SPDS SET 2: ASH-01

1.2] SUMMARY REPORT OF VALIDATION:

Specificity Very specific for Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy Withastramonolide, Withanolide A & Withanolide B with no interference from other compounds. Confirmed by the PDA spectra

Accuracy Acceptance criterion for recovery: 75 to 125% for assay > 0.1 and < 0.5%. 85 to 120% for assay > 0.1 and < 0.5%. 70 to 130% for assay < 0.1%. Compound

and the peak purity. Peak purity > 0.98

Recovery

Withanoside IV Withanoside V Withaferin A RSD 12-Deoxy Withastramonolide

94.6 to 101.8% 95.3 to 101.2% 95.9 to 100.7% 91.2 to 101.6%

Ruggedness

One sample, 2 different HPLC columns, 2 different conditions and 2 different instruments Acceptance criterion: RSD = < 6.0% Actual RSD% = 5.0%

Withanolide A Withanolide B

93.7 to 99.6% 74.0 to 106.0%

Precision (of Concentration, Retention time and Reproducibility) 7 concentrations of each, 5 replicates of each Acceptance criterion: RSD = < 2.5%

Compound

RSD

Retention time

Peak area

Withanoside IV Withanoside V

0.62 0.55 0.49 0.38 0.34 0.21

0.5%

0.86% 0.5% 0.6% 1.5% 0.9%

Withaferin A

12-Deoxy Withastramonolide

Withanolide A Withanolide B

Range of quantification The following limits are considered as a better working range based on linearity and closeness (within ± 5%) to mean response factor. Compound Concentration (mcg/ml) Withanoside IV 20.64 – 330.3 Withanoside V 18.76 – 300.15 Withaferin A 23.05 – 184.39 12-Deoxy Withastramonolide 22.21 – 177.65 Withanolide A 21.59 – 172.76 Withanolide B 21.09 – 168.79 Linearity 7 concentrations of each, 5 replicates of each Acceptance criterion: Correlation coefficient (r 2 ) for all compounds to be >0.98 Compound r 2

Stability of Sample & Standard Solutions

Sample solution tested after 24 hrs. The variation in assay found to be + 3.2%

The solution is stable for 24 hours. Standard solution is also stable for 24 hours.

System suitability

1) The resolution is >3 for Withanoside V and Withaferin A in the standard mix. 2) The asymmetric factor (tailing factor) should be < 1.5 3) The precision of injection (with minimum of 3 replicates) RSD should be < 2.5%. 4) Relative retention time of Withanoside IV - 0.7 Withanoside V - 0.89 Withaferin A - 0.92 12-Deoxy Withastramonolide - 0.96 Withanolide A - 1.0 Withanolide B - 1.15

Concentration range(mcg/ml)

Withanoside IV Withanoside V

0.999 0.999 0.996 0.990

20.0 to 1321 18.7 to 1200 23.0 to 737 22.0 to 355

Withaferin A

12-Deoxy Withastramonolide

Withanolide A Withanolide B

0.992 0.984

21.0 to 345 21.0 to 337

Conclusion: The HPLC method [NR/QCD/APM04 WI(17)] for the estimation of Withania somnifera is validated.

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3DJH RI