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3DJH RI
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:
Peak integration
:
Base to base
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