Simultaneous high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) analysis of cyanide and thiocyanate from swine plasma
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An analytical procedure for the simultaneous determination of cyanide and thiocyanate in swine plasma was developed and validated. Cyanide and thiocyanate were simultaneously analyzed by high-performance liquid chromatography tandem mass spectrometry in negative ionization mode after rapid and simple sample preparation. Isotopically labeled internal standards, Na13C15N and NaS13C15N, were mixed with swine plasma (spiked and nonspiked), proteins were precipitated with acetone, the samples were centrifuged, and the supernatant was removed and dried. The dried samples were reconstituted in 10 mM ammonium formate. Cyanide was reacted with naphthalene-2,3-dicarboxaldehyde and taurine to form N-substituted 1-cyano[f]benzoisoindole, while thiocyanate was chemically modified with monobromobimane to form an SCN-bimane product. The method produced dynamic ranges of 0.1–50 and 0.2–50 μM for cyanide and thiocyanate, respectively, with limits of detection of 10 nM for cyanide and 50 nM for thiocyanate. For quality control standards, the precision, as measured by percent relative standard deviation, was below 8 %, and the accuracy was within ±10 % of the nominal concentration. Following validation, the analytical procedure successfully detected cyanide and thiocyanate simultaneously from the plasma of cyanide-exposed swine.
KeywordsBioanalysis Method validation Chemical warfare agent Monobromobimane Naphthalene-2,3-dicarboxaldehyde
The research was supported by the CounterACT Program, National Institutes of Health Office of the Director, and the National Institute of Allergy and Infectious Diseases, Interagency agreement numbers Y1-OD-0690-01/A-120-B.P2010-01, Y1-OD-1561-01/A120-B.P2011-01, and AOD12060-001-00000/A120-B.P2012-01 and the US Army Medical Research Institute of Chemical Defense (USAMRICD) under the auspices of the US Army Research Office of Scientific Services program contract no. W911NF-11-D-0001 administered by Battelle (Delivery order 0079, contract no. TCN 11077). We thank the National Science Foundation Major Research Instrumentation Program (grant no. CHE-0922816) and the State of South Dakota for funding the purchase of the AB SCIEX QTRAP 5500 LC-MS-MS. The LC-MS-MS instrumentation was housed in the South Dakota State University Campus Mass Spectrometry Facility which was supported by the National Science Foundation/EPSCoR grant no. 0091948 and the State of South Dakota. The authors would also like to acknowledge Dr. George Perry, Animal and Range Science (South Dakota State University) for providing swine plasma. Furthermore, the authors are thankful to Dr. Vikhyat Bebarta, Susan M. Boudreau, RN, BSN, Maria G. Castaneda, MS, Toni E. Vargas, PA-C, MHS, and Patricia Dixon, MHS from the Clinical Research Division, Wilford Hall Medical Center (Lackland Air Force Base, San Antonio, TX) for providing potassium cyanide-exposed swine 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 Department of the Army, the National Institutes of Health, the Department of Defense, the National Science Foundation, or the State of South Dakota.
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