The use of SAX-HPLC–CD as a heparin screening strategy

Abstract

Heparin, a heterogeneous polysaccharide, has been widely used as an anticoagulant for decades. Recently, however, international events involving the sudden onset of allergic-type reactions following heparin administration led to numerous fatalities, and demanded the use of multiple laborious, time consuming techniques to identify an economically motivated adulterant. Using these methods cooperatively, the semi-synthetic molecule known as oversulfated chondroitin sulfate (OSCS), was found to be present at significant concentrations. Since the discovery of this adulterant, several analytical methods have been put forth or updated to advance the process of screening pharmaceutical heparins; of these, strong anion exchange high performance liquid chromatography (SAX-HPLC) methods have now become routine. In this preliminary work, we report the use of circular dichroism (CD) detection in conjunction with existing SAX-HPLC methods to quantitate various sulfated polysaccharides. The proposed strategy exploits the selectivity associated with CD detection of heparin and heparin-like polysaccharides, while taking advantage of the method's insensitivity to the use of mobile phase additives and programmed gradients. The limit of detection of heparin by CD was found to be ∼0.22 mg/mL, whereas traditional UV/Vis detection yielded a detection limit of ∼1.09 mg/mL. The success of CD detection varied for other polymers, however no significant modifications were made to the separations method to capitalize on the advantages of CD detection.