title: Development and validation of a highly sensitive LC-MS/MS method for quantitation of bivalirudin in human plasma: application to a human pharmacokinetic study.
abstract: A sensitive, specific and simple LC-MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API-4000 LC-MS/MS was operated under multiple-reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C18 100 × 2.1 mm column with a mobile phase of water-methanol-0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple-reaction monitoring mode were m/z 1091 → 650 for bivalirudin (at 2.70 min) and m/z 285 → 193 for diazepam (at 3.85 min). The developed method was validated in human plasma with a lower limit of quantitation of 20 µg/L for bivalirudin. A linear response function was established for the range of concentrations 20-10,000 µg/L (r > 0.998) for bivalirudin. The intra- and inter-day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench-top, freeze-thaw cycles and long-term stability. The developed assay method was applied to an intravenous administration study in humans.
authors.affiliation: Pharmaceutical Care Center, Chinese PLA General Hospital, Beijing, 100853, China; Department of Pharmacy, Hainan Branch of PLA General Hospital, Sanya, 572013, Hainan Province, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Chai
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.forename: Nan
authors.initials: N
authors.lastname: Bai
authors.forename: Yun
authors.initials: Y
authors.lastname: Cai
authors.forename: Beibei
authors.initials: B
authors.lastname: Liang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D005260:Female; D006629:Hirudins / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D006801:Humans; D008297:Male; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C074619:bivalirudin
keywords: LC-MS/MS; bivalirudin; human plasma; method validation; pharmacokinetics