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K
oshy
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
6, 2016
1445
Table 3. Sample materials used in the validation study
Sample
Standardized to
Batch No.
Raw material
Total withanolides >0.2% by HPLC RD/1162
Raw material
Total withanolides >0.2% by HPLC ERH-046
Water extract
Total withanolides >0.15% by HPLC WS/05Lot20
Hydroalcoholic
extract
Total withanolides >1.0% by HPLC WS/05Lot21
Methanolic
extract
Total withanolides >1.5% by HPLC RD/1045
Methanolic
extract
Total withanolides >2.5% by HPLC WS/06Lot08
Methanolic
extract
Total withanolides >2.5% by HPLC WS/06Lot10
Methanolic
extract
Total withanolides >2.5% by HPLC RD/1170
[Applicable to the determination of withanoside IV,
withanoside V, withaferin A, 12-deoxywithastromonolide,
withanolide A, and withanolide B content in raw material
(roots) and dried extracts.]
Caution
: This method uses common-use solvents and
reagents. Refer to an adequate manual or Material
Safety Data Sheets (MSDSs) to ensure that safety
guidelines are applied before using chemicals. Store
solvents in a flammable-liquid storage cabinet. The
solvents and reagents used herein are harmful if
inhaled, swallowed, or absorbed through the skin.
Use appropriate personal protective equipment,
such as a laboratory coat, safety glasses, rubber
gloves, and a fume hood. Dispose of all materials
according to federal, state, and local regulations.
A. Principle
Withanolides are compounds specific to
Withania somnifera
.
This method comprises extraction of withanolides from the
sample matrix using methanol and separation of the compounds
using gradient LC on a C18 column with UV detection at 227 nm.
B. Apparatus
(a)
LC system
.—Shimadzu HPLC system equipped with an
LC10A pump with an SPD-M 10Avp photodiode array (PDA)
or UV detector in combination with CLASS-VP software
(Shimadzu) or an LC-2010A and LC-2010HT integrated
system (Shimadzu) equipped with a quaternary gradient and
autoinjector in combination with laboratory solution software;
or any other suitable HPLC system with a similar configuration.
(b)
Column
.—Phenomenex Luna C18(2), 250×4.6 mm,
with 5 μm particle size (Part No. 00G-4252- E0; Phenomenex,
Torrance, CA;
http://www.phenomenex.com); or equivalent.
(c)
Analytical balance
.—Accuracy to 0.1 mg.
(d)
Filtration apparatus
.—0.45 μm nylon filter.
(e)
Ultrasonic bath
.
(f)
Syringe filter
.—0.45 μm polyethersulfone filter.
C. Reagents
(a)
Degassed mobile phase
.—(
1
)
Solvent
A
.—Dissolve
0.136 g anhydrous potassium dihydrogen orthophosphate
(KH
2
PO
4
) in 900 mL HPLC grade water (Milli-Q Water
purification system; Millipore) and add 0.5 mL orthophosphoric
acid. Dilute to 1000 mLvolume with water, filter through 0.45 μm
membrane, and degas in a sonicator for 3 min (solvent A).
(
2
)
Solvent B
.—Acetonitrile.
(b)
Diluent
.—Methanol.
(c)
Individual withanolide standards
.—Natural Remedies
Private Ltd (Bangalore, India;
www.naturalremedy.com), or other
suppliers.—(
1
)
Withanoside IV
.—CAS No. 362472-81-9.
(
2
)
Withanoside V
.—CAS No. 256520-90-8.
(
3
)
Withaferin A
.—CAS No. 5119-48-2.
(
4
)
12-Deoxywithastromonolide
.—CAS No. 60124-17-6.
(
5
)
Withanolide A
.—CAS No. 32911-62-9.
(
6
)
Withanolide B
.—CAS No. 56973-41-2.
D. Preparation of Mixed Standards
Accurately weigh 10 mg each of withanoside IV, withanoside V,
withaferin A, 12-deoxywithastromonolide, withanolide A, and
withanolide B reference standards in a 50 mL volumetric flask,
dissolve in 10 mL methanol with the aid of gentle heating, cool,
and dilute to a 50 mL volume with methanol to yield 200 μg/mL of
each standard. Suitably prepare three additional concentrations of
withanolides to obtain concentrations of 150, 100, and 50 μg/mL.
E. Preparation of Test Solutions
(a)
Raw material
.—Accurately weigh a sample quantity
of
W. somnifera
raw material equivalent to 5 mg (~2.5 g is
sufficient) of withanoside IV, withanoside V, withaferin A,
12-deoxywithastromonolide, withanolide A, and withanolide B
in a 250 mL beaker. Extract raw material with 100 mL methanol
on a boiling water bath for 10–15 min, and repeat this procedure
three to four times until the raw material has been completely
extracted or the extracts become colorless. Combine all extracts
and evaporate the methanol on a water bath or by using a vacuum
Table 2. Performance requirements for recovery,
repeatability, and reproducibility
Parameter
Range, ppm
10–100 >100–1000 >1000–10000 >10000
Recovery, % 80–110 90–107
95–105
97–103
Repeatability, % ≤7
≤6
≤4
≤1
Reproducibility, % ≤10
≤9
≤6
≤2
Table 1. Performance requirements for analytical range
and LOQ
Parameter
Total glycosides
Aglycones
Analytical range, ppm
10–250000
10–20000
LOQ, ppm
10
AOAC Official Method 2015.17
Withanolides (Withanoside IV, Withanoside V,
Withaferin A, 12-Deoxywithastromonolide, Withanolide A,
and Withanolide B) in
Withania somnifera
LC
First Action 2015
50