Simultaneous LC-MS/MS Analysis of the Plasma Concentrations of a Cocktail of 5 Cytochrome P450 Substrate Drugs and Their Metabolites

Abstract

A "cocktail" approach, which involves simultaneous administration of multiple CYP-specific probes, concurrently detects the activity of multiple CYP enzymes. We developed and validated a rapid and selective LC-MS/MS method for determining the plasma concentrations of 5 CYP probe drugs and metabolites (caffeine/paraxanthine, CYP1A2 substrate; losartan/losartan carboxylic acid (E3174), CYP2C9 substrate; omeprazole/5-hydroxyomeprazole, CYP2C19 substrate; dextromethorphan/dextrorphan, CYP2D6 substrate; and midazolam/1′-hydroxymidazolam, CYP3A4 substrate) by single-step extraction, followed by a single LC-MS/MS run. An Ostro 96-well plate was used for extraction of CYP substrates and metabolites from human plasma and urine. Following optimization of the chromatographic conditions, all the peaks were well separated, and retention times ranged between 4.4 and 11.7 min. The total run time for a single injection was within 13 min. The accuracy and precision values suggested that the assay had high accuracy and reliability in plasma and urine samples. No significant matrix interference was observed. To demonstrate the efficacy of this method, plasma and urine concentrations of 5 CYP probe substrates and their metabolites were determined after simultaneous oral administration of 5 drugs to 4 healthy volunteers. All the substrates and metabolites were detected over an 8 h period, and the plasma concentrations of each substrate at 8 h after administration were above the lower limit of quantification. Urine concentrations of drugs and their metabolic ratio were evaluated after the administration. In conclusion, the advantage of our cocktail approach is that it enables in vivo assessment of the activity of various drug-metabolizing enzymes in a single assay. Key words cytochrome P450; cocktail; LC-MS/MS; healthy volunteerThe CYP system, which constitutes the major drug-metabolizing machinery in humans, catalyzes the biotransformation of xenobiotics and has been reported to process over 90% of the currently marketed drugs.1,2) The drug-metabolizing activities of different CYP isoforms show high inter-and intraindividual variations; age, gender, genetics, environmental factors, dietary components, and endogenous mediators contribute to this variability. [3][4][5] In addition, certain medications can induce or inhibit the activity of drug-metabolizing enzymes, resulting in drug-drug interactions. Variation in CYP activity results in variation in the pharmacokinetics of its substrates, which in turn may alter the pharmacodynamics of these drugs. Thus, predicting the pharmacokinetics and pharmacodynamics of the drug substrates of CYP is important for predicting potential drug interactions and for CYP substrate dose optimization.A "cocktail" approach concurrently detects the activity of multiple CYP enzymes and involves simultaneous administration of multiple CYP-specific probes followed by the evaluation of their pharmacokinetics in a single experiment. Since the Pittsburgh cocktail was first introduced in 1997, 6) several cocktail...

Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes

“…However, the application of these methods was hindered by a number of defects. Methods involving the quantification of several metabolic reactions in human and rat plasma and urine, human liver microsomes, or hepatocytes were only used to evaluate the activity of a limited number of CYP isoforms . Additionally, a method that involved the quantification of eight metabolic reactions and one that quantified nine metabolic reactions in human liver microsomes were unable to comprehensively assess CYP3A4 activity because only one probe was used for CYP3A4.…”

“…Therefore, it is necessary to establish a fast and accurate method for simultaneous quantification of the above ten metabolic reactions to rapidly, accurately, and comprehensively evaluate the impact of drug candidates and other factors on the activity of CYP enzymes to guide rational drug use in clinical settings. Although thus far, several studies have reported analytical methods to detect the formation of multiple metabolites from probe substrates using LC–MS in biological samples, many disadvantages were associated with the proposed methods, including the facts that only a small number of metabolic reactions was detected and a single CYP 3A4‐specific probe was used to evaluate the activity of this isozyme in some of the methods [, ]. No rapid and accurate method currently exists that would be capable of simultaneous determination of ten metabolites to quantify ten different metabolic reactions in biological samples, which could thereby be used to evaluate the activities of multiple CYP isoforms.…”

High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability

“…Cell culture supernatant was collected at 0, 4, 24, and 48 h after exposure. LC-MS analysis of metabolites was performed by Janssen Pharmaceutics (Beerse, Belgium) in a similar way to what has been described earlier (Tanaka et al 2014).…”

The perspective of caffeine and caffeine derived compounds in therapy

“…In mammals,caffeine is metabolized by cytochromes P450 via oxidative demethylation into paraxanthine, theobromine and theofi line. The methyl moiety is split from the structure as formaldehyde (24).The most relevant pathway for caffeine detoxifi cation is based on oxidative demethylation by P450 isotype CYP1A2 to paraxanthine (25,26). Activity of CYP 1A2 is distinct in the population and formssuch as 1A, 1B, 1C, and 1F can be foundamonghumans; however, the ability to oxidize xanthine alkaloids is not signifi cantly different (27,28).…”