Simultaneous Determination of Methotrexate and 7-Hydroxy-Methotrexate by UHPLC-MS3 Assay Coupled With Multiple Stage Fragmentation to Enhance Sensitivity

IF 2.8 3区工程技术 Q2 CHEMISTRY, ANALYTICAL Journal of separation science Pub Date : 2024-11-08 DOI:10.1002/jssc.70020

Huaqiang Li, Xujian Duan, Feifei Wu, Lei Yin

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Abstract

A selective ultra-high performance liquid chromatography tandem mass spectrometry cubed (UHPLC/MS³) assay for simultaneous determination of methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) in human plasma was developed and validated. After protein precipitation with methanol, chromatographic separation of MTX, MTX-d₃, and 7-OH-MTX was performed on a Waters AcQuity UPLC-BEH column (2.1 × 50 mm I.D., 1.7 µm) with gradient elution. MRM³ transition was used for the detection of MTX (m/z 455.2→308.0→175.1) and 7-OH-MTX (m/z 471.3→191.0→148.1). The linear range of UHPLC/MS³ assay for the determination of MTX and 7-OH-MTX was 0.5–300 ng/mL (R² ≥ 0.99) and 5–1500 ng/mL (R² ≥ 0.99), respectively. The enhanced selectivity and sensitivity are the novelties of the developed UHPLC/MS³ assay. The analytical method was successfully applied to simultaneous determination of MTX and its major metabolite 7-OH-MTX in real human plasma samples.

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利用超高效液相色谱-质谱 3测定法同时测定甲氨蝶呤和 7-羟基甲氨蝶呤，并采用多级碎片化技术提高灵敏度

开发并验证了一种选择性超高效液相色谱-串联质谱立方（UHPLC/MS3）测定法，用于同时测定人体血浆中的甲氨蝶呤（MTX）和 7-羟基甲氨蝶呤（7-OH-MTX）。用甲醇沉淀蛋白后，在 Waters AcQuity UPLC-BEH 色谱柱（内径 2.1 × 50 mm，1.7 µm）上对 MTX、MTX-d3 和 7-OH-MTX 进行梯度洗脱。采用 MRM3 转换检测 MTX（m/z 455.2→308.0→175.1）和 7-OH-MTX（m/z 471.3→191.0→148.1）。超高效液相色谱/质谱 3 检测器测定 MTX 和 7-OH-MTX 的线性范围分别为 0.5-300 ng/mL（R2 ≥ 0.99）和 5-1500 ng/mL（R2 ≥ 0.99）。所开发的超高效液相色谱/质谱三重检测法具有更高的选择性和灵敏度。该分析方法成功地应用于实际人体血浆样品中MTX及其主要代谢物7-OH-MTX的同时测定。

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来源期刊

Journal of separation science 化学-分析化学

CiteScore

6.30

自引率

16.10%

发文量

408

审稿时长

1.8 months

期刊介绍： The Journal of Separation Science (JSS) is the most comprehensive source in separation science, since it covers all areas of chromatographic and electrophoretic separation methods in theory and practice, both in the analytical and in the preparative mode, solid phase extraction, sample preparation, and related techniques. Manuscripts on methodological or instrumental developments, including detection aspects, in particular mass spectrometry, as well as on innovative applications will also be published. Manuscripts on hyphenation, automation, and miniaturization are particularly welcome. Pre- and post-separation facets of a total analysis may be covered as well as the underlying logic of the development or application of a method.