Validation of a Sensitive UHPLC-MS/MS Method for Cytochrome P450 Probe Substrates Caffeine, Tolbutamide, Dextromethorphan, and Alprazolam in Human Serum Reveals Drug Contamination of Serum Used for Research

Publication date: Available online 10 November 2019Source: Journal of Pharmaceutical and Biomedical AnalysisAuthor(s): Luying Chen, Richard B. van BreemenAbstractTo evaluate the potential for interactions between botanical dietary supplements and drug metabolism, Phase I clinical pharmacokinetics studies are conducted using an oral cocktail of probe substrates of cytochrome P450 (CYP) enzymes. A sensitive, specific, and fast ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for determination of caffeine (probe of CYP1A2), tolbutamide (probe of CYP2C9), dextromethorphan (probe of CYP2D6), and alprazolam (probe of CYP3A4/5) in human serum. Stable isotope-labelled analogs were used as internal standards, and sample preparation involved only rapid protein precipitation and centrifugation. The method of standard addition was used for the measurement of caffeine, because commercially available pooled human serum contains caffeine. Out of 18 lots of pooled human serum tested, caffeine was detection in all lots, alprazolam was detected in 13 lots, 8 lots contained dextromethorphan, and no tolbutamide was detected. Only serum prepared from the blood of select individuals was determined to be drug-free. The analytical method was validated with respect to linearity, accuracy and precision, recovery, stability, and matrix effects. The calibration curves were linear over the range of 25-12,000 ng/mL for caffeine, 75-36...
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research