Alka Puri1, Bibhu P Panda2. 1. Microbial and Pharmaceutical Biotechnology Laboratory, Centre of Advance Research in Pharmaceutical Sciences, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India. 2. Microbial and Pharmaceutical Biotechnology Laboratory, Centre of Advance Research in Pharmaceutical Sciences, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India bibhu_panda31@rediffmail.com.
Abstract
A simple, accurate and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of three glycosidic isoflavones (daidzin, genistin and glycitin) in soybean (Glycine max L.) has been established and validated. Chromatography was performed on aluminum foil-backed silica gel 60 F254 HPTLC plates and found compact spots for daidzin, genistin and glycitin (Rf value of 0.39, 0.51 and 0.32, respectively) with mobile phase toluene : ethyl acetate : formic acid : acetic acid in the ratio of 1 : 8 : 1 : 0.5, v/v/v/v. Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 260 nm. The method was validated for precision, recovery, robustness, specificity, limit of detection (LOD) and limit of quantification (LOQ), in accordance with the ICH guidelines. The LOD (2.9, 19.3 and 3.5 µg mL(-1)), LOQ (9.03, 58.6 and 10.7 µg mL(-1)), recovery (95.9-106.66, 86.97-106.56 and 98.54-105.65%) and precision (≤2.12, ≤0.722 and ≤0.066) were satisfactory for glycosidic form of isoflavones daidzin, genistin and glycitin, respectively. Soybean variety Kh-09 bragg was found to have relatively higher amount of glycosidic isoflavones, namely daidzin, genistin and glycitin 278, 597.5 and 109.4 µg g(-1), respectively, and after fermentation the glycosidic isoflavones concentration in soybean fermented with Bacillus subtilis strain were decreased significantly after 24 h of incubation; conversely, aglycone isoflavones were increased significantly. The method for quantification of isoflavones in unfermented and fermented soybeans, with good resolution has been developed.
A simple, accurate and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of three glycosidic isoflavones (daidzin, genistin and glycitin) in soybean (Glycine max L.) has been established and validated. Chromatography was performed on aluminum foil-backed silica gel 60 F254 HPTLC plates and found compact spots for daidzin, genistin and glycitin (Rf value of 0.39, 0.51 and 0.32, respectively) with mobile phase toluene : ethyl acetate : formic acid : acetic acid in the ratio of 1 : 8 : 1 : 0.5, v/v/v/v. Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 260 nm. The method was validated for precision, recovery, robustness, specificity, limit of detection (LOD) and limit of quantification (LOQ), in accordance with the ICH guidelines. The LOD (2.9, 19.3 and 3.5 µg mL(-1)), LOQ (9.03, 58.6 and 10.7 µg mL(-1)), recovery (95.9-106.66, 86.97-106.56 and 98.54-105.65%) and precision (≤2.12, ≤0.722 and ≤0.066) were satisfactory for glycosidic form of isoflavonesdaidzin, genistin and glycitin, respectively. Soybean variety Kh-09 bragg was found to have relatively higher amount of glycosidic isoflavones, namely daidzin, genistin and glycitin 278, 597.5 and 109.4 µg g(-1), respectively, and after fermentation the glycosidic isoflavones concentration in soybean fermented with Bacillus subtilis strain were decreased significantly after 24 h of incubation; conversely, aglycone isoflavones were increased significantly. The method for quantification of isoflavones in unfermented and fermented soybeans, with good resolution has been developed.