Development and validation of high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) methods for the simultaneous determination of myricetin and quercetin in Manilkara hexandra

High-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) techniques are considered for the quantitative analysis of phytochemicals due to their simplicity, speed, and cost-effectiveness. The proposed study is deliberated with the aim of developing HPTLC and HPLC methods validated according to the International Council for Harmonisation (ICH) guidelines for estimating myricetin and quercetin in the leaf extract of Manilkara hexandra, a valuable Ayurvedic plant. Both flavonols were distinctly resolved on the HPTLC plates layered with silica gel 60 F₂₅₄ using the mobile phase toluene‒ethyl acetate‒formic acid (6:6:2.4, V/V) yielding linear range of myricetin 0.5–3 µg/band with an r² value of 0.99, while for quercetin 0.4–1.4 µg/band with r² value of 0.99. The limit of detection (LOD) was 0.13 µg/band for myricetin and 0.07 µg/band for quercetin, with limit of quantification (LOQ) of 0.40 µg/band and 0.21 µg/band, respectively. In the HPLC method developed using mobile phase 0.5% orthophosphoric acid and 100% methanol (2:3, V/V), the linear range of myricetin and quercetin ranged from 5 to 100 µg/mL exhibited an r² value of 0.99 with a retention time (RT) of 7.02 min and LOD and LOQ values of 1.41 µg/mL and 4.30 µg/mL, respectively. Quercetin demonstrated an r² value of 0.99 with an RT of 10.28 min, along with LOD and LOQ values of 1.60 µg/mL and 4.84 µg/mL, respectively. The significance of both proposed methods is evident from the data, based on which we claim that these methods can be used to separate and quantify the two flavonols, myricetin, and quercetin efficiently and accurately in M. hexandra leaf extract.