Simultaneous Determination of Methotrexate and 7‐Hydroxy‐Methotrexate by UHPLC‐MS<sup>3</sup> Assay Coupled With Multiple Stage Fragmentation to Enhance Sensitivity
A selective ultra-high performance liquid chromatography tandem mass spectrometry cubed (UHPLC/MS<sup>3</sup>) 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-d<sub>3</sub>, 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. MRM<sup>3</sup> 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/MS<sup>3</sup> assay for the determination of MTX and 7-OH-MTX was 0.5-300 ng/mL (R<sup>2</sup> ≥ 0.99) and 5-1500 ng/mL (R<sup>2</sup> ≥ 0.99), respectively. The enhanced selectivity and sensitivity are the novelties of the developed UHPLC/MS<sup>3</sup> assay. The analytical method was successfully applied to simultaneous determination of MTX and its major metabolite 7-OH-MTX in real human plasma samples.
Authors
- Kaichun Wu (ORCID: https://orcid.org/0000-0002-0699-9666)
- Huaqiang Li (ORCID: https://orcid.org/0000-0002-6096-3752)
- Xujian Duan
- Lei Yin (ORCID: https://orcid.org/0000-0002-8068-5999)
Institutions
- Dalian Ocean University (CN)
- Jilin University (CN)
- Dalian University of Technology (CN)
- First Hospital of Jilin University (CN)
- Guangdong University of Petrochemical Technology (CN)
- BYD (China) (CN)
Publication Details
- Journal
- Journal of Separation Science
- Published
- 2024-11-01
- DOI
- https://doi.org/10.1002/jssc.70020
- Citations
- 1
- Primary Topic
- Acute Lymphoblastic Leukemia research
- Type
- article
- Field-Weighted Citation Impact
- 0.54
Funders
- Guangdong University of Petrochemical Technology