Simultaneous quantification of withanolides in Withania somnifera by a validated high-performance thin-layer...

Area of Science:

• Phytochemistry
• Analytical Chemistry
• Pharmacognosy

Background:

• Ashwagandha (Withania somnifera) is a vital medicinal herb.
• Withanolides are key bioactive compounds in Ashwagandha.
• Accurate quantification of withanolides is crucial for quality control and therapeutic efficacy.

Purpose of the Study:

• To develop and validate a sensitive, selective, and robust densitometric HPTLC method.
• To simultaneously determine three major withanolides: withaferin-A, 12-deoxywithastramonolide, and withanolide-A.
• To ensure reliable quantification of withanolides in Ashwagandha plant samples.

Main Methods:

• High-Performance Thin-Layer Chromatography (HPTLC) using silica gel 60F254 plates.
• Mobile phase composition: dichloromethane-methanol-acetone-diethyl ether (15 + 1 + 1 + 1, v/v/v/v).
• Quantification by densitometry at 230 nm in reflection/absorption mode.

Main Results:

• Successful simultaneous determination of withaferin-A, 12-deoxywithastramonolide, and withanolide-A.
• Linear quantification range: 66-330 ng/band with high correlation coefficients (r² ≥ 0.996).
• Method validation confirmed high recovery, precision, accuracy, robustness, and specificity.

Conclusions:

• The developed HPTLC method is sensitive, selective, specific, robust, and validated for withanolide analysis.
• This method provides a reliable tool for the quality assessment of Ashwagandha raw materials and products.
• The validated method adheres to International Conference on Harmonization guidelines for analytical procedures.