J.C. Kraak

Simple and fast solvent extraction system for selective and quantitative isolation of adrenaline, noradrenaline and dopamine from plasma and urine

A very simple solvent extraction system for the selective and quantitative isolation of adrenaline, noradrenaline and dopamine from plasma and urine is described. The extraction system makes use of the complex formation, in alkaline medium, between diphenylborate and the diol group in the catecholamines in combination with ion-pair formation. The influence of various parameters on the distribution coefficient was investigated by analysis of the liquid phases by high-performance liquid chromatography with electro-chemical detection. From these results the optimal extraction conditions can be selected. With hexane + 1% n-octanol containing 0.25% (w/v) of tetraoctylammonium bromide as extraction solvent, the catecholamines can be quantitatively isolated from plasma and urine at pH 8.6 in the presence of 0.1% (w/v) of diphenylborate. For urine the recovery was 101.5 + 1.9% for adrenaline, 100.6 +/- 2.0% for noradrenaline and 99.9 +/- 1.5% for dopamine. For plasma the recoveries were, respectively, 101.8 +/- 3.3%, 100.5 +/- 2.6% and 92.9 +/- 3.5%. The recovery of dihydroxybenzylamine, included in the study as internal standard, was determined to be 96.3 +/- 1.6% for urine and 89.9 +/- 2.7% for plasma. The applicability of the developed extraction system as clean-up and concentration step for the analysis of catecholamines in plasma and urine by high-performance liquid chromatography with electrochemical detection is demonstrated.

Capillary zone electrophoretic separations of proteins in polyethylene glycol-modified capillaries

Abstract Fused-silica capillaries were wall modified with γ-glycidoxypropyltrimethoxysilane and polyethylene glycol 600 in order to decrease the influence of wall adsorption in capillary zone electrophoretic separations of proteins. It is shown that a significant decrease in adsorption is obtained and electro-osmotic flow is also diminished. For the proteins studied, symmetrical peaks were obtained in the pH range 3–5. However, some adsorption still occurs as the plate numbers are below theoretical expectations. At higher pH values appreciable peak deformations and drastic decreases in resolving power are observed. The procedure allows the rapid and efficient separation of protein mixtures suitable for separation in the indicated pH range, and the coating shows a good stability.

Comparison of five methods for the study of drug–protein binding in affinity capillary electrophoresis

The qualitative and quantitative aspects of capillary electrophoretic methods used to study drug-protein interactions, viz. the affinity capillary electrophoresis (ACE). Hummel-Dreyer (HD), frontal analysis (FA), vacancy peak (VP) and vacancy affinity capillary electrophoresis (VACE) methods have been investigated. In the ACE and the VACE methods the binding parameters can be calculated from the change in the electrophoretic mobility of the drug on complexation with a protein. In the frontal analysis and the vacancy peak method the free drug concentration is measured with UV detection. In the Hummel-Dreyer method the amount of drug bound is measured with UV detection. For the comparison of these five methods the warfarin-bovine serum albumin (BSA) system was used. Several factors that might influence the determination of association parameters were examined. With the FA, VP, HD and VACE methods the absolute numbers of the different binding sites involved in the complex formation can be determined, a major advantage in drug-binding studies.

Solvent-generated ion-exchange systems with anionic surfactants for rapid separations of amino acids.

The retention behaviour of amino acids in phase systems consisting of a hydrophobic solid support as the stationary phase and water-organic solvent mixtures containing a small amount of an anionic detergent as the mobile phase was investigated. Such phase systems are found to behave like conventional ion-exchange systems. The degree and order of retention of amino acids can be influenced by changing the temperature, the nature of the hydrophobic support, the pH and the nature and concentration of the anionic detergent, organic constituent and counter ion in the eluent. In many instances this solvent-generated (dynamic) ion-exchange chromatography shows a greater selectivity than conventional ion-exchange systems towards amino acids. The results obtained so far indicate that a complete separation of the 19 protein amino acids by applying solvent gradients or/and multi-column system is possible within 30 min.

Study of protein-drug binding using capillary zone electrophoresis

CApillary zone electrophoresis was tested for its suitability for studying protein-drug binding. Three methods were investigated, viz., the Hummel-Dreyer method, the vacancy peak method and frontal analysis. Frontal analysis appeared to be the preferred method.

Performance of a physically adsorbed high-molecular-mass polyethyleneimine layer as coating for the separation of basic proteins and peptides by capillary electrophoresis

A simple method for the preparation of a polyethyleneimine (PEI) coating on the inner surface of fused-silica capillaries for capillary electrophoresis (CE) is reported. The PEI layer can be coated on the silica surface by just flushing the capillary with a solution containing high-molecular-mass PEI. The physically adsorbed layer appears to be very stable and can be used in a pH range of 3-11. In comparison to described methods to fabricate an immobilized PEI layer, the proposed method does not require an immobilization step, is simple and the preparation time of the coating is less than two hours. Good reproducibilities of migration times of basic proteins and peptides were obtained on the same PEI-coated capillary as well as on different PEI-coated capillaries. For basic proteins efficiencies ranging from 300 000-500 000 plates per meter were normally found.

Chiral separations by complexation with proteins in capillary zone electrophoresis

Abstract The direct separation of enantiomeric drugs by capillary zone electrophoresis using proteins as chiral completing agents in the background electrolyte is described. The influence of protein type and organic modifiers added to the background electrolyte on the resolution of enantiomers was studied. Separations of the enantiomers of tryptophan, benzoin, pindolol, promethazine and warfarin are shown.

Performance of carbohydrate-modified fused-silica capillaries for the separation of proteins by zone electrophoresis

The walls of fused-silica capillaries were chemically modified with small carbohydrate moieties in order to diminish the wall adsorption of proteins in capillary zone electrophoresis. A diol-type coating, prepared by bonding of gamma-glycidoxy propyltrimethoxysilane to the wall followed by acidic hydrolysis, shows for proteins a similar electrophoretic behaviour as various pH values to a polyethylene glycol (PEG) coating tested previously. Although good peak shapes were obtained for proteins in the pH range 3-5, the efficiency on the diol coating is worse than that on the PEG coating. At higher pH values the peaks are deformed and the efficiency is lost. A maltose coating appears to shield the silica surface well for proteins up to pH 7. The peak shapes of proteins are acceptable, but the efficiency of the maltose coating is smaller than that on the diol coating. The diol coating is stable in the indicated pH range. However, with the maltose coating good stability is obtained only on adding an antimicrobial agent to the buffers.

Influence of thermal conditions on the efficiency of high-performance liquid chromatographic columns

Abstract The effect of thermal conditions on efficiency in high-performance liquid chromatography is investigated with special reference to the role of the viscous heat dissipation. In systems thermostatted by a water-bath or jacket it turns out to be advantageous to pre-cool the mobile phase when high flow-rates and small particle sizes are used. This is explained by a compensation of band broadening in the first and second part of the column. The wall region of the band moves faster in the first part, but it moves slower in the second part and it is eluted together with the solute from the core region. Our results indicate that a similar compensation effect occurs when the column is operated in still air. This is due to back-flow of heat through the stainless-steel column wall. For large diameter columns (larger than 3 mm), packed with small particles, a linear temperature profile imposed on the column wall may diminish the detrimental effects of viscous heat dissipation. Results obtained with such a system are promising.

Principles and limitations of methods available for the determination of binding constants with affinity capillary electrophoresis

Abstract At present there are five capillary zone electrophoresis (CZE) methods available for the measurement of binding (association) parameters viz. the frontal analysis method, the Hummel and Dreyer method, the affinity capillary electrophoresis, the vacancy peak and the vacancy affinity capillary electrophoresis methods. These methods exhibit their own advantages and limitations. In this paper the limitations of these five CZE methods will be explored with the aid of simulated concentration–position profiles of the interacting species. With the frontal analysis, the Hummel and Dreyer and the vacancy peak methods e.g., correct results for the binding parameters can only be obtained when the mobilities of e.g., a protein and the complex are equal. When the mobilities differ, the binding constants obtained with these methods will deviate systematically. The affinity capillary electrophoresis method on the other hand can only be performed when the mobility of the protein is not equal to the mobility of the complex. It is shown that this very necessary difference in the mobility between the free protein and the complex may lead to a deviation in the free ligand concentration and consequently in the binding constant.