SPDS SET 2: ASH-01

HPLC METHOD VALIDATION ESTIMATION OF Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A and Withanolide B WITHANIA SOMNIFERA (ROOTS) AND ITS EXTRACT

1.0] SUMMARY:

1.1] Introduction: The validation of an analytical procedure is the process of confirming the performance of the analytical procedure employed for the test. The performance of the analytical procedure is established by various kinds of validation characteristics such as Specificity, Linearity, Range of quantification, Accuracy, precision, Repeatability & System suitability. The validity of a proposed analytical procedure is shown by demonstrating experimentally that the validation characteristics of the analytical procedure satisfy the standards set up according to the acceptable limits of testing.

An HPLC method [NR/QCD/APM04 WI(17)] for estimation of Withanolides (sum of

Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A and Withanolide B) in Withania somnifera and its extract was validated using Shimadzu

High Performance Liquid Chromatography system, model: LC 2010A with system

controller unit, degasser unit, low pressure gradient unit up to 4 solvents, pump unit with seal unit mechanism mixer, auto sampler, column oven, UV-VIS detector, 220V 50Hz with LC solution software and another Shimadzu LC10A system equipped with binary LC10ADVP pump, with SIL10AD vp auto sampler, SPD-M 10A vp Photo Diode Array Detector, SCL 10AVP system controller, CTO 10AVP column oven in combination with Class VP software version 6.03.

Column containing octadecylsilane C18, 5 µ particle size was used to separate

Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A

and Withanolide B from the compounds present in Withania somnifera . A gradient mixture

of HPLC phosphate buffer and Acetonitrile (55 : 45) were used as mobile phase to elute the compounds. Chromatograms were recorded at 227nm using UV and SPD-M 10A vp

Photo diode Array Detector.

Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A and Withanolide B were separated from the other secondary metabolites present in the plant indicating specificity. Solutions of five different concentrations of Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy withastramonolide, Withanolide A and Withanolide-

B were prepared and injected to find out the linearity and reproducibility. Different samples

containing different concentrations of Withanoside IV, Withanoside V, Withaferin A, 12-

1$785$/ 5(0(',(6

3DJH RI