pharmacokinetics and bioequivalence of methotrexate in human plasma studied by liquid chromatography-mass spectrometry (lc-ms)

Background: 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 systemlymphoma(pcnsl), in which it is the cornerstone of chemotherapy. objectives: a selective and sensitive high-performance liquid chromatography-electrospray ionization-mass spectrometry method was developed for the determination of methotrexate in human plasma. materials and methods: methotrexate was extracted from plasma with acetonitrile. the mobile phase consisted of acetonitrile/ water/formic acid 74:25:1 (v/v/v), and 20µl of the sample was chromatographically analyzed using a repacked zorbax-xdb-ods c18 column (2.1x30 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. results: the limit of detection (lod) was 0.05 ng/ml. the mean (±sd) cmax, tmax, auc0-∞, and auc0–1values 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, and140.74 (±56.69) versus 155.80 (±65.11) ng/ml/h. themean(±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 onanova analysis for themeantest/reference ratios of cmax, auc0-1, and auc0-∞ of methotrexate, were in the bioequivalence range (96% - 101%). conclusions: 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.