Enhanced sensitivity electrochemical assay of low-molecular-weight heparins using rotating polyion-sensitive membrane electrodes

Abstract

Use of a novel rotating polycation-sensitive polymer membrane electrode yields sensors that can serve as simple potentiometric titration endpoint detectors for the determination of three FDA approved low-molecular-weight heparin (LMWH) anticoagulant drugs (Fragmin, Normiflo, and Lovenox). The rotating electrode configuration dramatically improves the reproducibility and increases the sensitivity for LMWH determinations by protamine titration. At a rotation speed of 3000 rpm, electrodes with optimized thin (50 µm) polymer membranes doped with dinonylnaphthalene sulfonate (DNNS) respond to low levels of protamine (<2 µg mL −1 ) with good precision (±1 mV, N =10), when protamine is infused continuously into a Tris-buffer solution, pH 7.4. When infusing protamine (at 5 µg min −1 ) continuously into solutions containing Fragmin, a clear endpoint is obtained, with the amount of protamine required to reach this endpoint proportional to the level of Fragmin present. A detection limit of less than 0.02 U mL −1 Fragmin can be obtained via this new method, approximately one order of magnitude lower than that previously reported based on a non-rotating polycation electrode. Similar low detection limits can be achieved for potentiometric titrations of Normiflo and Lovenox. Such titrations can also be carried out in undiluted plasma samples containing the various LMWH species. In this case, detection of the LMWHs at clinically relevant concentrations (>0.2 U mL −1 ) can be readily achieved.