8/22/2014
12
Enzymatic HPLC Method
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Modifications to method that may improve
performance/reproducibility between labs
I
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f Δdi 0S
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id k
•
ncreas ng retent on o
‐ away rom vo pea
(several labs did not achieve resolution)
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USP method uses anion exchange (SAX) column
•
Performing reaction in 2‐ mL or 5‐ mL conical
bottomed reaction vials to eliminate transfer step
•
For raw materials, dry sample in oven prior to
weighing, and place desiccant in balance chamber
Concerns Surrounding Enzymatic HPLC
Method
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Lack of availability and purity issues associated with
individual disaccharide reference standards
•
Adopting USP’s method of using a USP Chondroitin
Sulfate RM (or other well characterized/qualified
Chondroitin RM) verses the individual disaccharides
would address this issue
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Limited supply of enzyme
•
IBEX was only supplier for the past couple of years but
recently Sigma started offering Chondroitinase AC again
so availability is no longer an issue
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Expensive to run
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Technically Challenging
