title: Monitoring of lopinavir and ritonavir in peripheral blood mononuclear cells, plasma, and ultrafiltrate using a selective and highly sensitive LC/MS/MS assay.
abstract: For the determination of the HIV protease inhibitors lopinavir and ritonavir in human plasma, plasma ultrafiltrate, and peripheral blood mononuclear cells (PBMCs) a highly sensitive and selective method has been developed, validated, and applied to samples of a healthy volunteer. BD Vacutainer CPT and Amicon Centriplus centrifugal filter devices were used for separation of PBMCs and for ultrafiltrate generation, respectively. After liquid/liquid-extraction extracts were chromatographed isocratically within 6 min on a Jupiter Proteo column. The drugs were quantified using 2H5-saquinavir as internal standard and electrospray tandem mass spectrometry in the selected reaction monitoring mode. Limits of quantification for both analytes were 4.0 ng/mL in plasma, 0.2 ng/mL in ultrafiltrate, and 0.1 ng/cell pellet (approximately 3 x 10(6) cells) in PBMCs. The calibration ranges were linear over more than three logs with an over-all accuracy varying between 98.7% and 111.5% and an over-all precision ranging from 6.2% to 14.0% (SD batch-to-batch). After a regular oral dose of Kaletra (400 mg lopinavir, 100 mg ritonavir) analyte concentrations were detectable over a full dosing interval in plasma, ultrafiltrate, and PBMCs. The method is well suited for monitoring of free and total plasma, and intracellular lopinavir/ritonavir concentrations in samples from clinical trials.
authors.affiliation: Department of Internal Medicine VI, Clinical Pharmacology and Pharmacoepidemiology, Im Neuenheimer Feld 410, University of Heidelberg, D-69120 Heidelberg, Germany.
authors.forename: Manuela
authors.initials: M
authors.lastname: Ehrhardt
authors.forename: Marion
authors.initials: M
authors.lastname: Möck
authors.forename: Walter E
authors.initials: WE
authors.lastname: Haefeli
authors.forename: Gerd
authors.initials: G
authors.lastname: Mikus
authors.forename: Jürgen
authors.initials: J
authors.lastname: Burhenne
mesh_terms: D017320:HIV Protease Inhibitors / Q000097:blood*; D006801:Humans; D061466:Lopinavir; D011744:Pyrimidinones / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D019438:Ritonavir / Q000097:blood*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014462:Ultrafiltration
chemical_list: D017320:HIV Protease Inhibitors; D011744:Pyrimidinones; D061466:Lopinavir; D019438:Ritonavir