ABSTRACT
Atazanavir (Reyataz(®)) is an important member of the HIV protease inhibitor class. Because of the complexity of its chemical structure, metabolite identification and structural elucidation face serious challenges. So far, only seven non-conjugated metabolites in human plasma have been reported, and their structural elucidation is not complete, especially for the major metabolites produced by oxidations. To probe the exact sites of metabolism and to elucidate the relationship among in vivo metabolites of atazanavir, we designed and performed two sets of experiments. The first set of experiments was to determine atazanavir metabolites in human plasma by LC-MS, from which more than a dozen metabolites were discovered, including seven new ones that have not been reported. The second set involved deuterium labeling on potential metabolic sites to generate D-atazanavir analogs. D-atazanavir analogs were dosed to human in parallel with atazanavir. Metabolites of D-atazanavir were identified by the same LC-MS method, and the results were compared with those of atazanavir. A metabolite structure can be readily elucidated by comparing the results of the analogs and the pathway by which the metabolite is formed can be proposed with confidence. Experimental results demonstrated that oxidation is the most common metabolic pathway of atazanavir, resulting in the formation of six metabolites of monooxidation (M1, M2, M7, M8, M13, and M14) and four of dioxidation (M15, M16, M17, and M18). The second metabolic pathway is hydrolysis, and the third is N-dealkylation. Metabolites produced by hydrolysis include M3, M4, and M19. Metabolites formed by N-dealkylation are M5, M6a, and M6b.
Subject(s)
HIV Protease Inhibitors/blood , HIV Protease Inhibitors/metabolism , Oligopeptides/blood , Oligopeptides/metabolism , Pyridines/blood , Pyridines/metabolism , Atazanavir Sulfate , Chromatography, High Pressure Liquid/methods , Deuterium/analysis , Deuterium/metabolism , HIV/enzymology , HIV Infections/drug therapy , HIV Protease Inhibitors/administration & dosage , HIV Protease Inhibitors/chemistry , Humans , Hydrolysis , Metabolic Networks and Pathways , Oligopeptides/administration & dosage , Oligopeptides/chemistry , Oxidation-Reduction , Pyridines/administration & dosage , Pyridines/chemistry , Tandem Mass Spectrometry/methodsABSTRACT
A high throughput method was developed and validated for the quantitative determination of LAG078, a lipid-lowering compound, in dog plasma obtained during toxicokinetic studies. The method was based on reverse phase liquid chromatographic separation of the analyte from plasma extract followed by turbo-ionspray (TIS) in the negative ion mode and tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. Extraction was performed using a combination of protein precipitation and liquid-liquid extraction in the 96-well plate format to increase the throughput of the method. Optimized chromatographic separation under basic condition (pH approximately 10) in a short polymer based column (50 mm x 2.0 mm i.d.) coupled with MRM mode of detection yielded clean chromatograms with minimal signal suppression. The standard curve was linear (r = 0.997) within the concentration range of 0.05 (lower limit of quantification; LLOQ) to 50 ng/ml using only 0.1 ml of dog plasma. The accuracy of the method varied from 95 to 100% with a precision (CV) of 3.04-10.8% over the concentration range. The method was simple, rapid, and robust.
Subject(s)
Hydrocarbons, Fluorinated/blood , Hypolipidemic Agents/blood , Hypolipidemic Agents/pharmacokinetics , Sulfur Compounds/blood , Animals , Chromatography, Liquid/methods , Dogs , Hydrocarbons, Fluorinated/pharmacokinetics , Hydrocarbons, Fluorinated/toxicity , Hypolipidemic Agents/toxicity , Mass Spectrometry/methods , Sulfur Compounds/pharmacokinetics , Sulfur Compounds/toxicityABSTRACT
A high-throughput method was developed and validated for the quantitative determination of MMI270B, an inhibitor of matrix metalloprotease (MMP) enzymes, in human plasma. The method was based on reverse-phase chromatographic separation of the analyte from plasma extract followed by atmospheric pressure chemical ionization (APCI) and tandem mass spectrometry in the selected reaction monitoring mode (SRM). Extraction was performed using simple protein fi ltration in the 96-well plate format to increase the throughput of the method. Optimised chromatographic separation in a short column (30 x 4.6 mm i.d.) coupled with positive APCI mode of ionization followed by selective SRM mode of detection yielded clean chromatograms with minimal signal suppression. The chromatographic conditions resolved isobaric interference peaks observed in samples from patients dosed with MMI270B. The standard curve was linear (r = 0.997) within the concentration range of 1.04 (lower limit of quanti fi cation) to 1040 ng/mL using 0.1 mL of human plasma. The accuracy of the method varied from 93.6 to 103% with a precision of 2.17-6.71% over the concentration range. The method was simple, rapid, and robust with an analyte recovery of >98%.
