Analytical Sciences

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

Shenghui MEI,*,** Leting ZHU,* Xingang LI,*,** Jiaqing WANG,* Xueyun JIANG,** Haiyan CHEN,** Jiping HUO,* Li YANG,*,** Song LIN,*** and Zhigang ZHAO*,**

*Department of Pharmacy, Beijing Tiantan Hospital, Capital Medical University, 6 Tiantan Xili, Dongcheng District, Beijing 100050, P. R. China
**Department of Clinical Pharmacology, College of Pharmaceutical Sciences, Capital Medical University, Beijing 100045, P. R. China
***Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, 6 Tiantan Xili, Dongcheng District, Beijing 100050, P. R. China

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.

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