116
Anal. Chem. lB87, 59, 1 16-1 18
Determination of Trace Amounts of Organic and Inorganic Mercury in Biological Materials by Graphite Furnace Atomic Absorption Spectrometry and Organic Mercury Speciation by Gas Chromatography Marco Filippelli
Laboratorio Chimico di Igiene e Profilassi, I-19100 L a Spezia, Italy
A procedure for determinatbn of both organic and Inorganic mercury in Wobgkai materlals by graphlie furnace atomic absorption spectrometry (QFAAS) Is described. Organlc mercury Is extraded as a chbrkJe derivafhfeby berum and reextracted by a thlosdfate solution. Inorganic mercury is converted Into a methyl chloride derlvatlve by nwthandlc tetramethyltin pdor to extraction. A 20-pL allquot of the thiosutlate solutbn Is inJectedInto the graphlie furnace. The caHbratkn Is llnear up to 12 ng of tlg/20 pL inJected. The limit of daectkn of mercury Is 0.04 ng of Hg/20 pL InJected. The organic mercury thiosulfate extract was treated wtth CuCI,, reextracted In the benzene layer, and analyzed by gas chromatography (GLC) for speclatlon.
There is a need for the separate determination of organic and inorganic mercury species in biological materials. Of special toxicological interest is the proportion of mercury as methylmercury. Westoo (1-3), separated the inorganic and organic mercury forms by thin-layer chromatography. In untreated samples only inorganic mercury is reduced by tin(@ chloride under acidic conditions and the mercury vapors are detected by flameless atomic absorption spectrometry (4-8). The procedures generally used for organic mercury involve the addition of a halogenated acid to an aliquot of the sample, thereby causing the alkyl mercury originally bound to the biological material to form alkyl mercury halide. This is extracted with an organic solvent such as benzene. After the extract is purified, concentrated, and dried (if necessary), it is analyzed specifically by gas-liquid chromatography (9, l o ) , by spectrometric determination (1I ) , or by reextraction into the aqueous layer and determination as mercury (12,13). However, a more efficient and simple method that will allow large numbers of samples to be tested simultaneousely in a short time is desired. In this paper, such a method is described for the rapid determination of organic and inorganic mercury in biological samples. Organic mercury is directly detected by GFAAS whereas inorganic mercury is converted into methylmercury with tetramethyltin (14, 15) prior to its detection. For identification of organomercury species, a successive chromatographic separation step was used.
EXPERIMENTAL SECTION Apparatus. A Perkin-Elmer Model 372 atomic absorption spectrometer, equipped with a deuterium background corrector, a HGA-500 graphite furnace, and a mercury hollow-cathode lamp operated at 6 d (spectral band-paas = 0.2 nm) and at a resonance wavelength of 253.6 nm, and a Sargent Welch Model SRG recorder were used for mercury measurements. The optimized experimental furnace conditions were as follows: drying, 110 "C for 20 s, ramp time 5 s; ashing, 200 "C for 10 s, ramp time 5 s; atomization, 1000 "C for 5 s, ramp time 1s; cleaning, 2700 "C for 2 s, ramp time 1 s. 0003-2700/87/0359-0116$01.50/0
The nitrogen internal gas flow (40 mL/min) was stopped in the atomization step. A Carlo Erba Fractovap, Model 2350, gas chromatograph for separation of organomercury species was used, equipped with a B3Nielectron capture detector and a 2-m X 4-mm4.d. glass column packed with 2% Carbovax 20 M on Chromosorb W(HP) 100-120 mesh operated at 195 O C . The injector temperature was 200 "C, and the detector temperature was 250 "C. The nitrogen carrier gas flow rate was 85 mL/min. Reagents. Distilled, deionized water (DDW) was used. A 0.04 M methanolic tetramethyltin solution was prepared fresh daily by dissolving 544 pL of tetramethyltin in 10 mL of methanol. A 0.01 M solution of sodium thiosulfate (Na2SzO3.5H20) was prepared by dissolving 0.2482 g in 100 mL of DDW. Tetramethyltin and mercury compounds were obtained from Alfa Inorganics. Standards. Stock standard mercury(I1) chloride (0.1354 g), methylmercury(I1) chloride (0,1251 g), ethylmercury(I1) chloride (0.1321 g), and phenylmercury(I1) chloride (0.1561 g) were dissolved in 100 mL of ethanol and stored at -10 "C. Standard mercury working solutions 10 pg of Hg/mL of inorganic and organomercurycompounds, were prepared by appropriate dilutions of the stock solutions with ethanol and were stored at 5 "C. Sample Preparation of Homogenates. A 5-g portion of fiiely chopped sample and 5 mL of distilled water were homogenized in an omnimixer. Whole blood samples were used. Procedure. To 0.5 g (0.5 mL) of the homogenate in a 10-mL glass vial, 5 mL of 1%NaCl (w/v) in 1N hydrocloric acid and 1mL of benzene were added. The vial was stoppered, shaken in a horizontal shaker for 30 min, and then centrifuged for 5 min at 6000g. Thereafter, the benzene layer was transferred into a 10-mL vial. The operation was repeated three times with additional 1-mL aliquots of benzene. The mixture was then shaken for 5 min and centrifuged as before. Finally the organic layers were combined and stored in one glass vial for determination of total organic mercury. To the aqueous layer, 0.25 mL of methanolic 0.4 M tetramethyltin was added, and the vial was stoppered and placed in a beaker of boiling water for 5 min. Thereafter the vial wm shaken in a horizontal shaker for 5 min and cooled. Then 1mL of benzene was added to the vial and it was shaken again for 10 min. Finally the mixture was centrifuged for 5 rnin at 6000g, and the organic layer was transferred into a glass vial. The aqueous layer was extracted three times with new 1-mLaliquots of benzene, shaken for 5 min, and centrifuged as before. The benzene layers were collected in a glass vial for determination of inorganic mercury. One milliliter of 0.01 M Na2S20,solution was added to the benzene layers of organic and inorganic mercury and vortexzd for 30 s. Then, the benzene layer was discarded and the aqueous layer stored at 5 "C until the time of GFAAS measurement. For determination of organomercury compounds, 0.25 mL of 0.5 M CuCl, solution and 0.5 mL of benzene was added to a 0.5-mL aliquot of organic mercury thiosulfate extract in a glass vial and this mixture was vortexed for 30 s. A 1-FLaliquot of the benzene layer was injected directly into GLC.
RESULTS AND DISCUSSION The proposed method was compared with the extraction method of Cappon and Smith (15). The results in Table I show no significant differences for the three methods tested. 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
117
0.3
" W
0.2
z a
0
Time
m 0:
15
20
(min)
Flgure 2. Chromatogram of organic mercury: a, CH,HgCi (0.125 ng/pL); b, C2H5HgCi(0.125 ng/pL); c, C8H,HgCi (2.5 ng/pL).
0
cn m
a
10
5
01
I
30 0
200
I
400
500
T E M P E R A T U R E ("CI Figure 1. Variation of absorbance with charring temperature. An injection of 20 pL of 0.01 M sodium thiosulfate solution containing 0.5 pg/mL of organic mercury in the following forms was used: 0, methyimercury(I1) chloride; A,ethylmercury(I1) chloride; 0,phenylmercury(I I)chloride.
Table I. Concentration of Organic and Inorganic Mercury in Wet Tuna Flesh for Different Extraction Methods
organic Hg, pg/g
% coeff of
method" a b
0.585 0.550
C
0.570
6.5 5.2 4.1
variation
inorganic Hg, pg/g
% coeff of
0.065
4.8 5.7 3.5
0.075 0.070
variation
"The following methods were used a, alkaline digestion and Cappon procedure (15); b, aqueous homogenate and Cappon procedure; c, present method. The values are the mean of ten determinations. In addition, there is no emulsification during mercury extraction requiring high-speed centrifugation. The final thiosulfate solution is clear. Total Organic Mercury Determination. The possibility for direct detection of organic mercury by GFAAS was studied. T o this end, the absorbance values of methyl, ethyl, and
phenyl mercuric standards were measured as a function of temperature (Figure 1). No significant difference was found for these compounds a t 1300 "C. At an atomization temperature of 1000 "C, the mercuric species are atomized prior to vaporization of the sodium thiosulfate matrix, so there is no interference during mercury determinations. The high sensitivity obtained with GFAAS is of great analytical interest because it permits the detection of trace amounts of organic mercury. It is clear that only total organic mercury is measured. The calibration curve is linear over the range 2-600 ng/mL of mercury when 20-pL aliquots are injected into the furnace. To check the efficiency of the extraction, samples of biological material were spiked with known amounts of a standard mercury solution and measured by GFAAS; an equilibration time of 30 min permitted the mercury species to bind to active sites of biological samples. The results are reported in Table 11. Organic Mercury Speciation. If speciation of organic mercury is required, the water-soluble thiosulfate complex of organomercury compounds must be converted into the copper(I1)-thiosulfate complex by adding a CuClz solution. Then the liberated organic mercury can be easily extracted into the benzene layer. The benzene extract is injected directly into the gas chromatograph. Thiosulfate solutions containing three different organic mercury compounds a t various concentrations were extracted and the Hg concentration was determined. The calibration curve was linear up to 0.6 ng/pL for methyl- and ethylmercury and up to 24 ng/pL for phenylmercury. A typical chromatogram of a benzene solution is shown in Figure 2. The sensitivity for phenylmercury under these 'conditions was low and it was necessary to inject 10 pL of the benzene extract for its determination. In fact, for methyl- and ethylmercury chloride the limit of detection was 0.003 ng/ pL
Table 11. Recoveries of 0.25 pg Hg/g as Organomercury and Inorganic Mercury Compounds Added to Biological Samplesn % CH3HgC1
% coeff of
sample
recovered
variation
recovered
variation
recovered
variation
tuna in olive oil sardine, fresh anchovy, fresh oyster, dried freshwater fish meat, dried milk, dried banana, dried flour, wheat blood
105
5.4
97 100 103
6.2 5.4 3.1
105
100 100
4.5 8.1
102
96 102 97 105 98
5.6 3.7 5.1 2.7 3.3
100 100
5.2 3.6 4.1 3.8 6.2 4.4 5.2 3.8 5.1 3.2
3.2 97 2.7 100 4.8 105 4.1 96 3.5 97 2.8 102 3.1 98 4.5 100 3.5 a The values are the mean of ten determinations. 100
% C2H5HgC1 % coeff of
70 CGH5HgC1 % coeff of
102
99 98 105 98 100
70 HgCl, recovered
70coeff of variation
99
2.8
102
4.2
100 98 105 97 100
100 102 100
3.2
5.4 3.6 6.2 4.1 7.3 2.1
3.5
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
Table 111. Extraction Efficiency of Organomercury Compounds in Biological Samples
amt of organic mercury added, o.25
rgl g CHSHgC1 C2H5HgC1 C6H5HgCl
organic mercury extracted: % I I1 I11 IV 60.9 72.6 92.5
23.0 18.6 7.5
11.5 8.8
0
4.6
0 0
a Every extraction was carried out with 1 mL of benzene and the benzene layer was extracted with 1 mL of 0.01 M sodium thiosulfate. The values are means of different kinds of biological samdes (tuna, blood, meat. sardine. and ovster).
Table IV. Comparison of Organic and Inorganic Mercury Recoveries by GFAAS and GLC Methods for Wet Tuna Flesh Samples
mercury added CHSHgCI: 0.500 pg/g C2H5HgCI: 0.600 pg/g C6HbHgCI: 0.250 Mg/g HgCl,: 0.200 pg/g
recovery of mercury added, rg/g GFAAS GLC 0.490 0.625 0.255 0.193
0.482 0.603 0.260 0.186
ratio" GFAASJ GLC 1.01 1.03 0.98 1.04
"The values are the mean of three determinations. Table V. Comparison of Total Mercury with a Reference Sample
interlaboratory study no. of laboratories mean, ppm std dev % coeff of variation
94 0.955
0.099 10.4
this study 1.070 0.043 4.0
" The values are the mean of five determinations. and for phenylmercury chloride it was only 0.125 ng/pL. Inorganic Mercury Determination. For this determination it is important that all organic mercury be removed from the samples. Therefore particular attention was paid
to the extraction efficiency of organic mercury. four successive extractions were required for the complete removal of methyl, ethyl, and phenyl mercuric compounds (Table 111). The inorganic mercury can be converted quantitatively into CHSHgCl by methanolic tetramethyltin and extracted with benzene. recovery data for biological samples spiked with inorganic mercury are given in Table 11. Accuracy of the Method. In order to confirm that all the organic and inorganic mercury present in a biological material can be detected by GFAAS method, the data obtained by analysis of tuna samples spiked with organic and inorganic mercury were compared with those obtained with GLC method. The results of this comparison study, listed in Table IV, show good correlation for the two methods as evidenced by the ratio of the GFAAS/GLC values. Finally, Table V shows the good interlaboratory comparison obtained for the analysis of an IAEA mussel homogenate sample (16),with respect to total mercury. Registry No. Hg, 7439-97-6; MeHgC1, PhHgC1, 100-56-1.
115-09-3;
EtHgC1,
107-27-7;
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8)
(9) (10) (11) (12) (13) (14) (15) (16)
Westoo, G. Acta Chem. Scand. 1988, 20, 2131. Westoo, G. Acta Chem. Scand. 1987, 27, 1970. Westoo, G. Acta Chem. Scand. 1988, 22, 2277. Clarkson, T. W.; Greenwood, M. R. Anal. Biochem. 1970, 37, 236. Gage, J. C.; Warren, J. M. Ann. Occup. Hyg. 1970, 73, 115. Magos, L. Analyst (London) 1971, 96, 847. Kamada, T.; Hayashi, Y.; Kumamaru, T.; Yamamoto, Y. Bunseki Kagaku 1973, 22, 1481. Kacprzak, J. L.; Chrojka, R. J . Assoc. Off. Anal. Chem. 1976, 59, 153. Fishbeln, L. Chromatogr. Rev. 1970, 73, 83. Mushak, P. EHP Envlron. Health Perspect. 1973, 4, 55. Jones, P.;Nickless, G. Ana/yst(London) 1978, 703, 1121. Gage, J. C. Analyst (London) 1981, 86, 457. Davies, I. M.; Anal. Chlm. Acta 1978. 702, 189. Zarnegar, P.; Mushak, P. Anal. Chim. Acta 1979, 69, 389. Cappon, C. J.; Smith, J. C. Anal. Chem. 1977, 49, 365. Report No. 26 "Intercalibration of Analytical Methods on Marine Environmental Samples", Trace Elements Measurements on Mussel Homogenate (MA-MPITM); International Atomic Agency, Laboratory of Marine Radioactivity, Oceanographic Museum; Monaco; October 1985.
RECEIVED for review August 12, 1985. Resubmitted January 27, 1986. Accepted August 1, 1986.
