Development and validation of a highly sensitive LC-MS/MS method for quantitation of bivalirudin in human plasma: application to a human pharmacokinetic study

A sensitive, specific and simple LC-MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API-4000 LC-MS/MS was operated under multiple-reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C-18 100x2.1mm column with a mobile phase of water-methanol-0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple-reaction monitoring mode were m/z 1091650 for bivalirudin (at 2.70min) and m/z 285193 for diazepam (at 3.85min). The developed method was validated in human plasma with a lower limit of quantitation of 20 mu g/L for bivalirudin. A linear response function was established for the range of concentrations 20-10,000 mu g/L (r>0.998) for bivalirudin. The intra- and inter-day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench-top, freeze-thaw cycles and long-term stability. The developed assay method was applied to an intravenous administration study in humans. Copyright (c) 2013 John Wiley & Sons, Ltd.