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

A rapid and sensitive method based on liquid chromatographtandem 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 reextracted with dichloromethane, after which the analyte and triptorelin as an internal standard （IS） were separated on a 300SB-Cl8 column （150 mm x 4.6 mm i.d., 5 gm 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 multiplereaction monitoring （MRM） transitions of bivalirudin and IS were at m/z 1091.0-650.4 and m/z656.5 - 249.3, respectively. The lower limit of quantification （LLOQ） was 1 ng/mL for 100 ng/mL 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.

Determination of repaglinide in human plasma by high-performance liquid chromatography–tandem mass spectrometry

A rapid and sensitive method based on high-performance liquid chromatography–tandem mass spectrometry(LC–MS/MS)has been developed for the determination of repaglinide in human plasma.The analyte and internal standard(I.S.),diazepam,were extracted from plasma(25 mL)by liquid–liquid extraction with diethyl ether–dichloromethane(60:40,v/v)and separated on a XDB-C_(18) column using acetonitrile–ammonium acetate buffer(pH 6.8,0.01 mol/L)as mobile phase.The retention times of repaglinide and I.S.were 1.95 and 2.35 min,respectively.Detection was carried out using API 4000 mass spectrometer with an ESI interface operating in the multiple reaction monitoring(MRM)mode.The assay was linear over the concentration range 0.050–50 ng/mL with a limit of detection(LOD)of 0.010 ng/mL.Intra-and inter-day precisions(as relative standard deviation,R.S.D.)were ≤5.07%and ≤11.2%,respectively,and accuracy(as relative error,R.E.)was from-0.593%to -1.26%.The assay was successfully applied to a pharmacokinetic study involving a single oral administration of a tablet containing 2 mg repaglinide to each of 10 healthy volunteers.