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

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Abstract
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.