Department of Laboratory Medicine, The First Hospital of Jilin University, Jilin University, Xinmin Street, Changchun 130061, PR China; School of Life and Pharmaceutical Sciences, Dalian University of Technology, Panjin 124221, PR China.
Department of Laboratory Medicine, The First Hospital of Jilin University, Jilin University, Xinmin Street, Changchun 130061, PR China.
J Pharm Biomed Anal. 2021 Oct 25;205:114345. doi: 10.1016/j.jpba.2021.114345. Epub 2021 Aug 30.
A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry cubed (LC/MS) method was developed and validated for quantification of methotrexate in human plasma. The MS detection is a scanning mode of QTrap MS systems or ion trap MS systems. After simple protein precipitation with methanol, methotrexate and methotrexate-d were separated on an Agilent Poroshell 120 SB-C18 column (4.6 × 50 mm, 2.7 µm) using isocratic elution with a mobile phase consisting of 60% 0.1% formic acid in water and 40% 0.1% formic acid in acetonitrile. The flow rate is 0.8 mL/min. MS detection in positive ion mode used the MRM transitions at m/z 455.2→308.2→175.1 for quantification of methotrexate and m/z 458.2→311.2→175.1 for quantification of methotrexate-d. The total run time was only 3 min for each sample. The LC/MS assay was linear in the concentration range 10-3000 ng/mL(R ≥ 0.995) and the intra- and inter-day accuracies were< 3.72% and precisions were< 7.78% at all concentrations. The absolute recoveries (%) and matrix effect (%) for methotrexate in human plasma were between 92.6 and 114.3. The novelty of the presented methodology is the MS technique resulting in enhanced selectivity and sensitivity. The application of this LC-MS method was successfully completed on 46 human plasma samples and the quantitative results of identical human plasma samples were compared with another LC-MRM based method. Passing-Bablok regression coefficients demonstrated that there is no significant difference between the LC-MS method and LC-MRM method. Bland-Altman plots showed a concordant results, supporting the developed LC-MS method is a reliable and accurate assay for determination of methotrexate in human plasma. This work is also a proof of concept for using LC-MS technique to determination of chemicals in biological samples.
建立并验证了一种简单、高选择性和高通量的液相色谱串联质谱法（LC/MS），用于人血浆中甲氨蝶呤的定量分析。MS 检测采用 QTrap MS 系统或离子阱 MS 系统的扫描模式。经甲醇简单蛋白沉淀后，在 Agilent Poroshell 120 SB-C18 柱（4.6×50mm，2.7μm）上，以含有 60%水和 40%乙腈的 0.1%甲酸的流动相进行等度洗脱，实现甲氨蝶呤和甲氨蝶呤-d 的分离。流速为 0.8mL/min。MS 检测采用正离子模式，甲氨蝶呤的定量分析采用 m/z 455.2→308.2→175.1 的 MRM 跃迁，甲氨蝶呤-d 的定量分析采用 m/z 458.2→311.2→175.1 的 MRM 跃迁。每个样品的总运行时间仅为 3 分钟。LC/MS 分析在 10-3000ng/mL 浓度范围内呈线性（R≥0.995），日内和日间准确度均<3.72%，精密度均<7.78%，所有浓度均如此。人血浆中甲氨蝶呤的绝对回收率（%）和基质效应（%）在 92.6%至 114.3%之间。本方法的新颖之处在于采用 MS 技术提高了选择性和灵敏度。该 LC-MS 方法已成功应用于 46 个人血浆样品，并将相同人血浆样品的定量结果与另一种基于 LC-MRM 的方法进行了比较。Passing-Bablok 回归系数表明，LC-MS 方法与 LC-MRM 方法之间无显著差异。Bland-Altman 图显示了一致的结果，支持所开发的 LC-MS 方法是一种可靠且准确的测定人血浆中甲氨蝶呤的方法。该工作也证明了 LC-MS 技术在生物样品中化学物质测定方面的概念验证。
