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AOAC Official Method 2015.17
Estimation of Withanolides
(Withanoside IV, Withanoside V, Withaferin A,
12-Deoxywithastromonolide, Withanolide A, Withanolide B)
in
Withania somnifera
Liquid Chromatography
First Action 2015
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 227 nm.
B. Apparatus
(
a
)
LC system
.—Shimadzu high performance liquid chromatographic system equipped with LC10A pump
with SPD-M 10Avp photodiode array detector or UV detector in combination with Class-VP software or
LC 2010 A and LC 2010HT integrated system equipped with quaternary gradient, autoinjector 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 µm particle size; Part No. 00G-4252-E0
(Phenomenex, Torrance, CA, USA;
www.phenomenex.com).
(c)
Analytical balance.—
Readability, 0.1 mg
.
(
d
)
Filtration apparatus
.—0.45 µm nylon filter.
(
e
)
Ultrasonic bath.
(f)
Syringe filter.–
0.45 µm PES filter.
C. Reagents
(
a
)
Degassed mobile phase
.—(1) Dissolve 0.136 g of anhydrous potassium dihydrogen orthophosphate
(KH
2
PO
4
) in 900 mL HPLC grade water (obtained from Millipore, Milli-Q Water purification system) and
add 0.5 mL orthophosphoric acid. Make up to 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
Candidates for 2016 Method of the Year
242