Development of an AQbD driven HPLC method for the simultaneous estimation of caffeine and misoprostol in nanoparticle formulations

Abstract

Orthodontic treatment involves the use of braces and clear aligners to correct the misalignment of teeth and jaws, thus enhancing functionality, aesthetics and overall dental health. The significant drawback of this treatment is its lengthy duration. Caffeine and misoprostol have exhibited the potential to enhance orthodontic tooth movement and decrease treatment time. However, no combined formulation of these two agents is currently available and a validated method for their simultaneous estimation has not been yet established. To address this gap, our research group is actively working on developing a novel formulation incorporating both caffeine and misoprostol for orthodontic applications. As a critical first step toward this goal the present study focuses on employing the AQbD approach using a Box–Behnken design for the simultaneous estimation of caffeine and misoprostol. The optimized mobile phase consisted of acetonitrile, HPLC-grade water and 1.25 × 10⁻⁴ M orthophosphoric acid solution in a ratio of 69.5 : 30 : 0.5 (v/v/v). Key parameters of the method were optimized, which included a flow rate of 0.9 ml min⁻¹, an injection volume of 20 µl and a column temperature of 40 °C. Under these chromatographic conditions the retention times for caffeine and misoprostol were 2.8 and 4.9 minutes, respectively, with both exhibiting an acceptable tailing factor of 1.1. Calibration plots demonstrated excellent linearity for both compounds. The method's sensitivity was assessed with the LOD and LOQ determined to be 0.51 µg ml⁻¹ and 1.57 µg ml⁻¹ for caffeine and 0.40 µg ml⁻¹ and 1.23 µg ml⁻¹ for misoprostol, respectively. The method was then validated according to ICH Q2(R2) guidelines. Also, the quantitative analysis of lab developed nanoparticles demonstrated a recovery of no less than 98% with a relative standard deviation (RSD) of no more than 2%. This validated method provides a reliable analytical approach for the simultaneous estimation of caffeine and misoprostol in pharmaceutical formulations, thereby strengthening quality control in orthodontic treatment formulations.