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3DJH RI
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
Concentrations (mcg/ml)
Sl. No.
Reference standard
UV detector
PDA detector
1.
Withanoside IV
330.30
310.0
2.
Withanoside V
300.15
360.0
3.
Withaferin A
368.78
210.0
4.
12-Deoxy withastramonolide
355.30
300.0
5.
Withanolide A
345.51
240.0
6.
Withanolide B
337.59
350.0
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