title: Simultaneous quantification of withanolides in Withania somnifera by a validated high-performance thin-layer chromatographic method.
abstract: This paper describes a sensitive, selective, specific, robust, and validated densitometric high-performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of 3 key withanolides, namely, withaferin-A, 12-deoxywithastramonolide, and withanolide-A, in Ashwagandha (Withania somnifera) plant samples. The separation was performed on aluminum-backed silica gel 60F254 HPTLC plates using dichloromethane-methanol-acetone-diethyl ether (15 + 1 + 1 + 1, v/v/v/v) as the mobile phase. The withanolides were quantified by densitometry in the reflection/absorption mode at 230 nm. Precise and accurate quantification could be performed in the linear working concentration range of 66-330 ng/band with good correlation (r2 = 0.997, 0.999, and 0.996, respectively). The method was validated for recovery, precision, accuracy, robustness, limit of detection, limit of quantitation, and specificity according to International Conference on Harmonization guidelines. Specificity of quantification was confirmed using retention factor (Rf) values, UV-Vis spectral correlation, and electrospray ionization mass spectra of marker compounds in sample tracks.
authors.affiliation: Central Institute of Medicinal and Aromatic Plants, Analytical Chemistry Division, Lucknow, India.
authors.forename: Pooja
authors.initials: P
authors.lastname: Srivastava
authors.forename: Neerja
authors.initials: N
authors.lastname: Tiwari
authors.forename: Akhilesh K
authors.initials: AK
authors.lastname: Yadav
authors.forename: Vijendra
authors.initials: V
authors.lastname: Kumar
authors.forename: Karuna
authors.initials: K
authors.lastname: Shanker
authors.forename: Ram K
authors.initials: RK
authors.lastname: Verma
authors.forename: Madan M
authors.initials: MM
authors.lastname: Gupta
authors.forename: Anil K
authors.initials: AK
authors.lastname: Gupta
authors.forename: Suman P S
authors.initials: SP
authors.lastname: Khanuja
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid; D002855:Chromatography, Thin Layer; D003720:Densitometry; D005374:Filtration; D007202:Indicators and Reagents; D012015:Reference Standards; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D014465:Ultrasonics; D032341:Withania / Q000737:chemistry*; D054358:Withanolides / Q000032:analysis*
chemical_list: D007202:Indicators and Reagents; D054358:Withanolides