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 (IS.). Chromatographic separation was performed on an Ultra C-18 column (150 mm x 2.1 mm, 5.0 mu m). The mixed mobile phase delivered at 300 mu l/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 1.5. were quite stable. This method is especially useful for the pharmacokinetic study of benvitimod. (C) 2012 Elsevier B.V. All rights reserved.