Simultaneous Quantification of Apremilast and Leflunomide Using Reversed Phase High‑Performance Liquid Chromatography Method in Nano‐Liposomal Convoy: Formulation Optimization, Force Degradation Studies, and Greenness Evaluation

The current explored research shows the developed RP‐HPLC analytical method and its validation for estimation of apremilast and leflunomide in nano‐liposomal convoy. Apremilast, a phosphodiesterase‐4 inhibitor approved for psoriatic arthritis, and leflunomide, a dihydroorotate dehydrogenase inhibitor approved for rheumatoid arthritis. The combination of these two drugs has been explored for synergistic anti‐inflammatory activity in rheumatoid arthritis and minimizing gastro‐intestinal/hepatic toxicity. The gradient method utilizes mobile phase of 10 mM ammonium acetate buffer (unadjusted pH) as aqueous phase and acetonitrile as organic solvent on reversed‐phase Xbridge C‐18 column (4.6 × 150 mm, 5 µm) at a wavelength of 232 and 260 nm for apremilast and leflunomide, respectively. The run time was 12 min at a flow speed of 0.8 mL/min. The validation of developed analytical method followed international council for harmonization guidelines, demonstrating notable selectivity, linearity specificity, and precision, with all values in acceptable limits. The developed analytical method was found linear in a concentration range of 5–150 µg/mL with a regression coefficient of 0.999. Force degradation studies were done to assess stability of apremilast and leflunomide across distinct conditions. Furthermore, apremilast and leflunomide‐loaded liposomes were formulated, optimized, and characterized. The particle size and poly dispersibility index for apremilast‐loaded liposomes were found to be 113.7 ± 8.83 nm and 0.187, whereas particle size and poly dispersibility index for leflunomide‐loaded liposomes were found to be 126.7 ± 5.98 nm and 0.187, respectively. The zeta potential values for apremilast and leflunomide‐loaded liposomes were −14.6 ± 5.6 mV and 0.0831 ± 0.012 mV. The entrapment efficiency and drug loading of apremilast and leflunomide‐loaded liposomes were also determined using developed analytical method. In final analysis, we confirmed eco‐friendliness of developed RP‐HPLC method by evaluating its greenness through analytical GREEnness metric, green analytical procedure index, and eco‐scale. The method was found to be excellent according to these metrics.