Probing Serum Albumins and Cyclodextrins as Binders of the Mycotoxin Metabolites Alternariol-3-Glucoside, Alternariol-9-Monomethylether-3-Glucoside, and Zearalenone-14-Glucuronide.

Mycotoxins are toxic metabolites of molds. Chronic exposure to alternariol, zearalenone, and their metabolites may cause the development of endocrine-disrupting and carcinogenic effects. Alternariol-3-glucoside (AG) and alternariol-9-monomethylether-3-glucoside (AMG) are masked derivatives of alternariol. Furthermore, in mammals, zearalenone-14-glucuronide (Z14Glr) is one of the most dominant metabolites of zearalenone. In this study, we examined serum albumins and cyclodextrins (CDs) as potential binders of AG, AMG, and Z14Glr. The most important results/conclusions were as follows: AG and AMG formed moderately strong complexes with human, bovine, porcine, and rat albumins. Rat albumin bound Z14Glr approximately 4.5-fold stronger than human albumin. AG-albumin and Z14Glr-albumin interactions were barely influenced by the environmental pH, while the formation of AMG-albumin complexes was strongly favored by alkaline conditions. Among the mycotoxin-CD complexes examined, AMG-sugammadex interaction proved to be the most stable. CD bead polymers decreased the mycotoxin content of aqueous solutions, with moderate removal of AG and AMG, while weak extraction of Z14Glr was observed. In conclusion, rat albumin is a relatively strong binder of Z14Glr, and albumin can form highly stable complexes with AMG at pH 8.5. Therefore, albumins can be considered as affinity proteins with regard to the latter mycotoxin metabolites.

The effect of column packing procedure on column end efficiency and on bed heterogeneity - Experiments with flow-reversal.

The effect of column end structure and bed heterogeneity of six commercially available reversed-phase chromatographic columns for fast liquid chromatography with different column packing materials - such as fully porous (Waters XBridge C18 with 1.7 µm particles) and superficially porous (Waters CORTECS C18 with 1.6 µm particles), with column dimension of 2.1 × 50, 100 or 150 mm were tested with flow-reversal method. The method includes arresting the flow when a non-retained marker (thiourea) has penetrated to a given distance into the column and then reversing the column. Hence, when the flow has been restarted, the sample is eluted at the same end of the column where it entered. The experiments showed that all columns are axially heterogeneous, and some differences could be observed between the two respective column ends. Furthermore, we can conclude that the shorter columns are axially more homogeneous than the longer ones, thus the column length is an influencing factor on the column packing procedure.

The effect of the frictional heat on retention and efficiency in thermostated or insulated chromatographic columns packed with sub-2-µm particles.

The mass-transfer properties of a core-shell packing material with 1.6 µm particle diameter, and that of a fully porous packing material with 1.7 µm particle diameter were investigated and compared. The first absolute and the second central moments of the peaks of the homologous series of alkylbenzenes, over a wide range of mobile phase velocities were measured and used for the calculation of the mass-transfer coefficients. For the evaluation of the band broadening caused by the thermal dissimilarities, the measurements were carried out under thermostated conditions and also at near adiabatic insulation of the columns.

Comparison of the kinetic performance of different columns for fast liquid chromatography, emphasizing the contributions of column end structure.

The kinetic performance of five chromatographic columns designed for fast liquid chromatography with different column packing materials - including fully porous (2.0 and 1.9µm particles), core-shell (2.6µm particles) or monolithic packings - with identical column dimensions (2.1×50mm) was tested. Since the tested monolithic column showed systematically better efficiency for early eluting compounds than the packed columns, an additional band broadening effect was suspected for the packed columns. The effects of the presence of the frits and the bed heterogeneity of the columns near the frits were characterized by a column-reversal method. It has been shown that significant differences - even 20-25% difference in efficiency - can exist between the two ends of the packed columns, while the monolithic column shows rather similar performance at either column end.

Performance of the same column in supercritical fluid chromatography and in liquid chromatography.

We have studied the chromatographic behavior of the homologous series of alkylbenzenes (ranging from octylbenzene to octadecylbenzene) on the same C18 reversed-phase column in supercritical fluid chromatography (SFC) and reversed phase liquid chromatography (RPLC) at various experimental conditions, such as different eluent compositions, flow-rates, and mobile phase densities. The first and the second moments of the peaks were used to estimate the overall mass-transfer processes in both chromatographic modes using the stochastic model of chromatography. The results confirm that in SFC - as the density of the mobile phase is influenced by the flow-rate - there is a broader variation of mass-transfer properties than in liquid chromatography. As expected, the optimum mobile phase velocity is higher in SFC, but there is no real difference in the minimum value of plate height, i.e. in the optimum efficiency.

Mass-transfer properties of insulin on core-shell and fully porous stationary phases.

The mass-transfer properties of three superficially-porous packing materials, with 2.6 and 3.6µm particle and 1.9, 2.6, and 3.2µm inner core diameter, respectively, were investigated and compared with those of fully porous packings with similar particle properties. Several sources of band spreading in the chromatographic bed have been identified and studied according to the general rate model of chromatography. Besides the axial dispersion in the stream of the mobile phase, and the external mass transfer resistance, the intraparticle diffusion was studied in depth. The first absolute and the second central moments of the peaks of human insulin, over a wide range of mobile phase velocities were measured and used for the calculation of the mass-transfer coefficients. The experimental data were also analyzed using the stochastic or molecular dynamic model of Giddings and Eyring. The dissimilarities of the mass-transfer observed in the different columns were identified and evaluated.