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© 2015 AOAC INTERNATIONAL
where P
2,4,6
= peak area of ΔDi-tri(2,4,6)S in sample chromatogram;
b
6
= y-intercept of calibration curve for disaccharide ΔDi-6S; m
6
=
slope of calibration curve for disaccharide ΔDi-6S; V = volume of
Test Solution 1 = 100 mL; W = sample weight, in g; D = dilution
factor = 50; and F = molecular weight conversion between ΔDi-6S
and ΔDi-tri(2,4,6)S = 1.380.
(
g
) The total amount of CS in μg/g in the sample is the sum of
ΔDi-0S, ΔDi-4S, ΔDi-6S, ΔDi-di(2,6)S, ΔDi-di(4,6)S, and ΔDi-
tri(2,4,6)S.
(
h
) % (w/w) is calculated from μg/g as follows:
(
i
) Milligrams per tablet (mg/tab) is calculated from μg/g as
follows:
where TW = the average tablet weight in grams.
(
j
) Milligrams per capsule (mg/cap) is calculated from μg/g as
follows:
where FW= the average capsule fill weight in grams.
(
k
) Milligrams per milliliter (mg/mL) is calculated from μg/g for
liquid samples as follows:
where SG = the specific gravity of the sample in g/mL.
G. Validation Design
(
a
)
Linearity
.—The five instrument calibration solutions were
injected at the beginning of each chromatographic injection
sequence, after every 20 sample injections, and at the end of
each sequence. A 5-point standard curve was generated for all
three analytes, and the slope, y-intercept, correlation coefficient,
and relative standard deviation (RSD) of the standard curve were
calculated for using the average peak areas at each calibration point
on each day.
(
b
)
Repeatability
.—Four replicates of each of the Materials 1–5
(Table
2015.11A
) representing a CS raw material, a hard-shell
capsule product containing CS, a tablet product containing CS, a
chewable product containing CS, and a liquid product containing
CS were prepared on each of 3 days, for a total of 12 replicate
preparations of each material. The within-day, between-day, and
total repeatability of the total CS content were calculated. The
HorRat value (1) for each material was also calculated. In addition,
four replicates of each of the Materials 6–9 were prepared on a
single day to demonstrate the applicability of the method to these
materials. The within-day repeatability was calculated for these
materials.
(
c
)
Accuracy
.—(
1
)
CS raw material
.—Heparin, a related
glycosaminoglycan (GAG), was used as a negative control. About
200 mg heparin was transferred into ten 100 mL volumetric flasks;
300 mg bovine trachea CS raw material used in the repeatability
study (Material 1 in Table
2015.11A
) was added to three of the
flasks, 200 mg of the same CS raw material was added to another
three of the flasks, 100 mg of the CS raw material was added to
another three of the flasks, and the 10th flask was used as a negative
control. Each of the spiked negative controls was prepared and
analyzed according to the method on 3 separate days.
(
2
)
Spike recovery of dietary supplement finished products
.—A
dietary supplement tablet product containing glucosamine HCl and
methyl sulfonylmethane (MSM) was used as a negative control
for spike recovery study of dietary supplement finished products.
The tablets were first ground to a powder and homogenized. About
500 mg of tablet negative control material was transferred into
ten 100 mL volumetric flasks. The tablet negative control was
then spiked with the bovine trachea CS raw material used in the
repeatability study using the same procedure as described for the
CS raw material spike recovery study. Each of the spiked negative
controls was prepared and analyzed according to the method on 3
separate days.
(
d
)
Ruggedness
.—AYouden ruggedness study was conducted on
the bovine trachea raw material, varying the seven factors presented
in Table
2015.11E
(2).
(
e
)
Selectivity
.—The selectivity of the method was demonstrated
by injecting solutions of non-CS ingredients typically found
in CS-containing dietary supplements, including glucosamine,
MSM, vitamins, and minerals, into the chromatographic system
after treatment with enzyme. In addition, possible contaminants
and/or adulterants, such as carrageenan, dermatan sulfate, and
heparin, were subjected to the same sample preparation procedure
and injected into the chromatographic system. The potential
chromatographic interference of hyaluronic acid (HA) was also
investigated.
(
f
)
Stability
.—The stabilities of the chondroitinase AC II enzyme
in solution and the sample solution were evaluated over the course
of the study.
(
1
)
Enzyme stability
.—A portion of the enzyme solution used
to prepare the precision samples from Day 1 was stored at –20°C.
Table 2015.11E. Youden ruggedness testing
Parameter
High value
Low value
Factor
Sonication time, min
A = 30
a = 15
0.45
Sample weight, mg CS
B = 200
b = 100
–0.45
Digestion temperature,
°
C
C = 42
c = 37
–0.8
Concentration of enzyme solution
D = 5 units/0.5 mL
d = 5 units/1.0 mL
–0.05
pH of TRIS buffer solution
E = 7.1
e = 7.5
0.8
Injection volume,
μ
L
F = 50
f = 30
1.3
Detector wavelength, nm
G = 240
g = 235
–0.6
Candidates for 2016 Method of the Year
133