Quantification of α-ketoglutarate cyanohydrin in swine plasma by ultra-high performance liquid chromatography tandem mass spectrometry
2013
Mitchell, Brendan L. | Rockwood, Gary A. | Logue, Brian A.
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.
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