245 / 363
(
3
)
Tailing.—
Calculate the tailing factor (F) as follows:
F
=
L + R
2L
where L is the width from start of the peak to the perpendicular from the peak apex at 5% of the peak
height; R is the width from the perpendicular from the peak apex to the peak end at 5% of the peak
height. The tailing factor must be
≤
1.5 for all individual withanolides in the linearity standard solution
chromatograms.
(
4
)
Determination coefficient
.—The r
2
for the regression line of peak area vs. concentration for each
withanolide must be
≥
0.998.
Procedure: Inject three times the standard preparation and calculate the mean area and the RSD. The
RSD should not be more than 2%. Inject 20 μL of sample preparation and record the chromatogram at
227 nm. Calculate the percentage of withanoside IV, withanoside V, withaferin A, 12-
deoxywithastramonolide, withanolide A and withanolide B content from the peak areas using the
formula:
Peak integration: Base to base
Individual withanolide (%w/w)
=
Area of the sample x
Weight of standard
(mg)
x
Sample dilution
x
Purity of
standard %
Area of the standard
Standard dilution
Sample weight (mg)
Compound name
CAS No.
Withanoside IV
362472-81-9
Withanoside V
256520-90-8
Withaferin A
5119-48-2
12-Deoxywithastramonolide 60124-17-6
Withanolide A
32911-62-9
Withanolide B
56973-41-2
References: AOAC SMPR 2015.007
J. AOAC Int
.
98
, 1104(2015)
DOI: 10.5740/jaoac.int.SMPR2015.007
J. AOAC Int
. (future issue)
Posted: January 19, 2016
Candidates for 2016 Method of the Year
244