Indirect UV detection as a non-selective detection method in the qualitative and quantitative analysis of heparin fragments by high-performance capillary electrophoresis.

The application of capillary electrophoresis (CE) in combination with indirect UV detection for the qualitative and quantitative analysis of synthetic low-molecular-mass heparin fragments, at low pH, is described. It is demonstrated that, in contrast to direct UV detection, with indirect UV detection the signal obtained for various synthetic heparin pentasaccharides is nearly independent of their molecular structure. Moreover, the sensitivity of indirect UV detection is at least one order of magnitude higher than that of direct UV detection. CE-indirect UV detection for the qualitative and quantitative analysis of low-molecular-mass glycosaminoglycans was achieved by using 5 mM 5-sulphosalisylic acid, pH 3 or 5 mM 1,2,4-tricarboxybenzoic acid, pH 3.5 as electrophoresis buffer and chromophore. The technique is exemplified by the analysis of three pharmaceutical preparations of synthetic heparin pentasaccharides. The method employing indirect UV detection was validated with respect to repeatability, limit of detection, limit of quantitation, linearity, accuracy and ruggedness. In the indirect detection mode, the limit of detection for synthetic pentasaccharides is below 5 fmol, whereas the limit of quantitation is about 25 fmol. The method shows excellent repeatability and is linear in the femtomole-picomole range. Finally, it is demonstrated that the method is suitable for the analysis of various types of glycosaminoglycans.

Separation of natural and synthetic heparin fragments by high-performance capillary electrophoresis.

The application of capillary electrophoresis (CE) for the analysis of natural and synthetic low-molecular-mass heparin fragments at low pH is described. It is demonstrated that under the applied conditions the separation is based on charge, charge distribution and molecular mass of the heparin molecules, yielding a high resolution. It is shown that the presence of sodium chloride in the sample solution has hardly any effect on the CE performance. However, the pH of the electrophoresis buffer is a critical parameter. The resolutions obtained with CE and high-performance anion-exchange chromatography (HPAEC) are compared for various heparin fragments and it is concluded that, at least for this type of molecule, CE forms an attractive alternative to HPAEC.