Development and Validation of a New RP-HPLC Method for Simultaneous Analysis of Cyclosporine A and α-Linolenic Acid in Pharmaceuticals

To test the synergistic therapeutic impact of the synthetic combination of cyclosporine A and
-linolenic acid (ALA), an appropriate isocratic reverse-phase HPLC technique was created. Cyclosporine A is used as an immunosuppressant drug in post-organ transplantation to prevent rejection. It has been used widely for the treatment of certain autoimmune diseases such as severe rheumatoid, arthritis, psoriasis, and inflammatory skin conditions. The reversed phase HPLC chromatographic method was developed to obtain two separate peaks for both drugs within a short retention period (30 min run time), without any interference from excipient peaks. The chromatographic separation was achieved on the C18 column (5 µm, 4.6 × 250 mm) at 50 ± 0.3 ºC. The analytes were eluted at a flow rate of 0.8 mL/min using an ultraviolet detector at 210 nm with a mobile phase of 1% v/v orthophosphoric acid in water: acetonitrile:2-propanol (25:60:25 v/v/v). The method was validated for specificity, precision, linearity, accuracy, and system suitability. The average retention time for cyclosporine and ALA was found to be 16.1 minutes and 18.6 minutes respectively. The accuracy of the assay was within ± 2% of the true value and the method was found to be linear from 1.5 g/ml to 4.5 g/ml for cyclosporine and 5.0 µg/ml to 15
g/ml for ALA. The method provided selectivity based on resolution among peaks and the recovery was 98% to 102%. The new RP-HPLC method successfully separated two peaks from the interfering chromatographic peaks of the excipients. The high resolution obtained at a comparatively lower temperature (50°C) makes this method more promising and useful. All validation parameters were within the acceptable range.