title: Quantification of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in peripheral blood mononuclear cell lysate using liquid chromatography coupled with tandem mass spectrometry.
abstract: For pharmacokinetic monitoring, measurement of antiretroviral agents in plasma is the gold standard. However, human immunodeficiency virus protease inhibitors (PIs) or non-nucleoside reverse transcriptase inhibitors (NNRTIs) exert their action within the infected cell. Cell-associated concentrations may therefore more adequately reflect therapy outcome. Therefore, for the quantification of nine PIs (amprenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir), 1 active PI metabolite (nelfinavir M8) and 2 NNRTIs (efavirenz and nevirapine) in lysate of peripheral blood mononuclear cells (PBMCs) an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Analytes were extracted from a PBMC pellet by means of a one-step extraction with 50% methanol containing the internal standards D6-indinavir, D5-saquinavir, 13C6-efavirenz and dibenzepine. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. The analytical run time was 10min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 1-500ng/mL in PBMC lysate for all analytes. The method was proven to be specific, accurate, precise and robust. The mean precision and accuracy was less than +/-12% at all concentration levels. Using the developed assay and a previously developed assay for these analytes in plasma, the relationship between plasma and intracellular pharmacokinetics and their relationship with therapy outcome can now be determined.
authors.affiliation: Slotervaart Hospital, Department of Pharmacy & Pharmacology, Louwesweg 6, 1066EC Amsterdam, The Netherlands. rob.terheine@slz.nl
authors.forename: R
authors.initials: R
authors.lastname: ter Heine
authors.forename: M
authors.initials: M
authors.lastname: Davids
authors.forename: H
authors.initials: H
authors.lastname: Rosing
authors.forename: E C M
authors.initials: EC
authors.lastname: van Gorp
authors.forename: J W
authors.initials: JW
authors.lastname: Mulder
authors.forename: Y T
authors.initials: YT
authors.lastname: van der Heide
authors.forename: J H
authors.initials: JH
authors.lastname: Beijnen
authors.forename: A D R
authors.initials: AD
authors.lastname: Huitema
mesh_terms: D000998:Antiviral Agents / Q000097:blood*; D001772:Blood Cell Count; D002457:Cell Extracts / Q000737:chemistry*; D002853:Chromatography, Liquid; D004355:Drug Stability; D017320:HIV Protease Inhibitors / Q000097:blood*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D009705:Nucleosides / Q000097:blood*; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000998:Antiviral Agents; D002457:Cell Extracts; D017320:HIV Protease Inhibitors; D009705:Nucleosides; D018894:Reverse Transcriptase Inhibitors