LC-MS/MS method for determination of r-RGD-hirudin in human plasma and its application in pharmacokinetic study

Abstract

ObjectiveThis study aimed to develop a simple, sensitive, and selective Liquid chromatography with a Mass spectroscopic method for simultaneous quantification of a recombinant bifunctional hirudin (r-RGD-Hirudin, Bifunctional Hirudin, BFH) in human plasma and verify its effectiveness. MethodsThe analytes and the internal standards from human plasma were extracted using the solid-phase extraction technique. The reconstituted samples were chromatographed on Waters C18 column (BEH 50 × 2.1 mm, 1.7 μm) using a mixture of 0.1% formic acid/acetonitrile (85%/15%, v/v) with gradient elution as the initial mobile phase at a flow rate of 0.3 mL/min. ResultsThe effectiveness of the proposed method was verified over the concentration range of 10–2000 ng/mL for r-RGD-Hirudin. A linear calibration curve was obtained. The precision and accuracy of BFH in the intra- and inter-day runs fell within the range of ±15% at LQC, GMQC, MQC and HQC concentrations. The extraction recoveries and matrix effect at two quality control (QC) levels for BFH were confirmed to conform to the relevant requirement. ConclusionThe proposed method was successfully adapted to examine the pharmacokinetics of BFH in 40 Chinese healthy volunteers, respectively.