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CHON-002

Capillary zone electrophoresis (CZE) was used for the analysis

of unsaturated CS disaccharides. Response factors were used

for the disaccharides (Di-0S, Di-4S, and Di-6S), for quantitation

of disaccharides in the samples. CS was calculated as the

amount of unsaturated disaccharides released by

chondroitinase ABC divided by the ratio of 1.036 (ratio of MW

of disaccharides and MW of disaccharide unit in CS). The

method was run ay 60C to avoid peak splitting of the

disaccharides.

Size exclusion chromatography (SEC) was used to analyze CS

using an RI detector. Dextrans (MW: 10K, 20K, 40K, 70K, and

150K g/mol) were used for calibration standards. The

relationship of the respective dextran and log MW was

evaluated. The separation was carried out using 3 columns in

series. Chondroitinase ABC was used to determine the

composition and content of the disaccharides. The authors

state they followed a published method with modifications.

The authors state the digestion time and inactivation method

used was sufficient but did not provide any supporting data.

The mixtures were then analyzed using CZE.

Safety Review

Analytical Range:

The range of the method was not defined by the authors. The

authors used statistical regression for linearity. Calibration

curve was evaluated for CS-A (20-200 mg/L). Routine samples

were evaluated from 20-100 mg/L. There is no mention of why

the curve range was varied.

LOQ:

Calculated as 3 times the LOD. LOD was calculated from the

calibration equation. LOD corresponds to the concentration of

CS which gives the minimum detectable zone length equal to

one second. The authors report LOQ at 9mg/mL, I calculated

the LOQ at 8 mg/mL based on the information given by the

authors. 8 mg/mL is at the 1% level. Its unclear if the LOD

concentration was properly evaluated since the calibration

curve used to extrapolate LOD concentration was much higher

(20-200 mg/L) than the lowest standard concentration (LOD

estimate implied at 3 mg/L). P. 59

Accuracy/Recovery

Assessed by the average of three replicates of sample spiked

with CS-A at the 50% and 100% level. 20 tablets composited,

weight equivalent to one tablet was used. Accuracy was

reported as 99.7 +/- 2.6% (97.1 – 102.3); therefore, not

meeting the SMPR requirement of 98-103% for >10% w/w.

Precision (RSDr)

Assessed by determination of six replicates in two different

samples. 20 tablets composited, weight equivalent to one