1976 Part 3 - Analytical Determination of Ethyl Mercury and Methyl Mercury in Blood Samples Martina Logar1, Milena Horvat1, Nuša Horvat1, Miha Benedik2, Andreja Marn-Pernat2, Rafael Ponikvar2, Joško Osredkar3 1 Department of Environmental Sciences, Jožef Stefan Institute, Ljubljana, Slovenia; martina.logar@ijs.si 2 Department of Nephrology, University Medical Centre, Ljubljana, Slovenia; Q Institute of Clinical Chemistry and Biochemistry, University Medical Centre, Ljubljana, Slovenia Abstract: Thiomersal is a mercury-containing organic compound, commonly used as a preservative in topical pharmaceutical preparations, cosmetics, and biological products such as vaccines, as well as a disinfectant during dialysis treatment. The purpose of the present work was to develop a sensitive and accurate method for determination of ethyl mercury (EtHg) and methyl mercury (MeHg) in whole blood of dialysis patients. Key words: ethyl mercury, methyl mercury, blood, speciation Introduction Current mercury investigations have focused primarily on MeHg from fish contamination and from rare occupational or catastrophic events. Recently, another source of exposure has been identified. Thiomersal, a preservative with both bactericidal and fungicidal action utilized in the production of biological and pharmaceutical products, contains 49.6 % EtHg by weight. [1] Because of an increasing awareness of the theoretical potential for neurotoxicity of even low levels of organomercurials, development of analytical techniques for determination of low concentrations of EtHg and MeHg is required. Results and discussion Metal speciation is impossible without the use of modern hyphenated techniques, in which highly sensitive and selective elemental detection systems are coupled to modern chromatographic separation systems. One of the most widely used separation method is gas chromatography which however requires volatile species. The main disadvantage of the commonly used derivatization reagent sodium tetraethylborate (NaEtRB) is that the important ethylmercury species cannot be distinguished from inorganic Hg after ethylation. [2] In this work sodium tetra(n-propyl)borate (NaPrRB) was tested for simultaneous determination of EtHg and MeHg in blood samples. The method proposed is based on acid leaching (5 % H2SO4/18 % KBr/lM CuSO4), extraction of EtHgBr and MeHgBr into an organic solvent (CH2Cl2), followed by back extraction into Milli-Q water, subsequent propylation with a 1 % solution of NaPr4B, room temperature precollection on Tenax, isothermal gas chromatographic separation RMZ-M&G 2004, SI 7th International Conference on Mercury as a Global Pollutant_ 1977 (80 °C), pyrolysis (600 °C) and cold vapour atomic fluorescence spectrometric detection (CV AFS). [3-4] Optimization of the method was performed on a number of different blood samples of patients before and after dialysis treatment. The concentrations of MeHg were comparable to concentrations of MeHg in the normal healthy population and showed similar values before and after dialysis. The concentration of EtHg were much elevated after treatment and reached up to 4 ng/g. Some of the results are shown in Table I. Table I. MeHg and EtHg results in blood samples obtained using NaPrRB as a derivatization regent. Sample EtHg (as Hg) ng/g MeHg (as Hg) ng/g 1 BT 0.09±0.00 0.36±0.04 AT 1.74±0.07 0.33±0.05 2 BT 1.69±0.00 0.20±0.01 AT 4.23±0.12 0.23±0.02 3 BT 0.05±0.01 0.15±0.02 AT 1.54±0.14 0.11±0.01 4 BT <0.01 0.18±0.01 AT 1.06±0.08 0.17±0.01 BT- before dialysis treatment; AT - after dialysis treatment The performance of NaEtRB and NaPrRB as derivatization reagents was checked. Comparison of the results in Table 2 shows that the values for MeHg obtained by the two derivatization reagents are in good agreement. Table 2. Comparison of MeHg results obtained by NaEtRB and NaPrRB as derivatization reagents. Sample MeHg (as Hg) ng/g NaPr4B NaEttB 1 0.45±0.01 0.47±0.01 2 1.13±0.04 0.92±0.05 CRM IAEA 405 5.26±0.49 5.02 IAEA 405 Estuarine sediment: certified value 4.96-6.02 ng/g RMZ-M&G 2004, SI The limit of detection calculated on the basis of three times the standard deviation of the repeatability of the results was about 5-10 % for EtHg and 5-15 % for MeHg. Recoveries were between 90-110 % for both species. A certified reference material was tested to check the accuracy of MeHg determination, but for EtHg no CRM was available. Conclusions The analytical procedure developed was found to be a suitable and appropriate method for determination of low concentrations of EtHg and MeHg in blood samples. It also shows great potential for determination of both species in other biological samples influenced by thiomersal. Acknowledgements This work was supported by the Ministry of Education, Science and Sport of the Republic of Slovenia through a programme P-0143. 1976 Part 3 - Analytical References [1] Redwood, L., Bernard, S., Brown, D. (2001): Pre- dicted mercury concentrations in hair from infant immunizations: Cause for concern. NeuroToxicology; Vol. 22, pp. 691-697. [2] De Smaele, T., Moens, L., Dams, R., Sandra, P., Van der Eycken, J., Vandyck, J. (1998): Sodium tetra(n-propyl)borate: a novel aqueous in situ derivatization reagent for the simultaneous determination of organomercury, -lead and -tin compounds with capillary gas chromatography - inductively coupled plasma mass spectrometry. J. Chromatogr. A; Vol. 793, pp. 99-106. [3] Liang, L., Horvat, M., Bloom, N.S. (1994): An im- proved method for speciation of mercury by aqueous phase ethylation, room temperature precollection, GC separation and CV AFS detection. Talanta; Vol. 41, pp. 371-379. [4] Logar, M., Horvat, M., Falnoga, I., Stibilj, V. (1999): A methodological study of mercury spe-ciation using Dogfish liver CRM (DOLT-2). Presenius J. Anal. Chem.; Vol. 366. pp. 4S2-460. RMZ-M&G 2004, SI