Quantification of α-ketoglutarate cyanohydrin in swine plasma by ultra-high performance liquid chromatography tandem mass spectrometry

Abstract

Determination of exposure to cyanide can be accomplished by direct cyanide analysis or indirectly by analysis of cyanide detoxification products, such as thiocyanate and 2-amino-2-thiazoline-4-carboxylic acid. A potentially important marker and detoxification product of cyanide exposure, α-ketoglutarate cyanohydrin (α-KgCN), is produced by the reaction of cyanide and α-ketoglutarate. Therefore, an ultra high-performance liquid chromatography tandem mass spectrometry method to determine α-KgCN in plasma was developed. Swine plasma was spiked with α-KgCN and α-KgCN-d4 (internal standard) and proteins were precipitated with 1% formic acid in acetonitrile. After centrifugation, the supernatant was dried, reconstituted, separated by reversed phase high performance liquid chromatography and analyzed by tandem mass spectrometry. The method produced a dynamic range of 0.3-50μM and a detection limit of 200nM for α-KgCN. Furthermore, the method produced a %RSD of less than 13% for all intra- and inter-assay analyses. The stability of α-KgCN was poor for most storage conditions tested, except for -80°C, which produced stable concentrations of α-KgCN for the 30days tested. The validated method was tested by analysis of α-KgCN in the plasma of cyanide-exposed swine. α-KgCN was not detected pre-exposure, but was detected in all post-exposure plasma samples tested. To our knowledge, this method is the first reported analytical method for detecting α-KgCN in any matrix.