Fine structural characterization of sulodexide.

Sulodexide is a heparinoid which combines the properties of its components heparin and dermatan sulfate and is used not only for the prophylaxis and treatment of thromboembolic diseases but also for the treatment of diabetic nephropathy. Despite many clinical studies have been conducted to investigate its activity and safety, no data are available on the fine chemical characterization of its components. In this work, the in-depth investigation on the structural features of both the whole mixture and the isolated components was accomplished, involving the analysis of molecular weight distribution and of their mono, di and oligosaccharide composition by HP-SEC/TDA, 2D-NMR and HPLC-MS techniques. Moreover, also the separation of fractions endowed of graded affinity to antithrombin was achieved followed again by detailed structural analysis. The combination of different techniques permits to profile in depth the structural features of such a drug and offers a useful tool for possible analysis of batch production.

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.

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.

Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array.

The determination of molecular weight (M) and molecular weight distribution (MD) of heparins by a novel approach, consisting of a high performance size exclusion chromatography (HP-SEC) combined with a triple detector array (TDA) is described. HP-SEC/TDA permits the evaluation of MD of polymeric samples through a combined and simultaneous action of three on-line detectors, right-angle laser light scattering (RALLS), refractometer (RI), and viscometer. The method does not require any chromatographic column calibration, thus overcoming also the difficulty to obtain adequate reference standards. It permits the size determination also of small molecules, even when scattering dissimmetry is not observable. Unfractionated heparins, eight fractions of a size fractionated heparin, and dermatan sulfates were analyzed by HP-SEC/TDA. The M values found for the heparin fractions were used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing unfractionated heparin samples with both HP-SEC/TDA and HP-SEC were in excellent agreement, suggesting the possibility to use the TDA data to generate standard samples with known MD and intrinsic viscosity [eta]. Moreover, HP-SEC/TDA can successfully be employed also for the determination of the Mark-Houwink a and k parameters.