Development and Validation of a Rapid and High Sensitive Sax- Hplc Method for Analysis of Impurities in Heparin Sodium Using Ammonium Perchlorate (in Situ) Mobile Phase

A simple, rapid, sensitive and high resolution SAX HPLC method for the determination of Dermatan sulphate (an impurity) and Oversulphated chondroitin sulphate (a contaminant) in Heparin sodium drug substance has been developed. This method was developed to address the issues of very long runtime, poor resolution between heparin, DS & OSCS, broad peaks, high drift in the baseline and lesser sensitivity for the impurities with the existing method for determination of impurities in Heparin sodium. The newly developed analytical method employs a hydrophilic polymer resin-based strong anion exchange column which yielded a high resolution between dermatan sulphate and Heparin. Chromatographic separation was achieved using a linear gradient elution from 15% to 70% of 1.0M ammonium perchlorate (prepared in situ) containing 5mM sodium dihydrogen phosphate at pH 3.0 over a period of 22 minutes with UV detection at 202 nm. The method has been validated and the limit of detection for DS and OSCS were found to be 0.03% & 0.06% respectively. 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 of 40 mg/mL. The precision, accuracy, specificity, robustness and ruggedness of the method were also established. The stability of standard and sample solutions was also determined.