Accidental or intentional cyanide poisoning is a serious health risk. The current suite of FDA approved antidotes, including hydroxocobalamin, sodium nitrite, and sodium thiosulfate is effective, but each antidote has specific major limitations, such as large effective dosage or delayed onset of action. Therefore, next generation cyanide antidotes are being investigated to mitigate these limitations. One such antidote, 3-mercaptopyruvate (3-MP), detoxifies cyanide by acting as a sulfur donor to convert cyanide into thiocyanate, a relatively nontoxic cyanide metabolite. An analytical method capable of detecting 3-MP in biological fluids is essential for the development of 3-MP as a potential antidote. Therefore, a high performance liquid chromatography tandem mass spectrometry (HPLC-MS-MS) method was established to analyze 3-MP from rabbit plasma. Sample preparation consisted of spiking the plasma with an internal standard (13C3-3-MP), precipitation of plasma proteins, and reaction with monobromobimane to inhibit the characteristic dimerization of 3-MP. The method produced a limit of detection of 0.1 µM, a linear dynamic range of 0.5–100 µM, along with excellent linearity (R2 ≥ 0.999), accuracy (±9% of the nominal concentration) and precision (<7% relative standard deviation). The optimized HPLC-MS-MS method was capable of detecting 3-MP in rabbits that were administered sulfanegen, a prodrug of 3-MP, following cyanide exposure. Considering the excellent performance of this method, it will be utilized for further investigations of this promising cyanide antidote.
Poisoning by cyanide can be verified by analysis of the cyanide detoxification product, α-ketoglutarate cyanohydrin (α-KgCN), which is produced from the reaction of cyanide and endogenous α-ketoglutarate. Although α-KgCN can potentially be used to verify cyanide exposure, limited toxicokinetic data in cyanide-poisoned animals are available. We, therefore, studied the toxicokinetics of α-KgCN and compared its behavior to other cyanide metabolites, thiocyanate and 2-amino-2-thiazoline-4-carboxylic acid (ATCA), in the plasma of 31 Yorkshire pigs that received KCN (4mg/mL) intravenously (IV) (0.17 mg/kg/min). α-KgCN concentrations rose rapidly during KCN administration until the onset of apnea, and then decreased over time in all groups with a half-life of 15 min. The maximum concentrations of α-KgCN and cyanide were 2.35 and 30.18 μM, respectively, suggesting that only a small fraction of the administered cyanide is converted to α-KgCN. Although this is the case, the α-KgCN concentration increased >100-fold over endogenous concentrations compared to only a three-fold increase for cyanide and ATCA. The plasma profile of α-KgCN was similar to that of cyanide, ATCA, and thiocyanate. The results of this study suggest that the use of α-KgCN as a biomarker for cyanide exposure is best suited immediately following exposure for instances of acute, high-dose cyanide poisoning.
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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