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 0.5 μM and 0.2 μM, respectively. The linear ranges were 2–500 μM for 3-MP and 0.5–50 μM for Cbi. The accuracy and precision for 3-MP were 100 ± 9% and \u3c8.3% relative standard deviation (RSD), respectively. For Cbi(CN)2, the accuracy was 100 ± 13% and the precision was \u3c9.5% 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

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% and \u3c7% relative standard deviation (RSD), respectively. The accuracy and precision for the 3-MP with Cbi(CN)2 method were 100 ± 9% and \u3c5.5% RSD for 3-MP. For Cbi(CN)2, the accuracy was 100 ± 13% and \u3c9.5% RSD, respectively. The methods were able to detect 3-MP in rabbits treated with sulfanegen and rats treated with cobinamide. The development of the validated analytical methods for detection of next generation antidotes will facilitate potential FDA approval, and ultimately, use of the therapeutics for cyanide poisoning. To further determine the effectiveness of 3-MP as a cyanide antidote, its pharmacokinetic properties were evaluated in rabbits. Plasma 3-MP concentrations were monitored using liquid chromatography-tandem mass spectrometry following IV administration of sulfanegen to rabbits. Concentrations of 3-MP rapidly increased during sulfanegen administration, indicating rapid absorption and distribution of 3-MP throughout the body. Elimination of 3-MP was also relatively rapid; the calculated halflife of sulfanegen was ~262 mins, which is comparable to current FDA approved cyanide antidotes. A one-compartment model with first-order elimination was used to describe the pharmacokinetic behavior of 3-MP. The results of this study indicate that sulfanegen may be useful to rapidly combat the effects of cyanide poisoning

Determination of 3-mercaptopyruvate in Plasma by High Performance Liquid Chromatography Tandem Mass Spectrometry

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 (\u3c7% 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

Pharmacokinetics of Next Generation Cyanide Antidote Sulfanegen in Rabbits

Aim: Sulfanegen has been shown to be an effective next generation cyanide antidote in multiple animal studies. Sulfanegen detoxifies cyanide by acting as a sulfur donor, converting cyanide to thiocyanate through the enzyme 3-mercaptopyruvate (3-MP) sulfurtransferase. The current study was performed to determine the PK behavior of sulfanegen in rabbits and compare it to current US FDA-approved cyanide therapeutics. Methods: Plasma sulfanegen concentrations, as 3-MP (i.e., sulfanegen is a prodrug that converts to the active sulfur donor, 3-MP, upon administration), were monitored using LC–MS/MS following intramuscular administration of sulfanegen in rabbits. Results: Concentrations of 3-MP rapidly increased following sulfanegen administration, indicating rapid absorption and distribution of 3-MP throughout the body. Elimination of 3-MP was also relatively rapid; the calculated half-life was approximately 114 min. A one-compartment model with first-order distribution and elimination was used to describe the PK behavior of 3-MP. Conclusion: Overall, the PK characteristics of sulfanegen were found to be well suited for the rapid treatment of cyanide poisoning