TECHNICAL NOTE

Mercury speciation analysis in human hairby species-specific isotope-dilution using GC–ICP–MS

Laure Laffont & Laurence Maurice & David Amouroux &

Patricia Navarro & Mathilde Monperrus &

Jeroen E. Sonke & Philippe Behra

Received: 13 March 2012 /Revised: 11 May 2012 /Accepted: 15 May 2012# Springer-Verlag 2012

Abstract We optimized a mercury (Hg) speciation extrac-tion method for human hair in combination with species-specific isotope-dilution analysis by gas chromatography–inductively coupled plasma–mass spectrometry (GC–ICP–MS). The method was validated on human hair referencematerial RM (IAEA-086), which is recommended for anal-ysis of monomethylmercury (MMHg) and inorganic mercu-ry (IHg). Three reagents, hydrochloric acid (HCl), nitric acid(HNO3), and tetramethylammonium hydroxide (TMAH),and three extraction procedures, at ambient temperature for12 h, microwave-assisted at 75 °C for 6 min, and oven

heated at 80 °C for 2 h were tested. Extraction efficiency,recovery, and potential species transformations were evalu-ated for each method. The most efficient procedures, withrecovery of ~90 % for each species with limited demethy-lation (<5 %) and methylation (0 %), were HNO3 digestion,irrespective of temperature, and microwave-assisted TMAHextraction. Acidic extraction with HCl induces significantdemethylation, with production of artifacts. To correct forpotential demethylation artifacts we recommend spikingwith isotopically enriched standards before the extractionstep.

Published in the topical collection Isotope Ratio Measurements: NewDevelopments and Applications with guest editors Klaus G. Heumannand Torsten C. Schmidt.

L. Laffont : L. Maurice : J. E. SonkeObservatoire Midi-Pyrénées, Laboratoire GéosciencesEnvironnement Toulouse, Université Paul Sabatier Toulouse III,14 avenue Edouard Belin,31400 Toulouse, France

L. Laffont : J. E. SonkeCNRS; GET,31400 Toulouse, France

L. MauriceIRD; GET,31400 Toulouse, France

D. Amouroux : P. Navarro :M. MonperrusLaboratoire de Chimie Analytique Bio-Inorganiqueet Environnement, Insitut des Sciences Analytiqueset de Physico-Chimie pour l’Environnement et les Matériaux,Université de Pau et des Pays de l’Adour, Hélioparc Pau Pyrénées,2 Avenue Pierre Angot,64053 PAU Cedex 9, France

D. Amouroux (*)CNRS; LCABIE,64053 Pau Cedex 9, Francee-mail: david.amouroux@univ-pau.fr

M. MonperrusUPPA; LCABIE,64053 Pau Cedex 9, France

P. BehraUniversité de Toulouse; INPT,LCA (Laboratoire de Chimie AgroIndustrielle), ENSIACET,4 allée Emile Monso,31030 Toulouse Cedex 4, France

P. BehraINRA; LCA (Laboratoire de Chimie AgroIndustrielle),31030 Toulouse Cedex 4, Francee-mail: laure.laffont@ensiacet.fr

Present Address:L. Laffont (*)INRA; LCA (Laboratoire de Chimie AgroIndustrielle),31030 Toulouse Cedex 4, Francee-mail: laure.laffont@ensiacet.fr

Anal Bioanal ChemDOI 10.1007/s00216-012-6116-2

Keywords Metals/heavy metals . Organometals .

Speciation . Mass spectrometry/ICP–MS . Biologicalsamples

Introduction

Mercury (Hg) is a well known toxic element, especially inthe form of monomethylmercury (MMHg), which is mainlyabsorbed via dietary exposure [1]. Usually MMHg exposureis evaluated by measuring the total Hg concentration in hair.However, people can also be exposed to inorganic forms ofHg (IHg) via dental amalgams, urban air pollution, or occu-pational exposure, for example gold or silver mining [2, 3].

Unraveling the simultaneous exposure of humans toMMHg and IHg requires species-specific analysis of Hg inhair. Several methods have been developed for analysis ofMMHg and/or IHg in human hair: neutron-activation anal-ysis [4–7], high-performance liquid chromatography cou-pled with ICP–MS [8–10], electrothermal atomic-absorption spectrometry [11], and gas chromatography cou-pled with cold-vapor atomic fluorescence spectroscopy[12–14] or electron-capture detection [15].

GC–ICP–MS using a double isotopic-dilution method(ID-GC–ICP–MS) enables analysis of both MMHg andIHg with high precision [16–20]. In particular the methodenables evaluation and correction of possible MMHg deme-thylation or IHg methylation artifacts that could occur dur-ing sample preparation [17]. Several methods for acidicextraction of Hg species from hair samples before chromato-graphic (GC or HPLC) separation have been reported in theliterature: water bath mainly at 100 °C [10, 12, 14, 15, 21],microwave-assisted [10], and stirring at ambient tempera-ture [9, 10] with diverse concentrations of acid reagents(HCl, HNO3, H2SO4, CH3COOH) and diverse reactiontimes (from 5 min to 1 night). Basic reactants have beenused for extraction of Hg species from hair before analysisby neutron-activation analysis. Bases such as KOH or tetra-methylammonium hydroxide (TMAH) have also been usedfor extraction of Hg species from biotissues, for examplefish and shellfish, with open-focused microwaves [19,22–24] for analysis by GC–ICP–MS. As far as we areaware, no validated extraction method for GC–ICP–MSanalysis of Hg species in human hair has yet been published.

The choice of human hair reference material for methodvalidation is fundamental because it must be as similar aspossible to real hair samples. In most previous studies,IAEA-85 reference material from the International AtomicEnergy Agency (Vienna, Austria) has been used to validatespecies-specific analysis of Hg [4–8, 10, 11, 15]. The use ofthis reference material is questionable, because it containsboth endogenousMMHg and artificially addedMMHgwhichcould have different chemical behavior during sample

preparation, more specifically during extraction. SpikedMMHg has not been incorporated in the hair fiber during itsgrowth and is probably easier to extract from the hair surfacethan endogenous MMHg. Consequently, transfer to naturalsamples of methods validated with this spiked MMHg humanhair is arguable. Other authors have used BCR-397 from theInstitute for Reference Materials and Measurements (Geel,Belgium) and GBW-07601 from the Institute of Geophysicaland Geochemical Exploration (Lanfang, China); these certi-fied for total Hg only and cannot reasonably be used tovalidate a species-specific analytical method [9, 11]. For suchmethod validation, only two reference materials are adequate:NIES-13 from the National Institute for Environmental Stud-ies (Tsukuba, Japan), with up to 90 % MMHg, and IAEA-86from the International Atomic Energy Agency (Vienna, Aus-tria) with ~45 % MMHg [4–8, 10, 11, 14, 15, 25], bothcontaining natural Hg. Because the difficulty of species-specific analyses of Hg in hair is to desorb IHg withoutdegrading the MMHg form, we chose, in this study, CRMIAEA-86, which contains more IHg than NIES-13.

The main objective of this work was to develop anaccurate analytical method for analysis of both MMHg andIHg species in real hair samples by simple and doubleisotopic dilution and GC–ICP–MS. Isotopic dilution hasbeen demonstrated to be more accurate than external cali-bration for GC–ICP–MS analysis [26]. Double isotopicdilution could enable evaluation of potential methylation anddemethylation artifacts to obtain corrected concentrations.

For this purpose several extraction methods and differentreagents were tested, as was the extract volume derivatized,the addition of the enriched species, and the calculationapproach used. The method was finally validated by analy-sis of reference material IAEA-86 and applied to hair sam-ples taken from Bolivian gold-miners.

Experimental

Reagents and reference materials

The isotopically enriched species used were purchased fromISC Science (Oviedo, Spain). IHg is enriched in 199Hg(91 %) and MMHg in 201Hg (96.5 %). MMHg chlorideand IHg chloride standards with natural isotopic composi-tion were obtained from Strem Chemicals (Newburyport,MA, USA). All working standard solutions were prepareddaily in 1 % HCl and kept in the dark at –20 °C. Hydro-chloric acid (HCl, 33–36 %, Instra-analyzed) and nitric acid(HNO3, 67–69 %, Instra-analyzed) were purchased fromJ.T. Baker (Phillipsburg, NJ, USA). Tetramethylammoniumhydroxide (TMAH, 25 % in water) was purchased fromFluka (Steinheim, Germany). The reagent used for derivati-zation was sodium tetrapropylborate NaBPr4 (98 %, packed

L. Laffont et al.

under argon) purchased from GALAB (Geesthacht, Ger-many); 3 % (w/v) solution was prepared daily in Milli-Qwater and kept in the dark at –20 °C. Acetate buffer solution(0.1 mol L−1; pH 4) was prepared by dissolving sodiumacetate and glacial acetic acid in Milli-Q water. Analyticalreagent grade isooctane, glacial acetic acid, and sodiumacetate were obtained from Sigma–Aldrich (Saint-Louis,MO, USA). The reference material used for method valida-tion was natural human hair IAEA-86 from the InternationalAtomic Energy Agency (Vienna, Austria) [25].

Instrumentation

In some experiments extraction of Hg forms from hair wasconducted with an Explorer focused microwave system fromCEM Corporation (Mathews, NC, USA). A commercial GC–ICP–MS interface (Silcosteel, 0.5 m length, inner i.d. 0.28 mmand o.d. 0.53 mm, outer i.d. 1.0 mm and o.d. 1.6 mm; ThermoFisher Scientific, Franklin, MA, USA) was used to couple aThermo Electron gas chromatograph (Trace) to a Thermo X2series ICP–MS (Thermo Fisher Scientific, Waltham, MA,USA). The GC column was an MTX-1 Silcosteel (30 m×0.53 mm×1 μm) containing a Crossbond 100 % dimethylpo-lysiloxane stationary phase (Restek, Bellefonte, PA, USA). Thesample (2 μL) was introduced in splitless mode at 250 °C. Thetemperature program used for the chromatographic separationwas: 1 min at 60 °C, temperature gradient from 60 °C to 250 °Cat 60 °min–1, and 1min at 250 °C. Carrier gas was helium, witha flow of 25mLmin–1, andmake-up gas was argon, with a flowof 300 mL min–1. ICP–MS conditions used for analysis were:nebulizer, plasma, and auxiliary flows 0.6, 1.5, and 0.9 Lmin–1 respectively, plasma power 1250 W, Hg isotopes198, 199, 200, 201, and 202 with dwell time of 25 ms,and Tl isotopes 203 and 205 with dwell time of 5 ms.ICP–MS optimization was conducted with an In stan-dard solution from Analytika (Prague, Czech Republic).Simultaneous introduction of Tl enabled checking ofplasma stability and mass bias during analysis.

Total Hg (THg) concentration in hair was measured byAAS (DMA-80 Milestone) by combustion with gold-trappre-concentration and atomic-absorption detection. Relativereproducibility of DMA-80, evaluated by use of differentCRMs, is 8 % for total Hg concentration.

Methods

Hair samples preparation procedure

The procedure can be divided into three steps: spiking,extraction, and derivatization. Several procedures were test-ed and compared for each step (Fig. 1).

Two spiking procedures were used: enriched species199IHg or 201MMHg were added either after or before theextraction procedure. IAEA-86 (10, 20, or 50 mg) wasdigested with 5 mL of three different reactants: TMAH25 %, HNO3 6 mol L−1, and HCl 6 mol L−1. Three extractionmethods, modified from published methods, were tested:

& no heating control, ambient temperature for 12 h witheach reagent [9, 10];

& use of focused microwave heating, with stirring, at 75 °Cfor 6 min with TMAH [19, 22, 24, 27]; and

& oven heating at 80 °C for 2 h with HNO3 6 mol L−1 orHCl 6 mol L−1 [10, 12, 14, 15].

Combined methods, for example TMAH in an oven oracidic digestion in a microwave oven, were not tested,because the two ovens are not equipped with a gas extractor.Indeed, decomposition of TMAH gives CO, a highly toxicgas, and acids corrode the microwave oven.

The derivatization procedure was optimized by testingseveral reagents and extract volumes. After cooling, 250–2000 μL of the extracts of each sample, were weighed in4 mL of acetate buffer solution pH 4. After pH adjustmentto 4 with HCl, 100 μL NaBPr4 at 20 % or 200 μL at 3 % wasadded with 2 to 6 mL isooctane to derivatize Hg species(Fig. 1). Previous tests on CRMs have shown that the deriv-atization reagent NaBPr4 gives more accurate concentrationscompared with certified values than NaBEt4 [22]. The organicphase was recovered after 5 min of manual shaking andanalyzed in triplicate by GC–ICP–MS.

Microwaves75 ° C 6 min

Oven80 ° C 2 h

Ambienttemperature

1 night

1) Method

TMAH

HNO3

HCl

2) Reactant

10 mg

20 mg

50 mg

3) IAEA-86 mass

4) Extract volume

250 µL

Optimisation of extraction procedures (spike added before extraction)a

b

Fig. 1 Schematic representation of experiments conducted to optimizeand validate the sample-preparation procedure for species-specificanalysis of Hg in hair by GC–ICP–MS and isotopic dilution: (a)extraction procedure; (b) derivatization procedure

Mercury speciation analysis in human hair

Analysis and calculation methods

Concentrations of the different forms of Hg were calculatedby averaging isotope dilution calibration with two enrichedspecies: 199IHg and 201MMHg. In order to study the chem-ical behavior of both Hg forms during the sample-preparation procedure and to determine methylation and/ordemethylation transformation factors, two enriched species199IHg or 201MMHg ([199IHg]0[201MMHg]≈10 ng g–1)were added before or after the extraction.

Endogenous IHg and MMHg concentrations were thencalculated by means of two different calculation methods:simple isotope dilution analysis (S-IDA) [17] and isotopepattern deconvolution (IPD) with double spiking isotopicdilution [19, 20, 28]. S-IDA calculation is based on inde-pendent Hg species determination and enables correction fornon-quantitative reactions and/or losses only whereas IPDcalculation, in addition, enables determination of demethy-lation (D) and methylation (M) transformation factors dur-ing sample preparation and calculation of methylation and/or demethylation-corrected concentrations [20]. Quantifica-tion of D and M factors is helpful for choosing the optimumsample-preparation procedure generating the lowest deme-thylation and methylation artifacts.

Experimental strategy

To optimize the overall speciation method, several condi-tions used for extraction, enriched species addition, andderivatization procedures were investigated (Fig. 1). Effi-ciency of the extraction procedure was first optimized bytesting:

1. the extraction method (microwave, oven, or ambienttemperature);

2. the extraction reagent (TMAH, HNO3, or HCl); and3. the ratio of mass of hair sample to volume of extraction

reagent (10 mg:5 mL, 20 mg:5 mL, or 50 mg:5 mL)(Fig. 1a).

The two best extraction procedures were chosen for op-timization of the last step, the derivatization procedure.

This step was optimized by testing different volumes ofextract derivatized with the same amount of derivatizationreagent (NaBPr4) (Fig. 1b). The spiking procedure afterextraction was evaluated for practical and routine laboratoryapplications: first, to avoid any cross-contamination in thelaboratory by the use of enriched spike during sample prep-aration when performed in the same way (oven for example)compared with determination of natural isotopes; and, sec-ond, to evaluate the method without uncertainties related todifferent sample-preparation procedures and eventual prob-lems linked to endogenous and spiked Hg species equilibra-tion between a solid sample and a liquid spiking solution.

The spiking procedure, before or after extraction, was usedto discriminate when potential demethylation or methylationtransformations occurred: during the extraction procedure orduring the derivatization procedure. Finally, the stability ofthe Hg species in the extract spiked before the extraction(2 months and 6 months) was also checked for the bestmethod. In the first step, to evaluate extraction method, eachsample was extracted once and each extract was analyzedthree times. In the second step, to evaluate derivatizationprocedure, each sample were extracted three times and eachextract was analyzed three times. Standard deviation (SD)was calculated for extraction replicates and standard error(SE) was calculated on injection replicates.

Results and discussion

Evaluation of the extraction procedure

All the results were evaluated by comparison with theMMHg and THg concentration values given for RMIAEA-86: 258±11 ng g–1 for MMHg and 315±20 ng g–1

for IHg. As shown in Table 1, no significant differenceswere noticed in determination of MMHg concentrationsbetween the different extractions procedures tested. MMHgrecovery relative to the recommended value ranged between96 and 114 % (S-IDA calculations), indicating that MMHgextraction is efficient, irrespective of the extraction proce-dure used. Recovery of IHg, relative to the value calculatedby subtracting MMHg from total Hg recommended value,ranged between 77 and 113 % (S-IDA calculations). Theextraction procedure at ambient temperature with two reac-tants (HNO3 and TMAH) and the microwave-assisted ex-traction with TMAH did not result in sufficient recovery ofIHg, with values averaging 88±4 %, 81±3 % (n03; SD) and83±3 % (n02; SD), respectively (for S-IDA calculations).The “10 mg” value was not integrated in the average calcu-lation because the recovery of 113 % is questionable. Atambient temperature, only HCl with hair masses of 20 and50 mg and HNO3 with a hair mass of 50 mg gave goodrecovery—92 %, 91 %, and 93 % respectively. The extrac-tion procedure in the oven at 80 °C seems the most effective,irrespective of the reagent used (HNO3 or HCl), giving IHgrecovery of 91±8 % and 92±7 % (n03; SD) for HNO3 andHCl, respectively (S-IDA calculations). Moreover, addingboth isotopically enriched Hg species before the extractionstep with IPD calculation enables determination of the meth-ylation and/or demethylation factors during the extractionstep and correction of the Hg species concentrations. Nosignificant methylation artifact was observed, whatever ofthe extraction procedure used. Demethylation factors (D)were found to have the highest values for extraction proce-dures using HCl under both temperature conditions—4±

L. Laffont et al.

Tab

le1

Valuesof

MMHgandinorganicHgconcentrations

(ngg–

1)andmethy

latio

nM

(%)anddemethy

latio

nD(%

)factorsob

tained

with

differentextractionmetho

dsforapprox

imately10

,20,

and50

mgIA

EA-86referencematerialforhu

man

hairHgspeciesdeterm

ination.

Stand

arderror(SE)iscalculated

fortriplicateanalysis.R

ecov

eryiscalculated

relativ

eto

recommendedvalues

ofIA

EA-86:

258±11

ngg–

1forMMHgand31

5±20

ngg–

1forIH

g

S-IDA

calculation

IPD

calculation

Recov

eries(%

)

Mean

SE

Mean

SE

Mean

SE

Mean

SE

Mean

SE

Mean

SE

Reactant

Sam

ple

mass

Massof

extract

[MMHg]

[MMHg]

[IHg]

[IHg]

[MMHg]

[MMHg]

[IHg]

[IHg]

MM

DD

MMHg

IHg

MMHg

IHg

Metho

d(g)

(g)

(ngg-1)

(ngg-1)

(ngg-1)

(ngg-1)

(ngg-1)

(ngg-1)

(ngg-1)

(ngg-1)

(%)

(%)

(%)

(%)

(S-IDA)

(S-IDA)

(IPD)

(IPD)

Microwaves

TMAH

0.011

4.07

254

2035

413

266

1641

911

-1.3

0.4

2.5

0.5

99113

103

133

75°C

0.02

54.05

255

525

47

253

1527

013

0.6

1.2

2.0

1.1

9981

9886

0.05

33.98

260

626

82

255

727

25

0.6

0.4

0.6

0.5

101

8599

86

Oven

HNO36M

0.011

4.74

293

1025

86

287

1330

86

-0.1

1.7

1.2

0.6

114

82111

98

80°C

0.02

24.74

264

931

07

269

635

016

-0.3

0.8

1.2

0.4

102

9810

4111

0.05

34.72

255

229

05

257

629

65

-0.1

0.6

3.5

1.2

9992

9994

HCI6M

0.01

34.35

260

626

54

260

428

56

-0.6

0.6

7.8

1.5

101

8410

190

0.02

14.39

254

530

617

254

1232

311

0.1

1.3

5.3

0.6

9997

9810

3

0.05

24.35

252

530

03

251

630

12

0.1

0.4

6.2

0.1

9895

9796

Ambient

temperature

HNO36M

0.01

24.74

275

1527

115

288

1031

810

-0.3

0.9

0.0

1.3

107

86112

101

0.02

14.75

250

227

211

261

5311

10-0.7

0.8

-0.6

0.1

9786

101

99

0.05

24.74

248

829

22

252

730

89

0.0

0.7

0.0

0.5

9693

9898

HCI6M

0.011

4.35

254

826

69

262

530

519

0.8

0.1

2.8

2.0

9884

101

97

0.01

94.34

259

528

910

264

9311

30.1

0.4

4.2

0.7

101

9210

299

0.04

94.35

251

728

76

253

728

612

0.2

0.4

4.2

1.1

9791

9891

TMAH

0.011

4.07

248

1624

45

269

1729

623

-0.8

0.7

1.5

0.7

9677

104

94

0.02

14.06

259

525

613

266

1328

211

0.0

1.6

0.8

1.6

101

8110

389

0.05

14.06

251

426

55

254

427

312

-0.1

1.1

1.5

0.6

9784

9887

Mercury speciation analysis in human hair

1 % and 7±1 % (n03; SD) at ambient temperature and 80 °C,respectively. The extraction procedures using HNO3 andTMAH induce fewer demethylation artifacts (D≈1–2 %).

In contrast, MMHg and IHg recovery calculated byIPD combined with both enriched species addition be-fore extraction were satisfactory for all the extractionprocedures considering standard deviation on recom-mended values and analysis uncertainty. The precisionof the MMHg and IHg concentrations calculated byIPD is slightly higher than that calculated by S-IDA,with average RSE of 3.5 and 3.9 % and 2.5 and 2.5 %,respectively.

The mass of hair sample extracted did not result in anysignificant differences, either in MMHg and IHg concentra-tions or in transformation factors. Even when using very lowsample mass (10 mg) the results are consistent with therecommended values but lower precision for the concentra-tions (Table 1) was observed, probably because of the un-certainty of the weight using an analytical balance (massbalance reproducibility is 0.2 mg, inducing weighing errorof 2 % and 1 % for 10 and 20 mg, respectively). Thus, aminimum sample mass of 20 mg must be used for accurateanalysis and was selected for performing subsequentexperiments.

Consequently, according to the MMHg and IHg recoverycalculated by S-IDA and demethylation factors calculatedby IPD, the best extraction procedure is oven extractionusing HNO3 at 80 °C. However, TMAH is mainly usedfor extraction of MMHg and IHg from biological tissuesbecause:

1. MMHg is probably more stable in TMAH than in HNO3

whereas MMHg could be demethylated; and2. hair is completely mineralized in TMAH whereas resi-

dues remain in HNO3, because bases are more effectivethan acids at dissolving proteins.

Consequently, subsequent tests were also performed withmicrowave-assisted extraction using TMAH.

Evaluation of the derivatization step

To assess when addition of the isotopically enriched Hgspecies is more effective in terms of non-quantitative reac-tions, losses, and transformations correction, the labeledanalogues were added before and after extraction of theRM IAEA-86 and were extracted by the two previouslychosen procedures. No significant difference was noticedbetween MMHg or IHg concentrations among the valuesobtained when the enriched species were added before orafter extraction. However, demethylation factor (D) forHNO3 extraction was 5±2 % when enriched standards werespiked before extraction and 0±1 % when they were spikedafter extraction (Table 2). Consequently, demethylationoccurs during the extraction process and not during deriva-tization of the samples. In the TMAH extraction procedure,demethylation artifacts are similar (4±2 % and 4±1 % be-fore and after extraction, respectively) meaning that deme-thylation occurs during derivatization. For analysis ofhuman hair samples, the two extraction procedures can beused if enriched species are added before extraction. How-ever, if addition is not possible before extraction for practi-cal reasons, as explained above, the TMAH extractionprocedure must be chosen to ensure the best accuracy ofthe measured concentrations. This supports the conclusionsreached in previous work [28, 29] that the accuracy of Hganalysis by species-specific isotope dilution depends on thebehavior of the enriched Hg species during the procedure,which must be as close as possible to that of the endogenousspecies.

To evaluate the effect of the extract volume on derivati-zation efficiency, different masses of TMAH and HNO3

extracts (250, 500, 1000, and 2000 mg) were tested usingthe same amount of NaBPr4 reagent. Recovery of Hg spe-cies calculated by S-IDA vs. mass of extract is reported inFig. 2. Recovery of MMHg is not affected by the extractmass used, whereas IHg recovery decreases when the massof the extract is increased. Similar trends were observed

Table 2 Mean values of MMHg and inorganic Hg concentrations(ng g–1) and methylation M (%) and demethylation D (%) factorsobtained when spiking enriched standards 199IHg and 201MMHg be-fore or after sample extraction by TMAH with microwave assistance or

HNO3 at 80 °C in an oven for a same mass of IAEA-86 (Fig. 1b).Standard deviation (SD) is calculated for triplicate extraction of thesamples, each analyzed in triplicate. Recommended values of IAEA-86are: 258±11 ng g–1 for MMHg and 315±20 ng g–1 for IHg

S-IDA calculation IPD calculation

Method Spikingprocedure

Mean SD Mean SD Mean SD Mean SD M M D D[MMHg] [MMHg] [IHg] [IHg] [MMHg] [MMHg] [IHg] [IHg] (%) SD (%) SD(ng g-1) (ng g1) (ng g-1) (ng g-1) (ng g1) (ng g-1) (ng g1) (ng g-1) (%) (%)

HNO3 80 °C before 254 29 276 27 246 30 261 18 -0.1 1.3 4.6 2.5

after 228 10 284 10 227 9 292 13 -0.1 0.5 -1.1 1.5

TMAH before 250 5 290 2 252 5 292 5 0.5 1 4.2 1.7

after 253 16 306 41 256 17 311 40 -0.1 0.5 4.2 0.8

L. Laffont et al.

with double isotopic dilution and IPD calculation. Conse-quently, labile enriched and endogenous Hg species are notequilibrated and matrix effects are thus not properly cor-rected, irrespective of the calculation method used.

To minimize the matrix effect in human hair samples forIHg determination a constant extract volume for all the sam-ples, i.e. here 250 mg for the best recovery, irrespective of thereactant, must be used. This requires that variation of the massof hair used for the extraction does not induce artifacts. Pre-viously, we have shown that no artifacts were induced by suchvariations. Another option could be addition of cysteine tosaturate the solution with a matrix close to that of the samples,as proposed previously by Point et al. [29].

Stability of Hg species in the TMAH extract

For assessment of the Hg species stability in the extract,samples spiked before extraction with TMAH under

microwave irradiation were re-analyzed after 2 and 6 months.MMHg and IHg concentrations calculated by S-IDA and IPDare reported in Fig. 3a, b. IHg concentration is not significant-ly different after 6 months but MMHg concentration de-creased slightly as a function of time. MMHg degradationrelative to t00 was calculated. At t02 months, MMHg deg-radation was, approximately, between 1 and 2 % dependingon the calculation method. Considering the analyticaluncertainty of approximately 4 %, as shown previously,MMHg is stable over 2 months in the TMAH extract.At t06 months, MMHg degradation of approximately6 % was observed, irrespective of the calculation meth-od used. This degradation cannot be corrected withdouble isotopic dilution and IPD calculation. MMHg degra-dation of 6 % should induce approximately 5 % IHg enrich-ment which is consistent with IHg enrichment of 4 % att06 months relative to t00.

Fig. 2 Recovery of MMHg and IHg relative to recommended valuesfor IAEA-86 vs. mass of extract used for derivatization. Two extractionprocedures were investigated: (a) TMAH with microwave-assistedheating and (b) HNO3 in an oven. MMHg and IHg concentrationswere evaluated by means of the S-IDA calculation method

Fig. 3 Evaluation of Hg species stability in TMAH extracts, withspiking before extraction: (a) MMHg and (b) inorganic Hg concen-trations analyzed after t00, t02 months, and t06 months calculated byS-IDA and IPD. Error bars were calculated as standard deviation (SD)for n02 extraction replicates, each analyzed in triplicate

Mercury speciation analysis in human hair

Consequently, digested samples in TMAH are stable at4 °C in the dark for 2 months which is sufficient for routineenvironmental and/or health monitoring.

Analytical performance and application

Evaluation of the analytical performances of the method, i.e.repeatability and accuracy, was carried out on human hairsample reference material (IAEA-86) extracted aftermicrowave-assisted digestion using TMAH and addition ofthe isotopically labeled Hg species before extraction.

The relative repeatability, calculated as the standard errorfor triplicate injection of six IAEA-86 samples at 200 pg g–1

for MMHg and 250 pg g–1 for IHg in isooctane, was be-tween 1–2.5 % and 0.5–1.8 % by S-IDA calculation andbetween 1–2.5 % and 1–2.5 % by double isotopic dilutionand IPD calculation, respectively. As shown in the literature,double isotopic dilution and IPD calculation leads to lowerrepeatability than S-IDA calculation [19, 22].

Accuracy was calculated as the ratio of the measured tothe recommended values of eight IAEA-86 samples ana-lyzed in triplicate. MMHg and IHg average recovery was 98

and 88 % with S-IDA calculation and 98 and 90 % with IPDcalculation, respectively. Certified reference materials ofmarine biotissues (DOLT-4, BCR 464, TORT-2, NIST-2977) have previously been analyzed by Clemens et al.and Navarro et al. [19, 22] by the same extraction andanalysis method. The average accuracy was 100 % forMMHg and 111 % for IHg (n06) by S-IDA calculationand 99 % for MMHg and 96 % for IHg (n06) by IPDcalculation [19, 22]. Therefore, double isotopic dilution withIPD calculation gives more accurate results than S-IDAcalculation according to the accuracy obtained in this workand published in the literature for other biological tissue.

The limits of detection (LOD) and quantification (LOQ)were calculated as the sum of spiked TMAH blank averagevalues analyzed in triplicate (n02) plus three times thestandard deviation (SD) of this blank for LD or ten timesfor LQ (IUPAC). For extraction of 20 mg of hair in 5 mLTMAH, LOD is 27 ng g–1 and 12 ng g–1 and LOQ is37 ng g–1 and 20 ng g–1 for IHg and MMHg, respectively.

The validated TMAH microwave-assisted extractionmethod was applied to real hair samples of gold-minersworking in Bolivia (n023) [30]. These people were exposed

Table 3 Mean values of MMHg and inorganic Hg concentrations(ng g–1) and standard error for triplicate injection calculated by S-IDA. THg concentration (ng g–1) was measured by DMA-80, standard

deviation (SD) is calculated for n02 analyses. Recovery is the sum ofMMHg and IHg concentrations analyzed by GC–ICP–MS relative toTHg analyzed by DMA-80

Sample Mean [MMHg](ng g−1)

SE [MMHg](ng g−1)

Mean [IHg](ng g−1)

SE [IHg](ng g−1)

Mean [THg] DMA(ng g−1)

SD [THg](ng g−1)

Recovery(%)

FY7 121 9 750 3 1121 137 78

FY3 314 24 591 11 1047 82 86

FY1 352 16 1520 9 2134 181 88

FY2 131 1 1055 16 1467 34 81

FY8 466 12 591 0 1161 15 91

T116 841 49 1025 21 2086 5 89

SU1 78 5 1786 22 1987 30 94

SU2 240 3 419 9 676 10 97

SU3 183 6 437 11 608 25 102

SU4 373 2 112 4 435 19 111

SU5 215 20 323 2 505 4 107

SU6 203 4 84 6 258 8 111

SU7 165 6 149 14 290 4 108

SU8 320 4 90 2 385 13 106

SU9 368 2 1881 1 2106 98 107

SU10 700 1 297 1 1045 20 95

SJ1 387 2 96 9 495 1 98

SJ2 488 1 250 3 723 37 102

SJ3 134 2 119 8 269 2 94

SJ4 432 2 338 4 612 6 126

SJ5 1004 3 942 2 2252 117 86

SJ7 110 0 109 7 231 8 94

HU2 129 0 60 15 168 4 112

L. Laffont et al.

to both inorganic and organic species of Hg depending ontheir work and dietary habits. The hair contains differentproportions of MMHg and IHg, because of the workingenvironment and diet of the gold-miners [30]. Values ofMMHg and IHg concentrations are reported in Table 3.The results obtained were compared with total Hg concen-tration analysis by DMA-80. The recovery of each samplewas calculated as the ratio between the sum of MMHg andIHg measured by GC–ICP–MS and the total Hg (THg)concentration measured by DMA-80 (Table 3). The relativerepeatability of MMHg and IHg concentrations calculatedfor triplicate injection ranged from 0 to 9 % and from 0 to10 %, respectively. Recovery of total Hg ranged between 78and 128 % and is acceptable, assuming a DMA-80 repro-ducibility of 8 %.

Acknowledgements We wish to thank Interregional Midi-Pyrénées-Aquitaine and ERC-StG-20091028 for funding the RIMNES Projectand L. Laffont post-doctoral contracts, and the French National Re-search Agency (ANR CES) within the IDEA project for support ofanalytical development and analytical costs. P. Navarro is grateful tothe Basque Government for her postdoctoral fellowship.

References

1. Mergler D, Anderson HA, Chan LHM, Mahaffey KR, Murray M,Sakamoto M, Stern AH (2007) Methylmercury exposure andhealth effects in humans: A worldwide concern. Ambio 36:3–11

2. Feng X, Li P, Qiu G, Wang S, Li G, Shang L, Meng B, Jiang H,Bai W, Li Z, Fu X (2007) Human Exposure To Methylmercurythrough Rice Intake in Mercury Mining Areas, Guizhou Province,China. Environ Sci Technol 42:326–332

3. Lee R, Middleton D, Caldwell K, Dearwent S, Jones S, Lewis B,Monteilh C, Mortensen ME, Nickle R, Orloff K, Reger M, RisherJ, Rogers HS, Watters M (2010) A review of events that exposechildren to elemental mercury in the United States. Cien SaudeColetiva 15:585–598

4. Feng WY, Chai CF, Qian QF (1996) A new neutron activationtechnique for simultaneous determination of inorganic and totalmercury contents in human hair. J Radioanal Nucl Chem 212:61–68

5. Sarmani SB, Alakili I (2004) Application of neutron activationanalysis for mercury species determination in scalp hair samplesfrom Malaysia. Libya and Jordan. J Radioanal Nucl Chem 262:43–48

6. Sarmani SB, Alakili I (2004) Determination of total mercury andmethylmercury in hair samples from residents of Kuala Lumpur,Malaysia by neutron activation analysis. J Radioanal Nucl Chem259:261–264

7. Sarmani SB, Hassan RB, Abdullah MP, Hamzah A (1997)Determination of mercury and methylmercury in hair samples byneutron activation. J Radioanal Nucl Chem 216:25–27

8. de Souza SS, Rodrigues JL, Souza VCD, Barbosa F, de OliveiraSouza VC, Barbosa F Jr (2010) A fast sample preparation proce-dure for mercury speciation in hair samples by high-performanceliquid chromatography coupled to ICP–MS. J Anal At Spectrom25:79–83

9. Morton J, Carolan V, Gardiner PHE (2002) The speciation ofinorganic and methylmercury in human hair by high-performance

liquid chromatography coupled with inductively coupled plasmamass spectrometry. J Anal At Spectrom 17:377–381

10. Rahman GMM, Fahrenholz T, Kingston HM (2009) Application ofspeciated isotope dilution mass spectrometry to evaluate methodsfor efficiencies, recoveries, and quantification of mercury spe-cies transformations in human hair. J Anal At Spectrom 24:83–92

11. Bermejo-Barrera P, Verdura-Constenla EM, Moreda-Pineiro A(1999) Rapid acid leaching and slurry sampling procedures forthe determination of methyl-mercury and total mercury in humanhair by electrothermal atomic absorption spectrometry. Anal ChimActa 398:263–272

12. Diez S, Bayona JM (2002) Determination of methylmercury inhuman hair by ethylation followed by headspace solid-phasemicroextraction-gas chromatography-cold-vapour atomic fluores-cence spectrometry. J Chromatogr A 963:345–351

13. Liang L, Horvat M, Bloom NS (1994) An improved speciationmethod for mercury by GC/CVAFS after aqueous phase ethylationand room temperature precollection. Talanta 41:371–379

14. Montuori P, Jover E, Alzaga R, Diez S, Bayona JM (2004)Improvements in the methylmercury extraction from human hairby headspace solid-phase microextraction followed by gas-chromatography cold-vapour atomic fluorescence spectrometry. JChromatogr A 1025:71–75

15. Kehrig HA, Malm O, Akagi H (1997) Methylmercury in hairsamples from different riverine groups, Amazon, Brazil. WaterAir Soil Pollut 97:17–29

16. Rodrıguez-Gonzalez P, Marchante-Gayon JM, Garcıa-Alonso JI,Sanz-Medel A (2005) Isotope dilution analysis for elemental spe-ciation: a tutorial review. Spectrochim Acta B 60:151–207

17. Monperrus M, Rodriguez-Gonzalez P, Amouroux D, Garcia-Alonso JI, Donard OFX (2008) Evaluating the potential and lim-itations of double-spiking species-specific isotope dilution analysisfor the accurate quantification of mercury species in differentenvironmental matrices. Anal Bioanal Chem 390:655–666

18. Rodríguez-González P, García Alonso JI (2010) Recent advancesin isotope dilution analysis for elemental speciation. J Anal AtSpectrom 25:239–259

19. Clémens S, Monperrus M, Donard OFX, Amouroux D, Guérin T(2011) Mercury speciation analysis in seafood by species-specificisotope dilution: method validation and occurrence data. AnalBioanal Chem 401:2699–2711

20. Clémens S, Monperrus M, Donard OFX, Amouroux D, Guérin T(2012) Mercury speciation in seafood using isotope dilution anal-ysis: a (critical) review. Talanta 89:12–20

21. Akagi H, Malm O, Branches FJP, Kinjo Y, Kashima Y, GuimarãesJRD, Oliveira RB, Haraguchi K, Pfeiffer WC, Takizawa Y, Kato H(1995) Human exposure to mercury due to goldmining in theTapajos river basin, Amazon, Brazil: Speciation of mercury inhuman hair, blood and urine. Water Air Soil Pollut 80:85–94

22. Navarro P, Clémens S, Perrot V, Bolliet V, Tabouret H, Guérin T,Monperrus M, Amouroux D (2011) Simultaneous determination ofmercury and butyltin species using a multiple species-specificisotope dilution methodology on the European, Anguilla anguillaglass eel and yellow eel. Int J Environ Anal Chem: 1–17

23. Rodriguez Martín-Doimeadios RC, Krupp E, Amouroux D,Donard OFX (2002) Application of isotopically labeled methyl-mercury for isotope dilution analysis of biological samples usinggas chromatography/ICPMS. Anal Chem 74:2505–2512

24. Tseng CM, Garraud H, Amouroux D, Diego AD (1998) Openfocused microwave-assisted sample preparation for rapid totaland mercury species determination in environmental solid sam-ples. J Autom Chem 20:99–108

25. Heller-Zeisler SF, Parr RM, Zeisler R (1998) Certification of twohuman hair reference materials issued by the International AtomicEnergy Agency. Fresenius J Anal Chem 360:419–422

Mercury speciation analysis in human hair

26. Monperrus M, Rodriguez Martin-Doimeadios RC, Scancar J,Amouroux D, Donard OFX (2003) Simultaneous SamplePreparation and Species-Specific Isotope Dilution MassSpectrometry Analysis of Monomethylmercury and Tributyltinin a Certified Oyster Tissue. Anal Chem 75:4095–4102

27. Rodriguez Martin-Doimeadios RC, Tessier E, Amouroux D,Guyoneaud R, Duran R, Caumette P, Donard OFX (2004)Mercury methylation/demethylation and volatilization pathwaysin estuarine sediment slurries using species-specific enriched sta-ble isotopes. Mar Chem 90:107–123

28. Rodríguez-González P, Rodríguez-Cea A, Garcia-Alonso JI, Sanz-Medel A (2005) Species-specific isotope dilution analysis and

isotope pattern deconvolution for butyltin compounds metabolisminvestigations. Anal Chem 77:7724–7734

29. Point D, Garcia Alonso JI, Clay Davis W, Christopher SJ,Guichard A, Donard OFX, Becker PR, Turk GC, Wise S (2008)Consideration and influence of complexed forms of mercury spe-cies on the reactivity patterns determined by speciated isotopedilution model approaches: A case for natural biological referencematerials. J Anal At Spectrom 23:385–396

30. Laffont L, Sonke JE, Maurice L, Monrroy SL, Chincheros J,Amouroux D, Behra P (2011) Hg speciation and stable isotopesignatures in human hair as a tracer for dietary and occupationalexposure to mercury. Environ Sci Technol 45:9910–9916

L. Laffont et al.