Microscale Isolation and Analysis of Heparin from Plasma using an Anion Exchange Spin Column

Abstract

Heparin, a linear highly sulfated polysaccharide, is a member of the glycosaminoglycan (GAG)1 family [1,2]. Since the 1930s, heparin has been widely used as a clinical anticoagulant [3,4]. More recently, low-molecular weight heparin (LMWH) derivatives have come into widespread use as anticoagulant and antithrombotic drugs [5]. Although heparin’s clinical use predated the establishment of the US. Food and Drug Administration (FDA), the approval of LMWH required a number of pharmacological studies on these new drugs. The pharmacologically relevant concentration of heparin and LMWH in plasma is between 0.1 and 1.0 anti-factor XaU/ml, corresponding to approximately 1–10 µg/ml of drug [6]. Plasma is a complex mixture, making the direct analysis of heparin or LMWH at these concentrations exceedingly challenging. Thus, most of the pharmacological studies of heparin and LMWH have relied on bioassays such as anti-factor Xa activity to evaluate these agents, providing only pharmacodynamic data. This inability to obtain pharmacokinetic data on LMWH is a serious concern because the plasma anti-factor Xa activity of LMWH does not correlate well with its therapeutic efficacy [7].