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

Michael W. Stutelberg, Chakravarthy V. Vinnakota, Brendan L. Mitchell, Alexandre R. Monteil, Steven E. Patterson, Brian A. Logue

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


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 0.1μM, a linear dynamic range of 0.5-100μM, 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.

Original languageEnglish (US)
Pages (from-to)94-98
Number of pages5
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
StatePublished - Apr 2014

Bibliographical note

Funding Information:
We gratefully acknowledge the support from a U.S. Dept. of Education, Graduate Assistance in Areas of National Need (GAANN) award to the Department of Chemistry & Biochemistry ( P200A100103 ). We thank the National Science Foundation Major Research Instrumentation Program (Grant Number CHE-0922816 ) for funding the AB SCIEX QTRAP 5500 LC-MS-MS. We also would like to acknowledge the support by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD) , and the National Institute of Neurological Disorders and Stroke (NINDS) , (Grant Number U01NS058087-06 ). In addition, the LC-MS-MS instrumentation in the South Dakota State University Campus Mass Spectrometry Facility used in this study was obtained with support from the National Science Foundation/EPSCoR (Grant No. 0091948 ) and the State of South Dakota . Furthermore, the authors are thankful to Dr. Sari B. Mahon from the Beckman Laser Institute and Dr. Matthew Brenner from the Division of Pulmonary and Critical Care (University of California-Irvine, Irvine, CA) for providing sulfanegen treated rabbit plasma. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the National Science Foundation, the National Institutes of Health or the CounterACT Program.


  • 3-Mercaptopyruvate
  • Cyanide antidote
  • Liquid chromatography-tandem mass spectrometry
  • Sulfanegen


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