A pH-GRADIENT SAX-HPLC METHOD FOR DETERMINATION OF TRACE LEVELS OF OVER SULFATED CHONDROITIN SULFATE AND DERMATAN SULFATE IN HEPARIN SODIUM

A rapid and sensitive pH-gradient SAX HPLC method for the determination of Dermatan sulphate (DS) and trace levels of Oversulphated chondroitin sulphate (OSCS) in Heparin sodium was developed and validated. A pH gradient was opted to strike a balance between resolution and sensitivity because the response of OSCS was found to be better at neutral pH and the resolution between DS and Heparin was good at acidic pH. The use of commercially available lithium perchlorate was avoided and an in situ preparation of the same was opted to decrease the baseline drift which is a major concern for gradient methods using perchlorate mobile phases for compounds being analyzed at lower wavelengths. Chromatographic separation was achieved using a hydrophilic polymer resin-based SAX column, a linear gradientelution from 15% to 70% of solution-B which is 1.0M lithium perchlorate containing 5mM sodium dihydrogen phosphate at pH 6.0 and solution-A being 5mM sodium dihydrogen phosphate at pH 3.0 and a UV detection at 202 nm. The method has been validated and the limit of detection for DS and OSCS were found to be 15µg/ml and 20µg/ml respectively which is 0.03% &0.04% respectively with respect to the method concentration of 50 mg/mL. The method was found to be linear over the range of 50-150% of the specifications of the impurities with respect to the working sample concentration. The precision, accuracy, specificity robustness and solution stability were determined.