Investigation of the sorption properties and pharmaceutical application of alpha1-acid glycoprotein by liquid chromatography

The separation and quantitation of the enantiomers and also the determination of the enantiomeric purity are all current and indispensable tasks for the pharmaceutical analysis. The aim of this work was to study the sorption properties of the alpha 1-acid glycoprotein (AGP) based stationary phase, which may have crucial importance for the enantioselectivity. New binding data are presented for the mechanism of the chromatographic separation, which has not clarified in the literature yet. On the basis of these new findings the composition of the mobile phase for a given analytical separation can be designed, including the preparative separation, as well. Binding data for dioxane, known as a solvent with low dielectric constant, are determined at first time. It was found that the sorption of both acetonitrile and dioxane is pH-dependent: as at pH 7.2 the binding can be characterized by a saturation curve, while at pH 4.0 by a twostep, monotone curve. The pH-dependence of the adsorption has been explained by conformational changes of the selector, which were confirmed by CD-spectroscopic and fluorescence quenching study of the native AGP. In accordance with the binding study by increasing the acetonitrile percentage at pH 4.0 a tipical alpha-helical peak was observed in the CD-spectrum. Opposite that at pH 7.2 the increase in the acetonitrile content does not result in any changes in the spectra. Effective dynamic quenching constants of AGP have been determined at given acetonitrile concentrations using 2,2,2-trichloroethanol as fluorescence quencher. In agreement with the results of the CD measurements at pH 4.0 it was found that the degree of quenching enhanced by increasing the amount of the acetonitrile, that can be explained by enhanced exposure of the buried tryptophan residues. Taking these results into account, new optimized and validated HPLC methods have been developed for compendial control in testing the enantiomeric purity of an acidic (ibuprophen), a basic (propranolol) and a neutral (norgestrel) drug molecule.