SPDS SET 2: ASH-01

4.0] STEP-1: VALIDATION OF INSTRUMENTAL PARAMETERS

4.1) Specificity:

4.1.1) Definition: The specificity is the ability of an analytical procedure to measure accurately an analyte in the presence of components that may be expected to be present in the sample

matrix.

4.1.2) Assessment: It was confirmed by spectral similarity, determining the resolution between

each peak, by checking the peak purity using PDA detector.

4.1.3) Acceptance Criteria: The compound of interest should be well separated and resolved from the rest. The resolution should be > 2.0. PDA spectrum of sample peak should be

similar to the standard peak; peak purity should be > 0.98.

4.1.4) Experiment: a) Standard solution preparation:

The following concentrations of withanolides were

prepared in methanol.

Table-1 Sl. No.

Reference standard

Concentrations (mcg/ml)

UV detector

PDA detector

1.

330.30

310.0

Withanoside IV

2.

300.15

360.0

Withanoside V

3.

368.78

210.0

Withaferin A

4.

355.30

300.0

12-Deoxy withastramonolide

5.

345.51

240.0

Withanolide A

6.

337.59

350.0

Withanolide B

b) Sample preparation: The sample solution was prepared by weighing the quantity shown

in the Table-1 in a 250 ml beaker, extracted with 100ml of methanol, boiling on water bath for 10-15 minutes and repeated the procedure 3-4 times till the material is completely

extracted or till the extracts turn colourless. Combined all the fractions, concentrated and

made up the volume to 50ml with methanol. Filtered through 0.45 microns membrane filter paper.

Table-2

Batch No

Quantity (mg)

UV detector

PDA detector

WS/06Lot10

750 mg

1520.4 mg

ERH-46

4091mg

4091.0 mg

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

3DJH RI