Validation of LC-MS/MS Method for Determination of Bivalirudin in Human Plasma: Application to a Pharmacokinetic Study

ABSTRACT

BACKGROUND: Bivalirudin is a direct thrombin inhibitor for patients with unstable angina undergoing percutaneous coronary intervention. METHODS: A sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of bivalirudin, in human plasma using nafarelin as internal standard (IS). Chromatographic separation was performed using a Shiseido MG3 mm column (2.0 x 50 mm) with a gradient mobile phase consisting of water and acetonitrile containing 0.1 % formic acid at a flow rate of 0.4 mL/min, and total run time was within 5 min. Detection and quantification was performed by the mass spectrometer using a multiple reaction-monitoring mode at m/z 1091.0 --> 650.3 for bivalirudin, and m/z 662.1 --> 249.3 for IS. RESULTS: The assay was linear over a concentration range of 10 - 10000 ng/mL with a lower limit of quantification of 10 ng/mL in human plasma. CONCLUSION: This method was successfully applied for pharmacokinetics study after intravenous administration of bivalirudin to healthy Korean male volunteers.