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Temperature
: Maintained at a constant temperature between 20 to 30°C (preferably 27°C)
Detector
: SPD-M 10Avp Photo diode array detector or UV Detector
Wave length
: 227 nm
Flow rate
: 1.5ml/ min
Run time
: 45minutes.
(
b
)
Retention time relative
.—
Relative retention time of
Withanoside IV - 0.7
Withanoside V - 0.89
Withaferin A - 0.92
12-Deoxywithastramonolide - 0.96
Withanolide A - 1.0
Withanolide B - 1.15
(
c
)
System suitability
.—(
1
)
Repeatability.—
The RSD of each of the individual withanolides peak area for at least 5 consecutive
injections of the level 4 linearity standard solution must be
%.
(
2
)
Resolution
.—Calculate the resolution between Withanoside V and Withaferin A peaks as follows:
R
=
2
x
T2 - T1
W1 + W2
Where T1 and T2 are the retention times of Withanoside V and Withaferin A respectively and W1 and W2 is their peak widths
measured at the base line between tangents drawn to the peak sides. The resolution between ephedrine and pseudoephedrine in
should be
3.0.
(
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 20μl of sample preparation and record the chromatogram at 227nm. 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