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analytical lab news1 News #1: Chondroitin
analytical lab news 2 News #2: Glucosamine
analytical lab news3 News #3: L-Carnitine
analytical lab new4 News #4: Chondroitin & Glucosamine in Pet Food
analytical lab new5 News #5: Single Lab Validation
analytical lab news6 News #6: Identification and Quantification of Oversulfated Chondroitins
   
   
   
Activities
Member of AOAC method Committee for Dietary Supplement
Active participant in Dietary Supplement Task Force
Official contractor for NIH through BPA program (Blanket Purchase Agreement)
 
Completed Single Lab Validation of Enzymatic-HPLC Method for Chondroitin Sulfate Assay funded by NIH, and the paper is going to be published on Journal of AOAC
 
Developed Pre-Column Derivatization Method for Glucosamine Assay funded by NIH, and the paper have been published on Journal of AOAC
 
 
 
 
 
Enzymatic - HPLC Method for Chondroitin Sulfate Testing

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).

testing data diagram

testing data diagram

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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