title: Application of a rapid and selective method for the simultaneous determination of protease inhibitors, lopinavir and ritonavir in human plasma by UPLC-ESI-MS/MS for bioequivalence study in Indian subjects.
abstract: A high throughput and rugged ultra performance liquid chromatography tandem mass spectrometry (UPLC-ESI-MS/MS) method is developed and validated for the selective determination of protease inhibitors -- lopinavir (LPV) and ritonavir (RTV) in human plasma. Plasma samples were prepared by solid phase extraction of the analytes and their deuterated analogs as internal standard (IS) using Waters Oasis HLB cartridges. The chromatographic separation was achieved in a run time of 1.2 min on Waters Acquity UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 microm) under isocratic conditions. The mobile phase consisted of 10 mM ammonium formate, pH 4.0 adjusted with formic acid and methanol (10:90, v/v). The protonated precursor --> product ion transitions for lopinavir, ritonavir, d(8)-lopinavir and d(6)-ritonavir were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. A linear dynamic range of 2.9-1452 ng/mL and 29.6-14379 ng/mL was established for ritonavir and lopinavir respectively using 0.1 mL human plasma. The mean relative recovery of lopinavir (96.6%), ritonavir (97.5%), d(8)-lopinavir (85.5%) and d(6)-ritonavir (86.3%) from spiked plasma samples was consistent and reproducible. The method was successfully applied to a bioequivalence study of [200(lopinavir)+50(ritonavir)]mg tablet formulation in 36 healthy human subjects under fasting conditions.
authors.affiliation: Bioanalytical Research Department, Veeda Clinical Research, Ambawadi, Ahmedabad, India.
authors.forename: Manish
authors.initials: M
authors.lastname: Yadav
authors.forename: Rajasekhar
authors.initials: R
authors.lastname: Rao
authors.forename: Hemal
authors.initials: H
authors.lastname: Kurani
authors.forename: Puran
authors.initials: P
authors.lastname: Singhal
authors.forename: Sailendra
authors.initials: S
authors.lastname: Goswami
authors.forename: Pranav S
authors.initials: PS
authors.lastname: Shrivastav
mesh_terms: D000328:Adult; D019540:Area Under Curve; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D018592:Cross-Over Studies; D017320:HIV Protease Inhibitors / Q000097:blood* / Q000493:pharmacokinetics; D006207:Half-Life; D006801:Humans; D007194:India; D061466:Lopinavir; D008297:Male; D011744:Pyrimidinones / Q000097:blood* / Q000493:pharmacokinetics; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D019438:Ritonavir / Q000097:blood*; D021241:Spectrometry, Mass, Electrospray Ionization; D013810:Therapeutic Equivalency
chemical_list: D017320:HIV Protease Inhibitors; D011744:Pyrimidinones; D061466:Lopinavir; D019438:Ritonavir