RP-HPLC method development and validation for the simultaneous estimation of Piceatannol and -Arginine in pharmaceutical formulations

Dental caries results from microbial dysbiosis, making microbiota balance a superior strategy to antibiotics, which can disrupt oral flora and promote resistance. The Piceatannol–L-Arginine combination helps prevent caries by inhibiting biofilms and supporting beneficial microbes that provide competition to pathogenic microbes. A novel analytical method is needed to simultaneously quantify Piceatannol and L-Arginine, addressing the current lack of techniques capable of accurately measuring both compounds together. The study aims to validate a newly developed RP-HPLC method for the accurate and precise quantification of Piceatannol and L-Arginine, in accordance with ICH Q2(R1) guidelines. An efficient RP-HPLC method was developed, wherein acceptable chromatographic separation was achieved using a Shimadzu C18 column (250 × 4.6 mm × 5 µm) with a mobile phase of 0.1% Orthophosphoric Acid (in Water): acetonitrile (70:30). The column temperature was maintained at 25 0 C with an injection volume of 10 µl. UV detection was performed at 215 nm using a photodiode-array detector. Validation of the developed method demonstrated accuracy, precision, linearity, robustness, and solution stability. The retention times of Piceatannol and L-Arginine were observed at 1.931 and 7.699 mins, respectively. For L-Arginine (100 µg/ml to 600 µg/ml) and Piceatannol (1 µg/ml to 6 µg/ml), calibration curves were linear, with correlation coefficients of 0.9912 and 0.9917, respectively. The developed RP-HPLC method demonstrated good resolution, as both peaks were well separated, with an analysis time of 12 mins. It was subsequently validated and employed to quantify the PIC and L-Arg in the developed flexi-liposomal gel precisely.