# SPDS SET 2: ASH-01

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

R

=

2

x

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

F

=

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 %

=

x

x

x

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

12-Deoxywithastramonolide

Withanolide A Withanolide B

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