5 / 9
1326
B
rown
et al
.
:
J
ournal of
AOAC I
nternational
V
ol
. 97, N
o
. 5, 2014
(
b
)
Linearity
.—Linearity was evaluated using a seven-point
standard calibration curve of aloin A. Calibration curves were
plotted and linear regression was used to determine the slope and
y-intercept. Linearity was also confirmed visually by generating
trendlines using a Microsoft Excel program (Microsoft,
Mississauga, ON, Canada).
(
c
)
LODand LOQ
.—The LODand LOQ for the quantification
of aloin A and aloin B were determined using the International
Union of Pure and Applied Chemistry (IUPAC) method. This
method, recommended byAOAC INTERNATIONAL, uses data
sets from at least seven replicate injections of the blank matrix.
The LOD is defined as the mean response plus 3x the SD, and the
LOQ is the mean plus 10x the SD.
(
d
)
Precision (repeatability; precision study)
.—Precision was
evaluated by analyzing multiple replicates of each test sample.
Four replicate preparations of each test material were prepared
and analyzed on 3 separate days, thereby having 12 replicates of
each test material prepared.
The within-day, between-day, and total SDs for aloin A and
aloin B for each of the nine test materials were calculated and
consequently used to generate HorRat values by using the
Horwitz formula for each of the test material to summarize the
precision of the validation study (19). Acceptable HorRat values
are 0.1 to 2.0.
(
e
)
Accuracy
.—Accuracy of the method was assessed using
both solid and liquid aloe test samples that were determined
not to contain any detectable aloins. Stock solutions of known
concentrations were spiked in both liquid and solid materials
in triplicate at three levels that correspond to 10% (low), 100%
(medium), and 200% (high) of aloin A and aloin B expected in
typical aloin raw materials and finished products.
For the solid matrix recovery study, a commercial spray-dried
A.
vera
powder was spiked to expected concentrations of 4,
40, and 80 µg/mL. An
A. vera
inner fillet juice was spiked to
expected concentrations of 1, 10, and 20 µg/mL for the liquid
matrix. The samples were processed as per the
Preparation of
Test Samples
section above, taking into account the volume of
spike solution added.
(
f
)
Stability
.—The stability of the aloin A and aloin B was
determined by comparing the concentration of the reference
standards either acidified with 0.1% acetic acid or nonacidified at
room temperature over time. Each aloin was evaluated by storing
the solutions at room temperature (18–22°C). Identical reference
standards were aliquotted in two sets having four replicates each:
one set was acidified and the other set nonacidified and analyzed
at a given time (t) point (t = 0, 4, 8, 24, 48, and 240 h). Stability
was determined by comparing the peak areas to time = 0 where
the assumption of no degradation was made.
Results and Discussion
Optimization Studies
Prior to the method validation, the extraction and
chromatography parameters were investigated through
optimization studies. For the extraction optimization, parameters
such as extraction solvent, the addition of acid, sonication time,
and the effect of heat were explored. For the chromatography
method, parameters such as analytical column length and
type, mobile phase composition, gradient versus isocratic
elution, peak resolution, and run time were factors that were
examined to provide the best resolution and shortest run time for
the separation of aloin A and aloin B. The optimization of the
method was carried out using both aloin reference standards and
A.
vera
test materials.
Method Validation Results–Performance
Characteristics
Identification of aloin A and aloin B in the test materials
was possible by comparing the retention times of the aloins
from the reference standards. An isocratic elution was used for
the analysis of aloin A and aloin B, where the retention order
is aloin B followed by aloin A. Representative chromatograms
of the reference standards and test material are displayed in
Figures 1 and 2.
Quantification of the analytes was carried out by a simple
linear regression analysis using quadruplicate samples prepared
on three separate days at seven concentration levels. The
analytical range used for the following aloins are listed above
in the section on
Preparation of Calibration and QC Solutions
.
Table 2. The LOD and LOQ by the IUPAC method
Analyte
LOD, µg/mL
LOQ, µg/mL
Aloin A
0.09
0.23
Aloin B
0.09
0.21
Figure 1.
10
2
3
4
5
6
7
8
9
Absorbance (mAU)
0
5
10
15
20
25
30
35
Aloin B
Aloin A
Time (min)
Figure 1. Chromatogram of an aloin A and aloin B reference
standard.
Figure 2.
10
2
3
4
5
6
7
8
9
Absorbance (mAU)
0
5
10
15
20
25
30
35
Aloin A
Aloin B
Time (min)
Figure 2. Chromatogram of an untreated
A. vera
juice product.