School of Pharmaceutical Sciences of Ribeirão Preto, Department of Clinical Analysis, Toxicology and Food Science, University of São Paulo, Brazil.
Ribeirão Preto Medical School, Department of Obstetrics and Gynecology, University of São Paulo, Brazil.
J Pharm Biomed Anal. 2019 Feb 5;164:430-441. doi: 10.1016/j.jpba.2018.10.029. Epub 2018 Oct 28.
Drug transporters and CYP enzymes are important sources of pharmacokinetics (PK) variability in drug responses and can cause various pharmacological and toxicological consequences, leading to either toxicity or an insufficient pharmacological effect. In recent years, the cocktail approach was developed to determine in vivo CYP and transporters activities, but these approaches are somewhat limited. We described the development and validation of three sensitive and specific LC-MS/MS assays for the determination of P-gp and major human CYP isoenzyme activities following oral administration of a drug cocktail of subtherapeutic doses (lower than 10 times) of caffeine (CAF), omeprazole (OME), losartan (LOS), midazolam (MDZ), metoprolol (METO) and fexofenadine (FEX) in healthy volunteers. The three validated methods were selective for all tested analytes. No interference or matrix effect was observed for the mass transition and retention times for all compounds monitored. Additionally, assays were linear over a wide range, and limits of quantification varied between 0.01-5 ng/mL plasma. The coefficients of variation obtained in the precision studies and the inter- and intra-assay accuracies were less than 15%, guaranteeing the reproducibility and repeatability of the results. All substrates and metabolites were stable in plasma during freeze-thaw cycles. Three healthy volunteers were selected based on genotyping for CYP2C9, CYP2C19 and CYP2D6. One volunteer was genotyped as an extensive metabolizer (EM) for all tested CYP isoforms, one volunteer was genotyped as a poor metabolizer (PM) for the CYP2C9 isoform (CYP2C9*3/3), and one volunteer was genotyped as a PM for the CYP2D6 isoform (CYP2D64/*4). The methods allowed the quantification of all analytes over the entire sampling period (12 h) in all studied genotypes. Thus, the analytical methods described here were sufficiently sensitive for use in low-dose pharmacokinetic studies.
药物转运体和 CYP 酶是药物反应中药代动力学（PK）变异性的重要来源，可导致各种药理和毒理后果，导致毒性或药理作用不足。近年来，开发了鸡尾酒方法来确定体内 CYP 和转运体的活性，但这些方法存在一定的局限性。我们描述了一种灵敏和特异的 LC-MS/MS 分析方法的开发和验证，用于测定口服低于治疗剂量（低于 10 倍）的药物鸡尾酒（咖啡因（CAF）、奥美拉唑（OME）、洛沙坦（LOS）、咪达唑仑（MDZ）、美托洛尔（METO）和非索非那定（FEX））后，健康志愿者的 P-糖蛋白和主要人 CYP 同工酶活性。三种经验证的方法对所有测试的分析物均具有选择性。对于所有监测的化合物，质量转换和保留时间均未观察到干扰或基质效应。此外，该测定法具有宽线性范围，定量下限在 0.01-5ng/mL 血浆之间变化。在精密度研究中获得的变异系数和内、日间准确度均小于 15%，保证了结果的重现性和可重复性。在冻融循环期间，所有基质和代谢物在血浆中均稳定。根据 CYP2C9、CYP2C19 和 CYP2D6 的基因分型，选择了 3 名健康志愿者。一名志愿者被基因分型为所有测试 CYP 同工酶的广泛代谢者（EM），一名志愿者被基因分型为 CYP2C9 同工酶（CYP2C9*3/3）的弱代谢者（PM），一名志愿者被基因分型为 CYP2D6 同工酶（CYP2D64/*4）的 PM。这些方法允许在所有研究的基因型中在整个采样时间（12 小时）内定量分析所有分析物。因此，这里描述的分析方法对于使用低剂量药代动力学研究来说足够灵敏。
