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B
rown
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
97, N
o
. 5, 2014
1323
Determination of Aloin A and Aloin B in
Aloe vera
Raw Materials and Finished Products by High-Performance
Liquid Chromatography: Single-Laboratory Validation
P
aula
N. B
rown
, R
onan
Y
u
, C
hiow
H
ui
K
uan
, J
amie
F
inley
, and
E
lizabeth
M. M
udge
BC Institute of Technology, Centre for Applied Research and Innovation, Department of Natural Health and Food Products
Research, 3700 Willingdon Ave, Burnaby, BC, V5G 3H2
S
teven
D
entali
Herbalife International of America, Inc., 990 W. 190th St, Suite 650, Torrance, CA 90502
Received January 27, 2013. Accepted by APApril 10, 2014.
Corresponding author’s e-mail:
paula_brown@bcit.caDOI: 10.5740/jaoacint.13-028
DIETARY SUPPLEMENTS
A single-laboratory validation (SLV) was conducted
on an HPLC method for the detection and
quantification of aloin A and aloin B in
Aloe vera
raw materials and finished products. An extraction
procedure using sonication with an acidified solvent
was used for solid test materials while liquid test
materials only required dilution, if necessary, prior
to filtration and analysis. Separation was achieved
using a fused core C
18
column in 18 min under
isocratic elution conditions allowing for a single
analyte (aloin A) calibration curve to quantify both
aloins. Adequate chromatographic resolution
(Rs >1) was achieved for aloin A and aloin B. The
calibration curves for aloin A exhibited coefficients
of determination (r
2
) of >99.9% over the linear range
of 0.3–50 µg/mL. The LOD values were 0.092 and
0.087 µg/mL, and LOQ 0.23 and 0.21 µg/mL for aloin
A and aloin B, respectively. Repeatability studies
were performed on nine test materials on each
of 3 separate days, with five of the test materials
determined to be above the LOQ having repeatability
RSD (RSD
r
) values ranging from 0.61 to 6.30%.
Method accuracy was determined through a spike
recovery study on both liquid and solid matrixes at
three different levels: low, medium, and high. For
both aloins, the recovery in the liquid matrix ranged
from 92.7 to 106.3% with an RSD
r
of 0.15 to 4.30%,
while for the solid matrix, the recovery ranged from
84.4 to 108.9% with an RSD
r
of 0.23 to 3.84%. Based
on the results of the SLV study, it is recommended
that this method be evaluated for reproducibility
through a collaborative study.
T
he genus
Aloe
comprises more than 100
species of
semitropical perennial flowering plants. The most
common of these is
A. vera
(L.) Burm. F. (
A. barbadensis
Mill.), more commonly known as
A.
vera.
The plant is a native
of southern and eastern Africa and was subsequently introduced
to the northern part of Africa, the Arabian Peninsula, China,
Mediterranean countries, and the West Indies (1).
Historically,
A. vera
plants were cultivated for the treatment
of burns, dermatitis, and fungal infections (2). Furthermore,
they have been used as an ingredient in cosmetics, and the latex
of this and other species is generally known for its laxative
properties (3). The hydroxyanthrone derivates or anthraquinones
are active ingredients known to be responsible for this cathartic
effect (4). The anthraquinones, mainly aloins (15–40%), are
a mixture of aloin A (barbaloin) and aloin B (isobarbaloin),
which are mainly present in the latex of
A.
vera
(5). When
ingested, aloins are hydrolyzed and reduced to the active
metabolite (aloe-emodin-9-anthrone), which acts as an irritant
in the gastrointestinal tract, producing the purging and cleansing
reaction (6).
Pharmacological studies have shown that the consumption
of aloin-containing products causes acute and chronic adverse
reactions such as abdominal pain, electrolyte disturbances,
and even hepatitis (7–9). In 2002, the U.S. Food and Drug
Administration recommended that aloe laxatives no longer be
considered generally recognized as safe over-the-counter drugs
and further pharmacological and toxicological investigations are
necessary. In early 2011,
the National Toxicological Program
concluded a 2-year animal study on the toxic and carcinogenic
effects of nondecolorized whole leaf
A.
vera
juice in drinking
water and found clear evidence of carcinogenic activity in male
and female rodents (10).
In North America, the International Science Aloe Council has
set an industry guideline of less than or equal to 10 ppm total
aloins at single-strength concentrations to be considered safe
for ingredients in products intended for oral consumption (11).
In order to meet this standard, commercial producers and
manufacturers follow manufacturing procedures that process
A.
vera
leaf to remove the latex either through physical leaf rind
removal and/or decolorization. In this way, finished products can
be certified to contain minute to nondetectable levels of aloins.
To support a program of this nature, a validated analytical
method that is reliable and suitable for its intended purpose
of determining and quantifying aloin A and aloin B in
A.
vera
juice and finished products is essential. Although there are
several analytical methods available (12–16) that can detect
and quantify aloins, none of these methods have addressed
performance characteristics such as accuracy, sensitivity,
specificity, reproducibility, LOD, LOQ, linearity, and range of
the test method using AOAC INTERNATIONAL guidelines. By
having a validated method,
A.
vera
ingredients and products can
be examined with a degree of confidence that they contain no
more than the permissible aloin level set as an industry standard.