PubMed · 13137972
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P GRONBAEK, C RUD, N S SORENSEN. 1953. Cation-exchanging resins.. https://pubmed.ncbi.nlm.nih.gov/13137972/
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A simple and rapid fully automated bio-analytical method for the liquid chromatographic (LC) determination of sotalol in human plasma has been described. The method is based on the use of a new kind of porous silica restricted access material (RAM) with cation exchange properties for sample clean-up. 100 microl of plasma samples were directly injected into the precolumn coupled on-line to a reversed-phase column (RP-Select B) by means of column switching system. The plasma matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate and methanol (97:3; v/v). By rotation of the switching valve, the analytes were then eluted in back-flush mode for 2 min and transferred to the analytical column by the LC mobile phase constituted of a mixture of methanol and 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM 1-octanesulphonic acid sodium salt (20:80; v/v). The flow-rate was 1.0 ml/min and sotalol was detected using fluorescence detection at 235 and 300 nm as excitation and emission wavelengths, respectively. The method was then validated using a new approach based on accuracy profile over a concentration range from 5 to 500 ng/ml. The limit of quantitation (LOQ) was 5 ng/ml and the total analysis time was 19 min.
Ion chromatographic analysis of ions in samples containing complex matrix composition strongly depends on the on-column co-processes caused by sample matrix components. In the present publication studies of different separation phenomena in cation-exchange chromatography are described. The studies were performed at 'non-linear' chromatographic conditions, when the concentration of matrix (interfering) ions significantly exceeded the concentration of the eluent ions. During the research work, the processes already identified in anion-exchange chromatography, i.e. self-elution, on-column change of the eluent and sample-induced micro-gradient elution were used to explain the chromatographic behavior of alkaline and earth-alkaline cations when samples with high matrix cation concentration were analyzed. When present at higher concentrations, the ammonium cation was found to cause self-elution (NH4+ fraction) as well as on-column eluent neutralization due to its ability to diffuse into/back from the porous core of the stationary phase (NH3 fraction). Co-elution of a matrix component and analytes of interest caused spectroscopic interferences that additionally influenced the peak shape of each individual analyte.
N-Glycans in glycoprotein can be liberated either from glycoproteins or from their glycopeptides with glycoamidases. The latter approach is preferable, because it requires a smaller amount of the enzyme, and yields N-glycans in excellent yields. Moreover it alleviates the necessity of removing from the reaction mixture the detergents needed to denature the glycoproteins. On the other hand, this approach necessitates removal of interfering peptidic materials, because some of the peptide peaks often overlap with the peaks of carbohydrate chains in high-performance anion-exchange chromatography (HPAEC). These peptidic materials also hinder labeling of N-glycans by reductive amination. We have tried to remove the interfering peptidic materials by several different methods--octadecyl (C18) silica cartridge, cation-exchange resin column, and graphitized carbon cartridge. Unfortunately, none of these could completely remove the interfering peptidic materials. Therefore, we resorted to modify the amino groups of the peptidic materials with sodium 2,4,6-trinitro-benzene-1-sulfonate (TNBS) to render them more hydrophobic, so that they can be retained more strongly on the C18 or graphitized carbon cartridges. In the model study presented here, we were able to obtain N-glycans for HPAEC analyses without any interfering materials by a combination of TNBS reaction and graphitized carbon treatment.