Ghareeb Mwlod, Leggio Lorenzo, El-Kattan Ayman, Akhlaghi Fatemeh
Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 495A College of Pharmacy, 7 Greenhouse Road, Kingston, RI, 02881, USA.
Anal Bioanal Chem. 2015 Jul;407(19):5603-13. doi: 10.1007/s00216-015-8730-2. Epub 2015 May 7.
PF-5190457 is a ghrelin receptor inverse agonist that is currently undergoing clinical development for the treatment of alcoholism. Our aim was to develop and validate a simple and sensitive assay for quantitative analysis of PF-5190457 in human or rat plasma and rat brain using liquid chromatography-tandem mass spectrometry. The analyte and stable isotope internal standard were extracted from 50 μL plasma or rat brain homogenate by protein precipitation using 0.1% formic acid in acetonitrile. Chromatography was carried out on an Acquity UPLC BEH C18 (2.1 mm × 50 mm) column with 1.7 μm particle size and 130 Å pore size. The flow rate was 0.5 mL/min and total chromatographic run time was 2.2 min. The mobile phase consisted of a gradient mixture of water: acetonitrile 95:5% (v/v) containing 0.1% formic acid (solvent A) and 100% acetonitrile containing 0.1% formic acid (solvent B). Multiple reaction monitoring was carried out in positive electro-spray ionization mode using m/z 513.35 → 209.30 for PF-5190457 and m/z 518.47 → 214.43 for the internal standard. The recovery ranged from 102 to 118% with coefficient of variation (CV) less than 6% for all matrices. The calibration curves for all matrices were linear over the studied concentration range (R(2) ≥ 0.998, n = 3). The lower limit of quantification was 1 ng/mL in rat or human plasma and 0.75 ng/g in rat brain. Intra- and inter-run mean percent accuracies were between 85 and 115% and percent imprecision was ≤15%. The assays were successfully utilized to measure the concentration of PF-5190457 in pre-clinical and clinical pharmacology studies of the compound.
PF-5190457是一种胃饥饿素受体反向激动剂，目前正处于治疗酒精中毒的临床开发阶段。我们的目标是开发并验证一种简单且灵敏的检测方法，用于使用液相色谱-串联质谱法定量分析人或大鼠血浆及大鼠脑中的PF-5190457。通过用含0.1%甲酸的乙腈进行蛋白沉淀，从50 μL血浆或大鼠脑匀浆中提取分析物和稳定同位素内标。色谱分析在粒径为1.7 μm、孔径为130 Å的Acquity UPLC BEH C18（2.1 mm×50 mm）柱上进行。流速为0.5 mL/min，总色谱运行时间为2.2分钟。流动相由含0.1%甲酸的水：乙腈95:5%（v/v）（溶剂A）和含0.1%甲酸（溶剂B）的100%乙腈的梯度混合物组成。在正电喷雾电离模式下进行多反应监测，PF-5190457的质荷比为m/z 513.35→209.30，内标的质荷比为m/z 518.47→214.43。所有基质的回收率在102%至118%之间，变异系数（CV）小于6%。所有基质的校准曲线在研究的浓度范围内呈线性（R(2)≥0.998，n = 3）。大鼠或人血浆中的定量下限为1 ng/mL，大鼠脑中为0.75 ng/g。批内和批间平均准确度百分比在85%至115%之间，不精密度百分比≤15%。这些检测方法已成功用于该化合物临床前和临床药理学研究中PF-5190457浓度的测定。
