Development and validation of HPTLC method for the estimation of diosgenin in In Vitroculture and rhizome of Dioscorea deltoidea

Abstract

A new simple, accurate, selective, precise, economical and stability-indicating high-performance thin layer chromatographic method for the analysis of diosgenin in callus and rhizome of Dioscorea deltoidea was developed and validated. The method was developed on TLC aluminium plates precoated with silica gel 60F254 using solvent system petroleum ether-isopropanol (12:1, v/v), which gives a compact spot of diosgenin (RF value 0.76 ± 0.02). Densitometric analysis of diosgenin was carried out in the absorbance mode at 366 nm after spraying with methanolic sulphuric acid. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.991 and 0.995 for diosgenin with respect to peak height and peak area, respectively, in the concentration range of 100-1000 ng per spot. The limits of detection and quantification for diosgenin were 16.58 and 50.25 ng per spot. The proposed method was applied for determination of diosgenin in rhizome of D. deltoidea (0.047% w/w) as well as in in vitro culture (callus) (0.092% w/w). Statistical analysis proves that the method is repeatable, selective and accurate for the estimation of diosgenin in D. deltoidea. The developed method effectively resolved the diosgenin in D. deltoidea; hence, it can be employed for routine analysis as a stability indicating method.