A Highly Sensitive Spectrophotometric Method with Solid-Phase

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Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, ... The Lambert-Beer ... ical membranes such as the blood/brain barrier and the pla- ... thors (1,3,4) have reviewed methods for the determination of ... (GC-ICP-MS) (10), and neutron activation analysis (11,12).
Journal of Analytical Toxicology, Vol. 24, November/December 2000

A Highly SensitiveSpectrophotometricMethod with Solid-Phase Extraction for the Determination of Methylmercury in Human Hair Hua-Bin Li1,2, Feng Chen 1,*, and Xiang-Rong Xu 3 IDepartmentof Botany, The University of Hong Kong, PokfulamRoad, Hong Kong; 2StateKey Laboratoryof Environmental Aquatic Chemistry, ResearchCenterfor Eco-EnvironmentalSciences, ChineseAcademyof Sciences, Beijing, 100085,China; and 3Instituteof EnvironmentalHealth and Engineering,ChineseAcademyof PreventiveMedicines, Beijing, 100050,China

I Abstrac, Methylmercury is the most toxic among the mercury species. In order to provide a quick method to screen samples for methylmercury, a highly sensitive spectrophotometric method was established. The method was based on formation of the red complex of methylmercury with thio-Michler's ketone, which can be extracted with n-butanol. The organic layer was determined at a maximum absorption wavelength of 564 nm. The Lambert-Beer law was obeyed for methylmercury from 1.00 x 10 -7 mol/L to 2.00 x 10-s mol/L with a correlation coefficient of 0.9992. The detection limit was 3 x 10 -8 m o l / L The method was used to determine methylmercury in human hair. The recovery was from 94% to 102%, and the relative standard deviation was 2.5%. The results agreed with those obtained by gas chromatography with electron capture detection.

Introduction Methylmercury is the most toxic among the mercury species because of its volatility and its ability to pass through biological membranes such as the blood/brain barrier and the placenta. It can cause central nervous system irreversible damage and fetal Minarnata disease. Particularly disastrous were the widespread methylmercuric compound poisoning cases of Minamata Bay, Japan, from which the name "Minarnata disease" was derived to describe methylmercury poisoning (1). Inorganic mercury can be converted into methylmercury under anaerobic conditions in biological systems, not only through microbial activity but also through chemical methylation in the absence of bacteria. Once methylmercury is formed it enters the food chain by rapid diffusion and tight binding to proteins in aquatic biota and attains its highest concentrations in * Author to whom correspondence should be addressed

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the tissues of fish at the top of the aquatic food chain because of biomagnification through trophic levels. Then, methylmercury poisons humans through their diet, which puts them at the top of the food chain. Because of the higher level of metal concentrations in hair than in blood or urine, and the advantages of these kinds of samples related to their disposability or for being easily transported and stored, recent studies propose hair samples to determine the index of exposure to toxic metals (2). Therefore, the determination of methylmercury in human hair is of much importance in medicine, public health, and environmental protection. In the literature, a number of methods have been proposed for the determination of methylmercury. Recently, several authors (1,3,4) have reviewed methods for the determination of methylmercury, and their advantages and disadvantages have been indicated. Gas chromatography-electron capture detection (GC-ECD) was used widely for the determination of methylmercury (5-8), but the lack of selectivity of ECD determined the need to resort to laborious cleanup processes of the extract in the organic phase used (1). In an attempt to overcome the numerous drawbacks associated with the GC determination of methylmercury, high-performance liquid chromatography (HPLC) techniques have been used. However, HPLC techniques are usually less sensitive for detection (1). In online HPLC separation and cold vapor atomic absorption spectrometric determination of methylmercury, the detection limit was only 13.2 ng/mL (9). Gas chromatographyatomic fluorescence spectrometry (GC-AFS), gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) (10), and neutron activation analysis (11,12) have also been proposed for the determination of methylmercury. Although these techniques have the required sensitivity and accuracy, they are not easily accessible because of the high level of specialization needed and the high cost involved. The spectrophotometric method is inexpensive and easy to operate. It has been widely used, but its sensitivity is usually low

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Journal of Analytical Toxicok)gy, Vol. 24, November/December 2000

compared to other methods such as fluorimetry. On the other hand, methylmercurycontent in biological materials is usually very low. Therefore, the development of a sensitive spectrophotometric method is of great importance for quick screening samples for methylmercury. To our knowledge, no data about determination of methylmercury by direct spectrophotometric method are available in the literature. In this paper, we establish a highly sensitive spectrophotometric method for the direct determination of methylmercury. The method was applied to the determination of methylmercury in human hair with a solid-phase extraction technique.

Experimental Apparatus Absorption spectra were obtained with a Beckman model DU-650 spectrophotometer (Beckman, Fullerton, CA). For measurements at a single wavelength, a Shimadzu model UV120-02 spectrophotometer (Shimadzu, Kyoto,Japan) was used. Measurements were made in matched 1-cm quartz cell. All pH measurements were made with a Rex model pH S-3C pH meter (Rex, Shanghai, China). For comparison, an HP 5890A GC (Hewlett-Packard, Palo Alto, CA)with electron capture detection and an HP 3396A integrator was used with a Supelco SPB TM 1701 fused-silicacapillary column (15 m x 0.53-mm i.d., 1.00-1Jmfilm thickness). Reagents All reagents were of analytical reagent grade. Double-distilled water was used. Stock solution at a concentration of 1.000 x 10-2 mol/L of methylmercury chloride (Merck, Darmstadt, Germany) was prepared in a water/ethanol mixture (50:50, v/v) and stored in the dark at 4~ Working standards were prepared fresh daily and handled in brown bottles that were washed with nitric acid and double-distilled water before use. The thio-Michler'sketone (Aldrich,Milwaukee,WI) solution in n-butanol (BDH, Poole, U.K.) was at 1.50 x 10-4 mol/L. The concentrations of the hydrochloric acid (Merck)solution and the sodium hydroxide (BDH) solution were 2.00 mol/L and 0.500 tool/L, respectively. The acetic acid (Merck)/sodium acetate (Aldrich)buffer solution was prepared by the addition of 0.100 mol/L acetic acid and 0.100 mol/L sodium acetate to pH 4.7. The sulphydryl cotton fiber adsorbent was synthesized according to the procedure described in the literature (13,14).

Methods A certain volume of standard methylmercurychloride solution was transferred into a 25-mL graduated tube with glass stopper; 2.00 mL of acetic acid/sodium acetate buffer solution (pH 4.7) and 5.00 mL of 1.50 x 10-4 mol/L thio-Michler's ketone solution in n-butanol were added. Then the solution was diluted to 25 mL with water and immediatelyshaken for 2 min. After the solution was allowed to stand for 10 rain and centrifuged if necessary, the organic layer was transferred to a 1-cm cell and the absorbance measured at 564 nm against the corresponding reagent blank prepared concurrently. Human hair samples were cut to less than 5-mm long pieces

with stainless steel scissors, washed with water and acetone according to the procedure recommended by IAEAand WHO (15), and homogenized by mixing. Pretreatment of samples was carried out according to the literature (16,17). NaOH (1.40 mL of 10.0 mol/L) was added to a human hair sample (about 1 g) in a centrifuge tube. The tube was kept in a thermostat at 90-95~ for 30 rain. Water (5.00 mL) was added to the dissolved sample, and its pH value was adjusted to pH 3.0 using concentrated H2SO4. The solution was cooled to room temperature and then allowed to pass through the column of sulphydryl cotton-fiber adsorbent (13,14). Methylmercury was desorbed from the sulphydryl cotton fiber by 2.00 mL of 2.00 mol/L HCI, and the pH value of the eluate was adjusted to pH 4.5 using NaOH solution. Other procedures for human hair were the same as for standard methylmercury chloride solution as described here previously.

Results and Discussion

Absorption spectra The absorption spectra of the complex in the presence of methylmercury ion and of the thio-Michler's ketone (TMK) alone were obtained, as shown in Figure 1. The contrast (A2~) was 109 nm. The absorption maximum of 564 nm was chosen for the determination of methylmercury. Effect of solvent Carbon tetrachloride, n-hexane, methenyl chloride, and n-butanol were examined as possible extractants for the methylmercury-TMK complex. The TMK is only slightly soluble in carbon tetrachloride or n-hexane. For methenyl chloride, absorbance of organic layer was not changed in the presence of methylmercurychloride. Only n-butanol was able to extract the methylmercury-TMK complex. The optimum amount of n-butanol was 5 mL.

0.8

~ 0.6

,"'"",

"~ 0.4 0.2

320

380

440 500 560 Wavelength (rim)

620

680

Figure 1. Absorption spectra of thio-Michler's ketone-methylmercury complex. Thio-Michler's ketone reagent against water blank ( .... ) and thio-Michler's ketone-methylmercury complex against water blank (--). Concentration of methylmercury chloride, 1.00 x 10-5 mol/L; concentration of thio-Michler's ketone, 2.00 x 10-5 mol/L; pH = 4.7. 705

Journal of Analytical Toxicology, Vol. 24, November/December 2000

Effect of pH The optimal pH range for the color reaction of methylmercury with TMK was 4.0-6.0, as shown in Figure 2. Therefore, a pH of 4.7, maintained with acetate buffer solution, was used. In this study, the optimum amount of buffer solution was 2 mL. Effect of thio-Michler's ketone concentration The effect of TMK concentration was studied. The result is shown in Figure 3. A maximum and stable absorbance was attained with 1.20-2.00 mL of 5.00 x 10-4 mol/L TMK solution. Therefore, 1.50 mL of 5.00 x 10-4 TMK solution was chosen.

Characteristics of the color reaction The TMK color reaction with methylmercury was rapid, reaching its maximum absorbance immediately. The formed complex and its absorbance were found to be stable at ]east for 100 min. The ratio of methylmercury to TMK in the complex was 1:2 by the conventional continuous variations and mole ratio methods. Thus, the complex formed had the empirical composition MeHg(TMK)2 (where MeHg represented the methylmercury cation).

'l ~ 0.4 0.2 i

o~ 0

2

4

6

i

i

8

10

pH Figure2, Effectof pH on absorbar~ce.Concer~tratioTlof methylmercury chloride, 1.00 x 10-s tool/L;concentrationof thio-Michler'sketone, 2.00x 10 ~mol/L.

1.2 -

0.8-

.J

0.4

The present method was applied to the determination of methy]mercury in human hair. In order to assess the reproducibility of the method, eight aliquots were taken from one mixed human hair sample containing 0.19 mg/g methylmercury, and the whole procedure was applied to each sample. The relative standard deviation was 2.5%. Six human hair samples were determined. As shown in Table [, the recovery was from 94% to 102%. Results obtained with present method agreed with those obtained by GC with electron capture detection (13,14). The t-test indicated that no significant difference between the proposed method and the reference method was observed at the 95% probability ]evel. The methy]mercury in certified reference material of human hair ([AEA-086) was also determined by the proposed method. The certified va]ue of human hair is 0.258 mg/kg. The result obtained by the present method was 0.254 mg/kg with an RSD (n = 8) of 1.8%.

Sample Amountof no. MeHg(pg)

0.4-

0.8

1.2

1.6

2

2.4

TMK solution (mL)

Figure3. Effectof thio-Michler'sketoneconcentrationon absorbance. Concentrationof methylmercurychloride,1.00x 10-5mol/L;pH = 4.7. 706

The effect of various cations and anions on the determination of methylmercury was examined by spiking 1.00 x 10-6 mo]/L of methylmercury chloride with known quantities of foreign materials and analyzing it by the present method. Under the present conditions of determination, no interference (relative error less than + 5%) was observed at ratios (m/m) of 1000:1 for K+, Na +, NH4+, Ca2+, Mg2+, Zn2ยง Hg 2+, AI3+, NO3-, CI-, I-, S042-, and C032-, and 100:1 for Fe3+, Fe 2+, Pb2+, Cd 2+, and Cu 2+. Only a few kinds of ions such asAg+ and Sn2+ (at ratios of 10:1) affected the determination of methylmercury. Thus, the solid-phase extraction technique was adopted for preconcentration and eliminating interference.

Table I. Determination of Methylmercury in Human Hair*

0.6-

~

Effect of foreign ions

Determination of methylmercury in human hair

=~ 0.6 0.8 t

1

Lambert-Beer's law was obeyed for methylmercury from 1.00 x 10-7 mol/L to 2.00 x 10-5 mol/L, and the linear regression equation was given by A = 1.05 x 105 C + 0.086 with a correlation coefficient of 0.9992 (n = 8). The molar absorptivity (r of the complex was 1.05 x 105 L/mol/cm. The detection limit [signal-to-noise ratio 3:1 (18)] was 3 x 104 mol/L (6 ng/mL as Hg) based on 10 blank determinations. The relative standard deviation (n = 12) was 1.8% for determination of 1.00 x 10-6 mol/L of methylmercury.

I 2 3 4 S 6

0.27 0.38 0.]5 0.23 0.34 0.18

MeHgadded Recovery Amountof (pg) (%) MeHg(pg)t 0.30 0.30 0.30 0.30 0.30 0.30

9 Each result was the average of three determinations. t Result was obtained by gas chromatography.

101 94 98 102 95 97

0.29 0.36 0.15 0.24 0.32 0.19

Journal of Analytical Toxicology, Vol. 24, November/December2000

Conclusions The proposed method has very high sensitivity and good selectivity. The instrument used is inexpensive and easy to operate, and the method can be employed as a quick method to screen samples for the methylmercury in human hair.

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9. S. Rio-Segade and C. Bendicho. Online high performance liquid chromatographic separation and cold vapor atomic absorption spectrometric determination of methylmercury and inorganic mercury. Talanta 48:477484 (1999). 10. H.E.L. Armstrong, W.T. Corns, P.B. Stockwell, G. O'Connor, L. Ebdon, and E.H. Evans. Comparison of AFS and ICP MS detection coupled with gas chromatography for the determination of methylmercury in marine samples. Anal. Chim. Acta 390: 245-253 (1999). 11. S.F. Heller-Zeisler, M.K. Donais, and R. Zeisler. Instrumental neutron activation analysis for quality assurance of a hair reference material for mercury speciation. J. Radioanal. Nucl. Chem. 233: 55-57 (1998). 12. S.B. Sarmani, R.B. Hassan, M.P. Abdullah, and A. Hamzah. Determination of mercury and methylmercury in hair samples by neutron activation. J. Radioanal. Nucl. Chem. 216:25-27 (1997). 13. Y.H. Lee and J. Mowrer. Determination of methylmercury in natural waters at the sub-nanograms per litre level by capillary gas chromatography after adsorbent preconcentration. Anal. Chim. Acta 221:259-268 (1989). 14. H.B. Li and W.H. Wang. Interface exchange of methylmercury in water, soil and air. Chin. J. Environ. Sci. 21:81-83 (2000). 15. UNEP---WHO--IAEA. Reference Method for Marine Pollution Studies No. 46. United Nations Environment Programme (UNEP), Regional Seas Programme Activity Center, Geneva, Switzerland, 1987. 16. K. Kratzer, P. Benes, V. Spevackova, D. Kolihova, and J. Zilkova. Determination of chemical forms of mercury in human hair by acid leaching and atomic absorption spectrometry. J. Anal. At. Spectrom. 9:303-306 (1994). 17. K. Kratzer, E Benes, and V. Spevackova. Separation of methylmercury from human hair by solvent extraction. Int. J. Environ. Anal. Chem. 57:91-98 (1994). 18. K.K. Verma, A. Jain, and A. Verma. Determination of iodide by high performance liquid chromatography after precolumn derivatization. Anal. Chem. 64:1484-1489 (1992). Manuscript received January 18, 2000; revision received April 10, 2000.

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