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© 2015 AOAC INTERNATIONAL
(
c
)
CS control solution
.—Accurately weigh about 100 mg CS
control sample into a 50 mL volumetric flask. Add about 30 mL
water, and sonicate until the sample is completely dissolved (about
15 min). Dilute to volume with water and mix well. Label Control
Solution 1.
(
d
)
Sample test solutions
.—(
1
)
Raw materials
.—Accurately
weigh about 200 mg CS raw material into a 100 mL volumetric
flask. Add 60 mL water, and sonicate until the sample is completely
dissolved (about 15 min). Dilute to volume with water and mix
well. Label Test Solution 1.
(
2
)
Tablets
.—Determine the average tablet weight by weighing
20 tablets and calculating the average weight of one tablet. Grind
the 20 tablets to a powder and mix. Accurately weigh a test portion
containing the equivalent of about 200 mg CS into a 100 mL
volumetric flask. Add about 60 mL water and sonicate for 15 min.
Dilute to volume with water and mix thoroughly. Filter ca 1–2 mL
Test Solution 1 through a 0.2 μm PTFE syringe filter. Label Test
Solution 1.
(
3
)
Capsules
.—Determine the average capsule content weight by
weighing 20 capsules. Record the weight. Empty and combine the
capsule contents. Thoroughly clean the capsule shells using a swab
and/or compressed air. Weigh and record the weight of the empty
capsule shells:
Average capsule fill weight, g =
where C = total weight of 20 capsules and S = total weight of
20 capsule shells. Proceed as directed in (
d
)(
2
) for tablets.
(
4
)
Liquid formulations
.—Thoroughly mix the sample.
Accurately weigh an amount of test portion containing the
equivalent of about 200 mg into a 100 mL volumetric flask.
Dissolve in and dilute to volume with water. Mix thoroughly. Label
Test Solution 1.
(
e
)
Enzymatic hydrolysis of control solution and test solution
.—
Pipet 20 μLTRIS buffer solution, 30 μL enzyme solution, and 20 μL
Control Solution 1 or Test Solution 1 into a 2 mL LC injection vial
with a 200 μL insert. Place the vial in a 37°C dry bath or water
bath for 3 h. Allow to cool room temperature. Using an automatic
pipettor or gas-tight syringe, carefully transfer the solution into
an LC vial. Rinse the 200 μL insert with exactly 100 μL mobile
phase A using a calibrated automatic pipettor or gas-tight syringe,
and quantitatively transfer this into the LC vial. Dilute to 1.00 mL
by adding 830 μL mobile phase A to the LC vial. Mix well. Label
Control Solution 2 or Test Solution 2–Treated.
E. Determination
(
a
)
Mobile phase gradient program
.—Elute the analytes with
the linear gradient program of mobile phases A and B shown in
Table
2015.11D
.
(
b
)
System suitability tests
.—Equilibrate the LC system with the
mobile phases for at least 30 min until a stable baseline is obtained.
Inject each of the five instrument calibration solutions. Use linear
regression to determine the slopes, y-intercepts, and correlation
coefficient (r
2
) of the calibration lines for ΔDi-0S, ΔDi-4S, and
ΔDi-6S. The correlation coefficient of the calibration line for each
component must be >0.998 (for Di-0S >0.995). The tailing factor
for all the components in the linearity standards must be between
0.80 and 1.5. Inject Control Solution 2–Treated and calculate the
total amount of CS in the control material,
F
(
a
)–(
h
). The recovery
should be within ±3% of the specification.
(
c
)
Injection
.—Make single injections of each standard and
test solution. After every 20 sample injections, and after all of the
sample injections are completed, make a single injection of each
standard solution.
(
d
)
Retention times
.—The approximate retention times for each
analyte are presented in Figure
2015.11A
.
(
e
)
Chromatograms
.—Representative standard and sample
chromatograms are presented in Figures
2015.11A
–
C
.
Table 2015.11B. Reference standards
Name
Abbreviation
Supplier
2-Acetamido-2-deoxy-3-
O
-(
β
-D-gluco-4-enepyranosyluronic acid)-D-galactose
Δ
Di-0S
Sigma
2-Acetamido-2-deoxy-3-
O
(
β
-D-gluco-4-enepyranosyluronic acid)-4-
O
-sulfo-D-galactose
Δ
Di-4S
Sigma
2-Acetamido-2-deoxy-3-
O
-(
β
-D-gluco-4-enepyranosyluronic acid)-6-
O
-sulfo-D-galactose
Δ
Di-6S
Sigma
2-Acetamido-2-deoxy-3-
O
-(2-
O
-sulfo-
β
-D-gluco-4-enepyranosyluronic acid)-6-
O
-sulfo-D-galactose
Δ
Di-di(2,6)S
ICN
a
2-Acetamido-2-deoxy-3-
O
-(
β
-D-gluco-4-enepyranosyluronic acid)-4,6-di-
O
-sulfo-D-galactose
Δ
Di-di(4,6)S
ICN
2-Acetamido-2-deoxy-3-
O
-(2-O-sulfo-
β
-D-gluco-4-enepyranosyluronic acid)-4,6-di-
O
-sulfo-D-galactose
Δ
Di-tri(2,4,6)S
ICN
a
Now MP Biomedicals (Solon, OH, USA).
Table 2015.11D. Linear mobile phase gradient
a
Time, min
Mobile phase A, % Mobile phase B, %
0
80
20
0–7.0
35
65
7.0–12.0
35
65
12–12.5
80
20
a
The column should be reequilibrated at the starting mobile phase
conditions for at least 10 min after each injection.
Table 2015.11C. Preparation of instrument calibration
solutions
Calibration solutions
Volume of stock
pipetted, mL
Final volume
(flask size), mL
1
25
50
2
10
50
3
5
50
4
2
50
5
1
100
Candidates for 2016 Method of the Year
131