Nucleus of Toxicopharmacological Studies and Research - NEPET, Faculty of Pharmacy, Federal University of Goiás, Goiânia 74605-170, Brazil.
Nucleus of Bioequivalence and Clinical Trials - NuBEC, Federal University of São Paulo, São Paulo, CEP 04041-002, Brazil.
J Pharm Biomed Anal. 2020 Sep 5;188:113385. doi: 10.1016/j.jpba.2020.113385. Epub 2020 May 23.
A new, rapid, selective and sensitive UPLC-MS/MS method was developed and validated for the quantification of (-) - hydroxycitric acid (HCA) in human plasma, using DL-malic acid-2,3,3-d3 as internal standard (IS) and simple protein precipitation for the sample preparations. HCA is a highly polar compound make challenging its determination in biological fluids. A specific chromatography column Acquity UPLC HSS T3 (100 × 2.1 mm, 1.8 μm), eluted with mobile phase composed of acetonitrile/ammonium hydroxide 0,1 % (15:85, v/v) were applied for the HCA quantification. The bioanalytical method showed high-throughput achieving as fast chromatographic run as 1 min per sample. No matrix effect was observed with excellent mean chromatographic peak areas ratio of 0.98 ± 0.07 and CV% of 7.17 from normal, lipemic and hemolyzed plasma lots. Calibration curves range was linear at 0.05-10 μg/mL, presenting adequate mean correlation coefficient great than 0.99. Excellent intra-assay and inter-assay precision were achieved, ranging from 5.02-12.01 % (CV%) as well as great intra- and inter-assay accuracy from 0.29-9.20 % (RE%). UPLC-MS/MS bioanalytical method was efficiently applied to the HCA pharmacokinetic study analyzing more than 670 plasma samples.
建立并验证了一种新的、快速、选择性和灵敏的 UPLC-MS/MS 方法，用于人血浆中（-）-羟基柠檬酸（HCA）的定量分析，采用 DL-苹果酸-2,3,3-d3 作为内标（IS），并采用简单的蛋白沉淀进行样品制备。HCA 是一种高度极性化合物，使其在生物体液中的测定具有挑战性。特定的色谱柱 Acquity UPLC HSS T3（100×2.1mm，1.8μm），采用乙腈/氨水溶液 0.1%（15:85，v/v）作为流动相进行洗脱，用于 HCA 的定量分析。该生物分析方法具有高通量的特点，每个样品的色谱运行时间快达 1 分钟。在正常、脂血和溶血的血浆批次中，均观察到无基质效应，且平均色谱峰面积比为 0.98±0.07，CV%为 7.17，具有优异的重现性。校准曲线范围在 0.05-10μg/mL 内呈线性，平均相关系数大于 0.99。表现出良好的日内和日间精密度，范围为 5.02-12.01%（CV%），以及良好的日内和日间准确度，范围为 0.29-9.20%（RE%）。UPLC-MS/MS 生物分析方法成功地应用于 HCA 的药代动力学研究，分析了超过 670 个血浆样本。
