UPLC-MS/MS Analysis of Methotrexate in Human Plasma and Comparison with the Fluorescence Polarization Immunoassay

• Published in: Analytical Sciences 2017/06/10, Vol.33(6), pp.665-670
• Main Authors: MEI, Shenghui, ZHU, Leting, LI, Xingang, WANG, Jiaqing, JIANG, Xueyun, CHEN, Haiyan, HUO, Jiping, YANG, Li, LIN, Song, ZHAO, Zhigang
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Bland–Altman plot; Blood plasma; Chemistry; Chromatography, High Pressure Liquid; Correlation coefficients; Dosage; Drugs; Elution; Fluorescence; Fluorescence polarization; Fluorescence Polarization Immunoassay; Formic acid; human blood plasma; Humans; Immunoassay; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Methanol; method comparison; method development and validation; Methotrexate; Methotrexate - blood; Methotrexate - chemistry; Molecular Structure; Patients; Polarization; Separation; Tandem Mass Spectrometry; Therapy; UPLC-MS/MS
• ISSN: 0910-6340; 1348-2246
• DOI: 10.2116/analsci.33.665

Methotrexate (MTX) plasma concentration is routinely monitored to guide the dosage regimen of rescue drugs. This study aims to develop and validate an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for plasma MTX analysis, and to establish its agreement with the fluorescence polarization immunoassay (FPIA) in patients with high-dose MTX therapy. Separation was achieved by gradient elution with methanol and water (0.05% formic acid) at 40°C with a run time of 3 min. The intra- and inter-day inaccuracy and imprecision of the UPLC-MS/MS method were –4.25 to 3.1 and less than 7.63%, respectively. The IS-normalized recovery and matrix effect were 87.05 to 92.81 and 124.43 to 134.57%. The correlation coefficients between UPLC-MS/MS and FPIA were greater than 0.98. The UPLC-MS/MS method was in agreement with the FPIA at high levels of MTX (1.0 – 100 μmol/L), but not at low levels (0.01 – 1.0 μmol/L). Further studies are warranted to confirm these results.