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3DJH RI
2.4) Procedure:
The method is validated as per the protocol number NR/QCD/PRT/001 and
standard operating procedure (SOP No. NR/QCD/SOP/072/00).
2.5) HPLC method
Purpose:
To estimate the content of Withanoside IV, Withanoside V, Withaferin A, 12-Deoxy
withastramonolide, Withanolide A and Withanolide B in
Withania somnifera
by HPLC
.
Analysis:
Chromatographic system:
Shimadzu
High Performance Liquid Chromatographic system
equipped with LC10A pump with SPD-M 10A
vp
Photo diode Array Detector or UV detector in
combination with Class-VP software or LC 2010 A and LC 2010HT integrated system equipped
with Quaternary gradient, auto injector in combination with Lab solution software or any other
suitable HPLC system with similar configuration can be used.
Chromatographic conditions:
Mobile phase
:
1)
Dissolve 0.136 g of anhydrous potassium dihydrogen
orthophosphate (KH
2
PO
4
) in 900 ml of HPLC grade
water (obtained from Millipore, Milli-Q Water
purification system) and add 0.5ml of orthophosphoric
acid. Make upto 1000 ml with water, filter through
0.45
µ
membrane and degas in a sonicator for 3
minutes
(Solvent A).
2)
Acetonitrile
(Solvent B)
Time (min) Buffer Value Acetonitrile Value
0.01
A.Conc 95.0 B.Conc 5
18.0 A.Conc 55.0 B.Conc 45
25.0 A.Conc 20.0 B.Conc 80
28.0 A.Conc 20.0 B.Conc 80
35.0 A.Conc 55.0 B.Conc 45
40.0 A.Conc 95.0 B.Conc 5
45.0 A.Conc 95.0 B.Conc 5
Column
:
i) Col No: LiChrosphere RP-18e Catalogue No:1.50995
ii) Phenomenex- Luna 5
µ
C-18(2) Size: 250x4.60mm, S No.:417653
Column oven
Temperature :
Maintained at a constant temperature between 20 to 30°C(preferably 27°C)
Detector
:
SPD-M 10A
vp
Photo diode array detector or UV Detector
Wave length :
227 nm
Flow rate
:
1.5ml/ min
Run time
:
45
minutes
Injection volume:
20
µ
l