Development of a High Performance Liquid Chromatography Method for the Analysis of Next-generation Cyanide Antidote, 3-mercaptopyruvate, in Plasma

Although the current FDA approved cyanide antidotes (i.e., sodium nitrite, sodium thiosulfate, and hydoxocoboalamine) are effective for treating cyanide poisoning, each individual antidote has major limitations, including large effective dosage, delayed onset of action, or dependence on enzymes generally confined to specific organs. To overcome these current limitations, next-generation cyanide antidotes are being investigated, including 3-mercaptopyruvate (3-MP). Analytical methods capable of detecting 3-MP from plasma are essential for the development of 3-MP as a nextgeneration cyanide antidote. Although 3-MP has been analyzed by LC-MS-MS, this instrument is not widely available. Therefore, a high performance liquid chromatography (HPLC) method with fluorescence detection (FLD) was developed to analyze 3-MP from swine plasma such that more labs could potentially perform the method. Sample preparation included spiking the plasma with the internal standard (3-mercaptopropionic acid) and reacting the 3-MP and IS with monobromobimane to prevent the characteristic dimerization of 3-MP. The method produced a limit of detection of 0.5 nM, a large dynamic range, and good accuracy and precision. The solid phase mixed-mode anion exchange sample preparation protocol produced excellent selectivity for the method. The wide availability and affordability of the instrumentation and the simple of the implementation method presented should allow more labs to contribute to further investigations of 3-MP as a promising cyanide antidote