62 / 63
K
oshy
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
6, 2016
1457
Conclusions
The LC method demonstrated acceptable specificity,
precision, and recovery, meeting the recently established
SLV criteria of SMPR 2015.007 within the linear range of
the method. The AOAC ERP on Dietary Supplements ERP
approved the method for First Action
Official Method
status
on December 10, 2015. The ERP recommendations prior
to moving to Final Action status include improving baseline
separation of peak 1, optimizing the pH of the phosphate buffer,
optimizing the column temperature, investigating the heat
stability of the analytes during extraction, simplifying sample
preparation, reducing the number of reference standards to two
(one withanolide and one withanoside) with the use of relative
Table 11. Repeatability precision of withanolide content in sample WS/06Lot10 with replicate extractions of the sample at
five sample weights
Level
Trial
Amount extracted, mg
Withanolide content, % w/w
W-IV
W-V WF-A
12-D
W-A W-B Sum
A
1
751
0.802
0.681 0.605
0.263
0.209 0.064 2.625
2
754
0.821
0.697 0.617
0.268
0.212 0.066 2.680
3
747
0.828
0.696 0.626
0.272
0.221 0.066 2.709
4
755
0.821
0.684 0.615
0.265
0.218 0.064 2.667
B
1
1032
0.808
0.684 0.609
0.265
0.211 0.065 2.642
2
936
0.827
0.703 0.621
0.270
0.215 0.065 2.701
3
1073
0.818
0.684 0.612
0.265
0.217 0.064 2.661
4
1011
0.826
0.687 0.618
0.268
0.220 0.065 2.684
C
1
1660
0.813
0.685 0.608
0.265
0.214 0.064 2.649
2
1628
0.818
0.691 0.618
0.269
0.216 0.065 2.678
3
1503
0.818
0.690 0.616
0.268
0.221 0.065 2.677
4
1497
0.821
0.684 0.612
0.266
0.219 0.064 2.666
D
1
1959
0.806
0.686 0.607
0.265
0.210 0.065 2.638
2
2159
0.811
0.685 0.612
0.267
0.212 0.065 2.651
3
2100
0.822
0.686 0.616
0.266
0.223 0.065 2.677
4
2096
0.815
0.680 0.611
0.267
0.219 0.064 2.657
E
1
2762
0.800
0.677 0.601
0.264
0.211 0.064 2.617
2
2449
0.817
0.679
0.61
0.267
0.218 0.065 2.656
3
2509
0.818
0.679 0.612
0.268
0.221 0.065 2.663
Mean, % w/w
0.816
0.686 0.613
0.267
0.216 0.065 2.663
RSD
r
, %
0.98
0.98
0.96
0.83
2.01
1.01
0.88
Table 12. Recovery of withanolides after spiking samples prior to or after sample extraction
Batch No.
Spiking
Recovery, %
Withanoside IV Withanoside V Withaferin A
12-Deoxy
withastramonolide Withanolide A Withanolide B
WS/05Lot21
Prior to sample
extraction
94.9
95.0
97.5
92.2
92.2
104.7
WS/06Lot10
93.5
95.4
94.5
91.6
96.5
97.3
RD/1170
93.6
93.6
95.1
95.0
94.1
95.8
RD/1162
97.7
94.4
97.6
90.5
93.2
96.5
WS/06Lot08
After sample
extraction
95.2
94.6
94.6
95.5
92.1
71.1
WS/05Lot20
99.6
98.5
97.8
99.2
96.4
72.4
RD/1045
94.3
94.7
94.8
95.8
91.0
70.7
ERH-46
99.5
99.0
98.4
99.3
97.2
72.7
62