Hu Xinhua, Xu Yichao, Chen Jinliang, Shen Yuting, Yang Dandan, Hu Yin, Jiang Bo, Lou Honggang, Ruan Zourong
Center of Clinical Pharmacology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
Center of Clinical Pharmacology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Oct 15;1183:122954. doi: 10.1016/j.jchromb.2021.122954. Epub 2021 Sep 25.
A simple, selective, rapid, and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine CX3002 in human plasma using CX3002-d3 as the internal standard (IS). After a rapid protein precipitation with acetonitrile (3:1, v/v), the chromatographic separation of CX3002 and IS was performed on a Thermo Hypersil GOLD C18 column (2.1 mm × 50 mm, 1.9 μm) with gradient elution at a flow rate of 0.4 ml/min. Gradient elution was achieved with mobile phase A consisting of water containing 0.1% formic acid and 5 mmol/L ammonium formate and mobile phase B consisting of methanol containing 0.1% formic acid. The detection was performed on AB SCIEX QTRAP® 5500 tandem mass spectrometry in the positive ion mode. Multiple reactions monitoring (MRM) was used for quantitative analysis at transition of m/z 460.3 → 199.3 for CX3002 and m/z 463.3 → 202.3 m/z for IS. The method was fully validated and displayed good linearity over a concentration range of 0.2-400 ng/mL with the correlation coefficient above 0.997. The intra-run and inter-run precision (coefficient of variation, CV) ranged from 0.60%-16.46% and the accuracy bias ranged from -7.09%-9.75%. The mean IS-normalized extraction recovery ranged from 98.30% to 104.52%. The CV(%) of IS-normalized matrix factors at the low and high QC concentration were 4.09% and 1.68%, respectively. The storage stability under different conditions was in accordance with the bioanalytical guidelines. The method was successfully applied to the pharmacokinetic study of CX3002 (30 mg) in healthy Chinese subjects.
建立并验证了一种简单、选择性好、快速且可靠的超高效液相色谱-串联质谱法（UPLC-MS/MS），以氘代CX3002（CX3002-d3）作为内标物（IS）来测定人血浆中的CX3002。用乙腈（3:1，v/v）进行快速蛋白沉淀后，CX3002和内标物在Thermo Hypersil GOLD C18柱（2.1 mm×50 mm，1.9 µm）上进行色谱分离，采用梯度洗脱，流速为0.4 ml/min。流动相A由含0.1%甲酸和5 mmol/L甲酸铵的水组成，流动相B由含0.1%甲酸的甲醇组成，通过梯度洗脱实现分离。在AB SCIEX QTRAP® 5500串联质谱仪上以正离子模式进行检测。采用多反应监测（MRM）模式对CX3002进行定量分析，其质荷比（m/z）的跃迁为460.3→199.3，内标物质荷比的跃迁为463.3→202.3。该方法经过全面验证，在0.2 - 400 ng/mL的浓度范围内具有良好的线性，相关系数大于0.997。批内和批间精密度（变异系数，CV）范围为0.60% - 16.46%，准确度偏差范围为 -7.09% - 9.75%。平均内标归一化提取回收率范围为98.30%至104.52%。低、高质控浓度下内标归一化基质因子的CV（%）分别为4.09%和1.68%。不同条件下的储存稳定性符合生物分析指导原则。该方法成功应用于健康中国受试者中CX3002（30 mg）的药代动力学研究。
