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K
line
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
3, 2017
1
Quantitative Analysis of Aloins and Aloin-Emodin in Aloe
Vera Raw Materials and Finished Products Using High-
Performance Liquid Chromatography: Single-Laboratory
Validation, First Action 2016.09
D
avid
K
line
, V
icha
R
itruthai
,
and
S
ilva
B
abajanian
Herbalife International of America, 950 West 190th St, Torrance, CA 90502
Q
uanyin
G
ao
1
and
P
rashant
I
ngle
Herbalife Manufacturing, LLC, Quality Control Department, 20481 Crescent Bay Dr, Lake Forest, CA 92630
P
eter
C
hang
and
G
ary
S
wanson
Herbalife International of America, 990 West 190th St, Torrance, CA 90502
A single-laboratory validation study is described for
a method of quantitative analysis of aloins (aloins A
and B) and aloe-emodin in aloe vera raw materials
and finished products. This method used HPLC
coupled with UV detection at 380 nm for the aloins and
430 nm for aloe-emodin. The advantage of this test
method is that the target analytes are concentrated
from the sample matrix (either liquid or solid form)
using stepwise liquid–liquid extraction (water–ethyl
acetate–methanol), followed by solvent evaporation
and reconstitution. This sample preparation process
is suitable for different forms of products. The
concentrating step for aloins and aloe-emodin has
enhanced the method quantitation level to 20 parts
per billion (ppb). Reversed-phase chromatography
using a 250 × 4.6 mm column under gradient elution
conditions was used. Mobile phase A is 0.1% acetic
acid in water and mobile phase B is 0.1% acetic acid
in acetonitrile. The HPLC run starts with a 20% mobile
phase B that reaches 35% at 13 min. From 13 to
30 min, mobile phase B is increased from 35 to 100%.
From 30 to 40 min, mobile phase B is changed from
100% back to the initial condition of 20% for
re-equilibration. The flow rate is 1 mL/min, with a
100 µL injection volume. Baseline separation (R
s
> 2.0)
for aloins A and B and aloe-emodin was observed
under this chromatographic condition. This test
method was validated with raw materials of aloe
vera 5× (liquid) and aloe vera 200× (powder) and
finished products of aloe concentrate (liquid) and aloe
(powder). The linearity of the method was studied from
10 to 500 ppb for aloins A and B and aloe-emodin, with
correlation coefficients of 0.999964, 0.999957, and
0.999980, respectively. The test method was proven
to be specific, precise, accurate, rugged, and suitable
for the intended quantitative analysis of aloins and
aloe-emodin in raw materials and finished products.
The S/N for aloins A and B and aloe-emodin at 10 ppb
level were 12, 10, and 8, respectively, indicating our
conservative LOD level at 10 ppb (the typical LOD
level S/N is about 3). The S/N for aloins A and B and
aloe-emodin at the 20 ppb level were 17, 14, and 16,
respectively, indicating our conservative LOQ level
at 20 ppb (the typical LOQ level S/N is about 10). The
stock standard solution of a mixture of aloins and
aloe-emodin and a working standard solution were
found to be stable for at least 19 days when stored
refrigerated at 2–8°C, with a recovery of 100 ± 5%.
A
loe vera has long been used in health foods and dietary
supplements (1, 2). Aloin A (barbaloin), aloin B
(isobarbaloin), and aloe-emodin are natural components
in aloe vera and are referred to as anthraquinones (3). These
compounds are removed during the raw material manufacturing
process because recent literature indicates adverse effects if the
compounds are consumed in sufficient quantities (4). In 2011, the
International Aloe Science Council provided an industry guideline
limiting the amount of aloins present in aloe products for oral
consumption to less than 10 parts per million (ppm; Figure 1; 4).
In 2015,AOAC
StandardMethod Performance Requirements
called for “Methods for the Determination of Aloin A and Aloin
B in Dietary Supplement Products and Ingredients” (5) with a
low parts-per-billion (ppb) level quantitation limit. To support
this AOAC initiative and to QC raw materials and finished
products of aloe vera, it was essential to develop and validate a
method to quantitate aloins Aand B. Several analytical methods
are published in the literature for the detection and quantitation
of aloins; however, these lack the required performance
characteristics of accuracy, precision, specificity, linearity, and
LOD and LOQ (6–11). Our test method had conservative LOQ
levels at 20 ppb with nearly twice the response (S/N) of what
is usually required using a conventional-sized HPLC column
(250 × 4.6 mm) and HPLC instrument. In using an ultra-
performance LC system and smaller particle-size columns, it is
possible that the LOQ level may reach single-digit ppb levels.
DIETARY SUPPLEMENTS
Received November 16, 2016. Accepted by AP January 17, 2017.
This method was approved by the AOAC Expert Review Panel for
Aloin as First Action.
The Expert Review Panel for Aloin Methods invites method users
to provide feedback on the First Action methods. Feedback from
method users will help verify that the methods are fit-for-purpose
and are critical for gaining global recognition and acceptance of the
methods. Comments can be sent directly to the corresponding author
or
methodfeedback@aoac.org.1
Corresponding author’s e-mail:
quanying@herbalife.comDOI: 10.5740/jaoacint.16-0387
3