SPDS SET 2: ASH-01

2.0] Introduction

2.1) Objective: To ensure the method [(NR/QCD/APM04 WI(17) - estimation of Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A and Withanolide- B in Withania somnifera )] developed by Natural Remedies Private Limited provides high

degree of assurance that it will consistently produce result which is accurate, precise,

reproducible and robust when performed at Natural Remedies Private Limited or at any other laboratory by competent analyst using appropriate equipment, chemicals and

reference standards.

2.2) Scope: This method of estimation of Withanoside IV, Withanoside V, Withaferin A, 12- Deoxy withastramonolide, Withanolide A and Withanolide B, and total Withanolides using Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A and Withanolide B as reference standards applicable to Withania somnifera roots (raw material) and its extract. These compounds are ranging from 0.01 to 10%. It is applicable to all material having matrix similar to that of Withania somnifera roots (raw material) and extracts / products containing Withania somnifera extracts blended with excipients which 2.3) Purpose and application: Withania somnifera extract is prepared by extracting Withania somnifera roots with solvents like methanol, water etc. It is standardized to 1.5%, 2.5%, 3%, 5% Total withanolides. In general the content of Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A and Withanolide B would be 20 to 30% of Total withanolides. It is important to estimate the Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A and Withanolide- B contents to are insoluble in methanol and acetonitrile.

ensure the product meets the specification before its release to the market. Also the raw

material used for extraction to be estimated for withanolides content as the product quality

depends on raw material used for extraction. Withanolides (Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A and Withanolide- B) can be estimated only after clearly separating the various secondary metabolites present in the raw material or in the extract along with the withanolides. This could be achieved only by

employing chromatographic techniques. Hplc is more suitable for this purpose.

The purpose of this method validation is to establish documental evidence which provides adequate assurance that the method comply with the requirements of validation characteristics suggested in the protocol which was prepared based on international guidelines. The purpose is also to demonstrate that the method performs in accordance to

the claims made.

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