Determination of free cyano-cobinamide in swine and rabbit plasma by liquid chromatography tandem mass spectrometry.

In recent years, Cobinamide (Cbi) has shown promise as a therapeutic for cyanide poisoning. There are several forms of Cbi based on the identity of the ligands bound to the cobalt in Cbi and these different forms of Cbi have divergent behavior (e.g., the aquo and hydroxo forms of Cbi readily bind to proteins, limiting their distribution significantly, whereas [Cbi(CN)2] does not). While current analysis techniques only measure total Cbi, methods to elucidate the behavior of 'available' Cbi versus cyanide-complexed Cbi would be valuable for biomedical and pharmacokinetic studies. Therefore, a method was developed for the analysis of cyanide-complexed Cbi in plasma via liquid chromatography tandem mass spectrometry (LC-MS-MS). Plasma samples were prepared by denaturing proteins with 10% ammonium hydroxide in acetonitrile. The resulting mixture was centrifuged, and the supernatant was removed, dried, and reconstituted. Cyanide-complexed Cbi was then analyzed via LC-MS-MS. The limit of detection was 0.2 µM, and the linear dynamic range was between 1 and 200 µM. The accuracy was 100 ±â€¯17% and the precision, measured by relative standard deviation (%RSD), was ≤18.5%. Carryover, a severe problem when analyzing Cbi via liquid chromatography was eliminated using a polymeric-based stationary phase (PLRP-S) and a controlled washing protocol. The method allowed evaluation of the cyanide-bound and 'available' Cbi from treated animals and, when paired with a method for total Cbi analysis, allows for estimation of Cbi utilization when treating cyanide poisoning.

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 ((13)C3-3-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.