4.5 Article

Effective reversed-phase ion pair high-performance liquid chromatography method for the separation and characterization of intact low-molecular-weight heparins

Journal

ANALYTICAL BIOCHEMISTRY
Volume 387, Issue 1, Pages 113-121

Publisher

ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.01.007

Keywords

Reversed-phase ion pair high-performance liquid chromatography; Low-molecular-weight heparins; Enoxaparin; Dalteparin; Evaporative light scattering detector

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A simple, selective, and efficient reversed-phase ion pair high-performance liquid chromatography (RPIP-HPLC) method was developed for the separation of various commercially available intact low-molecular-weight heparins (LMWHs). The developed method uses a C-18 column (150 x 4.6 mm) with diode array detection at 230 rim, flow rate at 1.0 ml/min, and a mobile phase containing acetonitrile/water (32:68%), tetrabutylammonium hydroxide (15 mM), and ammonium acetate (50 niM) at pH 7.0. The performance of this method was assessed in terms of selectivity, linearity, intra- and interday precision, and accuracy. The novel application of RPIP-HPLC with evaporative light scattering detection (ELSD) for the analysis of intact LMWHs was demonstrated. Intact LMWHs were analyzed with superior resolution and peak shape. Different chromatographic profiles were obtained for different LMWHs showing significant structural diversity. This method clearly showed chemical changes that occurred to LMWH under the stress condition. This method can be applied for the separation, identification, characterization, and pharmaceutical stability analysis of various LMWHs. (C) 2009 Elsevier Inc. All rights reserved.

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