Development of HPTLC method for simultaneous determination of quercetin and kaempferol in leaf extract of Hibiscus mutabilis

The aim of this study was to develop and validate a densitometric High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous quantification of quercetin (Q) and kaempferol (K) in Hibiscus mutabilis leaf extracts. The analyses were performed on silica gel 60 F254 plates using a mobile phase composed of toluene, formic acid, and ethyl acetate (6:0.4:4, v/v/v). Detection was carried out at a wavelength of 272 nm using a deuterium and tungsten light source. The method exhibited excellent linearity over the concentration range of 100–600 ng/spot for quercetin and 500–3000 ng/spot for kaempferol, with determination coefficients (r²) of 0.9989 and 0.9973, respectively. The method showed no interferences from the plant matrix. The relative standard deviation (RSD) values for intra- and inter-day precision were less than 2% for both flavonoids. Recovery rates ranged from 97.69% to 99.20% for quercetin and from 89.91% to 95.87% for kaempferol. The limits of detection (LOD) were 190.23 ng/spot for quercetin and 187.23 ng/spot for kaempferol, while the limits of quantification (LOQ) were 570.10 ng/spot for quercetin and 566.12 ng/spot for kaempferol. This validated HPTLC method is reliable, precise, and accurate, making it suitable for the quality control of Hibiscus mutabilis leaf extracts. The study’s findings can be broadly applied to the quality control of herbal products, pharmacological research, and the development of nutraceuticals. The method’s ability to provide rapid and accurate quantification makes it an invaluable tool for researchers across various disciplines.