43. Evaluation of a Method for the Analysis of Hydroxocobalamin and Cyanocobalamin in Plasma by Liquid Chromatography Mass Spectrometry (LC/MS/MS)

Poster: Tue 2:00-3:00PM

Sandra Valtier Lackland AFB

Sandra Valtier, Clinical Research Division, Lackland AFB, TX Harvey A. Schwertner, Clinical Research Division, Lackland AFB, TX Vikhyat S. Bebarta, Emergency Medicine, Wilford Hall Medical Center, Lackland AFB, TX

The purpose of this study was to develop a method for analysis of plasma hydroxocobalamin (OHCbl) and cyanocobalamin (CNCbl) using tandem mass spectrometry. Hydroxocobalamin is used as a cyanide antidote. It has a high affinity for cyanide compounds and forms non-toxic CNCbl complexes with cyanide. In this study, we evaluated sensitivity, linearity, precision, interference and other related parameters associated with method validation. Standards spiked with concentrations ranging from 1-10 µM OHCbl and 0.1 – 10 µM CNCbl were prepared in mobile phase and in pig plasma. Plasma samples from cyanide toxic animals (n=14) were analyzed at various time points following OHCbl administration. An equal volume of plasma sample and acetonitrile were mixed and centrifuged to precipitate the proteins. A 10 µL aliquot of the supernatant was mixed with 990 µL of 5% acetonitrile - 0.1% formic acid. The LC mobile phase consisted of 2.5 mM ammonium acetate in 50:50 methanol:H2O, pH 4 and 10% acetonitrile with 0.1% formic acid; flow rate was set at 0.3 mL/minute. Injection volume set at 5 µL. The major ions of analytical interest were not based on the native ions but rather on the most intense ions present in the mass spectra for each compound. Due to easy loss of the OH functional group, the [Cbl + H]2+ (m/z 664.7) ion was monitored for OHCbl while the more intense doubly charged ion, [CNCbl +H]2+ (m/z 678.8) was monitored for CNCbl. The mass spectrometer was set in the ESI positive mode. Analysis was performed using 2 MRM transitions (664.7→147.3; 664.7→359.2) for OHCbl and 3 transitions (678.8→457.1; 678.8→359.1; 678.8→147.3) for CNCbl. The method produced acceptable results for both compounds, although transition 3 for CNCbl did not meet acceptance criteria at low concentrations. The study describes a validated LC/MS/MS procedure for analysis of OHCbl and CNCbl in plasma. The advantage of evaluating multi-transitions per compound is apparent by examination of ion chromatograms at low concentrations and aids in choosing the most viable ions for quantitation. A fundamental question remains regarding what is an acceptable identification and quantitation of an analyte by LC/MS/MS – Is evaluation of a single MRM transition sufficient for quantitative analysis?

Email: sandra.valtier@us.af.mil

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