Development and validation of a rapid and sensitive liquid chromatography–tandem mass spectrometry method for benvitimod quantification in human plasma

Benvitimod is a newly synthesized non-steroid small molecule being developed as a candidate drug for the treatment of inflammatory skin diseases. Here a rapid, sensitive and specific high performance liquid chromatography–tandem mass spectrometry (LC/ESI/MS/MS) method was developed for the determination of benvitimod in human plasma. The samples were alkalified with disodium tetraborate firstly, and then extracted by methyl tert-butyl ether. Fluorophenyl-benvitimod was used as internal standard (I.S.). Chromatographic separation was performed on an Ultra C18 column (150 mm × 2.1 mm, 5.0 μm). The mixed mobile phase delivered at 300 μl/min was CH3CN/H2O, 76.65:23.35 (v/v), containing 0.2 mmol/L NH4COOH. Detection and quantitation was performed by electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the negative ion mode. The most intense [M−H]− MRM transition of benvitimod at m/z 253.1→211.0 was used for benvitimod quantitation and the transition at m/z 270.9→229.2 was used to monitor I.S. The calibration curve was linear within the concentration range of 0.1–10.0 ng/mL (r > 0.99). The lower limit of quantification (LLOQ) was 0.1 ng/mL. The extraction recovery was above 80%. The accuracy expressed as relative error (RE) was less than 1.03%. The intra- and inter-day precisions were less than 11.81%. The freeze–thaw stability was also investigated and it was found that both benvitimod and the I.S. were quite stable. This method is especially useful for the pharmacokinetic study of benvitimod.