Determination of an investigational HIV integrase inhibitor in human plasma using high performance liquid chromatography with tandem mass spectrometric detection.
An HPLC-MS/MS assay for the determination of an HIV integrase inhibitor, 5-(1,1-dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (I) in human plasma has been developed and validated. Compound I and a stable isotope labeled internal standard (II) were isolated from 0.5 mL plasma samples by solid phase extraction using an Ansys SPEC C-8 96-well plate. Extracts were separated on a Hypersil BDS C-18 HPLC column (3.0 mmx50 mm, 3 microm) with a mobile phase consisting of 25 mM ammonium formate pH 3.0:acetonitrile (60:40) vol%/vol% pumped at 0.5 mL/min. A Sciex API 365 mass spectrometer equipped with an atmospheric pressure chemical ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 431-->109 (I) and m/z 437-->115 (II) used for quantitation. The assay was validated over the concentration range of 10-5000 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was 69%. The intra-day accuracy of the assay was within 4% of nominal and intra-day precision was better than 4% C.V. Following a 200 mg dose of the compound administered to human subjects, concentrations of I ranged from 21.1 to 1500 ng/mL in plasma samples collected up to 12 h after dosing. Inter-day accuracy and precision results for quality control samples run over a 3-month period alongside clinical samples showed mean accuracies of within 6% of nominal and precision better than 3.5% C.V.
Vallano
P T
PT
Bioanalytical Laboratory, Mylan Pharmaceuticals Inc., 3711 Collins Ferry Road, Morgantown, WV 26505, USA. patrick_vallano@merck.com
Woolf
E J
EJ
Matuszewski
B K
BK
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D005615:Freezing; D019428:HIV Integrase Inhibitors / Q000097:blood*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009287:Naphthyridines / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013843:Thiazines / Q000097:blood*
D019428:HIV Integrase Inhibitors; D009287:Naphthyridines; D013843:Thiazines; C502941:5-(1,1-dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide