HPLC method development and validation for simultaneous analysis of methotrexate and leflunomide in bulk

High performance liquid chromatography (HPLC) is one of the commonly used techniques for the separation and quantification of various pharmaceuticals, both individually as well as in combination. The current studies were aimed to develop and validate a method for the simultaneous determination of methotrexate and leflunomide. These drugs are being used effectively for the treatment of rheumatoid and psoriatic arthritis. For simultaneous estimation of methotrexate and leflunomide, Shimadzu binary liquid gradient HPLC system, equipped with SPD-M40 photo diode array detector, DGU-20A 5R degassing unit, LC-20AD solvent delivery system, C18 column (4.6 mm × 250 mm, 5 µm) and, CTO-20AC oven, was used. Detection and quantification of both drugs were performed at 285 nm wavelength, using mobile phase, consisted of acetonitrile: water (65:35 v/v) along with 10% triethanolamine having adjusted pH 4.0 with ortho-phosphoric acid at a flow rate of 1.0 mL min −1 . The methotrexate and leflunomide were eluted at 1.99 and 6.45 min respectively, with sharp and distinguishing peaks. The Shimadzu's lab solution & Microsoft Excel 2016 was used for statistical analysis. The developed method was validated in accordance with USP <1225> and ICH guidelines Q2 (R1), for different parameters, including system suitability, specificity, linearity, range, accuracy, precision, LOD, LOQ, robustness and solution stability. The outcomes have confirmed that the method was validated with respect to studied parameters, as all of them were well within the specified limits.