The efficiency of drug metabolism by a single
enzyme can be measured as the fractional metabolic clearance which can be used as a measure of whole body activity for that
enzyme. Measurement of activity of multiple
enzymes simultaneously is feasible using a cocktail approach, however, analytical approach using different assays for drug probes can be cumbersome. A quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based method for the rapid measurement of six
cytochrome P450 (CYP) probe drugs and their relevant metabolites is described. The six specific probe substrates/metabolites are
caffeine/
paraxanthine (
CYP1A2),
flurbiprofen/4'-hydroxyflurbiprofen (
CYP2C9),
mephenytoin/4'-hydroxymephenytoin (
CYP2C19),
debrisoquine/4-hydroxydebrisoquine (
CYP2D6),
chlorzoxazone/6'-hydroxychlorzoxazone (
CYP2E1) and
dapsone/N-
monoacetyldapsone (NAT2). These probes were quantified by stable
isotope dilution from plasma and urine. The present workflow provides a robust, fast and sensitive assay for the "Pittsburgh cocktail", and has been successfully applied to a clinical phenotyping study of
liver disease. A representative group of 17 controls and patients with chronic
liver disease were administered orally
caffeine (100 mg),
chlorzoxazone (250 mg),
debrisoquine (10 mg),
mephenytoin (100 mg),
flurbiprofen (50 mg) and
dapsone (100 mg). Urine (0 through 8 h) and plasma (4 and 8 h) samples were analyzed for drug/metabolite amounts by stable
isotope dilution UPLC-MS/MS. The phenotypic activity of drug metabolizing
enzymes was investigated with 17 patient samples. Selected reaction monitoring (SRM) was optimized for each drug and metabolite. In the method developed, analytes were resolved by reversed-phase by development of a gradient using a water/
methanol solvent system. SRM of each analyte was performed in duplicate on a triple quadrupole mass spectrometer utilizing an 8 min analytical method each, one with the source operating in the positive mode and one in the negative mode, using the same
solvent system. This method enabled quantification of each drug (
caffeine,
chlorzoxazone,
debrisoquine,
mephenytoin,
flurbiprofen, and
dapsone) and its resulting primary metabolite in urine or plasma in patient samples. The method developed and the data herein demonstrate a robust quantitative assay to examine changes in CYP
enzymes both independently or as part of a cocktail. The clinical use of a combination of probe drugs with UPLC-MS/MS is a highly efficient tool for the assessment of CYP
enzyme activity in
liver disease.