Determination of the Molecular Weight of Low-Molecular-Weight Heparins by Using High-Pressure Size Exclusion Chromatography on Line with a Triple Detector Array and Conventional Methods
by
Antonella Bisio 1,*, Alessandra Mantegazza 1,†, Davide Vecchietti 1,‡, Donata Bensi 2, Alessia Coppa 2, Giangiacomo Torri 1 and Sabrina Bertini 1
1
Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni, via G. Colombo 81, Milan 20133, Italy
2
Laboratori Derivati Organici, via M. Barozzi 4, Milan 20122, Italy
*
Author to whom correspondence should be addressed.
†
Current address: Agilent Technologies, via Gobetti 2/c, Cernusco sul Naviglio 20063, Milan, Italy
‡
Current address: Department of Life Sciences, Universit
Academic Editor: Vito Ferro
Molecules 2015, 20(3), 5085-5098; https://doi.org/10.3390/molecules20035085
Received: 30 January 2015 / Revised: 6 March 2015 / Accepted: 12 March 2015 / Published: 19 March 2015
(This article belongs to the Special Issue Glycosaminoglycans and Their Mimetics)
The evaluation of weight average molecular weight (Mw) and molecular weight distribution represents one of the most controversial aspects concerning the characterization of low molecular weight heparins (LMWHs). As the most commonly used method for the measurement of such parameters is high performance size exclusion chromatography (HP-SEC), the soundness of results mainly depends on the appropriate calibration of the chromatographic columns used. With the aim of meeting the requirement of proper Mw standards for LMWHs, in the present work the determination of molecular weight parameters (Mw and Mn) by HP-SEC combined with a triple detector array (TDA) was performed. The HP-SEC/TDA technique permits the evaluation of polymeric samples by exploiting the combined and simultaneous action of three on-line detectors: light scattering detectors (LALLS/RALLS); refractometer and viscometer. Three commercial LMWH samples, enoxaparin, tinzaparin and dalteparin, a γ-ray depolymerized heparin (γ-Hep) and its chromatographic fractions, and a synthetic pentasaccharide were analysed by HP-SEC/TDA. The same samples were analysed also with a conventional HP-SEC method employing refractive index (RI) and UV detectors and two different chromatographic column set, silica gel and polymeric gel columns. In both chromatographic systems, two different calibration curves were built up by using (i) γ-Hep chromatographic fractions and the corresponding Mw parameters obtained via HP-SEC/TDA; (ii) the whole γ-Hep preparation with broad Mw dispersion and the corresponding cumulative distribution function calculated via HP-SEC/TDA. In addition, also a chromatographic column calibration according to European Pharmacopoeia indication was built up. By comparing all the obtained results, some important differences among Mw and size distribution values of the three LMWHs were found with the five different calibration methods and with HP-SEC/TDA method. In particular, the detection of the lower molecular weight components turned out to be the most critical aspect. Whereas HP-SEC/TDA may underestimate species under 2 KDa when present in low concentration, other methods appeared to emphasize their content.
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MDPI and ACS Style
Bisio, A.; Mantegazza, A.; Vecchietti, D.; Bensi, D.; Coppa, A.; Torri, G.; Bertini, S. Determination of the Molecular Weight of Low-Molecular-Weight Heparins by Using High-Pressure Size Exclusion Chromatography on Line with a Triple Detector Array and Conventional Methods. Molecules 2015, 20, 5085-5098.
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