Simultaneous qualitative and quantitative analyses of ursolic acid and oleanolic acid in Punica granatum L. (Pomegranate) flowers by high-performance thin-layer chromatography

The purpose of this study was to develop a method for the simultaneous qualitative and quantitative analysis of ursolic acid (UA) and oleanolic acid (OA) in Punica granatum L. flowers. UA and OA were analyzed qualitatively and quantitatively by high-performance thin-layer chromatography (HPTLC). The thin layers chosen were high-performance thin-layer G plates. Sample spots were pretreated in situ with iodine–dichloromethane solution with cyclohexane–ethyl acetate–methanol (8.2:1.8:0.5, V/V) as developing agent upward. The plates were immersed in a 1% sulphuric acid–ethanol solution, dried and heated at 105 °C for 30 min to develop the color and then it was scanned at the wavelength of 530 nm. The results revealed that the RF values for UA and OA were 0.43 ± 0.05 and 0.62 ± 0.05, respectively, with a linear range of 0.2595–3.633 μg and 0.2505–1.503 μg, with correlation coefficients of 0.9980 and 0.9954, representing a sound linear relationship. The limits of detection (LOD) for UA and OA were 10 and 20 ng, with their limits of quantification (LOQ) of 33 and 67 ng, respectively. Average recoveries of 101.38% and 100.13% were obtained with relative standard deviations (RSD) of 2.55% and 2.74%. In summary, it can be concluded that UA and OA in P. granatum L. flowers could be completely separated by in situ pretreatment, and this chromatographic method could quickly and accurately determine the content of UA and OA in P. granatum L. flowers.