Bioanalytical Method Development and Validation for Simultaneous Estimation of Phytomarkers in Novel Anti-diabetic Herbal Formulation

A simple, rapid, precise, and accurate high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of Mangiferin and Quercetin in human serum using Naringin as the internal standard (IS). The analytes were extracted from 500 µL aliquots of human plasma samples by direct liquid-liquid precipitation technique using acetonitrile. Chromatographic separation was achieved using a Chromatopak C18 (250 mm × 4.6 mm i.d., 5 µm particle size) analytical column, with a mobile phase composed of acetonitrile and 0.1% orthophosphoric acid (45:55 v/v) at a flow rate of 1 mL/min and an injection volume of 10 µL. The effluents were monitored at 273 nm with a photodiode array (PDA) detector. The total run time was 10 minutes, with retention times of Mangiferin, Quercetin, and Naringin at 3.543 minutes, 7.263 minutes, and 8.512 minutes, respectively. Linearity was established over a concentration range of 6-16 µg/mL for both Mangiferin (r² = 0.9937) and Quercetin (r² = 0.9905). The lower limit of quantification (LLOQ) was found to be 2.97 µg/mL and 5.52 µg/mL for Mangiferin and Quercetin, respectively. The average percent recovery of the analyte ranged from 89.68% to 97.40% for Mangiferin and 88% to 101.23% for Quercetin. The method was validated according to ICH M10 and EMA guidelines, and the results were within the acceptance criteria. This method is robust and reliable for the simultaneous determination of Mangiferin and Quercetin in human plasma, making it suitable for bioanalytical applications in pharmacokinetic and pharmacodynamic studies of anti-diabetic herbal formulations.