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3DJH RI
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-