Determination of Teriflunomide Across a Wide Dynamic Concentration Range in Human Plasma by Lc–ms/MS

Abstract

Leflunomide is an antirheumatic drug with anti-inflammatory and antirheumatic properties, it is rapidly metabolized in the body to the active metabolite teriflunomide, which causes its pharmacological activity. At the usual 20-mg daily dosage of leflunomide, the expected teriflunomide plasma concentration is about 35 μg/ml. The pharmacokinetics of the drug is characterized by a large interindividual variability and a long half-life, which in combination with possible side effects creates the need to control the concentration of teriflunomide in the blood plasma. Teriflunomide may increase the risk of fetal death or teratogenic effects when administered to pregnant women. Teriflunomide plasma concentrations less than 0.02 μg/ml are preferred for patients anticipating pregnancy. In this study, a sensitive and high-performance method for analyzing teriflunomide in human blood plasma in a wide range of concentrations was developed and validated using a triple quadrupole liquid chromatography-mass spectrometer (HPLC-MS/MS). Sample preparation was performed by protein precipitation with acetonitrile, followed by chromatographic separation using an Acquity UPLC BEN C8 1.7 mm, 2.1 × 50 mm column (Waters, USA). D4-teriflunomide was used as an internal standard. The developed method was validated in the concentration range from 0.001 μg/ml to 200 μg/ml teriflunomide in plasma. Accuracy (%), defined as the difference between the nominal and measured concentration and reproducibility (coefficient of variation CV) ranged from -5.02% to 5.00% and from 0.47% to 9.30%, respectively, within the series and between series of samples. The developed method was successfully used to analyze volunteer blood plasma samples after taking 20 mg of leflunomide.