SPDS SET 2: ASH-01

4.6)

Ruggedness

4.6.1) Definition: Ruggedness of an analytical method is a measure of degree of reproducibility of results obtained under different environmental conditions such as different laboratory, different chemist and different instrument. Purpose of ensuring the method under validation provides the same results when analysed

in a different HPLC, different brand column and different conditions.

4.6.2) Assessment: Withanoside IV, Withanoside V, Withaferin A, 12-Doxy withastramonolide, Withanolide A and Withanolide B contents in extracts sample and raw material were

analysed by two different analysts at different dates using different instruments and C18

column from a different manufacturer. The results were compared and RSD was

calculated.

Table-64: Name of the instrument

Condition–1

Condition-2

HPLC

LC-2010A

LC10ATvp

C18 Column

Lichrospher 100 RP-18e(5 µ m) Col. No. 625364 Make: Merck (Hiber)

Phenomenex, Luna 5 µ C 18 (2)100 A, 250 X 4.60 mm, 5 microns Part No. :00G-4252- E0 No. 359208-12

Column oven temperature

25°C

30°C

Concentration of H 3

PO 4

0.5ml in 1000ml

1.5ml in 1000ml

4.6.3) Acceptance criteria: The RSD between the two results obtained two different analysts

using two different instruments on different date should be less than 6.0%.

4.6.4) Experiment:

Experiment-1 (Condition - 1)

a) Standard / sample preparation: Refer 4.3.11

Table-65:

Batch No.

Withanoside IV

Withanoside V

Withaferin A

12-Deoxy withastra monolide

Withanolide A

Withanolide B

Sum

WS/06Lot08

0.917

0.650

0.659

0.284

0.225

0.066

2.802

WS/06Lot10

0.911

0.621

0.616

0.267

0.205

0.062

2.681

WS/05Lot20

0.067

0.044

0.081

0.022

0.021

0.005

0.241

WS/05Lot21

0.478

0.310

0.410

0.125

0.102

0.029

1.453

RD/1170

0.848

0.594

0.700

0.332

0.229

0.076

2.779

RD/1045

0.606

0.364

0.402

0.248

0.131

0.042

1.793

RD/1162

0.152

0.090

0.100

0.021

0.025

0.004

0.391

ERH-46

0.184

0.148

0.145

0.041

0.041

0.010

0.570

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