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 (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 (<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.

Pharmacokinetics of Next Generation Cyanide Antidote Sulfanegen in Rabbits

“…PK behavior of 3-MP Figure 1 shows the calculated plasma concentrations of 3-MP from rabbits treated with sulfanegen. The inter-assay accuracy (100 ± 5%) and precision (<6%), and intra-assay accuracy (100 ± 9%) and precision (<7% RSD) for quality control samples were previously reported for the method [23]. As seen in Figure 1, the plasma 3-MP concentration rapidly increased to a maximum (C max ) of about 1.775 mM at approximately 60 min postadministration (t max ).…”

“…Plasma was prepared and analyzed for 3-MP using the previously established method of Stutelberg et al [23]. Briefly, isotopically labeled 3-MP internal standard (100 µl) was initially added to rabbit plasma.…”

Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations

“…From this analysis, the following estimates for 3-MP concentration in murine tissue were obtained: 0.8±0.3 µmol•kg tissue -1 in liver, 1.4±0.5 µmol•kg tissue -1 in kidney, and 0.4±0.4 µmol•kg tissue -1 in brain (Table 4). For comparison, the concentration of 3-MP in rabbit plasma was reported to be 0.05-0.1 µM (32).…”

Development of sulfanegen for mass cyanide casualties

“…The optimized HPLC-MS-MS method was capable of detecting 3-MP in rabbits that were administered sulfanegen. 45 Thus, this method is suitable for further pharmacokinetic investigations of this promising cyanide antidote.…”

“…This allowed for further optimization of a high‐performance liquid chromatography (HPLC)–MS–MS method suitable for detection of sulfanegen in rabbit plasma with a total run time of 5.1 min, a limit of detection of 0.1 μM, a linear dynamic range of 0.5–100 μM, 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 . Thus, this method is suitable for further pharmacokinetic investigations of this promising cyanide antidote.…”