SPDS SET 2: ASH-01

6.0] VALIDATION RESULT (CONCLUSIONS):

6.1) HPLC consisting of the following modules are least required for the performing the analysis. a) Pump

b) UV / PDA detector (227nm ± 3)

c) Auto injector

d) Column oven e) Data processor / Integrator / Control software

Any Octa Decyl Siliane (ODS) column 250 x 4.0 mm, 5 µ particle size, 100 A° pore size with

carbon content >10% and end capped is suitable. Particle size up to 3 µ and

dimension up to 15 cms and diameter up to 4.6mm can also be used. In most of the C18

column a peak which is eluting just before Withanoside IV may merge with it leading to higher content of Withanoside IV. Hence Merck Hibar Lichrosphere, Phenomenex, Luna

are the validated columns for this estimation.

6.2) Specificity: PDA spectra and the peak purity of the Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy Withastramonolide, Withanolide A, Withanolide B indicate the method is very specific for withanolides and there is no interference from any other derivatives present in the extract which is confirmed by the resolution of Withanoside IV,

Withanoside V, Withaferin A, 12-Deoxy Withastramonolide, Withanolide A, Withanolide B.

Permissible variation allowed in the mobile phase concentration is ± 3% of each solvent.

The column to be maintained constant temperature (a temperature of 20 – 35) is suitable.

6.3) Linearity: The Correlation Coefficient (r 2 ) for withanolides was found to be > 0.98 indicating the method is linear. Though the method is linear up to concentration 1000mcg, it is advisable to operate within

20-200mcg where the linearity is the best.

6.4) Precision: The RSD was found to be within the specified limits for area, retention time and for the precision of assay indicating the method is precise and reproducible.

The method is precise. The variation in the retention time is found to be within 1% RSD.

Hence the variation of the retention time is to be ± 3 σ of the mean of the retention time of

standard and reference extract. Number of injections of standard or reference extract used

for calculation of RT window should be ≥ 3 and to be done initially and at the end of the

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