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(
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 5 mg of withanoside IV, withanoside V, withaferin A, 12-
deoxywithastramonolide, withanolide A, and withanolide B reference standards to 50 mL volumetric
flask. Dissolve in 10 mL methanol with the aid of gently heating and cool then make up to 50 mL 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 250 mL beaker. Extract with 100 mL
methanol boiling on water bath for 10-15 minutes and repeat the procedure 3-4 times until the raw
material is completely extracted or till the extracts turn colorless. Combine all the fractions, concentrate
and make up the volume to 50 mL with methanol. Filter through 0.45 microns 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 250 mL beaker. Extract with 100 mL
methanol boiling on water bath for 10-15minutes and repeat the procedure 3-4 times until the raw
material is completely extracted or the extracts turn colorless. Combine all the fractions, concentrate
and make up the volume to 50 mL with methanol. Filter through 0.45 microns membrane filter paper.
F. Analysis
(
a
)
Chromatographic conditions
.—
Column
.—Phenomenex Luna C18(2), 250
×
4.6 mm with 5 µm particle
size; Part No. 00G-4252-E0.
Temperature
: Maintained at a constant temperature between 20 to 30°C
(preferably 27°C).
Detector
: SPD-M 10Avp photodiode array detector or UV detector.
Wavelength
: 227
nm.
Flow rate
: 1.5 mL/min.
Run time
: 45 minutes.
(
b
)
Retention time relative
.—Relative retention time of Withanoside IV, 0.7; withanoside V, 0.89;
withaferin A, 0.92; 12-deoxywithastramonolide, 0.96; withanolide A, 1.0; withanolide B, 1.15.
(
c
)
System suitability
.—(
1
)
Repeatability.—
The RSD of each of the individual withanolides peak area for
at least 5 consecutive injections of the level 4 linearity standard solution must be
≤
2.5%.
(
2
)
Resolution
.—Calculate the resolution between withanoside V andwWithaferin A peaks as follows:
R
=
2
x
T2 - T1
W1 + W2
where T1 and T2 are the retention times of withanoside V and withaferin A respectively and W1 and W2
are their peak widths measured at the baseline between tangents drawn to the peak sides. The
resolution between ephedrine and pseudoephedrine in should be
≥
3.0.
Candidates for 2016 Method of the Year
243