title: Simultaneous quantification of the new HIV protease inhibitors atazanavir and tipranavir in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.
abstract: We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS), for the quantification of the novel protease inhibitors (PIs) atazanavir and tipranavir. The sample pre-treatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl plasma for atazanavir and 50 microl for tipranavir. Chromatographic separation was achieved on an Inertsil ODS3 column (50 mm x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml/min. The analytical run time was 5.5 min. The triple quadrupole mass spectrometer operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for drug quantification. The assay was linear over a concentration range of 0.05-10 microg/ml for atazanavir and 0.1-75 microg/ml for tipranavir. Saquinavir-d5 was used as internal standard. The intra- and inter-day coefficients of variation were less than 3.8% for atazanavir and less than 10.4% for tipranavir. Accuracies were within +/-7.3 and +/-7.2% for atazanavir and tipranavir, respectively. Both drugs were stable under various relevant storage conditions. The validated concentration ranges proved to be adequate to measure concentrations of human immunodeficiency virus type-1 (HIV-1)-infected individuals. The developed method could easily be combined with a previously developed LC-MS/MS assay for the quantification of protease inhibitors.
authors.affiliation: Department of Pharmacy and Pharmacology, Slotervaart Hospital, Louwesweg 6, 1066 EC Amsterdam, The Netherlands. apkcr@slz.nl
authors.forename: K M L
authors.initials: KM
authors.lastname: Crommentuyn
authors.forename: H
authors.initials: H
authors.lastname: Rosing
authors.forename: M J X
authors.initials: MJ
authors.lastname: Hillebrand
authors.forename: A D R
authors.initials: AD
authors.lastname: Huitema
authors.forename: J H
authors.initials: JH
authors.lastname: Beijnen
mesh_terms: D000069446:Atazanavir Sulfate; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015658:HIV Infections / Q000097:blood; D017320:HIV Protease Inhibitors / Q000097:blood*; D006801:Humans; D009842:Oligopeptides / Q000097:blood*; D011725:Pyridines / Q000097:blood*; D011753:Pyrones / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013449:Sulfonamides
chemical_list: D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D011725:Pyridines; D011753:Pyrones; D013449:Sulfonamides; D000069446:Atazanavir Sulfate; C107201:tipranavir