3 / 9
1324
B
rown
et al
.
:
J
ournal of
AOAC I
nternational
V
ol
. 97, N
o
. 5, 2014
A validated method can also aid in future assessments regarding
safety, use, and application of
A.
vera
products.
Experimental
Principle
This method is used to detect and quantify aloin A and aloin B
in
A.
vera
leaf juice, dietary supplements, and finished products
by HPLC with a diode array detector (DAD). This method has
been summarized in the American Herbal Pharmacopeia (AHP)
monograph for
A.
vera
leaf, leaf juice, and inner leaf juice (17).
The focus of the validation study is the diastereomers
aloin A (barbaloin) and aloin B (isobarbaloin), although other
anthraquinone-type compounds are known to exist in
A.
vera
containing materials. These diastereomers are present in the
latex of the plant, which is removed during processing of raw
materials into juices and other finished products since recent
studies have shown adverse effects when they are consumed in
sufficient quantities.
Test Samples
The validation consisted of nine test materials.
(
a
)
A. vera inner leaf dry juice powder A
.—Obtained from
Tampa Bay Analytical Research (Tampa Bay, FL).
(
b
)
A. vera inner leaf dry juice powder B
.—Obtained from
Tampa Bay Analytical Research.
(
c
)
A. vera nondecolorized leaf dry juice powder
.—Obtained
fromAloecorp (Ontario, Canada).
(
d
)
A. vera decolorized inner leaf dry juice powder
.—
Obtained fromAloecorp.
(
e
)
A. vera leaf juice powder capsule
.—Obtained fromTampa
Bay Analytical Research.
(
f
)
A. vera nondecolorized leaf juice
.—Obtained from
Aloecorp.
(
g
)
A. vera decolorized leaf juice
.—Obtained fromAloecorp.
(
h
)
A. vera nondecolorized inner leaf juice
.—Obtained from
Aloecorp.
(
i
)
A. vera nondecolorized inner leaf juice concentrate
.—
Obtained fromAloecorp.
All solid test samples were stored in a desiccator at room
temperature, and liquid test samples were stored at 4°C. For
capsules, to minimize variability due to nonuniformity between
dosage units, contents of 20 capsules were combined and
homogenized prior to analysis.
Reagents and Supplies
(
a
)
Acetic acid, glacial
(C
2
H
4
O
2
).—Formula weight (FW):
60.05 g/mol; CAS No. 64.19-7, ACS reagent grade (Fisher
Scientific, Ottawa, ON, Canada) or equivalent.
(
b
)
Methanol
(CH
4
O).—FW: 32.04 g/mol; CAS No. 67-65-1,
HPLC grade (VWR International, Mississauga, ON, Canada) or
equivalent.
(
c
)
Acetonitrile
(C
2
H
3
N).—FW: 60.10 g/mol, CAS No. 75-
05-08, HPLC grade (VWR International) or equivalent.
(
d
)
Water
(H
2
O).—FW: 18.01 g/mol; CAS No. 7732-18-5,
HPLC grade (Fisher Scientific) or equivalent.
Solutions
(
a
)
Reference standard diluent
.—100% methanol.
(
b
)
Extraction solvent for high concentration test
materials
.—0.1% (v/v) acetic acid in methanol.
(
c
)
Extraction solvent for low concentration test
materials
.—0.1% (v/v) acetic acid in water.
(
d
)
Mobile phase A
.—0.1% (v/v) acetic acid in water (filtered
through a 0.2 µm nylon filter).
(
e
)
Mobile phase B
.—0.1% (v/v) acetic acid in acetonitrile.
Reference Standards
Separate stock solutions of 100 µg/mL aloin A and aloin B
are prepared by dissolving 2.83 mg aloin A in 25.0 mL reference
standard diluent and 2.74 mg aloin B in 25.0 mL reference
standard diluent. The stock solution of aloin A is then diluted to
appropriate concentrations for external calibration. The aloin B
stock solution is used as a QC sample.
(
a
)
Aloin A
(C
21
H
22
O
9
).—FW: 418.39 g/mol; CAS No. 8015-
61-0, purity 88.4%, acquired from Chromadex (Irvine, CA), Cat.
No. 1625.
(
b
)
Aloin B
(C
21
H
22
O
9
).—FW: 418.39 g/mol; CAS No. 8015-
61-0, purity 91.3%, acquired from Chromadex, Cat. No. 1626.
Reference standards are stored at 4°C until use.
Apparatus
(
a
)
Analytical balance
.—Capable of reading ±0.01 mg
(Mettler Toledo, Vancouver, BC, Canada) or equivalent.
(
b
)
HPLC vials
.—Clear, volume 300 µL (Phenomenex,
Torrance, CA).
(
c
)
Centrifuge
.—Eppendorf 5810 table top centrifuge (VWR
International) or equivalent.
(
d
)
Centrifuge tubes
.—15 mL polypropylene (VWR
International).
(
e
)
Micropipets
.—Eppendorf Reference series 100, 200, and
1000 µL (Eppendorf, Mississauga, ON, Canada) or equivalent.
(
f
)
Solvent bottles
.—100, 500, and 1000 mL.
(
g
)
Syringe filters
.—Luer-lok
®
type, 0.2 µm PTFE (VWR
International).
(
h
)
Disposable syringes
.—3 mL screw-type (VWR
International).
(
i
)
Sonicating water bath
.—Bransonic 3510 (VWR
International) or equivalent.
(
j
)
Vortex mixer
.—Thermo Scientific Maxi Mix1 (VWR
International) or equivalent.
(
k
)
Volumetric flasks
.—Class A; 2, 5, and 10 mL.
(
l
)
Volumetric pipets
.—Class A; 1, 2, 4, and 8 mL.
HPLC Conditions
(
a
)
Instrument
.—The HPLC system must be equipped with
a DAD or UV detector, sample injection system, pump capable
of delivering constant flow up to 400 bar, temperature-controlled
column compartment, and computing data processor.
(
b
)
Autosampler temperature
.—Room temperature.