Simultaneous quantification of four active metabolites in Psidium guajava L. by a validated high-performance thin-layer chromatography method

Abstract

A simple, rapid, sensitive and reproducible method was developed for the simultaneous quantification of four bioactive compounds, quercetin, gallic acid, eugenol and β-sitosterol, by high-performance thin-layer chromatography (HPTLC) in Psidium guajava L. HPTLC was performed on silica gel 60 F₂₅₄ by using toluene‒ethyl acetate‒formic acid (7:3:1, V/V) for quercetin and gallic acid and toluene‒ethyl actetate‒formic acid (9:1:0.5, V/V) for β-sitosterol and eugenol as the mobile phase, at retention factor values of 0.52 ± 0.06, 0.28 ± 0.05, 0.87 ± 0.02 and 0.59 ± 0.04 for quercetin, gallic acid, eugenol and β-sitosterol, respectively. The quantifications of gallic acid, quercetin, eugenol and β-sitosterol were carried out using a densitometric absorption mode at 254 nm, 370 nm, 570 nm and 600 nm. The developed method was validated and found to be specific, linear and accurate with precision and accuracy in the concentration range of 200‒1000 ng/mL for gallic acid, eugenol and β-sitosterol and 100‒500 ng/mL for quercetin. Precision studies (both inter-day and intra-day) are within the standard limit of RSD (%) less than 3% (Table 1). The contents of the marker compounds, viz., quercetin (0.0031), gallic acid (0.223), eugenol (0.0052%) and β-sitosterol (0.0466) % dry weight basis were detected in of Psidium guajava. The statistical data analysis showed that the method is reproducible and selective for the estimation of all the four bioactive compounds in extracts of Psidium guajava.

Table 1 Calibration parameter of standards (quercetin, gallic acid, eugenol, β-sitosterol)