SPDS SET 2: ASH-01

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 :

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).

1)

Acetonitrile (Solvent B)

2)

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

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

:

Column oven Temperature :

Maintained at a constant temperature between 20 to 30°C(preferably 27°C)

SPD-M 10A vp Photo diode array detector or UV Detector

Detector

:

227 nm

Wave length :

1.5ml/ min

Flow rate

:

45 minutes

Run time

:

20 µ l

Injection volume:

1$785$/ 5(0(',(6

3DJH RI