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A Double Isotope Dilution Method for Using Stable Selenium Isotopes in Metabolic Tracer Studies: Analysis by Gas Chromatography/Mass Spectrometry (GC/MS)¹

U.S. Department of Agriculture, Beltsville Human Nutrition Research Center, Bldg. 307, Rm. 215, Beltsville, MD 20705

Enriched stable isotopes of selenium are used for a double isotope dilution method employing rapid sample digestion, chelation and measurement by combined gas chromatography/mass spectrometry (GC/MS). A known quantity of an enriched selenium isotope is added as an internal standard, and samples are rapidly digested with HNO₃, H₃PO₄ and H₂O₂. Undigested lipids are extracted with chloroform, and any selenate is reduced to selenite with HCl. The selenite reacts with 4-nitro-o-phenylenediamine (NPD) to form 5-nitropiazselenol (Se-NPD), which is then extracted into chloroform for subsequent GC/MS analysis. By monitoring the ion peaks in the Se-NPD⁺ parent ion cluster, and by using isotope ratio measurements, it is possible in principle to use any two of the stable selenium isotopes as tracer and internal standard. The method described herein utilizes ⁷⁶Se as the tracer and ⁸²Se as the internal standard, compared to ⁸⁰Se naturally present in the sample. Selenium recoveries from the digestion-chelation steps were verified by using animal tissues endogenously radiolabeled with ⁷⁵Se. The method is precise, accurate, rapid and extremely specific, and should lend itself well to determining selenium in biological materials, and to following stable selenium isotopes as tracers in metabolic studies.

KEY WORDS: • stable isotopes • selenium • GC/MS analysis

¹ Reported in part at the 66th annual meeting of the Federation of American Societies for Experimental Biology, New Orleans, LA, April 1982. Veillon, C. and Reamer, D. C. (1982) Use of a volatile chelate to measure stable ⁷⁶Se, ⁸⁰Se and ⁸²Se isotopes by combined gas chromatography/mass spectrometry (GC/MS). Fed. Proc. 41, 707 (abs.).

² D.C.R. is a Research Associate, Children's Hospital, Boston, MA, and is supported in part by General Cooperative Agreement No. 58-32U4-0-127 with the U.S. Department of Agriculture.

³ Author to whom requests for reprints should be addressed.

Manuscript received 9 August 1982.