Liquid Chromatography-tandem Mass Spectrometry of Next-generation Cyanide Antidotes, 3-mercaptopyruvate and Cobinamide, with the Pharmacokinetic Analysis of 3-mercaptopyruvate

Although the current suite of FDA approved antidotes (i.e., sodium nitrite, sodium thiosulfate, and hydroxocobalamin) are effective for treating cyanide poisoning, each antidote individually has major limitations (e.g., large effective dosage or delayed onset of action). To mitigate these current limitations, next-generation cyanide antidotes are being investigated, including sulfanegen and cobinamide (Cbi). Analytical methods capable of detecting these therapeutics individually and simultaneously (for combination therapy) are essential for the development of sulfanegen and Cbi as potential antidotes. Therefore, two liquid chromatography-tandem mass-spectrometry (LC-MS-MS) methods were developed for 1) the analysis of 3-MP (i.e., sulfanegen converts to the active sulfur donor, 3-MP, upon administration into the blood) individually, and 2) the simultaneous analysis of 3-MP and Cbi. Sample preparation of 3-MP for both methods consisted of spiking plasma with an internal standard (13C3-3-MP), precipitation of plasma proteins, and derivatizing with monobromobimane to produce 3-mercaptopyruvate-bimane (3-MPB). Preparation of Cbi involved denaturation of plasma proteins and addition of excess cyanide to produce dicyanocobinamide (Cbi(CN)2) from each Cbi species. The LOD for the 3-MP method was 0.1 μM and the LODs for the 3-MP/Cbi method were 0.5 μM for 3-MP and 0.2 μM for Cbi(CN)2. For the analysis of 3-MP alone, the linear range was 0.5-100 μM. Linear ranges for the simultaneous method were 2-500 μM for 3-MP and 0.5-50 μM for Cbi. Accuracy and precision for the 3-MP method was 100 ± 9% an