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© 2015 AOAC INTERNATIONAL
AOAC Official Method 2015.11
Chondroitin Sulfate Content in Raw Materials
and Dietary Supplements
High-Performance Liquid Chromatography
with Ultraviolet Detection After Enzymatic Hydrolysis
First Action 2015
A. Materials
Chondroitin sulfate (CS) raw materials from bovine trachea,
porcine skin/cartilage, and shark cartilage, and CS control material
from bovine trachea were obtained from Bioiberica (Barcelona,
Spain). Dietary supplement products containing CS (hard-shell
capsules, tablets, chewables, softgels, and liquids) were obtained
from commercial suppliers. Descriptions of the dietary supplement
products used in the study are presented in Table
2015.11A
.
B. Apparatus
(
a
)
LC system
.—Beckman 126 dual high pressure mixing pumps
(Beckman Coulter, Fullerton, CA, USA), 168 diode array UV
detector, 507e autosampler, and 32 Karat software.
(
b
)
Operating conditions
.—Mobile phase flow rate,
1.1 mL/min; column temperature, ambient; injection volume,
30 μL; and detection, 240 nm.
(
c
)
LC column
.—Phenomenex Synergi Polar-RP, 4.6 × 150 mm,
4 μm particle size (Phenomenex, Torrance, CA, USA).
(
d
)
Analytical balance
.—Accu-124 (Fisher Scientific, Pittsburgh,
PA, USA), ±0.01 mg readability.
(
e
)
Ultrasonic bath
.—Model FS60H (Fisher Scientific).
(
f
)
pH meter
.—Model pH 500 (Oakton, Vernon Hills, IL, USA),
±0.01 pH unit readability.
(
g
)
Dry block heater
.—Isotemp Dry Bath Incubator (Fisher
Scientific), maintained at 37°C.
(
h
)
LC injection vials
.—2 mL, with caps and Teflon-coated septa.
(
i
)
Limited volume inserts
.—200 μL, for LC vials.
(
j
)
Syringes
.—25, 100, and 500 μL Luer-Lok.
C. Reagents
Note
: Chemicals from other suppliers meeting the specifications
may also be used.
(
a
)
Solvents
.—Acetonitrile, LC grade; water, LC grade;
hydrochloric acid, concentrated, ACS reagent grade.
(
b
)
Tetrabutylammonium bisulfate
.—Minimum 99.0% (Fluka,
St. Louis, MO, USA;
http://www.sigma-aldrich.com; Cat.
No. 86868).
(
c
)
Tris-(hydroxymethyl)aminomethane (TRIS)
.—Sigma (St.
Louis, MO, USA), Cat. No. T-1503.
(
d
)
Sodium acetate
.—Anhydrous (Sigma, Cat. No. S-8750).
(
e
)
Acetic acid
.—Glacial (Sigma, Cat. No. A-0808).
(
f
)
Sodium chloride
.—ACS reagent grade.
(
g
)
Bovine serum albumin
.—1x Crystallized, 97% (Sigma, Cat.
No. A-4378).
(
h
)
Chondroitinase AC II
.—Five units (Seikagaku America/
Associates of Cape Cod, East Falmouth, MA, USA; Cat.
No. 100335-1A,
http://www.acciusa.com).
(
i
)
Mobile phase A
.—Weigh 340 mg tetrabutylammonium
bisulfate and transfer into a 1000 mL volumetric flask. Dissolve
and dilute to volume with water. Degas.
(
j
)
Mobile phase B
.—Weigh 340 mg tetrabutylammonium
bisulfate and dissolve in 330 mL deionized (DI) water. Add
acetonitrile to 1000 mL. Sonicate and filter.
(
k
)
0.12 M HCl
.—Carefully add 1 mL concentrated HCl to
99 mL water and mix well.
(
l
)
6 M HCl
.—Carefully add 50 mL concentrated HCl to 50 mL
water and mix well.
(
m
)
TRIS buffer solution
.—Dissolve 3 g TRIS, 2.4 g sodium
acetate, 1.46 g sodium chloride, and 50 mg crystalline bovine
serum albumin in 100 mL of 0.12 M HCl. Adjust pH to 7.3 with
6 M HCl.
(
n
)
Enzyme solution
.—Dissolve 5 units of chondroitinase ACII
enzyme in 0.5 mL water. Store at <0°C when not in use.
(
o
)
Dilution solution
.—Prepare at least 20 mL of a solution
containing 80% mobile phase A and 20% mobile phase B.
(
p
)
Reference standards
.—
See
Table
2015.11B
. Purities were
obtained from the supplier’s certificate of analysis. These purities
were determined by chromatographic purity, water content, and
residual solvent content. No independent confirmation of the purity
was performed.
D. Preparation of Test Solutions
(
a
)
Preparation of standard solutions
.—Accurately weigh
about 2 mg ΔDi-0S and 10 mg each of ΔDi-4S and ΔDi-6S
(Table
2015.11B
), and transfer into a 50 mL volumetric flask.
Dissolve and dilute to volume with water. This is the stock
instrument calibration solution.
(
b
)
Instrument calibration solutions
.—Prepare serial dilutions of
the stock instrument calibration solution in water at concentrations
of about 2, 8, 20, 40, and 100 μg/mL ΔDi-4S and ΔDi-6S, and 0.4,
1.6, 4, 8, and 20 μg/mL ΔDi-0S. A possible dilution scheme is
shown in Table
2015.11C
.
Table 2015.11A. Materials
Material No.
Type
Composition
Claim
1
Raw material
CS from bovine trachea
Pure
2
Capsules
CS
250 mg CS/capsule
3
Chewables
CS + glucosamine, ascorbic acid
125 mg CS/wafer
4
Tablets
CS + glucosamine sulfate (from marine source)
400 mg CS/tablet
5
Softgels
CS + glucosamine
200 mg CS/softgel
6
Raw material
CS from porcine
Pure
7
Raw material
CS from shark cartilage
Pure
8
Tablets
CS + ascorbic acid, manganese, herbs
200 mg CS/tablet
9
Liquid
CS + glucosamine, MSM, ascorbic acid, manganese
800 mg CS/29.57 mL
Candidates for 2016 Method of the Year
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