(M1030-06-36) Simultaneous Determination of Metabolites from Seven Key Probe Substrates in CYP Inhibition Assay Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Purpose: The Cytochrome P450 (CYP) family of enzymes is commonly involved in significant clinical drug-drug interactions (DDIs). A change in the metabolic clearance of these drugs due to changes in the CYP activities can produce severe adverse reactions or a loss of efficacy when two or more drugs are co-administered. To avoid failures in the later stages of drug development or post-marketing, an evaluation of the effects of new molecular entities (NMEs) on CYP activities during the early stages of drug discovery is crucial. The FDA recommends an in vitro CYP inhibition assay for the seven major human hepatic CYP isoforms: CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A. Instead of investigating individual metabolites separately with a single probe, a cassette analysis assay using a "cocktail" approach can simultaneously detect multiple metabolites from probe substrates to improve efficiency and throughput. The metabolites acetaminophen (CYP1A2), hydroxyl-bupropion (CYP2B6), n-desethyl-amodiaquine (CYP2C8), 4'-hydroxy-diclofenac (CYP2C9), 4'-hydroxymephenytoin (CYP2C19), dextrorphan (CYP2D6), and 1'-hydroxy-midazolam (CYP3A4/5) were combined as 7-in -1 method. However, this approach poses several challenges, such as interference separation issues, the solvent effect of acetaminophen, and the sensitivity of 4'-hydroxymephenytoin. Therefore, we have developed a novel 7-in-1 LC-MS/MS analysis method, which rapidly detects these metabolites simultaneously. This method of accuracy and precision, linearity, carryover, and matrix effect met acceptance criteria, greatly facilitating its use to assess a compound's potential inhibition of the seven major CYPs in drug development settings.

Methods: Seven metabolites were extracted from human liver microsomes using acetonitrile including seven stable isotope labeled (SIL) internal standards. They were detected in MRM mode by SCIEX Triple Quad™ 6500+ coupled with Shimadzu Nexera UHPLC-40 and a CTC autosampler. The ion transitions were m/z 152.1 → 110.1, 256.4 → 238.0, 328.2 → 283.2, 312.0 → 231.0, 235.1 → 150.2, 258.2 → 157.2, and 342.1 → 203.1 for acetaminophen, hydroxyl-bupropion, n-desethyl-amodiaquine, 4'-hydroxy-diclofenac, 4'-hydroxymephenytoin, dextrorphan, and 1'-hydroxy-midazolam, respectively. The analysis was carried out using an ACQUITY HSS T3 column (2.1 x 50 mm) with mobile phases of 2% formic acid in water and 2% formic acid in acetonitrile. All metabolites were eluted using a gradient method with a flow rate of 0.7 mL/min.

Results: The MS and liquid chromatography conditions were optimized, including mass spectrometry parameters, column, mobile phase, and gradient. By adjusting the pH of the mobile phase and confirming the additive as 2% formic acid, good peak shapes were achieved for the seven metabolites and SIL internal standard under these conditions. After optimizing the mass spectrometry parameters, the signal-to-noise ratio of 4'-hydroxymephenytoin increased by ten times. After comparing it to other columns such as C18 and C8, T3 was ultimately chosen for this method. The selectivity of the method was evaluated, and the results demonstrated no interference in the analysis of the seven metabolites by any potential substances, including the seven substrates and the metabolites themselves. The mean response of seven metabolites in a double blank was less than the lower limit of quantification (LLOQ). All metabolites, acetaminophen, hydroxyl-bupropion, n-desethyl-amodiaquine, 4'-hydroxy-diclofenac, 4'-hydroxymephenytoin, dextrorphan, and 1'-hydroxy-midazolam, exhibited good linearity in the ranges of 15-4000 nM, 1.5-400 nM, 6-1600 nM, 30-8000 nM, 1.2-320 nM, 3-800 nM, and 3-800 nM, respectively. The calibration curve was linear with 1/x^2 as the weighting factor, and the regression coefficient (r) values were higher than 0.99. The results demonstrated that the within-run accuracy and precision met the acceptance criteria the within-run %CV was less than 20%, within-run %bias was within ± 25%.

Conclusion: In this study, a new reliable LC-MS/MS method was developed to simultaneously quantify 7 metabolites from key probe substrates in CYP inhibition assays. This analytical method has been optimized to 2.5 minutes, which is 3 times faster than the method reported in the literature. This method not only offers superior efficiency and convenience, but also facilitates the establishment of high-throughput analysis platforms during the early discovery phases of CYP inhibition assays.

References: Spaggiari D, Geiser L, Daali Y, Rudaz S. A cocktail approach for assessing the in vitro activity of human cytochrome P450s: an overview of current methodologies. J Pharm Biomed Anal. 2014 Dec;101:221-37

Acknowledgements: No funding was received from outside institutions or funding agencies.