hair decontamination procedure

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Hair decontamination procedure prior to

multi-class pesticide analysisRadu-Corneliu Duca, Emilie Hardy, Guillaume Salqubre andBrice M. R. Appenzeller*

Although increasing interest is being observed in hair analysis for the biomonitoring of human exposure to pesticides, somelimitations still have to be addressed for optimum use of this matrix in that specic context. One main possible issue concernsthe need to differentiate chemicals biologically incorporated into hair from those externally deposited on hair surface fromcontaminated air or dust. The present study focuses on the development of a washing procedure for the decontaminationof hair before analysis of pesticides from different chemical classes. For this purpose, three different procedures of articialcontamination (with silica, cellulose, and aqueous solution) were used to simulate pesticides deposition on hair surface. Sev-eral washing solvents (four organic: acetone, dichloromethane, methanol, acetonitrile; and four aqueous: water, phosphatebuffer, shampoo, sodium dodecylsulfate) were evaluated for their capacity to remove articially deposited pesticides fromhair surface. The most effective washing solvents were sodium dodecylsulfate and methanol for aqueous and organic solvents,respectively. Moreover, after a rst washing with sodium dodecylsulfate or methanol, the majority of externally depositedpesticides was removed and a steady-state was reached since signicantly lower amounts were removed by additional secondand third washings. Finally, the effectiveness of a decontamination procedure comprising washing with sodium dodecyl-sulfate and methanol was successively demonstrated. In parallel, it was determined that the nal procedure did not affectthe chemicals biologically incorporated, as hair strands naturally containing pesticides were used. Such a procedure appearsto remove in one-shot the fraction of chemicals located on hair surface and does not require repeated washing steps.Copyright 2014 John Wiley & Sons, Ltd.

Keywords:hair; decontamination; washing procedure; pesticides; multi-class analysis

Introduction

Although increasing interest is being observed in hair analysis for

the biomonitoring of human exposure to pesticides, some limita-

tions still have to be addressed for optimum use of this matrix in

that specic context. One main possible issue concerns the need

to differentiate chemicals biologically incorporated into hair from

those externally deposited on hair surface due to contaminated

air or dust. Chemicals incorporated by means of biological mech-

anisms, likely to be located in the whole hair structure, can be

interpreted as representative of the internal dose people have

undergone during the period of hair growth. On the contrary,

chemicals externally deposited only represent recent contamina-

tion and are therefore less biologically relevant. Ideal decontam-

ination procedure therefore has to remove external chemicals

(deposited on the cuticle surface) without affecting internallyincorporated compounds (present in the bulk of the matrix).

In forensic sciences, decontamination of hair prior to drug

analysis is an important step, since false positive samples might

have legal implications. Thus, decontamination has been

extensively documented and several washing procedures with

different degrees of complexity have been proposed.[1] Hair

decontamination before drug testing is generally performed with

organic solvents (e.g. dichloromethane, acetone, methanol,

acetonitrile) and/or with aqueous solutions of detergents (e.g.

sodium dodecylsulfate) or buffers (e.g. phosphate buffer).[16]

Unlike for drug testing, the decontamination with organic solvents

before environmental pollutants analysis was less used[711] and

more gentle decontamination solvents like water[1214] and water

with shampoo[1520] were preferred. Nevertheless, no standardized

washing procedure removing the totality of the external contami-

nation without affecting the drugs biologically incorporated into

hair is available yet.[21] Still, the importance of hair decontamination

was acknowledged by the Society of Hair Testing (SoHT) and rec-

ommendations for washing were made: both organic and aqueous

solvents should be used, the efciency of the washing procedure

should be tested and, if required, additional clean-up steps should

be taken.[21]

An important step in the development of a decontamination

procedure is to produce articially contaminated specimens to

be used for testing the efciency of the washing to remove

chemicals deposited on hair surface. The most classic contamina-

tion procedure is the utilization of a soaking aqueous solution

containing the compounds of interest.[4,6,22,23] Even if this arti-

cial contamination is effective, it reects neither the external de-position of compounds on hair surface by biological uids nor

the environmental contamination occurring mainly through air-

borne dust particles. Moreover, an important limitation associ-

ated with hair dipping in aqueous solutions is the possible

irreversible penetration of the compounds within the hair shaft.[6]

* Correspondence to: Brice M. R. Appenzeller, Laboratory of Analytical Human

Biomonitoring, Centre de Recherche Public de la Sant (CRP-Sant), 162A,

Avenue de la Faencerie, L-1511 Luxembourg, Luxembourg. E-mail: brice.

[email protected]

Laboratory of Analytical Human Biomonitoring, CRP-Sant, 162A avenue de laFaencerie, L-1511 Luxembourg, Luxembourg

Drug Test. Analysis2014,6, 5566 Copyright 2014 John Wiley & Sons, Ltd.

Research articleDrug Testing

and Analysis

Recei ved: 31 October 2013 Revi sed: 7 Februa ry 2014 Accepted: 24 Februa ry 2014 P ub lished on lin e i n Wiley On line Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1649


2/12

In the last decade, more realistic articial contamination proce-

dures were also used: hair incubation with spiked blood to dem-

onstrate postmortem external contamination,[24] hair briey

dipped in authentic urine from a methamphetamine user to in-

vestigate the contamination/decontamination of pubic hair,[25]

hair coating with drugs followed by sweat conditioning using

synthetic sweat to study drug contamination/decontamination

in the analysis of human hair, [6] and subjects hair own contamina-

tion using their hands powdered with a drug mixture.[26]

Although such procedures might be relevant to clinical or forensic

contexts, they are not representative of hair surface contamina-

tion by organic pollutants present in air or dust; specic

approaches have to be developed for the latter purpose.

The present study is therefore focused on the development of

a washing procedure applied to hair samples articially contami-

nated with a mixture of different organic pollutants (pesticides)

from different chemical classes and thus displaying various

physicochemical properties. For the articial contamination, three

different media were used: two types of solid particles (silica and

cellulose) previously contaminated with the pesticide mixture to

simulate contamination from dust or airborne particles, and aque-

ous solution of the target compounds used for comparison. Thewashing procedures applied to the contaminated hair were eval-

uated according to the criteria proposed within a review paper

previously published:[27]

1. A signicant part of the chemicals has to be removed dur-

ing the rst washing.

2. No more pesticides or signicantly lower amounts are re-

moved by additional washings.

3. The part of chemicals extracted from hair after pulverization of

the matrix should not be affected by the washing procedure.

For this purpose, a rst step consisted in evaluating several

solvents for their capacity to remove the articially deposited

pesticides from the hair surface. Four organic solvents (acetone,dichloromethane, methanol, acetonitrile) and four aqueous sol-

vents (water, phosphate buffer pH 7, shampoo, and 5% sodium

dodecylsulfate) were tested. In a second step, the most effective

washing solvents (organic and aqueous, respectively) were evalu-

ated to determine whether a steady-state was reached after the

rst washing, by doing three successive washings. The third step

consisted of testing the effectiveness of the nal washing proce-

dure chosen based on the results obtained from the previous

steps. Finally, hair samples naturally containing pesticides were

submitted to the washing procedure to determine whether

pesticide concentration was affected.

Materials and methods

Chemicals and reagents

Pesticides analytical standards with purities higher than 97%,

except for permethrin and cypermethrin (94% purity), were

purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany).

Individual stock solutions at 1 g L-1 of all standards were prepared

by exact weighing and dissolution in acetonitrile. A working solu-

tion containing the targeted molecules at 10 mg L-1 in acetonitrile

was prepared. Acetonitrile, methanol, dichloromethane, acetone

and hexane were purchased from Biosolve (Valkenswaard, The

Netherlands). A phosphate buffer solution (1 M, pH 7.0) was pre-

pared using monosodium phosphate and disodium phosphate.

Sodium dodecylsulfate (SDS), monosodium phosphate, disodium

phosphate, formic acid, silica and cellulose were provided by

Sigma-Aldrich (Bornem, Belgium). The shampoo used for hair

washing tests was Baby shampoo (Mustela, Laboratoires

Expanscience, Courbevoie, France). Ultrapure water was produced

using a Milli-Q gradient water system (Millipore, Brussels, Belgium).

Hair articial contamination

Hair specimens were collected from the Laboratory staff mem-

bers (Caucasian, male, average age of 31 years). The collected

samples were initially analyzed for pesticides content. Two hair

specimens (brown) were nally selected: one containing low

levels of pesticides was used for the preliminary selection of de-

contamination solvents (after articial contamination); and one

naturally containing several pesticides was used to test the nal

washing procedure. Except for individuals self-washing with

shampoo, no cosmetic treatment was reported.

To simulate pesticide deposition on hair surface, three

different contamination procedures were used: two dry contam-

inations using silica or cellulose particles, and one wet con-

tamination using an aqueous solution. The articial drycontamination procedures were used to simulate the natural

solid-solid transfer of pesticides from dust to the hair shaft. Silica

(particle size: 210300m), a hydrophilic inorganic support, was

spiked with a mixture of pesticides to reach a nal concentration

of 0.5 g g-1. Then, 1 g hair was mixed with 10g silica for 24 h at

25 rpm, using an end-over-end mixer (Intelli-Mixer RM-2 L, Elmi

Ltd, Riga, Latvia). Cellulose (particle size: 2560 m), a lipophilic

organic support, was spiked with a pesticide mixture to reach a

nal concentration of 1 g g-1. Five grams of cellulose were gently

mixed with 1 g hair sample for 24 h at 25 rpm, using an end-over-

end mixer. The articial wet contamination, classically used to test

the efciency of hair washing solvents,[4,6,22,23] was done using

aqueous solution containing the target pesticides at 0.2 g mL-1.

A hair sample (1 g) was dipped in 100 mL pesticides aqueous solu-

tion for 1 h. The amount of pesticides added to each media (silica,

cellulose, and water) was adjusted with regard to its density, in or-

der to ensure that for each assay, regardless of the contamination

path, 1 g of hair was in contact with 20g of each pesticide.

Hair decontamination

The present paper is dealing with the development of a washing

procedure prior to multi-residue pesticides determination in hair.

The washing efciency was calculated as the ratio of the pesti-

cide concentration between the washed and non-washed hair

samples, expressed as percentage of decontamination (Table 2).

Thenal procedure was as follow: hair (100 mg) was successivelywashed for 5 min. with sodium dodecylsulfate, followed by a sec-

ond 5 min. washing with methanol using an orbital shaker at

350 rpm (Incubator Shaker G25, New Brunswick, NJ, USA). Then

the sample was allowed to dry at room temperature prior to pul-

verization for pesticides analysis.

Pesticides analysis in hair samples

Hair samples were extracted and analyzed according to

Salqubreet al.,[11] with minor modication and adaptation. Fifty

mg of pulverized hair were extracted overnight at 40 C, using

1 mL of acetonitrile:water (80:20). The extract was centrifuged

for 10min at 5000xg. Four-hundred L supernatant were

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collected and diluted in 7.6mL phosphate buffer (1 M, pH7.0)

prior to solid phase micro extraction-gas chromatography-

tandem mass spectrometry (SPME-GC-MS/MS) analysis. The

remaining supernatant was dried under a gentle stream of ni-

trogen and recovered in a solution of water:acetonitrile:formic

acid (94.5/5/0.5, v/v/v) for ultra performance liquid chroma-

tography-tandem mass spectrometry (UPLC-MS/MS) analysis.

Stable-isotope-labelled derivatives were used as internal stan-

dards to compensate any possible extraction losses (Table 1).

SPME-GC-MS/MS analysis

A direct immersion SPME was carried out using a 65 m mixed

polydimethylsiloxane/divinylbenzene (PDMS/DVB) bre. The ex-

traction was performed at 60 C for 80 min. Thebre desorption

was done at 260 C for 15 min. GC analyses for pesticides were

performed using an Agilent Technologies GC system (7890A),

and separations were carried out using an HP-5MS 5% Phenyl

Methyl Silox capillary column (30 m x 250m, 0.25 m particle

size) from Agilent Technologies (Santa Clara, CA, USA). The

analytes were separated using helium as carrier gas at a ow rate

of 1.2 mL/min. The oven temperature program started at 70 C for

5 min, followed by three successive linear increases of 10 C/min

to 200 C, 2 C/min to 240 C, and 10 C/min to 300 C; tempera-

ture was held for 3 min. MS/MS analysis was carried out using

an Agilent GC-MS TQ 7000A tandem quadrupole mass spectrom-

eter (Agilent Technologies, Santa Clara, CA, USA). The mass spec-

trometer was operated in negative chemical ionization mode

using methane as the reagent gas. The specic MS/MS parame-

ters for the GC-amenable compounds are shown in Table 1. The

methods quantication limits (LOQ) for GC-amenable target

analytes ranged from 0.02 to 0.5pg/mg hair, with most LOQs

lower than 0.2 pg/mg hair.

UPLC-MS/MS analysis

LC analyses were performed using an Acquity UPLC system, and

separations were carried out using an Acquity UPLC BEH C18

Table 1. MS/MS parameters for pesticides determination

Compound Name RTWa Quantier Transit ion CEb (V) Qualier Transition CEb (V)

GC amenable molecules

Triuraline 10.0 - 20.1 335 305 9 335 46 14

Triuralin-D14 10.0 - 20.1 349 319 9

-HCH 10.0 - 20.1 255 35 5 253 35 5

-HCH 10.0 - 20.1 255 35 5 253 35 5

-HCH D6 10.0 - 20.1 261 35 5

Diazinon 10.0 - 20.1 169 95 18 169 141 13

Chlorpyriphos methyl 20.1 - 23.2 285 95 8 287 95 8

Parathion methyl 20.1 - 23.2 263 154 5 263 141 17

Aldrin 20.1 - 23.2 237 35 32 328 35 22

Chlorpyriphos ethyl 20.1 - 23.2 313

189 12 313

95 23Parathion ethyl 20.1 - 23.2 291 154 4 291 169 12

Fipronil 23.2 - 25.1 366 318 11 400 331 6

-Endosulfan 23.2 - 25.1 406 35 4 404 35 4

p,p-DDE 23.2 - 25.1 318 35 1 316 35 1

p,p-DDE D8 23.2 - 25.1 326 35 1

Dieldrin 25.1 - 29.0 380 35 3 378 35 3

-Endosulfan 25.1 - 29.0 406 35 6 404 35 6

-Endosulfan D4 25.1 - 29.0 410 35 6

p,p-DDT 29.0 - 35.0 281 35 15 71 35 8

p,p-DDT D8 29.0 - 35.0 71 35 8

Diufenican 29.0 - 35.0 394 231 13 394 161 12

Permethrin 35.0 - 47.0 207 35 8 209 37 8

Cypermethrin 35.0 - 47.0 207 35 8 209 37 8

LC amenable molecules

Thiamethoxam 1.97 - 2.37 292 181 21 292 211 12

Clothianidin 2.25 - 2.65 250 132 18 250 169 12

Clothianidin D3 2.25 - 2.65 253 172 12

Imidacloprid 2.40 - 2.80 256 175 20 256 209 14

Propoxur 3.64 - 4.04 211 111 14 211 168 7

Carbofuran 3.69 - 4.11 223 123 22 223 165 12

Tebuconazole 5.37 - 5.79 309 70 18 309 125 34

Tebuconazole D6 5.37 - 5.79 315 72 18

Propiconazole 5.69 - 6.13 343 159 28 345 161 28

aRetention time window.bCollision energy.


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column (100 2.1 mm, 1.7 m particle size) from Waters

(Manchester, UK). The C18 column was equilibrated at 40 C.

The analytes were separated using a mobile phase consisting of

water:acetonitrile:formic acid, 94.5/5/0.5,v/v/v(eluent A) and ace-

tonitrile:formic acid, 99.5/0.5, v/v (eluent B), at a ow rate of

0.6 mL/min. The chromatographic separation program started at

1% eluent B for 0.5 min, than increased linearly to 99% in

8.5 min; composition that was held for 0.4 min before returning

to the initial conditions and was followed by an equilibration

time of 0.5 min. The total run time was of 10 min. Tandem mass

analysis was carried out using a Waters Acquity TQD tandem

quadrupole mass spectrometer (Manchester, UK). The instrument

was operated using an electrospray source in positive mode. The

specic MS/MS parameters for the LC-amenable compounds are

shown in Table 1. The methods LOQs for LC-amenable target

analytes ranged from 1 to 5 pg/mg hair, values which are higher

than for GC-amenable compounds partially explained by SPME

advantage to use the integrality of sample extract.

Results and discussion

Preliminary selection of decontamination solvents

In the rst step of the washing procedure development, eight

washing solvents water, phosphate buffer pH7, shampoo, so-

dium dodecylsulfate, acetone, dichloromethane, methanol, and

acetonitrilewere evaluated for their ability to remove chemicals

from the surface of articially contaminated hair samples. For this

preliminary evaluation, 20 representative pesticides from several

chemical classes were targeted: organochlorines (p,p-DDE,

HCH-beta/gamma, dieldrin, endosulfan-alpha/beta), pyrethroids

(permethrin and cypermethrin), organophosphates (chlorpyri-

phos ethyl, parathion ethyl and diazinon), neonicotinoids

(clothianidin, imidacloprid, thiametoxam), azoles (tebuconazole

and propiconazole), carbamates (carbofuran and propoxur),

pronil and triuraline.One hundred mg hair samples, articially contaminated with

silica, cellulose and aqueous solution containing the 20 target

pesticides, were washed with each solvent separately. All the

samples have been treated simultaneously and in ve replicates.

Removing deposit chemicals from hair contaminated with silica

The efciency of the washing to remove the externally deposited

chemicals from hair contaminated with silica was depending on

the solvent (Figure 1). Using water, the part of chemicals re-

moved from hair ranged from 4% (p,p-DDE) to 90%

(thiametoxam), and was above 50% for ve compounds. The de-

contamination efciency obtained with phosphate buffer ranged

from 2% (triuraline) to 88% (thiametoxam), and was above 50%for nine compounds. Decontamination with shampoo removed

from 1% (permethrin) to 96% (thiametoxam) of deposited

chemicals, with eight compounds decreased by more than 50%.

Using sodium dodecylsulfate, the decontamination efciency

ranged from 12% (propiconazole) to 98% (thiametoxam and

clothianidin) removal, with a concentration decreased by more

than 50% for 15 compounds. Among the aqueous solvents, the

most efcient decontamination of hair samples articially con-

taminated with silica was achieved by washing with sodium

dodecylsulfate.

The decontamination efciency reached using acetone ranged

from 31% (permethrin and propoxur) to 85% (endosulfan-alpha

and triuraline), with 16 compounds whose concentration was

decreased by more than 50%. For dichloromethane, the part of

chemicals removed ranged from 7% (permethrin) to 92% (endo-

sulfan-alpha and triuraline), with 17 compounds decreased by

more than 50%. Using methanol, the decontamination removed

from 54% (propiconazole) to 96% (diazinon) of deposited

chemicals, and the concentration was decreased by more

than 50% for the totality of target compounds. The de-

contamination obtained with acetonitrile ranged from 10%

(carbofuran) to 83% (diazinon), and the concentration of 9

compounds was decreased by more than 50%. Methanol

was therefore found to be the most appropriate washing

organic solvent to remove external contamination from hair

contaminated with silica.

Removing deposit chemicals from hair contaminated with cellulose

The capacity of the washing to remove externally deposited

chemicals from hair contaminated with cellulose was also highly

depending on the solvent (Figure 2). The percentage of

chemicals removed using water ranged from 2% (permethrin)

to 100% (propiconazole), with 14 compounds out of the 20 target

pesticides having their concentration decreased by more than

50%. The decontamination percentages obtained using phos-

phate buffer ranged from 3% (permethrin) to 100% (propicona-

zole), and 13 compounds were decreased by more than 50%.

Decontamination with shampoo removed from 8% (endosulfan-

beta) to 100% (thiametoxam and propiconazole) of the deposited

chemicals, and removed more than 50% of 14 compounds. Using

sodium dodecylsulfate, the percentage of decontamination

ranged from 36% (permethrin) to 100% (clothianidin, thiameto-

xam and propiconazole), and the concentration of all the com-

pounds was decreased by more than 50%, except permethrin.

Among the aqueous solvents, the most appropriate decontami-

nation of hair samples articially contaminated with cellulose

containing the target pesticides was achieved using sodium

dodecylsulfate.The percentage of chemicals removed using acetone ranged

from 25% (permethrin) to 100% (chlorpyriphos ethyl, thiameto-

xam, clothianidin, tebuconazole and propiconazole), 4% to

100% for dichloromethane and 32% to 100% for acetonitrile. All

chemicals, except permethrin, had their concentration decreased

by more than 50% upon washing with acetone, dichloromethane

and acetonitrile. All the chemicals, including permethrin, were re-

moved from the articially contaminated hair upon washing with

methanol. The percentage of chemicals removed ranged from

69% (permethrin) to 100% (chlorpyriphos ethyl, clothianidin,

tebuconazole and propiconazole). Methanol was found to be

the most efcient organic solvent to remove external contamina-

tion from hair contaminated with cellulose.

Removing deposit chemicals from hair contaminated with aqueous

solution

The capacity of washing externally deposited chemicals from hair

contaminated by dipping in an aqueous solution also depended

on the solvent nature, but was signicantly less effective than for

silica- and cellulose-contaminated hair (Figure 3). The part of

chemicals removed using water as washing solvent ranged from

0% (p,p-DDE, endosulfan-beta, parathion ethyl and diazinon) to

37% (pronil). Phosphate buffer decontamination was even less

effective than water and ranged from 0% (p,p-DDE, endosulfan-

beta, chlorpyriphos ethyl, diazinon, imidacloprid and tebuco-

nazole) to 23% (cyphermetrin). The decontamination using

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shampoo as washing solvent ranged from 0% (endosulfan -alpha/-

beta and propiconazole) to 39% (pronil). Sodium dodecylsulfate

was found to have the highest decontamination capacity on

dipped hair, ranging from 0% (endosulfan-beta, parathion ethyl

and propiconazole) to 95% (cypermethrin), with 10 compounds

having their concentration decreased by more than 50%.

Figure 1. Solvents capacity of washing externally deposit chemicals from hair articially contaminated with silica. The percentage of externally de-posited pesticides that was not removed upon washing is presented for: A - Organochlorides; B - Pyrethroids and Organophosphates; C - Neonicotinoids;D - Azoles, Carbamates, Fipronil and Triuraline. The level of pesticides determined in hair articially contaminated with silica without washing was ar-bitrary set at 100% of hair external contamination and was presented as No Wash (red line).


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Using acetone, decontamination ranged from 0% (endosulfan -

alpha/-beta, parathion ethyl, tebuconazole, carbofuran and

propoxur) to 56% (clothianidin), and only two compounds were

decreased by more than 50%. Using dichloromethane, the de-

contamination ranged from 0% (dieldrin and endosulfan-alpha)

to 57% (cypermethrin), with again only two compounds having

their concentration decreased by more than 50%. The decontam-

ination using methanol as washing solvent was the most

effective among all the organic solvents and ranged from 0%

(endosulfan-alpha) to 94% (pronil), with nine compounds

Figure 2. Solvents capacity of washing externally deposited chemicals from hair articially contaminated with cellulose. The percentage of externallydeposited pesticides that was not removed upon washing is presented for: A - Organochlorides; B - Pyrethroids and Organophosphates; C -Neonicotinoids; D - Azoles, Carbamates, Fipronil and Triuraline. The level of pesticides determined in hair articially contaminated with cellulose with-out washing was set to100% of hair external contamination and was presented as No Wash(red line).

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Figure 3. Solvents capacity of washing externally deposited chemicals from hair articially contaminated by dipping. The percentage of externally de-posit pesticides that was not removed upon washing is presented for: A - Organochlorides; B - Pyrethroids and Organophosphates; C - Neonicotinoids; D- Azoles, Carbamates, Fipronil and Triuraline. The level of pesticides determined in hair articially contaminated by dipping without washing was ar-bitrary set to 100% of hair external contamination and was presented as No Wash(red line).


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whose concentration was decreased by more than 50%. Deco-

ntamination with acetonitrile was similar to acetone and

dichloromethane, and ranged from 0% (dieldrin, HCH-gamma,

endosulfan-alpha/-beta, diazinon, imidacloprid, propiconazole

and carbofuran) to 58% (cypermethrin), with two compounds de-

creased by more than 50%. Even if the percentage of chemicals

removed was lower than for hair samples contaminated with sil-

ica and cellulose, sodium dodecylsulfate and methanol were still

the most efcient solvents.

The hair contamination model based on hair dipping could be

considered inappropriate since not reecting natural external

contamination, which is mainly due to dry contamination from

dust present in the individualssurroundings. Moreover, the hair

dipping in aqueous solution has two main limitations that can ex-

plain the low percentages of chemical removal upon washing:

rst, the irreversible penetration of compounds within the hair

shaft,[6] and secondly, the important transfer of compounds to

the hair resulting in unrealistic levels of pesticides deposited

on hair shaft (about ten times higher than for the dry contamina-

tions). Nevertheless, the issue associated with wet contamination

should not be disregarded, since the dry contamination might

become aqueous in high-humidity environment or in presenceof perspiration. Further assays involving aqueous contamination

with lowernal levels of pesticides deposited on (or incorporated

in) hair might therefore provide relevant information and allow

assessing the decontamination in more realistic conditions.

For environmental exposure studies the complete certainty of

the source of pesticide (external vs. incorporated) may not be

as critical as in some other applications of hair analysis (e.g. foren-

sic determination of use of illicit drugs). As stated in a previous re-

view paper, the part of externally deposited compound that

might become unremovable (due to wet conditions for in-

stance) can eventually (and reasonably) be considered as repre-

sentative of the history of the exposure.[27] The possible risk of

misinterpretation is actually associated with the part of externally

deposited compound that remains easily removable (ERC for

easily removable compounds) and can thus be removed by

peoples self-hair washing with shampoo. Since samples from

different subjects have to be treated the same for reliable com-

parison, the time between hair sampling and individuals last

bath/shower can be a source of variability, since ERC might have

been removed or not.[27] A washing procedure prior to hair anal-

ysis is therefore a necessary precaution, in order to have samples

similarly treated.

Successive washing with sodium dodecylsulfate or methanol

As presented here, sodium dodecylsulfate and methanol were

found to be the most efcient solvents to remove pesticides fromhair, whatever the articial contamination (with silica, cellulose,

or aqueous solution). The key question at this stage of the devel-

opment was whether to use sodium dodecylsulfate or methanol,

or both of them. Sodium dodecylsulfate and methanol were

equivalent regarding the number of pesticides whose concentra-

tion was decreased by more than 50% as well as the intensity of

average decontamination. Nevertheless, differences in their

washing properties are to be noticed mainly concerning organo-

phosphates and neonicotinoids (Figures 1 and 2). Sodium

dodecylsulfate was more effective than methanol to remove

the neonicotinoids externally deposit on hair. On the contrary,

methanol was more effective than sodium dodecylsulfate to re-

move the organophosphates deposited on hair. These results

are in concordance with the physicochemical properties of

these pesticides. Indeed, imidacloprid (neonicotenoid) is

water soluble (610 mg/L) and has an octanol/water partition

coefcient (Log P) of 0.57. At the opposite, diazinon

(organophosphate) has lower water solubility (60 mg/L)

and a Log P of 3.69. [28]

Since the objective was to develop a general decontamination

procedure for multi-class pesticides analysis in hair, both sodium

dodecylsulfate and methanol were therefore necessary.

In the second step of the washing procedure development, it

was determined whether a steady-state was reached after a sin-

gle washing. Three successive washings were tested with either

sodium dodecylsulfate or methanol. All the samples were treated

simultaneously and in three replicates. Figure 4 presents the

removal of several representative pesticides from hair samples

contaminated with silica, upon repeated washings with sodium

dodecylsulfate or methanol.

Therst washing with SDS removed from 12% (propiconazole)

to 100% (clothianidin) of the pesticides and the average decon-

tamination percentage was 63%. The second wash with SDS re-

moved from 0% to 25% of the pesticides and the average

decontamination was of 8%. The third wash with SDS removedfrom 0% to 5% and the average decontamination was 0.5%.

The rst washing with methanol removed from 54%

(propiconazole) to 95% (triuraline) of the pesticides and the average

decontamination percentage was 80%. The second wash with meth-

anol removed from 0% to 17% of the pesticides and the average de-

contamination was 4%. The third wash with methanol removed 0%

to 6% and the average decontamination percentage was 1.3%.

These results are in concordance with the few studies

previously published which highlighted the relevance of hair

washing prior to pollutants analysis.[27] Those studies compared

unwashed hair with hair washed with common surfactants just

before analysis, and reported that the levels of dioxins,[15,29]

PCBs,

[16]

or some pesticides (e.g. organochlorines

[16]

andcarbamates[30]) were decreased in hair samples after washing.

Moreover, most of the decrease occurs after the rst washing

and additional washing has no further effect on the elimination

of chemicals.[15,16] In the same sense, Toriba et al.[8] studied the

effect of washing hair with organic solvents (i.e. methanol,

hexane and dichloromethane) prior to polycyclic aromatic hydro-

carbons (PAHs) analysis in hair. Although different results were

obtained depending on the compounds analyzed and the

solvents, they were in line with the results of the present study,

in that a part of the target molecules was removed by washing

and the most signicant part of the chemicals was removed after

therst washing, even if some chemicals were also removed dur-

ing the second and the third washing. The latter difference might

be explained by the fact that washing was carried out withoutagitation, contrary to the present study.

A single washing with sodium dodecylsulfate or methanol

therefore appeared to be sufcient to remove the majority of

the deposited pesticides and to reach a steady-state since no

more chemicals or signicantly lower amounts were removed

by additional washings.

Final washing procedure

In the third and nal step of the development, the efciency of a

washing procedure using both SDS and methanol was tested

R.-C. Ducaet al.

Drug Testing

and Analysis



9/12

(Table 2). Hair naturally containing pesticides of a non-occupationally

exposed person was used. For this nal step, the list of target com-

pounds was enlarged withve more pesticides: two organochlorines

(p,p-DDT and aldrin), two organophosphates (chlorpyriphos methyl

and parathion methyl), and one carboxamide (diufenican). Hair

strands were articially contaminated with silica, cellulose or aque-

ous solution containing pesticides. Hair samples (100 mg) were rst

washed with sodium dodecylsulfate for 5 min and then SDS was re-moved. In a second step, the samples were washed with methanol

for another 5 min. Hair bres were then gathered and allowed to

dry at room temperature prior to pulverization and extraction before

pesticides analysis. Washing hair samples with methanol after SDS

(and not before) allowed the entire removal of the latter solvent from

hair. This precaution is necessary as SDS is known to interfere

with the chromatographic separation and mass-spectrometric

determination.[31,32]

The amount of pesticides removed from the surface of hair

contaminated with silica, using SDS-methanol washing, ranged

from 61% to 100%. For hair contaminated with cellulose, the to-

tality of externally deposited pesticides was removed upon

SDS-methanol washing. Even for dipped hair, the amount of

pesticides removed from the hair surface was above 50% for all

the pesticides except for p,p-DDT, dieldrin, HCH-gamma. The lat-

ter differences might be explained by the inuence the articial

contamination procedure might have on hair structure, com-

pounds deposition on hair surface and/or pesticides irreversible

incorporation. For the articial contamination of hair by dipping

in aqueous solution, the possible irreversible penetration of some

compounds within the hair shaft has been previously described[6]

and is in concordance with the present study results. In the case

of the dry contamination procedures, the differences between sil-

ica and cellulose observed here could suggest that silica, as

tough mineral particle, could abrade hair surface during the con-

tamination process and hence allow pesticides to reach internal

areas of the hair bers and become less removable by washing.

At the opposite, cellulose, as soft organic particles, would limit

pesticide deposition on hair surface. A good illustration is provided

with-endosulfan, naturally present in hair at low levels of concen-

tration (0.41 0.10 pg/mg), for which upon SDS-methanol washing

of the dry contaminated hair samples, only for cellulose were

reached comparable concentration levels (1.00 0.10 pg/mg) and

a decontamination percentage close to 100%, whereas the articial

Figure 4. Articially contaminated hair (by pesticides in silica particles), submitted to successive washings with sodium dodecylsulfate (right) ormethanol (left). The percentage of externally deposited pesticides that was not removed upon successive washing is presented for: (A) Organochlorides;(B) Pyrethroids, Organophosphates and Neonicotinoids; (C) Azoles, Carbamates and Miscellaneous.


Drug Testing

and Analysis



10/12

Table2.

Efciencyofthenalwashingprocedureusingbothsodium

dodecylsulfate

andmethanoltoremoveexternallydepositchemicalsfrom

hairboth

naturally

andart

iciallycontaminated

Nocontamination

Drycontamination

Liquid

contamination

Silica

Cellulose

Aqueoussolution

Nowash

(pg/mg)

Doublewashed

(pg/mg)

Decont

(%)

Nowash

(pg/mg)

Doublewashed

(pg/mg)

Decont

(%)

Nowash

(pg/mg)

Doublewashed

(pg/mg)

Decont

(%)

Nowash

(pg/mg)

Doublewashed

(pg/mg)

Decont

(%)

ORGANOCHLORIDES

p,p

-DDT

3.0

90

.21

4.3

11

.08

-

61018

1437

77

1497

71

96

4173220

2897128

31

p,p

-DDE

n.d.

n.d.

-

143036

20614

86

2419

41

98

12079520

4195129

65

Aldrin

n.d.

n.d.

-

261

40

.7

85

663

n.d.

100

237980

69558

71

Dieldrin

n.d.

n.d.

-

2777

442

84

2356

20

.1

99

5112139

463083

9

HCH-beta

1.4

60

.22

0.9

20

.09

37.3

184941

45513

75

3076

60

.4

98

9903175

3999392

60

HCH-gamma

5.1

60

.22

4.8

80

.12

5.4

174939

52823

70

2513

90

.4

96

349367

215777

38

Endosulfan-alpha

0.0

80

.01

0.0

60

.01

27.6

40112

926

77

26610

20

.2

99

146931085

7002272

52

Endosulfan-beta

0.4

10

.10

0.3

70

.08

9.8

1253

171

87

2577

10

.1

100

350314648

152421165

56

PYRETHROIDS

Permethrin

24

.111

.62

9.2

00

.77

61.8

213

51

.5

74

277

122

57

45734

10411

77

Cypermethrin

n.d.

n.d.

-

17925

6928

61

359104

n.d.

100

155651332

3968365

75

ORGANOPHOSPHATES

ChlorpyriphosEthyl

0.0

60

.01

0.0

50

.01

12.9

201

30

.2

84

663

0.3

0

.02

100

3423151

146795

57

ParathionEthyl

n.d.

n.d.

-

60

.5

10

.1

91

473

0.1

0

.01

100

2582207

732101

72

Chlorpyriphosmethyl

n.d.

n.d.

-

49331

8810

82

77348

51

.5

99

612223255

16120889

74

Parathionmethyl

n.d.

n.d.

-

81

10

.04

92

523

0.1

0

.02

100

72780

27231

63

Diazinon

0.1

70

.03

0.0

80

.02

54.9

251

0.2

0

.03

99

26315

0.2

0

.03

100

207243

61252

70

NEONICOTINOIDS

Clothianidin

n.d.

n.d.

-

202

n.d.

100

422

n.d.

100

10412

243

77

Imidacloprid

n.d.

n.d.

-

23332

204

91

68551

91

.6

99

1497150

33836

77

Thiametoxam

n.d.

n.d.

-

825

0.7

0

.1

99

18013

n.d.

100

21329

263

88

AZOLES

Tebuconazole

0.2

90

.03

n.d.

100

581

1.0

0

.3

98

786

1.7

2

98

59731

16914

72

Propiconazole

5.9

80

.47

4.5

60

.34

23.6

1387

40

.5

97

46112

3.4

0

.7

99

6957274

983102

86

CARBAMATES

Carbofuran

n.d.

n.d.

-

67470

303

96

3885287

n.d.

100

6590383

69548

89

Propoxur

n.d.

n.d.

-

70583

10912

84

8507194

n.d.

100

10107511

124586

88

MISCELLANEOUS

Fipronil

26

.402

.56

6.1

00

.23

76.9

36024

90

.4

98

51510

6.7

0

.6

99

4910203

78592

84

Diufenican

0.6

50

.04

0.4

10

.03

36.7

451

50

.1

88

1195

0.7

0

.1

99

455179

2033119

55

Triuraline

0.2

50

.08

0.0

70

.01

72.6

2584

344

87

46219

20

.1

100

6996183

2252217

68

n.d.:

notdetected

.

R.-C. Ducaet al.

Drug Testing

and Analysis



11/12

contamination was higher in hair contaminated with cellulose

(257 7 pg/mg) than with silica (125 3 pg/mg). Nevertheless,

these results conrmed that a washing procedure using rst so-

dium dodecylsulfate and secondly methanol, efciently removes

the pesticides deposited on hair.

Native hair samples (not submitted to articial contamination)

were also washed with SDS and methanol successively. The per-

centage of pesticides removed from hair then ranged from 5.4%

(HCH-gamma) to 100% (tebuconazole) of the total amount of

pesticides (determined in hair samples without washing). The

part of pesticides removed in the latter experiment is therefore

likely to correspond to externally deposited compounds whereas

the amount of pesticides still detected after washing would

correspond to biological incorporation.

Taking all together, the results obtained on SDS-methanol wash-

ing of both articially contaminated and native hair demonstrated

that the pesticides biologically incorporated into hair are not af-

fected by the decontamination procedure. A good illustration is

provided with propiconazole, for which the concentrations deter-

mined upon hair wash with SDS-methanol were 4.6 0.3pg/mg

in native hair, 4.0 0.5 pg/mg in hair contaminated with silica and

3.4 0.7 pg/mg in hair contaminated with cellulose. Although thepresent study shall represent an important stepin the development

of a standardized decontamination procedure before pesticide

analysis in hair, the results presented here were obtained from

Caucasian subjects who did not use cosmetics. As hair structure

might inuence incorporation, further studies should be conducted

on hair samples collected from subjects of different origins (African,

Asian) as well as on samples collected from people applying cos-

metic treatment that might have altered structure.

Conclusions

Evaluating the efciency of a washing procedure of hair before

analysis is a complex task which may be signicantly inuencedby the protocol of articial contamination of the samples, as

demonstrated in the present work. Therefore, articial contami-

nation of samples has to be as close as possible to reality. Remov-

ing from hair surface compounds from different chemical classes

presents an additional challenge, as the efciency of a washing

solvent varies for different chemicals. It was demonstrated here

that washing hair with sodium dodecylsulfate and with methanol

successively appears to be the best procedure to remove

chemicals from different classes deposited on hair surface. Such

procedure appears to remove in a one-shot the fraction of

chemicals supposed to be located on hair surface and does not re-

quire repeated washing steps. Moreover, this decontamination

procedure does not seem to inuence the concentration of

chemicals located inside hair, which are considered being incor-

porated from biological pathways and representative of exposure.

Acknowledgements

This study was carried out in the framework of the call for re-

search project 2010 of the national program Environmental

and Occupational Health (PNR EST) of the French Agency for

Food, Environmental and Occupational Health Safety(ANSES), with

the nancial support of theOfce National de lEau et des Milieux

Aquatiques (ONEMA) supporting the implementation of the Plan

Ecophyto 2018, France. R.-C. Duca beneted from a postdoctoral

grant from the Fonds National de la Recherche (FNR) (AFR

1069412), Luxembourg.

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