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K
oshy
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
6, 2016
1449
in duplicate at three extraction weights, the repeatability was
<2.2% for peak area and <1% for retention time (Table 9).
Content.—
Repeatability precision based on the determination
of content (% w/w) of each analyte was evaluated in two
experiments. In the first experiment, each of the eight study
samples was extracted and the resulting sample solution
analyzed in triplicate. The results are shown in Table 10. Seven
of eight samples demonstrated a repeatability of <3% for
each of the six analytes, as well as the sum of withanolides,
meeting the acceptance criteria of SMPR 2015.007. In two
cases, WS/06Lot08 and RD/1170, the sum of withanolides was
>1% w/w and the repeatability slightly exceeded the acceptance
criterion of ≤1%. The eighth sample, WS/05Lot20, yielded
RSD
r
values as high as 10.8% for withanolide B and <7% for the
remaining five analytes and the sum of analytes. This sample is
the only water extract sample tested, and the withanolide content
is the lowest of all samples tested, including the raw materials.
Compared with the SMPR requirements, the repeatability
values of withanolide B and 12-deoxywithastramonolide are
greater than the allowed variability, but the four other analytes
were within the acceptance criteria.
In the second experiment, the full method including methanol
extraction was replicated three to four times at five sample
weights, and each sample extract was analyzed once (Table 11).
Not surprisingly, the RSD
r
(%) of the full method, including all
sample weights, was somewhat higher than the repeatability of
replicate injections of a single extract in the first experiment.
RSD
r
values varied from 0.83 to 2.01% across all analytes
and the sum of the analytes, meeting the acceptance criteria
of RSD
r
≤1% at >1% w/w, ≤4% at >0.1–1% w/w, and ≤6% at
>0.01–0.1% w/w.
Recovery
Recovery of total withanolide content after spiking is shown
in Table 12. Materials spiked prior to extraction (WS/05Lot21,
WS/06Lot10, RD/1170, and RD/1162) demonstrated recoveries
ranging from 90.5 to 104.7%. With total content of each
Figure 2. Chromatograms of methanolic extract (WS/06Lot10) sample using (A) UV detection and (B) PDA detection.
Figure 3. Chromatograms of raw material (ERH-046) sample using (A) UV detection and (B) PDA detection.
A
B
A
B
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