title: Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats.
abstract: A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.
authors.affiliation: Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China. dorothy_cheung@163.com <dorothy_cheung@163.com>
authors.forename: Shaoyu
authors.initials: S
authors.lastname: Zhang
authors.forename: Naining
authors.initials: N
authors.lastname: Song
authors.forename: Quansheng
authors.initials: Q
authors.lastname: Li
authors.forename: Huirong
authors.initials: H
authors.lastname: Fan
authors.forename: Changxiao
authors.initials: C
authors.lastname: Liu
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D019388:Cytochrome P-450 CYP1A2 / Q000032:analysis; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis; D051544:Cytochrome P-450 CYP3A / Q000032:analysis; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000235:genetics* / Q000652:urine; D003580:Cytochromes; D008297:Male; D008617:Mephenytoin / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism; D010615:Phenacetin / Q000378:metabolism; D010641:Phenotype; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014044:Tolbutamide / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D003580:Cytochromes; C006913:carboxytolbutamide; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; C507848:CYP2C9 protein, rat; C017170:Cyp1a2 protein, rat; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D010615:Phenacetin; D008617:Mephenytoin; D008874:Midazolam