A NOVEL RP-HPLC GRADIENT ELUTION TECHNIQUE FOR BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATING GALLIC ACID IN WISTAR RAT PLASMA

Abstract

Objective: Present study aimed to develop and validate a novel, unique, simple, quick, cost-effective, sensitive, specific, accurate, precise, rugged, and robust bioanalytical method for the quantification of gallic acid in rat plasma by reverse phase high-performance liquid chromatography (RP-HPLC) using gradient elution technique. Methods: The stationary phase was a Zorbax SB C18 5 µ (4.6*150) mm column, with the mobile phase being water with 0.1 percent formic acid (A): acetonitrile (ACN) with 0.08 percent formic acid (B). Gradient chromatographic method was used throughout this experiment from the point of view of the estimation of gallic acid from herbal formulations when present along with other phytoconstituents. So at the gradient method, all the present phytoconstituents has cleared off from the column and no any strongly adsorption of phytoconstituents occurred. The experiment was carried out at a flow rate of 1.0 ml/min at 30 °C utilising PDA detectors at 271 nm. The proposed method was validated for different parameters. Results: The approach was found to be linear in the concentration range of 0.5-100 µg/ml, with a r2 of 0.9998. There was not observed any interference of co-eluting peaks of endogenous compounds from the biological matrix at the same retention time (Rt) of gallic acid. The RSD (%) of intra and interday precision was found to be within acceptable limit. The overall % mean recovery was found to be 99.97%. LOD and LOQ were found to be 0.1 and 0.5 μg/ml, respectively. In terms of fluctuation in essential parameters and operating settings, the devised bioanalytical approach was shown to be rugged and resilient. Short-term, long-term, autosampler, bench-top, and freeze-thaw stability experiments revealed that gallic acid is stable. Conclusion: The developed method described in this report was found to be well within an acceptable range. Hence, in the future, this method can be used successfully for the estimation of gallic acid alone or in combination with another analyte or marker present in bulk or an extract containing various phytoconstituents in pharmacokinetic, bioequivalence, and therapeutic drug monitoring studies in clinical laboratories.