Determination of 3-mercaptopyruvate in rabbit plasma by high performance liquid chromatography tandem mass spectrometry

Abstract

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 ((13)C3-3-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 0.1μM, a linear dynamic range of 0.5-100μM, along with excellent linearity (R(2)≥0.999), accuracy (±9% of the nominal concentration) and precision (<7% 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.

Keywords: 3-Mercaptopyruvate; Cyanide antidote; Liquid chromatography-tandem mass spectrometry; Sulfanegen.

Fig. 1

3-MP in equilibrium with its dimer and its reaction with MBB to form a stable 3-MPB complex.

Fig. 2

Representative chromatograms of 3-MP spiked (20 µM) and nonspiked rabbit plasma, monitoring the 311 → 223.1 m/z transition. The internal standard 314 → 223.1 m/z spiked and nonspiked in rabbit plasma is also shown. 3-MPB elutes at approximately 2.75 min. A small endogenous concentration of 3-MP can be observed in the nonspiked rabbit plasma.

Fig. 3

The mass spectrum of the 3-MPB complex with tentative identification of the abundant ions. The 3-MPB ion at 311 m/z corresponds to [M–H]⁺.

Fig. 4

HPLC-MS-MS chromatograms of 3-MP spiked rabbit plasma, plasma from a sulfanegen treated rabbit and plasma from the same rabbit prior to sulfanegen treatment. The 3-MP signal in the sulfanegen treated rabbits corresponds to 18 µM.