Development of Validated High‑performance Thin‑layer Chromatography Method for Simultaneous Determination of Quercetin and Kaempferol in Thespesia populnea

Introduction: Thespesia populnea L. (Family: Malvaceae) is a well‑known medicinal plant distributed in tropical regions of the world and cultivated in South Gujarat and indicated to be useful in cutaneous affections, psoriasis, ringworm, and eczema. Bark and fruits are indicated in the diseases of skin, urethritis, and gonorrhea. The juice of fruits is employed in treating certain hepatic diseases. The plant is reported to contain flavonoids, quercetin, kaempferol, gossypetin, Kaempferol‑3‑monoglucoside, β‑sitosterol, kaempferol‑7‑glucoside, and gossypol. T. populnea is a common component of many herbal and Ayurvedic formulation such as Kamilari and Liv‑52. Objective: The present study aimed at developing validated and reliable high‑performance thin layer chromatography (HPTLC) method for the analysis of quercetin and kaempferol simultaneously in T. populnea. Method: The method employed thin‑layer chromatography aluminum sheets precoated with silica gel as the stationary phase and toluene: ethyl acetate: formic acid (6:4:0.3 v/v/v) as the mobile phase, which gave compact bands of quercetin and kaempferol. Result: Linear regression data for the calibration curves of standard quercetin and kaempferol showed a good linear relationship over a concentration range of 100–600 ng/spot and 500–3000 ng/spot with respect to the area and correlation coefficient (R2) was 0.9955 and 0.9967. The method was evaluated regarding accuracy, precision, selectivity, and robustness. Limits of detection and quantitation were recorded as 32.06 and 85.33 ng/spot and 74.055 and 243.72 ng/spot for quercetin and kaempferol, respectively. Conclusion: We concluded that this method employing HPTLC in the quantitative determination of quercetin and kaempferol is efficient, simple, accurate, and validated.