Temperature : Maintained at a constant temperature between 20 to 30°C (preferably 27°C) Detector : SPD-M 10Avp Photo diode array detector or UV Detector

Wave length : 227 nm Flow rate : 1.5ml/ min Run time : 45minutes.

( b ) Retention time relative .—

Relative retention time of

Withanoside IV - 0.7 Withanoside V - 0.89 Withaferin A - 0.92 12-Deoxywithastramonolide - 0.96

Withanolide A - 1.0 Withanolide B - 1.15

( c ) System suitability .—( 1 ) Repeatability.— The RSD of each of the individual withanolides peak area for at least 5 consecutive injections of the level 4 linearity standard solution must be  %.

( 2 ) Resolution .—Calculate the resolution between Withanoside V and Withaferin A peaks as follows:

T2 - T1 W1 + W2





Where T1 and T2 are the retention times of Withanoside V and Withaferin A respectively and W1 and W2 is their peak widths measured at the base line between tangents drawn to the peak sides. The resolution between ephedrine and pseudoephedrine in should be  3.0.

( 3 ) Tailing.— Calculate the tailing factor (F) as follows:

L + R 2L



where L is the width from start of the peak to the perpendicular from the peak apex at 5% of the peak height; R is the width from the perpendicular from the peak apex to the peak end at 5% of the peak height. The tailing factor must be  1.5 for all individual withanolides in the linearity standard solution chromatograms.

( 4 ) Determination coefficient .—the r 2 for the regression line of peak area vs. concentration for each withanolide must be  0.998.

Procedure: Inject three times the standard preparation and calculate the mean area and the RSD. The RSD should not be more than 2%. Inject 20μl of sample preparation and record the chromatogram at 227nm. Calculate the percentage of Withanoside IV, Withanoside V, Withaferin A, 12-Deoxywithastramonolide, Withanolide A and Withanolide B content from the peak areas using the formula:

Peak integration: Base to base

Individual Withanolide (%w/w)

Area of the sample

Weight of standard (mg)

Sample dilution

Purity of standard %





Area of the standard

Standard dilution

Sample weight (mg)

Compound name

CAS no.

Withanoside IV Withanoside V Withaferin A

362472-81-9 256520-90-8 5119-48-2 60124-17-6 32911-62-9 56973-41-2


Withanolide A Withanolide B

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