High-throughput approaches to the quantitative analysis of ketoconazole, a potent inhibitor of cytochrome P450 3A4, in human plasma.
Ketoconazole, an imidazole-piperazine compound, is an orally active antimycotic agent. In addition, ketoconazole is a specific inhibitor of cytochrome P450 3A4. As about 60% of oxidized drugs are biotransformed by this isoform, the potential effect of a concomitant administration of ketoconazole on drug disposition may be of interest during drug development. The present paper describes three different approaches (methods A, B, and C) to attain high-throughput sample preparation and analysis in the quantification of ketoconazole in human plasma. Method A consisted of acetonitrile precipitation in a 96-well plate, transfer of the supernatant via a Tomtec Quadra 96 Model 320, and subsequent injection onto a 50 x 4.6 mm (i.d.) Develosil Combi-RP-5 column (packed with C30 bonded silica particles). Method B consisted of an identical sample preparation to method A with the exception that a Michrom Magic Bullet(trade mark) column, 2.0 --> 0.50 mm (i.d., tapered bore) x25 mm length, was used. Lastly, in method C, a turbulent-flow chromatography (TurboFlow LC/APCI-MS/MS) module was used for the direct analysis of ketoconazole in human plasma. A Sciex API 3000 was used in methods A and B, while a Micromass Quattro LC was employed in method C. Based on the values obtained for the calibrator (standard) and quality control samples, all three protocols yielded satisfactory accuracy, precision, and reduced manual sample preparation time.
Ramos
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L
Department of Drug Metabolism & Pharmacokinetics, Novartis Institute for Biomedical Research, East Hanover, NJ 07936, USA.
Brignol
N
N
Bakhtiar
R
R
Ray
T
T
Mc Mahon
L M
LM
Tse
F L
FL
D000935:Antifungal Agents / Q000097:blood*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002853:Chromatography, Liquid; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors / Q000097:blood*; D006801:Humans; D007654:Ketoconazole / Q000097:blood*; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D013997:Time Factors
D000935:Antifungal Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D007654:Ketoconazole