Development and Validation of a Bioanalytical Method for Estimating Ellagic Acid in Wistar Rat Plasma by RP-HPLC Using a Gradient Elution Technique

Abstract

Background: Ellagic acid (EA) is a natural polyphenolic compound recognized for bioactive and pharmacologic properties that is found abundantly in various plant groups, particularly eudicotyledons. In the current study a novel, reliable, and cost-effective bioanalytical method was developed for quantifying EA in Wistar rat plasma using RP-HPLC. Methods: Zorbax SB C18 (5μ, 4.6150 mm) and Ascentis C18 (5μ, 1004.6 mm) columns were used in the stationary phase, while the mobile phase consisted of water with 0.1 % formic acid (A) and acetonitrile (ACN) with 0.08% formic acid (B). Results: Optimized parameters were as follows: temperature, 30°C; flow rate, 1.0 ml/min; and PDA detectors, 254 nm. The method exhibited high linearity (r2 = 0.9993) between 5 and 300 μg/ml. Precision, both intra- and inter-day, was within acceptable limits (relative standard deviation <2%), and the mean recovery was 99.73%. The LOD and LOQ were 1.564 ± 0.026 μg/ml and 5.015 ± 0.025 μg/ml, respectively. Stability tests, including short- and long-term evaluations, demonstrated stability under various conditions. Conclusion: The developed method met the necessary criteria and holds promise for application in clinical laboratories for assessing EA levels, either alone or with analytes.