Development and Validation of a Simultaneous Bioanalytical Method for Methotrexate, Sulfasalazine and Hydroxychloroquine in Rat Plasma following Single Step Protein Precipitation Technique

Background: Triple therapy of methotrexate, sulfasalazine and hydroxychloroquine is widely used in the treatment of rheumatoid arthritis. Different studies reported the superior efficacy of combination of these three drugs in the improvement of this disease state. However, there is still a lot of scopes remains for preclinical study of this combination in future. Establishment of bioanalytical method is essential for quantitating these analytes in the samples from such types of studies. Therefore, in this study, we aim to develop a simultaneous method for quantification of methotrexate, sulfasalazine and hydroxychloroquine in rat plasma through a single run. Materials and Methods: The method includes a simple single step protein precipitation technique for extraction of all the three analytes from rat plasma with more than 84% recovery. The method was validated according to the USFDA guideline for a calibration range of 0.50-10μg/mL. Results: Overall, the method showed acceptable accuracy (92.14-116.12%) and precision (%coefficient of variation; 0.79-11.94%) at lower limit of quantification and three quality control levels. Analytes were found to be stable in all the tested stability study conditions. Conclusion: We have established a simultaneous bioanalytical method for methotrexate, sulfasalazine and hydroxychloroquine in rat plasma. Applicability of the method has been established through an oral pharmacokinetic study of the combination in rat.