Pharmacokinetics and Bioequivalence of Methotrexate in Human Plasma Studied by Liquid Chromatography-Mass Spectrometry (LC-MS)

Methotrexate is a folic acid antagonist that has been in clinical use for five decades. Its use in patients with brain tumors is primarily confined to primary central nervous system lymphoma (PCNSL), in which it is the cornerstone of chemotherapy..
A selective and sensitive high-performance liquid chromatography-electrospray ionization-mass spectrometry method was developed for the determination of methotrexate in human plasma..
Methotrexate was extracted from plasma with acetonitrile. The mobile phase consisted of acetonitrile/water/formic acid 74:25:1 (v/v/v), and 20 µL of the sample was chromatographically analyzed using a repacked ZORBAX-XDB-ODS C18 column (2.1 × 30 mm, 3.5 microns). The mode of mass spectrometry was selected-ion monitoring (SIM). The standard curve was linear (r = 0.998) over a concentration range of 0.1 - 100.0 ng/mL, and showed suitable accuracy and precision..
The limit of detection (LOD) was 0.05 ng/mL. The mean (± SD) Cmax, Tmax, AUC0-t, and AUC0–∞ values after administration of the test and reference formulations, respectively, were as follows: 13.94 (± 4.36) versus 13.49 (± 3.67) ng/mL, 2.63 (± 1.45) versus 2.75 (± 1.74) hours, 122.57 (± 54.34) versus 125.94 (± 53.09) ng/mL/h, and 140.74 (± 56.69) versus 155.80 (± 65.11) ng/mL/h. The mean (± SD) t1/2 was 5.32 (± 2.01) hours for the test formulation and 5.34 (± 2.13) hours for the reference formulation. No statistical differences were shown for Cmax or the area under the plasma concentration-time curve for the test and reference tablets. The calculated 90% confidence intervals, based on ANOVA analysis for the mean test/reference ratios of Cmax, AUC0-∞, and AUC0-th of methotrexate, were in the bioequivalence range (96% - 101%)..
The developed LC-MS method is quick, easy, stable, and precise for the partition, assignment, pharmacokinetic, and bioavailability evaluation of methotrexate in healthy Iranian adult male volunteers..