SPDS SET 2: ASH-01

analysis. If the variation in retention time is more than the limit, the result should not be

reported.

The highest variation observed in the reproducibility in the area is 1.5%. Hence mean of duplicate injection of sample and mean of triplicate injection of standard is recommended for calculation. The variation found in reproducibility of result for total withanolides is RSD <1.5%. Hence duplicate result obtained with two trials of sample with same concentration

should be < 2% otherwise the analysis need to be repeated.

6.5) System suitability: The following system suitability parameters are determined for this

method: - 1) Standard solution and reference extract solution to be injected

2) Peaks of all the withanolides are well separated and the resolution is >3 for Withanoside & Withaferin A in the standard solution. 3) The asymmetric factor (tailing factor) for Withanolides should be < 1.5. 4) The relative retention time of Withanoside IV is about 0.70, Withanoside V is about 0.89, Withaferin A is about 0.92, 12-Deoxy withastramonolide is about 0.96 and Withanolide B is about 1.15. 5) The precision of injection (with minimum of 3 replicates) RSD should be <1.0% for

standard and sample solution.

6) Retention time window ± 3 σ of mean retention time of the standard or reference extract.

7) Precision in result (The result of QC check sample (reference extract) shall be within

± 10% of the labelled result).

If the system suitability parameters are not met, achieve the same by changing the

composition of the mobile phase within permissible range or by using new column. The analysis is to be proceeded further only after the system suitability parameters are met.

6.6) Accuracy: Spike recovery is >90% indicating the method is accurate.

6.7) Ruggedness: Relative standard deviations for same batch sample when analysed in two

different instruments found to be < 5.0% indicating the method is rugged.

6.8) Final Conclusion: On the basis of above results the HPLC method [NR/QCD/APM04 WI(17)] for the estimation of Withania somnifera is considered as validated.

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