Simultaneous determination of 3-mercaptopyruvate and cobinamide in plasma by liquid chromatography-tandem mass spectrometry

The current suite of Food and Drug Administration (FDA) approved antidotes (i.e., sodium nitrite, sodium thiosulfate, and hydroxocobalamin) are effective for treating cyanide poisoning, but individually, each antidote has major limitations (e.g., large effective dosage or delayed onset of action). To mitigate these limitations, next-generation cyanide antidotes are being investigated, including 3-mercaptopyruvate (3-MP) and cobinamide (Cbi). Analytical methods capable of detecting these therapeutics individually and simultaneously (for combination therapy) are essential for the development of 3-MP and Cbi as potential cyanide antidotes. Therefore, a liquid chromatography-tandem mass-spectrometry method for the simultaneous analysis of 3-MP and Cbi was developed. Sample preparation of 3-MP consisted of spiking plasma with an internal standard (¹³C₃-3-MP), precipitation of plasma proteins, and derivatizing 3-MP with monobromobimane to produce 3-mercaptopyruvate-bimane. Preparation of Cbi involved denaturing plasma proteins with simultaneous addition of excess cyanide to convert each Cbi species to dicyanocobinamide (Cbi(CN)₂). The limits of detection for 3-MP and Cbi were 0.5μM and 0.2μM, respectively. The linear ranges were 2-500μM for 3-MP and 0.5-50μM for Cbi. The accuracy and precision for 3-MP were 100±9% and <8.3% relative standard deviation (RSD), respectively. For Cbi(CN)₂, the accuracy was 100±13% and the precision was <9.5% RSD. The method presented here was used to determine 3-MP and Cbi from treated animals and may ultimately facilitate FDA approval of these antidotes for treatment of cyanide poisoning.