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J Pharm Biomed Anal. 2020 Feb 5;179:112983. doi: 10.1016/j.jpba.2019.112983. Epub 2019 Nov 10.

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.

Author information

1
Linus Pauling Institute and Department of Pharmaceutical Sciences, Oregon State University, 2900 SW Campus Way, Corvallis, OR, 97331 USA; UIC/NIH Center for Botanical Dietary Supplements Research, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612 USA.
2
Linus Pauling Institute and Department of Pharmaceutical Sciences, Oregon State University, 2900 SW Campus Way, Corvallis, OR, 97331 USA; UIC/NIH Center for Botanical Dietary Supplements Research, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612 USA. Electronic address: richard.vanbreemen@oregonstate.edu.

Abstract

To 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, 75-36,000 ng/mL for tolbutamide, 0.05-30 ng/mL for dextromethorphan, and 0.1-60 ng/mL for alprazolam. The intra-assay and inter-assay coefficients of variation (%CV) and %Bias were <13 % (<17 % at the lower limit of quantitation). The recovery of each probe substrate ranged from 84.2%-98.5 %. All analytes were stable during sample storage and handling. Matrix effects were minimized by using stable isotope-labeled internal standards. The method was successfully applied to clinical studies investigating the pharmacokinetic alterations of probe substrates caused by chronic consumption of botanical dietary supplements.

KEYWORDS:

Cytochrome P450; Liquid chromatography; Mass spectrometry; Probe substrates

PMID:
31744669
PMCID:
PMC6943185
[Available on 2021-02-05]
DOI:
10.1016/j.jpba.2019.112983

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