title: Cocktail approach for in vivo phenotyping of 5 major CYP450 isoenzymes: development of an effective sampling, extraction, and analytical procedure and pilot study with comparative genotyping.
abstract: In this study, the authors developed a phenotyping method for CYP1A2, 2C9, 2C19, 2D6, and 3A4 using a cocktail of 100 mg caffeine, 125 mg tolbutamide, 20 mg omeprazole, 30 mg dextromethorphan, and 2 mg midazolam. A simple sampling scheme was established collecting 3 blood samples at 0, 4, and 24 hours followed by solid-phase extraction and liquid chromatography/tandem mass spectrometry analysis. After addition of 8 deuterated internal standards and extraction, the analytes were separated using gradient elution with ammonium acetate and methanol. Data acquisition was performed on a triple quadrupole linear ion trap mass spectrometer in multiple-reaction monitoring mode with positive electrospray ionization. The assay was validated according to international guidelines: limits of quantification (LOQs) were between 0.25 and 1.0 ng/mL for all analytes, except for paraxanthine and caffeine (20 ng/mL). Extraction efficiencies ranged between 77% and 103% and matrix effects between 23% and 95%; precision and accuracy data fulfilled accepted criteria. Calibration curves from LOQ to 1000 ng/mL were established for undiluted and 1:10 diluted plasma (r > 0.998). The method was tested in a pilot study with 14 volunteers. Additional genotyping of the probands generally demonstrated good accordance with the measured phenotyping indices but also disclosed certain contradictory results.
authors: Institute of Forensic Medicine, University Medical Center Freiburg, Albertstraße 9, 79104 Freiburg, Germany. ariane.wohlfarth@uniklinik-freiburg.de
authors: Ariane
authors: A
authors: Wohlfarth
authors: Jana
authors: J
authors: Naue
authors: Sabine
authors: S
authors: Lutz-Bonengel
authors: Sebastian
authors: S
authors: Dresen
authors: Volker
authors: V
authors: Auwärter
mesh_terms: D000328:Adult; D002110:Caffeine / Q000008:administration & dosage / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000097:blood* / Q000737:chemistry* / Q000235:genetics; D003915:Dextromethorphan / Q000008:administration & dosage / Q000097:blood; D005260:Female; D005838:Genotype; D006801:Humans; D007527:Isoenzymes; D008297:Male; D008874:Midazolam / Q000008:administration & dosage / Q000097:blood; D009853:Omeprazole / Q000008:administration & dosage / Q000097:blood; D010641:Phenotype; D010865:Pilot Projects; D053719:Tandem Mass Spectrometry / Q000379:methods; D014044:Tolbutamide / Q000008:administration & dosage / Q000097:blood; D055815:Young Adult
chemical_list: D007527:Isoenzymes; D002110:Caffeine; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D009853:Omeprazole; D008874:Midazolam