114 – 112 – 10 – 10 – 74p,p′-DDT 0.9996 2 10 – 122 – 110 – 0 – 14 – 82Tebuconazole 0.9995 2 10 – 113 – 96 – 9 – 14 – 72Diflufenican 0.9984 0.5 2 – 106 – 93 – 14 – 18 – 73Molecules not detected: pentachlorophenol, �-cyhalothrin, trans-permethrin, cypermethrin, fenvalerate, deltamethrin

a Injection mode retained according to validation parameters.b n = 6.c n = 12.d nd, not determined.

high temperature (90◦) allowed the detection of 17 out of the 22(Table 3). The general tendency was that for the most water-solubleanalytes, best sensitivity was obtained at low temperature. Forinstance, tebuconazole and pentachlorophenol that display solu-bility in water of 36 and 14 mg L−1 respectively were only detectedat low temperature. To a lower extent, trifluralin, HCHs andoxadiazon, with solubility in water between 0.2 and 7 mg L−1, were

detected both at 30 ◦C and 90 ◦C, but better sensitivity was reachedat low temperature. DDEs/DDTs and diflufenican, which displaysolubility in water close to 0.1 mg L−1, had comparable limits ofdetection at 30 ◦C and 90 ◦C. On the contrary, pyrethroids, whichare almost insoluble in water (below 0.01 mg L−1) were clearlymore easily detected when SPME was performed at high temper-ature. Dichlorvos was the only compound that was not detected



when using SPME. This could be explained by its highly hydrophiliccharacter (solubility in water: 8000 mg L−1; log P = 1.47) that avoidsefficient partitioning into the SPME fiber. It was then decided tocombine successively DI-SPME at 30 ◦C for 90 min and DI-SPME at90 ◦C for another 90 min on the same extract. This approach allowedthe detection of 21 different pesticides. Each analyte was quantifiedusing the most appropriate temperature. Fig. 2(A and B) presentschromatograms obtained from the analysis of hair supplementedwith standard solution, with DI-SPME at 30 ◦C and 90 ◦C.

Phosphate buffer pH had no significant influence on the analyti-cal response, but since degradation was observed for some analytesunder alkaline and acidic conditions, pH 7 was adopted. The influ-ence of buffer ionic strength during the immersion of the SPMEfiber tested by adding NaCl demonstrated that no influence wasobserved above 2% of saturation (corresponding to 0.12 M). Sincephosphate buffer concentration was 1 M, ionic strength was con-sidered sufficient and no further NaCl was added. The effect of themodification of ionic strength by salt addition and increased extrac-tion due to the “salting-out effect” has been widely discussed and isstill a matter of debate. In a recent work that investigated pesticidedetection in milk extracts using DI-SPME, a positive effect of saltaddition on extraction was observed when working at 50 ◦C, butbetter response was obtained without salt addition when workingat 100 ◦C [12]. Another study also performed upon milk reportedthat using HS-SPME, better sensitivity was achieved when 0.1 g ofsalt was added to the sample, but observed better results withoutadding salt when DI-SPME was used [13]. In a work conducted onrainwater samples tested for pesticides using DI-SPME, the authorsobserved that addition of salt (NaCl) improved the recovery for themost polar compounds but decreased the response for organophos-phorus and nonpolar compounds. The authors finally chose to use100% saturation since it allowed the extraction of compounds thatwere not extracted under normal conditions [14]. In general, directimmersion seems to be less affected by ionic strength modifica-tion than headspace. However, optimal conditions remain difficultto determine when facing multi-class analysis including analyteswith opposite physicochemical properties, and it seems that com-promises are necessary in each case.

In order to detect the analytes that were not detected with SPME,liquid injection (direct injection without SPME) was also tested.Liquid injection allowed the detection of 16 analytes, includingdichlorvos that was not detected with DI-SPME (Fig. 2C, Table 3).

In view of the above-mentioned results, the following protocolwas adopted for field sample analysis: after extraction of pesticidesfrom hair with acetonitrile, 20 �L was collected for liquid injectionand the remaining extract was added to 7 mL of phosphate buffer(pH 7, 1 M) with 20 �L acetic anhydride. The mixture was then sub-mitted to 2 successive steps of DI-SPME, at 30 ◦C for 90 min and at90 ◦C for another 90 min.

3.3. Analytical performances

Calibration curves using stable-isotope-labeled derivates asinternal standards were obtained by fortifying aliquots of 50 mg ofpulverized hair with standard mixtures, at 9 concentration levels(not including the blank matrix), 3 replicates, which were sub-mitted to the complete analytical procedure. The correspondingconcentrations of pesticides were 0, 0.05, 0.1, 0.2, 1, 2, 10, 20, 40and 100 pg mg−1 hair. The quality of the model (linear fit) used forthe calibration curve was assessed by the coefficient of determina-tion (R2) (1/x weighting factor) (Table 3). Calibration curves werelinear in the concentration range tested and r2 were higher than0.99 for all the compounds presented in Table 3 except for trans-permethrin. The latter result was explained by the fact that it wasnot possible to obtain a blank matrix without high concentration oftrans-permethrin. As a result, no LOD and LOQ were determined for

this analyte and the results concerning field sample analysis wereconsidered semi-quantitative.

The limit of detection (LOD) and limit of quantification (LOQ)were determined by analyzing hair samples fortified with pesti-cide standard solutions. LOD was determined as the concentrationwith signal-to-noise ratio of at least 3 and LOQ was the low-est concentration with a signal-to-noise ratio of at least 10 andacceptable accuracy and precision (% of target and relative stan-dard deviation within 25%). LOD and LOQ were determined usingthe lowest accessible concentration level of the calibration curveand confirmed using the two levels above. Although some analyteswere detected with both DI-SPME and liquid injection, DI-SPMEalways allowed reaching better sensitivity. The increase in sensi-tivity (LOD) ranged from ×2 for dieldrin up to ×400 for �-HCH.LOD and LOQ obtained here stood in the range of values recentlyreported for the analysis of pesticides in milk and serum usingSPME and gas chromatography coupled with tandem mass spec-trometry or with electron capture detection [12,13,24]. The degreeof sensitivity reached in the present study is furthermore satisfac-tory when considering the small amount of hair used for analysis(50 mg) compared to the volume of sample used in studies that ana-lyzed other matrices (from 0.5 mL for serum up to 1 or 10 mL formilk). Among the different studies which investigated pesticides inhair, none used SPME. The limits of detection observed were mostof the time significantly higher than those obtained in the presentstudy (e.g. up to ×10,000 for the same analytes) [4,18,19,21,25,26].As expected, the highest LODs (30–245 pg mg−1) were observedin studies that investigated pesticides from different chemicalclasses at the same time (organophosphate, organochlorine, car-bamate, pyrethroids) [26]. More recently, LODs in the range of2–5 pg mg−1 were obtained in a study that investigated organochlo-rine and organophosphate pesticides simultaneously [18]. Untilnow, LOD below 1 pg mg−1 was only obtained for studies thatinvestigated molecules with similar physicochemical properties(HCHs, DDTs, PCBs) [4,19,25] and using a sample amount of 500 mg,which is ten times higher than the amount used in the presentstudy.

Precision and accuracy were evaluated with hair samples spikedat two different levels of concentration (1 pg mg−1 and 20 pg mg−1)of each analyte. Results are expressed as percentage of target valueand relative standard deviation (RSD) for each injection mode(Table 3). Except for trans-permethrin, the intraday precision (n = 6)obtained with the most suitable injection mode (SPME at 30 ◦C,at 90 ◦C or liquid injection) was always below 20% (and oftenbelow 10%) for all the analytes. Interday precision (n = 12) wasmost of the time below 20% and was always below 25%. Except fortrans-permethrin, accuracy was within 80–120% for all the analytestested when they were analyzed with the most suitable injectionmode. Even though 20% is generally favored, precision up to 25%was considered acceptable according to the low levels of concen-tration reached here. Such tolerance on validation parameters isreported to be accepted to accommodate the complex compositionof solid biological matrices (e.g. meconium) [27].

Recovery from the matrix was determined at two concentrationlevels (1 and 20 pg mg−1) for each analyte. For the determinationof recovery, pulverized hair samples (50 mg) were supplementedwith control solution before and after extraction with acetonitrile.Recovery was expressed as the mean analyte area of samples withcontrol added before extraction (n = 4) divided by the mean ana-lyte area of samples with control added after extraction (n = 4). Asobserved in Table 3, the recovery obtained with the most suitableinjection mode for each analyte ranged from 72 to 84% except fordichlorvos, which displayed a recovery of 42% that can probably beexplained by partial degradation during samples incubation.

The selectivity of the method was ensured by the use of tan-dem mass spectrometry which is among the most reliable tools



Table 4Analysis of hair samples from human volunteers.

Compound Injection Number of values above Concentration (pg/mg) [range] mediana

LOD LOQ

Trifluraline SPME 30 ◦C 5 5 [0.1–0.8] 0.22SPME 90 ◦C 2 0 <LOQL.I. 0 0 Not detected

�-HCH SPME 30 ◦C 11 4 [<LOQ–0.6] 0.11SPME 90 ◦C 0 0 Not detectedL.I. 0 0 Not detected

�-HCH SPME 30 ◦C 14 14 [0.3–10.3] 2.3SPME 90 ◦C 13 12 [<LOQ–11.9] 3.8L.I. 11 1 [<LOQ–13.2] 13.2

�-HCH SPME 30 ◦C 14 14 [0.3–8.9] 2.1SPME 90 ◦C 14 11 [<LOQ–8.0] 2.9L.I. 6 0 <LOQ

Pentachloro-phenol SPME 30 ◦C 3 3 [18.1–244] 30.2SPME 90 ◦C 0 0 Not detectedL.I. 0 0 Not detected

o,p′-DDE SPME 30 ◦C 3 3 [0.26–0.28] 0.27SPME 90 ◦C 7 3 [<LOQ–0.4] 0.30L.I. 0 0 Not detected

�-Endosulfan SPME 30 ◦C 6 2 [<LOQ–1.3] 1.2SPME 90 ◦C 0 0 Not detectedL.I. 0 0 Not detected

p,p′-DDE SPME 30 ◦C 14 14 [0.3–5.1] 1.5SPME 90 ◦C 14 14 [0.3–4.2] 1.5L.I. 9 1 [<LOQ–5.4] 5.4

o,p′-DDT SPME 30 ◦C 9 4 [<LOQ–3.7] 1.6SPME 90 ◦C 9 1 [<LOQ–2.4] 2.4L.I. 2 0 <LOQ

p,p′-DDT SPME 30 ◦C 6 3 [<LOQ–8.20] 4.5SPME 90 ◦C 10 6 [<LOQ–5.6] 5.1L.I. 1 0 <LOQ

Tebuconazole SPME 30 ◦C 2 2 [20.0–65.6] 42.8SPME 90 ◦C 0 0 Not detectedL.I. 2 2 [17.4–56.7] 37.0

Diflufenican SPME 30 ◦C 1 1 1.9SPME 90 ◦C 1 1 2.6L.I. 1 1 2.1

btrans-Permethrin SPME 30 ◦C 14 14 [0.6–250] 34SPME 90 ◦C 14 14 [3.2–240] 33L.I. 0 0 Not detected

a For values above LOQ only.b Semi-quantitative results.

currently available for discriminating between background noisedue to matrix interferences and signal from analytes effectivelyinvestigated. In the present work, along with the quantificationtransition, 2 transitions were used for confirmation for each ofthe analytes tested (Table 1). The variability in the ratio quantifi-cation transition/confirmation transition had to be within 20% toconfirm the presence of analyte. No influence of the matrix effect(e.g. ion suppression/enhancement) was observed here. Indeed, thematrix effect rather has significant influence in liquid chromatog-raphy analysis (particularly electrospray ionization) [28] but is notconsidered to affect gas chromatography analysis and is thus gen-erally not evaluated for the latter. The use of stable-isotope-labeled(deuterium or 13C in this work) analogues as internal standard is afurther precaution to compensate for any potential matrix effect.

Finally, even though some analytes were detectable using twoor the three injection modes (e.g. HCHs), the validation parametersobtained with each of the injection modes tested here were signif-icantly different. For each analyte, the injection mode retained forthe analysis of field samples was the one showing the best valida-tion parameters.

3.4. Applications

The analysis of the 14 field samples displayed the presence ofpesticides in all of them (from 4 to 11 different analytes in eachsample) (Table 4). Fig. 3 presents the results obtained from theanalysis of the hair sample which contained 11 target analytes.Altogether, 13 different pesticides were detected at concentrationabove the LOQ. The most frequently detected analytes were �-HCH, �-HCH, p,p′-DDE and trans-permethrin that were present inall the samples tested here (Table 3). Organochlorines were themost frequently investigated pesticides in studies aiming to eval-uate human exposure using hair analysis [4,18–21,25,26,29]. Therate of positive detection reported in the different studies rangedfrom 0 to 100%. Although the differences observed between studiesmay be partially attributable to geographical disparities in humanexposure or to lifestyle and occupation (occupational exposure vsenvironmental exposure), the results may definitely have also beenaffected by differences in the sensitivity of the analytical meth-ods that were used. In studies which reported a level of frequencyof positive detection close to 0 [26,30–32], the analytical methods



Fig. 3. GC–MS/MS chromatogram obtained from the analysis of an extract from an authentic human hair sample. Each of the analytes presented on the chromatogram hasbeen quantified with the most suitable injection mode indicated in Table 3. 1: dichlorvos-D6; 3: trifluralin-D14; 4: trifluralin; 5: �-HCH-D6; 6: �-HCH; 7: �-HCH; 8: �-HCH-D6;9: �-HCH; 10: pentachlorophenol-13C6; 11: pentachlorophenol; 12: aldrin; 15: p,p′-DDE-D8; 16: p,p′-DDE; 17: dieldrin; 19: �-endosulfan-D4; 21: o,p′-DDT; 22: p,p′-DDT-D8;23: p,p′-DDT; 24: tebuconazol-D6; 28 and 28′: trans-permethrin; 29: trans-permethrin-D6. Asterisk (*) indicates internal standards.

that were used had significantly higher limits of detection (lowersensitivity) than in the studies where the reported level of fre-quency of positive detection was close to 100% [4,19,25,33]. Onlyfew studies included pyrethroids in the list of the analytes thatwere tested in hair [26,30–32], but trans-permethrin had neverbeen tested. Actually, only bioallethrin has been detected and therate of positive detection ranged from 0 to 16.7% [26,30–32]. Noneof the analytes �-endosulfan, trifluralin, pentachlorophenol, tebu-conazole and diflufenican that were detected in the present studyhad hitherto been investigated in hair.

The two samples that contained white hair shafts (obtained from60- and 36-year-old men) contained 11 and 7 different analytesrespectively. The participant who reported hair bleaching testedpositive for 5 different analytes. The concentration of the analytesdetected in these 3 samples was comparable to what was observedfor the other participants.

As the injection mode was found to significantly influence thesensitivity of the method, it also dramatically influenced the resultsobtained from the analysis of the field samples (the number ofsamples with pesticide concentration above the LOD and LOQ).The most suitable injection mode for each analyte, as determinedaccording to the validation parameters, was confirmed by the anal-ysis of the field samples. In fact, except for p,p′-DDE, the number ofsamples displaying a level of concentration above the LOD and LOQwas always greater when using the injection mode retained on thebasis of the validation parameters (as mentioned in Table 3).

4. Conclusions

Solid phase microextraction coupled with gas chromatography-tandem (triple quadrupole) mass spectrometry is particularlyuseful to the analysis of multi-class pesticides in human hair. Foreach analyte, the sensitivity of the method was proven to signifi-cantly depend on the injection mode. Besides, the combination oftwo successive steps of direct immersion-SPME, performed at low(30 ◦C) and high (90 ◦C) temperature on the same extract along withliquid injection was finally necessary to cope with the differencesin physical–chemical properties of the analytes investigated here.

The combined approach developed here allowed achievingparticularly low limits of detection and satisfactory validationparameters for all the analytes analyzed. The application of the

validated method to the analysis of field samples demonstrated towhat extent losses in sensitivity may dramatically influence resultsobtained from the same sample and confirmed the need for verysensitive methods when analyzing hair from non-occupationallyexposed subjects.

Acknowledgment

This work was supported in part by the French Agency for Food,Environmental and Occupational Health Safety, in the frameworkof the Pesticide Residue Observatory action.

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