Current HPLC Method - Quantification of
Withanolide Glycosides and Withaferin-A
Withaferin-A reference standard, developed in-house, is used for
both aglycone as well as glycoside estimation
The peak appearing at tR 7.5 min is considered that of
With f i A a er n-
Peaks appearing at tR 2.0-7.4 min are considered those of
withanolide glycosides.
U
1 20
1.40
1.60
1.80
2.00
2.20
2.40
2.865
43
A
0.00
0.20
0.40
0.60
0.80
1.00
.
Minutes
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Peak2 - 3.581
4.854
5.668
7.559
8.672
10.339
Current HPLC Method - Quantification of
Oligosaccharides
HPLC Conditions:
Equipment
Waters HPLC system consisting RI detector Waters 2414, pump Waters 515,
evaluation with Empower software.
C l
C b h d t A l i C l
[ t ] 300 3 9 P t N WAT 084038
o umn
ar o y ra e na ys s o umn wa ers
x . mm, ar o.
Column
Temperature
Ambient
Eluent
Acetonitrile: Water in the ratio of 80:20 (v/v)
Run Time
10 min
Flow rate
2 ml/min
Gradient
Programme
Isocratic
Injection volume
20μl (with a loop injector)
Sensitivity
16
Reagents
Pdt. No. UN-1648 Acetonitrile (Merck)
Pdt. No. 93956 Water for HPLC (Merck)
Evaluation
Method with external standard
44
.
External
Standard
Oligosaccharide isolated from Withania somnifera by multiple
column chromatographies was used as external standard.
Oligosaccharide 10 mg was weighed and dissolved in 1 ml of
water. Four different concentrations (10
g, 50
g, 100
g and
200
g in 20
l) were applied to HPLC. Calibration curve was
plotted between concentration and area. The linear regression
equation was obtained as follows.
Y = 6620.5 X+ 72099 with a co-relation coefficient of 0.996
where Y is the area and X is the concentration in
g.
The area of peaks appearing between 4-10 minutes is added
and contents of oligosaccharides are calculated using linear
regression equation
