title: Simultaneous determination of probe drugs, metabolites, inhibitors and inducer in human plasma by liquid chromatography/tandem mass spectrometry and its application to pharmacokinetic study.
abstract: Cytochrome P450 3A4 (CYP3A4) and UDP-glucuronosyltransferase 1A1 (UGT1A1) are important enzymes responsible for the metabolism of many xenobiotics. To investigate their induction and inhibition properties, administering probe drugs and monitoring their concentration in plasma under the effects of inducers/inhibitors is the gold standard method. A rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed for simultaneous quantification of midazolam, raltegravir (probe drugs for CYP3A4 and UGT1A1), their major metabolites, 1'-hydroxymidazolam, 1'-hydroxymidazolam glucuronide and raltegravir glucuronide, rifampicin (inducer), ritonavir and ketoconazole (inhibitors). Analytes were extracted from 100μl of plasma using solid-phase extraction followed by chromatographic separation on a reversed-phase C18 column (50mm×2.1mm, particle size 1.8μm). The mass spectrometer was operated under positive ionization mode. Excellent linearity (r(2)≥0.995) was achieved for all. The method was validated and found to be accurate (88-111%), precise (CV%<13) and selective. Matrix effect was acceptable (88-118%) and analytes recovery was reproducible (60-95%). Analytes in plasma were also found to be stable in the autosampler (6°C for 48h) and after two freeze-thaw cycles. We have developed a robust analytical method to simultaneously quantify probes, inducer and inhibitor of important drug metabolism enzymes. The method was successfully applied in a clinical study to investigate the degree of induction and inhibition of CYP3A4 and UGT1A1 among ethnic groups in Singapore.
authors.affiliation: National University Health System, 1E Kent Ridge Road, Singapore 119228, Singapore; National University of Singapore, Level 9 6 Science Drive 2, Singapore 117546, Singapore.
authors.forename: Kim H
authors.initials: KH
authors.lastname: Hee
authors.forename: Zhangyan
authors.initials: Z
authors.lastname: Yao
authors.forename: Lawrence S
authors.initials: LS
authors.lastname: Lee
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid; D018592:Cross-Over Studies; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry; D005615:Freezing; D014453:Glucuronosyltransferase / Q000737:chemistry; D064368:Healthy Volunteers; D006801:Humans; D007654:Ketoconazole / Q000032:analysis* / Q000097:blood; D008874:Midazolam / Q000032:analysis* / Q000097:blood; D010316:Particle Size; D011760:Pyrrolidinones / Q000032:analysis* / Q000097:blood; D011786:Quality Control; D000068898:Raltegravir Potassium; D015203:Reproducibility of Results; D012293:Rifampin / Q000032:analysis* / Q000097:blood; D019438:Ritonavir / Q000032:analysis* / Q000097:blood; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry; D015262:Xenobiotics / Q000737:chemistry; D000099148:UGT1A1 Enzyme
chemical_list: D051544:Cytochrome P-450 CYP3A; D014453:Glucuronosyltransferase; D007654:Ketoconazole; D008874:Midazolam; D011760:Pyrrolidinones; D000068898:Raltegravir Potassium; D012293:Rifampin; D019438:Ritonavir; D015262:Xenobiotics; D000099148:UGT1A1 Enzyme
keywords: Ketoconazole; Midazolam; Raltegravir; Rifampin; Ritonavir