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Estimation of Withanolides (Withanoside IV, Withanoside V, Withaferin A, 12-Deoxywithastromonolide,
Withanolide A, Withanolide B) in
Withania somnifera
Liquid Chromatography
A. Principle
Withanolides are compounds specific to
Withania somnifera
. The method proposes extraction of withanolides from the sample
matrix using methanol and separating the compounds using gradient high performance liquid chromatography on a C-18 column
and measure in UV at 227nm.
B. Apparatus
(
a
)
LC system
.—
Shimadzu High Performance Liquid Chromatographic system equipped with LC10A pump with SPD-M 10Avp
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.
(
b
)
Column
.— Phenomenex- Luna C18(2), 250
4.6 mm with 5
m particle size; Part No. : 00G-4252- E0 (Phenomenex,
Torrance, CA, USA;
www.phenomenex.com).
(c)
Analytical balance.—Read ability, 0.1 mg.
(
e
)
Filtration apparatus
.—0.45
m nylon filter.
(
f
)
Ultrasonic bath
(g)
Syringe filter – 0.45µm PES filter
C. Reagents
(
a
)
Degassed mobile phase
.—
1)
Dissolve 0.136 g of anhydrous potassium dihydrogen orthophosphate (KH2PO4) 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)
Solvent A Conc Solvent B Conc
0.01
95.0
5.0
18.0
55.0
45.0
25.0
20.0
80.0
28.0
20.0
80.0
35.0
55.0
45.0
40.0
95.0
5.0
45.0
95.0
5.0
(
b
)
Diluent
.—Methanol.
(
c
) Individual
Withanolide standards. —
M/s Natural Remedies Pvt. Ltd. (Bangalore, KA, India;
www.phytocompounds.com), or
other suppliers.
D. Standards
Weigh accurately each 5mg of Withanoside IV, Withanoside V Withaferin A, 12-Deoxywithastramonolide, Withanolide-A and
Withanolide-B reference standards to 50 ml volumetric flask. Dissolve in 10ml of methanol with the aid of gently heating and
cool then make up to 50ml with methanol.
E. Preparation of Test Solutions
(
a
)
Raw material
. — Weigh accurately a sample quantity of
Withania somnifera
raw material equivalent 5 mg (about 2.5 g will
be sufficient) of Withanoside IV, Withanoside V, Withaferin A, 12-Deoxywithastramonolide, Withanolide A and Withanolide B
in a 250ml beaker, Extract with 100 ml of methanol boiling on water bath for 10-15minutes and repeat the procedure 3 - 4 times
till the raw material is completely extracted or till the extracts turn colorless. Combine all the fractions, concentrate and make up
the volume to 50ml with methanol. Filter through 0.45microns membrane filter paper.
(
b
)
Standardized (common) extract
. — Weigh accurately a sample quantity of
Withania somnifera
extract equivalent 5 mg (about
0.5 g will be sufficient) of Withanoside IV, Withanoside V, Withaferin A, 12-Deoxywithastramonolide, Withanolide A and
Withanolide B in a 250ml beaker, Extract with 100 ml of methanol boiling on water bath for 10-15minutes and repeat the
procedure 3 - 4 times till the raw material is completely extracted or till the extracts turn colorless. Combine all the fractions,
concentrate and make up the volume to 50ml with methanol. Filter through 0.45microns membrane filter paper.
F. Analysis
(
a
)
Chromatographic conditions
.—
Column
.— Phenomenex- Luna C18(2), 250
4.6 mm with 5
m particle size; Part No. : 00G-4252- E0 (Phenomenex, Torrance,
CA, USA;
www.phenomenex.com).