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Simultaneous Quantification of Withanolides in Withania somnifera by a Validated High-Performance Thin-Layer Chromatographic Method

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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 dichloromethanemethanolacetonediethyl 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 66330 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.
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Document Type: Research Article

Affiliations: 1: Central Institute of Medicinal and Aromatic Plants, Analytical Chemistry Division, Lucknow226015, India. 2: Central Institute of Medicinal and Aromatic Plants, Genetic Resource and Biotechnology Division, Lucknow226015, India.

Publication date: 2008-09-01

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  • The Journal of AOAC INTERNATIONAL publishes refereed papers and reviews in the fields of chemical, biological and toxicological analytical chemistry for the purpose of showcasing the most precise, accurate and sensitive methods for analysis of foods, food additives, supplements and contaminants, cosmetics, drugs, toxins, hazardous substances, pesticides, feeds, fertilizers and the environment available at that point in time. The scope of the Journal includes unpublished original research describing new analytical methods, techniques and applications; improved approaches to sampling, both in the field and the laboratory; better methods of preparing samples for analysis; collaborative studies substantiating the performance of a given method; statistical techniques for evaluating data. The Journal will also publish other articles of general interest to its audience, e.g., technical communications; cautionary notes; comments on techniques, apparatus, and reagents.
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