Fachi Mariana Millan, Vilhena Raquel Oliveira, Boger Beatriz, Domingos Eric Luiz, Dos Santos Josiane Marlei Müller Fernandes, Junkert Allan Michael, de Fátima Cobre Alexandre, Momade Danilo Raul Ossufo, Beraldi-Magalhães Francisco, de Liz Marcus Vinicius, Cordeiro-Santos Marcelo, Pontarolo Roberto
Department of Pharmacy, Federal University of Paraná, Curitiba, PR, Brazil.
Department of Internal Medicine, Federal University of Paraná, Curitiba, PR, Brazil.
Biomed Chromatogr. 2020 May;34(5):e4812. doi: 10.1002/bmc.4812. Epub 2020 Mar 2.
In this research, we developed and validated a liquid chromatography coupled to mass spectrometry (LC-QToF-MS) method for simultaneous quantification of the anti-tuberculosis drugs ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma. Plasma samples spiked with cimetidine (internal standard) were extracted using protein precipitation with acetonitrile containing 1% formic acid. Separation was performed using a C column under flow gradient conditions with water and acetonitrile, both containing 5 mm ammonium formate and 0.1% formic acid. The method was validated according to the ANVISA and US Food and Drug Administration guidelines for bioanalytical method validation. The calibration curve was linear over a concentration range of 0.2-5 μg ml for ethambutol, 0.2-7.5 μg ml for isoniazid, 1-40 μg ml for pyrazinamide and 0.25-2 μg ml for rifampicin, all with adequate precision and accuracy. The method was reproducible, selective and free of carryover and matrix effects. The validated LC-QToF-MS method was successfully applied to real samples and shown to be applicable to future therapeutic and pharmacokinetic monitoring studies.
在本研究中，我们开发并验证了一种液相色谱-质谱联用（LC-QToF-MS）方法，用于同时定量测定人血浆中的抗结核药物乙胺丁醇、异烟肼、吡嗪酰胺和利福平。用含有西咪替丁（内标）的血浆样品，采用含1%甲酸的乙腈进行蛋白沉淀提取。使用C柱在流动梯度条件下，以均含有5 mM甲酸铵和0.1%甲酸的水和乙腈进行分离。该方法根据巴西国家卫生监督局（ANVISA）和美国食品药品监督管理局的生物分析方法验证指南进行验证。乙胺丁醇在0.2 - 5 μg/ml浓度范围内、异烟肼在0.2 - 7.5 μg/ml浓度范围内、吡嗪酰胺在1 - 40 μg/ml浓度范围内、利福平在0.25 - 2 μg/ml浓度范围内校准曲线呈线性，且均具有足够的精密度和准确度。该方法具有可重复性、选择性，无残留和基质效应。经过验证的LC-QToF-MS方法成功应用于实际样品，并显示适用于未来的治疗和药代动力学监测研究。
