Chromatographic separation and sensitive determination of teriflunomide, an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 878; no. 24; pp. 2217 - 2225
• Main Authors: Parekh, Jignesh M., Vaghela, Rajendrasinh N., Sutariya, Dipen K., Sanyal, Mallika, Yadav, Manish, Shrivastav, Pranav S.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.08.2010; Elsevier
• Subjects: Acetates; Adult; Analysis; Analytical, structural and metabolic biochemistry; Bioequivalence study; Biological and medical sciences; Chromatography; Chromatography, Liquid - methods; Computational efficiency; Computing time; Crotonates - blood; Crotonates - chemistry; Crotonates - metabolism; Crotonates - pharmacokinetics; Drug Stability; Fundamental and applied biological sciences. Psychology; General pharmacology; High-Throughput Screening Assays - methods; Human; Human plasma; Humans; Isoxazoles - administration & dosage; Isoxazoles - chemistry; Isoxazoles - metabolism; Isoxazoles - pharmacokinetics; LC-ESI-MS; LC–ESI-MS/MS; Least-Squares Analysis; Liquid chromatography; Male; Mass spectrometry; Medical sciences; Metabolites; Middle Aged; Pharmacology. Drug treatments; Reproducibility of Results; Run time (computers); Sensitivity and Specificity; Separation; Spectrometry, Mass, Electrospray Ionization - methods; Tandem Mass Spectrometry - methods; Teriflunomide; Therapeutic Equivalency; Toluidines - blood; Toluidines - chemistry; Toluidines - metabolism; Toluidines - pharmacokinetics; Teriflunomide; Bioequivalence study; Human plasma; LC–ESI-MS/MS; Human; Prostaglandin-endoperoxide synthase; Biological fluid; Isoxazole derivatives; Bioequivalence; Enzyme; Metabolite; Enzyme inhibitor; HPLC chromatography; Determination; Leflunomide; Electrospray; Blood plasma; Non steroidal antiinflammatory agent; LC―ESI-MS/MS; Oxidoreductases; Antirheumatic agent; Immunosuppressive agent; Pharmacokinetics; Quantitative analysis
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2010.06.028

A sensitive, selective and high throughput liquid chromatography tandem mass spectrometry (LC–ESI-MS/MS) method has been developed for the determination of teriflunomide, an active metabolite of leflunomide in human plasma. Plasma samples were prepared by liquid–liquid extraction of teriflunomide and valsartan as internal standard (IS) in ethyl acetate from 200 μL human plasma. The chromatographic separation was achieved on an Inertsil ODS-3 C18 (50 mm × 4.6 mm, 3 μm) analytical column using isocratic mobile phase, consisting of 20 mM ammonium acetate–methanol (25:75, v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for teriflunomide ( m/ z 269.0 → 82.0) and IS ( m/ z 434.1 → 350.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over a wide dynamic concentration range of 10.1–4001 ng/mL. Matrix effect was assessed by post-column infusion experiment and the mean process efficiency were 91.7% and 88.2% for teriflunomide and IS respectively. The method was rugged and rapid with a total run time of 2.0 min and is applied to a bioequivalence study of 20 mg leflunomide (test and reference) tablet formulation in 12 healthy Indian male subjects under fasting condition.