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

This 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. The 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. The 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. The proposed method was successfully adapted to examine the pharmacokinetics of BFH in 40 Chinese healthy volunteers, respectively. [Display omitted] •This method adopts isotope internal standard and solid phase extraction method, and there is no relevant literature report.•The method has good accuracy, high sensitivity, simple operation, and the linear range is 10 ng/mL∼2000 ng/mL.•The single sample analysis time of this method is 1.8min, and the analysis time is short, which is suitable for the analysis of large batches of samples.•The effectiveness of the method is demonstrated by studying the plasma concentration and pharmacokinetic of recombinant bifunctional hirudin in the healthy volunteers.