Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island Kingston, RI, USA.
Section on Clinical Psychoneuroendocrinology and Neuropsychopharmacology, National Institute on Alcohol Abuse and Alcoholism Division of Intramural Clinical and Biological Research and National Institute on Drug Abuse Intramural Research Program, Bethesda, MD, USA; Medication Development Program, National Institute on Drug Abuse Intramural Research Program, Baltimore, MD, USA; Center for Alcohol and Addiction Studies, Department of Behavioral and Social Sciences, Brown University, Providence, RI, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Nov 1;1130-1131:121820. doi: 10.1016/j.jchromb.2019.121820. Epub 2019 Oct 8.
PF-5190457 is a selective and potent ghrelin receptor inverse agonist presently undergoing clinical trials to treat alcohol use disorder (AUD). We describe the development and validation of a selective and sensitive liquid chromatography-tandem mass spectrometry-based method for quantification of PF-5190457 and its recently discovered hydroxy metabolite PF-6870961 in human plasma. Analytes were extracted after simple protein precipitation using methanol (2.5 ng mL tacrine as an internal standard). A gradient liquid chromatography method was used to separate the analytes on an Acquity UPLC BEH C18 analytical column. The separation was achieved at a flow rate of 0.25 mL min and the total chromatographic runtime was 11.30 min. Positive electrospray ionization and multiple reaction monitoring mode were used for the quantification of all the analytes. The calibration curves from six validation runs were linear with a correlation coefficient of ≥0.996 for the concentration range of 1-1000 ng mL and 2-250 ng mL for PF-5190457 and PF-6870961, respectively. The retention time for PF-5190457, PF-6870961 and tacrine were 4.4, 3.8, and 4.6 min, respectively. The lower limit of quantification for PF-5190457 and PF-6870961 was 1 and 2 ng mL, respectively. The inter-assay precision and accuracy results obtained were within the Food and Drug Administration recommended ±15% limit of nominal values. All the analytes were found to be stable under varied stability conditions. The recovery of PF-5190457 and PF-6870961 ranged from 95 to 103%. Further, the application of the method was demonstrated by measuring the concentration of PF-5190457 and its hydroxy metabolite in patient plasma samples from 100 mg dose.
PF-5190457 是一种选择性和有效的胃饥饿素受体反向激动剂，目前正在进行临床试验，以治疗酒精使用障碍（AUD）。我们描述了一种选择性和灵敏的基于液相色谱-串联质谱的方法的开发和验证，用于定量人血浆中的 PF-5190457 及其最近发现的羟基代谢物 PF-6870961。分析物在用甲醇（2.5ng/mL 他克林作为内标）进行简单的蛋白质沉淀后提取。使用 Acquity UPLC BEH C18 分析柱进行梯度液相色谱法分离分析物。在流速为 0.25mL/min 的情况下实现分离，总色谱运行时间为 11.30min。采用正电喷雾电离和多重反应监测模式对所有分析物进行定量。从六个验证运行获得的校准曲线呈线性，浓度范围为 1-1000ng/mL 和 2-250ng/mL 时，PF-5190457 和 PF-6870961 的相关系数均≥0.996。PF-5190457、PF-6870961 和他克林的保留时间分别为 4.4、3.8 和 4.6min。PF-5190457 和 PF-6870961 的定量下限分别为 1 和 2ng/mL。获得的批内精密度和准确度结果均在食品和药物管理局推荐的±15%名义值范围内。在各种稳定性条件下，所有分析物均被发现稳定。PF-5190457 和 PF-6870961 的回收率范围为 95-103%。此外，通过测量 100mg 剂量患者血浆样品中 PF-5190457 及其羟基代谢物的浓度，证明了该方法的应用。
