New HPLC Method
Reference standards of Glycosides (Withanoside-IV,
Withanoside-V, Withanolide-A, Withanoside-UV active)
and Aglycones (Withaferin-A, Withastramonolide,
Withanone) were obtained from ChromaDex.
Withanoside IV is used to calculate the total withanolide
glycoside content
Withaferin-A is used to calculate the total aglycone content
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ONFIDENTIAL
New HPLC Method – HPLC Conditions
HPLC conditions:
System : Alliance-Water (PDA 2996)
System number : NAT/HPLC/03
Method : Sensoril Gradient system
Column
: Lichrocart (250 × 4 mm, 5 μm Merck) [NI/LC/RPC18/005[250×4]]
Solvent system
: Gradient;
(A) Acetonitrile (B) Water
Detection : 225 nm (PDA detector)
Flow rate : 0.5 ml/min
Run time
: 30 min
HPLC programming for mobile phase is given below:
Table 1: Gradient systemof solvent-A and solvent-B
Time (Min.) Solvent A (%) Solvent B (%)
0
10
90
4.0
20
80
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7.0
80
20
10.0
70
30
15.0
60
40
17.0
100
0
22.0
100
0
25.0
20
80
30.0
10
90
