ASH-001_AOAC_Withania_Submission

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1.2] SUMMARY REPORT OF VALIDATION:
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 Recovery
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
and the peak purity.
Peak purity > 0.98
Withanoside IV 94.6 to 101.8%
Withanoside V 95.3 to 101.2%
Withaferin A RSD 95.9 to 100.7%
12-Deoxy
Withastramonolide
91.2 to 101.6%
Withanolide A 93.7 to 99.6%
Ruggedness
One sample, 2 different HPLC columns, 2 different
conditions and 2 different instruments
Acceptance criterion: RSD = < 6.0%
Actual RSD% = 5.0% Withanolide B 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%
RSD Compound
Retention time Peak area
Withanoside IV 0.62 0.5%
Withanoside V 0.55 0.86%
Withaferin A 0.49 0.5%
12-Deoxy Withastramonolide 0.38 0.6%
Withanolide A 0.34 1.5%
Withanolide B 0.21 0.9%
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
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.
Linearity
7 concentrations of each, 5 replicates of each
Acceptance criterion: Correlation coefficient (r2) for all
compounds to be >0.98
Compound r2 Concentration
range(mcg/ml)
Withanoside IV 0.999 20.0 to 1321
Withanoside V 0.999 18.7 to 1200
Withaferin A 0.996 23.0 to 737
12-Deoxy
Withastramonolide
0.990 22.0 to 355
Withanolide A 0.992 21.0 to 345
Withanolide B 0.984 21.0 to 337
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
Conclusion: The HPLC method [NR/QCD/APM04 WI(17)] for the estimation of Withania
somnifera is validated.