Simultaneous Determination of Five Cytochrome P450 Probe Substrates and Their Metabolites and Organic Anion Transporting Polypeptide Probe Substrate in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

Abstract

A rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of organic anion transporting polypeptide 1B1 (OATP1B1) and cytochrome P450 (P450) probe substrates and their phase I metabolites in human plasma was developed. The OATP1B1 (pitavastatin) and five P450 probe substrates, caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A) and their metabolites were extracted from human plasma (50 µL) using methanol. Analytes were separated on a C18 column followed by selected reaction monitoring detection using MS/MS. All analytes were separated simultaneously within a 9 min run time. The developed method was fully validated over the expected clinical concentration range for all analytes tested. The intra- and inter-day precisions for all analytes were lower than 11.3% and 8.82%, respectively, and accuracy was 88.5–117.3% and 96.1–109.2%, respectively. The lower limit of quantitation was 0.05 ng/mL for dextromethorphan, dextrorphan, midazolam, and 1′-hydroxymidazolam; 0.5 ng/mL for losartan, EXP-3174, omeprazole, 5′-hydroxyomeprazole, and pitavastatin; and 5 ng/mL for caffeine and paraxanthine. The method was successfully used in a pharmacokinetic study in healthy subjects after oral doses of five P450 and OATP1B1 probes. This analytical method provides a simple, sensitive, and accurate tool for the determination of OATP1B1 and five major P450 activities in vivo drug interaction studies.

Influence of serum inflammatory cytokines on cytochrome P450 drug metabolising activity during breast cancer chemotherapy: a patient feasibility study

“…Therefore, in order to validate the findings from this feasibility study and assess the impact of other confounding factors on circulating inflammation and in vivo CYP activity, another larger and more comprehensive patient study is warranted. Despite being an exploratory study, the current study numbers align with the number of participants investigated in previous ‘Inje’ cocktail studies, which ranged from four to twenty-two participants 44 – 48 , 51 . However, the lack of statistical power associated with this study should be considered when interpreting the results of the statistical comparisons.…”

“…concurrently assesses activity of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 using a single time point blood and urine sample collection following cocktail administration, with no notable probe-drug interactions or drug-associated adverse events observed 51 . Previous studies have used variations of the Inje cocktail to assess CYP activity in healthy human subjects 44 – 48 . However, the current study is the first to use this tool to assess in vivo activity of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a BC population while receiving chemotherapy.…”

“…Previous studies in healthy participants have shown that the Inje cocktail (caffeine, losartan, omeprazole, dextromethorphan, and midazolam) can be used to simultaneously assess in vivo activity of CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) responsible for most CYP mediated drug metabolism 44 – 48 . The use of the Inje cocktail to assess the effects of drugs or diseases on CYP activity has not yet been carried out in cancer patients receiving chemotherapy.…”

The Development and Validation of a Novel “Dual Cocktail” Probe for Cytochrome P450s and Transporter Functions to Evaluate Pharmacokinetic Drug-Drug and Herb-Drug Interactions

“…For the dual cocktail set development in rats, we applied a simultaneous determination of 15 substrates and metabolites in the rat plasma using an LC-MS/MS system based on the product ion scan results of authentic standards and previously published reports [ 13 , 43 , 44 , 45 , 46 , 47 , 48 , 49 ]. Representative multiple reaction-monitoring chromatograms for the selected probe substrates and metabolites of CYP enzymes and substrates for the transporters ( Figure 2 ) showed that all the 15 probe substrates and metabolites were well separated with no interfering peaks at their respective retention times, suggesting that the 15 probe substrates and metabolites were simultaneously quantified in the rat plasma samples following the concomitant administration of 10 probe substrates.…”

“…The concentrations of 15 probe substrates and metabolites for CYP isoforms and transporters were simultaneously analyzed using an Agilent 6470 Triple Quadrupole LC-MS/MS system (Agilent, Wilmington, DE, USA). The ionization mode and mass transition for Q1 to Q3 were selected based on the product ion scan results of authentic standards and previously published reports [ 13 , 43 , 44 , 45 , 46 , 47 , 48 , 49 ]. The selected probe substrates and metabolites for CYP isoforms and transporters and their mass conditions are summarized in Table 2 , including the ionization mode, mass transition, and collision energy.…”