lc-ms/ms method for the quantification of teriflunomide in human plasma: application to patients with multiple sclerosis

Teriflunomide (tf) is a disease modifying antirheumatic drug of the isoxazole class which is primarily used for the treatment of rheumatoid arthritis and multiple sclerosis [1]. tf has a long half-life and the plasma clearance of this drug in plasma is very slow. moreover, a non-teratogenic plasma concentration of tf was considered 20 ng/ml based on the animal data. therefore, in case of safe pregnancy, the tf plasma concentration must reach below the medical decision point of 20 ng/ml through fast elimination methods [2,3]. therefore, we aimed to develop and validate an lc–ms/ms method for the measurement of tf in human plasma. the chromatographic separation was achieved on a ymc ods-am column (150 mm × 3.0 mm, 3 µm) using a gradient mobile phase consisting of solvent a (water, containing 0.1% formic acid) and mobile phase b (acetonitrile: methanol: water, 18:1:1 v/v, containing 0.1% formic acid) with a run time of 12.0 min at a flow rate of 0.4 ml/min. detection was carried out by a triple–quadrupole tandem mass spectrometer in multiple reaction monitoring (mrm) in negative mode (at m/z 269.1/160.0 and 269.1/82.0). the method has been fully validated based on the guideline on bioanalytical method validation. the weighted calibration curves were linear over the range of 4- 400 µg l-1 in plasma with relative standard deviations and relative errors lower than 15%. intra- and inter-day precision and accuracy using quality control (qc) samples were within the generally accepted criteria for bioanalytical method. method selectivity, specificity, matrix-effect, and autosampler stability also have been studied. the developed method was successfully applied to determine the tf plasma concentration in patients with multiple sclerosis who have eliminated the drug to get pregnant.