Quantitation of bivalirudin, a novel anticoagulant peptide, in human plasma by LC-MS/MS: Method development, validation and application to pharmacokinetics

Abstract

A rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of a novel anticoagulant peptide bivalirudin in human plasma has been developed and validated. Plasma samples were precipitated protein with acetonitrile and re-extracted with dichloromethane, after which the analyte and triptorelin as an internal standard (IS) were separated on a 300SB-C18 column (150 mm×4.6 mm i.d., 5 μm particle size) using 0.1% formic acid:methanol (45:55, v/v) as mobile phase. The triple-quadrupole mass spectrometer, equipped with electrospray ionization (ESI) interface, was operated in the positive ion mode, and the multiple-reaction monitoring (MRM) transitions of bivalirudin and IS were at m/z 1091.0→650.4 and m/z 656.5→249.3, respectively. The lower limit of quantification (LLOQ) was 1 ng/mL for 100 μL plasma sample and the assay was linear over the concentration range 1-1000 ng/mL. The accuracy was within a range from -0.4% to 0.5% in terms of relative error (RE) and the intra- and inter-day precisions in terms of relative standard deviation (RSD) were ≤2.92 and ≤3.36, respectively. The method was successfully applied to a pharmacokinetic study involving intravenous administration of bivalirudin (0.5 mg/kg) to Chinese volunteers.

Keywords: Anticoagulant; Bivalirudin; Human plasma; LC–MS/MS; Pharmacokinetics.

Fig. 1

Q1 MS full-scan spectra of bivalirudin (A) and triptorelin (B).

Fig. 2

Full-scan product ion spectra of [M+2H]²⁺ ions for bivalirudin (A) and triptorelin (B).

Fig. 3

MRM chromatograms for bivalirudin[(A), (B), (C)] and triptorelin(IS)[(A′), (B′), (C′)] in human plasma samples. (A), (A′): a blank plasma sample; (B), (B′): a blank sample spiked with bivalirudin at the LLOQ (1 ng/mL) and IS (500 ng/mL); (C), (C′): a plasma sample from a human volunteer 1 h after intravenous administration of bivalirudin (0.5 mg/kg). Max is the maximum of the relative intensity.

Fig. 4

Mean plasma concentration–time profile of bivalirudin after a single intravenous dose of 0.5 mg/kg (Data are mean±SD, n=9.).