SPDS SET 2: ASH-01

Standard Preparation: • Weigh accurately each 5mg of Withanoside IV, Withanoside V Withaferin A, 12 Deoxy Withastramonolide, Withanolide-A and Withanolide-B reference standards to a separate 10 ml volumetric flask.

• Dissolve in 10ml of methanol gently heating and cool then make up to 50ml with methanol.

Sample preparation: Extract: • Weigh accurately a sample quantity of Withania somnifera extract equivalent 10mg (about 5 g will be sufficient) of Withanoside IV, Withanoside V, Withaferin A, 12 Deoxy Withastramonolide, Withanolide A and Withanolide B in a 250ml beaker, • Extract with 100 ml of methanol boiling on water bath for 10-15minutes and repeat the procedure 3 - 4 times till the raw material is completely extracted or till the extracts turn

colorless.

• Combine all the fractions, concentrate and make up the volume to 50ml with methanol. Filter

through 0.45microns membrane filter paper.

Procedure: Inject three times the standard preparation and calculate the mean area and the RSD.

The RSD should not be more than 2%. Inject 20 µ l of sample preparation and record the

chromatogram at 227nm. Calculate the percentage of Withanoside IV, Withanoside V, Withaferin A, 12 Deoxy Withastramonolide, Withanolide A and Withanolide B content from the peak areas

using the formula:

Base to base

Peak integration

:

Area of the sample x Weight of standard in mg x Sample dilution x Purity of standard Area of the standard Standard dilution Sample weight in mg

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