Chondroitin Sulfate(CS): CS is naturally
found in many living organisms. This macromolecule
naturally co-exists with several other known mucopolysaccharides
e.g. Keratan Sulfate, heparin, heparan sulfate,
dermatan sulfate etc. The extraction and purification
of CS is a process to selectively concentrate the
CS to the desired purity level. Many methods have
been used to verify the purity of CS products. The
capability and shortfalls of several methods are
compared below:
- Carbozole-This method is based on
the principle of strong acid hydrolysis to break
the components of a disaccharide into its monosaccharides
(hexuronic acid and hexosamine). The glucuronic
acid is then measured by a color reaction. The
method is easy to use, low cost and relatively
rugged. However, it is not specific. Other mucopolysaccharides
containing glucuronic acid, such as Heparin
or free glucuronic acid itself, if gone through
the same testing procedure, would give a similar
response as to CS.
- CPC Titration-This method is based
on formation of turbidity when a reagent (called
CPC=Cetyl Pyridinium Chloride) reacts with organic
anions such as sulfate or carboxylate ion under
slightly basic condition. The key word here
is ORGANIC ANION. The method is not specific
for CS. Other known or unknown mucopolysaccharides
would react to the reagent in the same way that
CS does. Even carboxylic acid groups on proteins
would react the same way as CS. Turbidity can
be measured by either auto- or manual-titrator.
This method must be combined with an array of
other techniques in order to obtain reliable
confirmation of the purity and identity of Chondroitin
sulfate.
- Reversed Phase HPLC-The lack of chromophores
and retention of this polymeric molecule results
in peaks typically<3.0 min on a RP column,
near the solvent front when UV detection at
200-205 nm is used. Again, this is not a stand-alone
method, because it demonstrates neither separation
nor specific UV absorption.
- Enzymatic HPLC-This method is based
on specific enzymatic hydrolysis to produce
disaccharides Di-4S, Di-6S and Di-0S that make
up the Chondroitin Sulfate A and C and non-sulfated
Chondroitin. The resulting disaccharides are
then measured by HPLC with a UV detector at
240 nm. The method is specific (virtually free
of interference) because of the selective reaction
of enzyme Chondroitinase ABC. It uses a standard
HPLC-with UV detector. It is rugged, robust
and accurate. Typical performance characteristics
are described below:
Method detection limit is 1.0ug.
Within the linear range 1.0ug -1000ug/ml,
r2 >0.999 for all three disaccharides.
Spiking with CS of known purity and A/C ratio,
recoveries never fall below 95%.
Spiking with disaccharides reference standards,
recoveries ranges 94 to 99%.
Ruggedness & Robustness: method has been
in use since July 1999. Control sample [fig.2
(A)] is run along with every batch of samples
tested in this lab.
SQC (Statistical Quality Control) chart for
9 month period shows a variation range <+
0.98%.
In addition to the excellent performance characteristics
listed above, the enzymatic method also provides
accurate information on the Chondroitin sulfate
A & C ratio that no other method can provide.
Figure 1 shows authentic bovine trachea CS, where
A/C ratio is typically around 1.5, comparing to
CS from other sources where the A/C ratio ranged
from 2 to 5.5 typically.
Fig. 1. Typical chromatograms of authentic bovine
trachea (A) vs. CS from other origin (B). A/C
ratio = 1.48 in (A), and A/C ratio= 4.2 in (B).
The therapeutic significance of a higher Chondroitin
sulfate C is still under debate. However, one
brand of CS product that has proved its efficacy
again and again through clinical trials uses only
the CS that has the A/C ratio at 1.5. This enzymatic
HPLC method has been developed and used on raw
materials, dosage forms and animal feed containing
as low as 0.01% CS at Analytical
Laboratories In Anaheim, Inc
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