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K
oshy
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
6, 2016
1447
withanolide A, and withanolide B were prepared in methanol at
the concentrations shown in Table 4.
Dietary ingredient samples, including raw material (plant
root, Batch No. ERH-046, 4091 mg) and a standardized dried
methanolic extract (Batch No. WS/06Lot10, 750 or 1520 mg)
were extracted with hot methanol according to the method.
Each reference standard, a mixed standard solution, and the
sample solutions were analyzed according to the method on
two different LC systems—a Shimadzu LC 2010A with a UV-
Vis detector and a Shimadzu LC 10A with a PDA detector—on
different days.
Linearity
The reference stock standard solution was prepared in
methanol at ~1.2–1.5 mg/mL after correction for purity. Six
2-fold serial dilutions of the stock standard solution were then
prepared in methanol. Five replicates of the stock and each
dilution were analyzed.
Precision
Peak areas and retention times—
The data from the stock
standard and serial dilutions for the linearity study were
analyzed for repeatability based on the peak areas and retention
times for each analyte. A standardized dried methanolic
extract of
W. somnifera
(Batch No. WS/06Lot10, >2.5% total
withanolides by the candidate LC method) was tested at three
levels—754, 1503, and 2096 mg—each extracted and analyzed
in duplicate according to the method.
Precision
Content—
Standards were prepared at ~0.2–0.4 mg/mL in
methanol and analyzed in triplicate. Eight materials (1552 mg
WS/06Lot08, 1536 mg WS/06Lot10, 5048 mg WS/05Lot20,
1527 mg WS/05Lot21, 1503 mg RD/1170, 1653 mg RD/1045,
4336 mg RD/1162, and 4229 mg ERH-46) were extracted and
analyzed in triplicate. Withanolide content was calculated for
each analyte.
Recovery
Four materials (2613mgWS/05Lot21, 1520mgWS/06Lot10,
1633 mg RD/1170, and 4004 mg RD/1162) were spiked with
pure standards (W-IV, WF-A, 12-D, W-A, and W-B) using
the third serial dilution of the stock standard solution from the
linearity study. A sample material containing 36% W-V was
used for the W-V spike. The spiked materials were extracted
and analyzed for total (endogenous + spike) withanolide content
according to the candidate method.
In a separate experiment, four materials (1714 mg
WS/06Lot08, 5030 mg WS/05Lot20, 1640 mg RD/1045, and
4091 mg ERH-46) were extracted according to the candidate
method. The extracts were spiked with pure standards using
the third serial dilution of the stock standard solution from
the linearity study. The spiked extracts were analyzed for total
(endogenous + spike) withanolide content according to the
candidate method.
Ruggedness
The ruggedness evaluation was conducted as an evaluation
of intermediate precision. All eight materials were tested under
two sets of conditions, including different analysts on different
days using different LC equipment, column temperatures, and
concentrations of H
3
PO
4
in the LC mobile phase (Table 5). Each
material was extracted, and the extracts analyzed under each of
the two conditions without replication.
Stability of Sample Solution
Material WS/05Lot21 (2613 mg) was extracted and filtered
according to the method and placed into an autoinjector vial.
The LC system and autosampler were in a room maintained
at 25±2°C. The extracted sample and linearity standards were
analyzed immediately and after 24 h. The difference in results
between 0 and 24 h was determined.
System Suitability
In addition to the standards and samples injected with
every run, a QC-check sample was developed for use during
validation. Batch No. WS/06Lot10 was sampled (~500 g),
homogenized, and stored in an airtight container for use
throughout the validation study. An initial test portion was
extracted according to the method and tested in duplicate
by two analysts on different days on multiple days by
multiple analysts to establish a “true” value. If the RSD of
the obtained results was <2.5%, then the mean value of the
obtained results was accepted as the true value. The QC-
check sample was then included with every run during the
validation study.
Table 4. Reference standard concentrations
Reference standard
Concn, μg/mL
UV detector
PDA detector
Withanoside IV
330.30
310.0
Withanoside V
300.15
360.0
Withaferin A
368.78
210.0
12-Deoxywithastramonolide
355.30
300.0
Withanolide A
345.51
240.0
Withanolide B
337.59
350.0
Table 5. Conditions for ruggedness evaluation
Parameter
Condition 1
Condition 2
Analyst
A
B
Day
1
2
HPLC
Shimadzu LC-2010A Shimadzu LC-10A
Detector
UV-Vis
PDA
C18 column
Merck LiChrospher 100
a
Phenomenex Luna
RP-18e (5 μm) Hibar RT 5 μm C18(2), 100 å
250×4.6 mm
250×4.6 mm
Column temperature, °C
25
30
H
3
PO
4
concn
0.5 mL in 1000 mL 1.5 mL in 1000 mL
a
EMD Millipore, Billerica, MA.
52