Stig Allenmark

Direct liquid chromatographic separation of enantiomers on immobilized protein stationary phases : IV. Molecular interaction forces and retention behaviour in chromatography on bovine serum albumin as a stationary phase☆

Chromatography with the use of immobilized bovine serum albumin as a stationary phase and aqueous buffer systems as eluents has proved to be a highly selective method, capable of separating structurally very closely related compounds. Retention can be effectively regulated by changes in at least three independent parameters of the mobile phase, which may be used for an optimization of separation factors. Particularly, the enantioselective properties of the chiral stationary phase have been demonstrated to be useful for the analytical resolution of a variety of racemates into enantiomers. From the variation of the retention behaviour with substituent effects, as well as the mobile phase composition, some indications regarding the molecular interaction forces regulating the substrate-protein equilibria have been obtained.

A selective postcolumn o-phthalaldehyde-derivatization system for the determination of histamine in biological material by high-performance liquid chromatography.

Based on studies of the reaction between histamine and o-phthalaldehyde in alkaline solution, a method optimized for the determination of histamine in biological samples by means of HPLC and postcolumn o-phthalaldehyde derivatization has been developed. The method permits determination of histamine even at low-picomolar levels. By means of a valve, placed immediately after the column outlet, the eluent stream can be switched between the fluorimetric and an electrochemical detector system whereby electroactive biogenic amines may also be studied under the same chromatographic conditions.

Direct optical resolution of a series of pharmacologically active racemic sulfoxides by high-performance liquid affinity chromatography

A series of pharmacologically active racemic sulfoxides was investigated with respect to optical resolution by an affinity chromatographic technique based on enantioselective interactions with immobilized bovine serum albumin. The results show that very small changes in the molecular structure of a drug, at a large distance from the asymmetric center, can drastically alter not only its affinity for the albumin molecule but also the enantioselectivity in the reversible binding process. Apart from being an excellent, rapid method for studies of drug binding to and transport by serum albumins, it has a large potential application as a chromatographic technique for the determination of enantiomeric purity as well as stereoselective uptake processes of drugs.

Microanalysis of catecholamines in human plasma by high-performance liquid chromatography with amperometric detection as compared with a radioenzymatic method

Abstract A high-performance liquid chromatographic procedure is described for the quantitative determination of epinephrine, norepinephrine, and dopamine in human plasma. The method, which is based on adsorption of the catecholamines to alumina and, after liberation, separation on a microparticulate bonded strong cation-exchange resin and amperometric detection, has been optimized to give complete baseline separation of the substances of interest. Dihydroxybenzylamine, a nonendogenous catecholamine, is used as the internal standard. The detection limit is about 0.1 pmol for dopamine. Analysis of data obtained for norepinephrine and epinephrine from a total of 59 plasma samples showed a good correlation to the corresponding values obtained with a radioenzymatic method. Some results from normal and pathological conditions are compared.

Recent advances in methods of direct optical resolution

Very great advances have been made in the field of direct optical resolution of organic compounds by chromatographic techniques. Chiral capillary gas chromatography now permits a determination of the enantiomeric composition of a few nanograms of a compound present in a mixture of many others. Coupled with high resolution mass spectrometry the technique will additionally permit structural elucidation; of great interest in pheromone research and related areas. Analytical separations of enantiomers are now also carried out by high-performance liquid chromatography (HPLC) methods based on a variety of principles. Basically, two main types are used, differing as to whether the mobile phase has to be a chiral medium or not. Two-dimensional HPLC, whereby compounds separated on a non-chiral column are progressively and automatically transferred to a chiral column for optical resolution, has been used successsfully for chiral amino acid separations. Many different chiral sorbents for preparative LC and HPLC resolutions have been prepared; some of these are now used in columns capable of producing pure enantiomers from a given racemate at a rate of the order of one gram/hour in continuous, automatic HPLC procedures. Apart from all important applications of these results of optical resolution technology, an increased knowledge of the underlying chiral recognition phenomena responsible for enantioselection has also been achieved.

Combined paper and reversed-phase high-performance liquid chromatography method for the study of pregnenolone and progesterone metabolites

Abstract A method for the separation and quantitation of steroid metabolites, obtained from enzymatic conversions of tritiated pregnenolone or progesterone, is described. As the first step, paper chromatography is used and the different zones obtained, as monitored by radiochromatogram scanning, are then eluted separately from the paper. The final resolution is achieved by reversed-phase high-performance liquid chromatography in 35% acetonitrile with UV detection at 215 nm. The method has been successfully applied to the study of metabolite patterns obtained from steroid conversion produced by testicular biopsy material under conditions of in vitro incubations.

Organ distribution and protein binding of cadmium in autopsy material from heavy smokers

Male heavy smokers were autopsied within 3 days postmortem. Samples from kidney, liver, and lung were taken for analysis of cadmium levels and degree of protein binding within the cytosolic fraction. The levels in lung, liver, and kidney were 0.50 +/- 0.35 (means +/- SEM), 2.21 +/- 0.63, and 17.4 +/- 8.8 micrograms cadmium/g wet weight tissue, respectively. In liver and kidney, approximately 75% was bound to a low-molecular-weight protein whereas the corresponding figure for the lung cytosolic fraction was 56%, a difference being statistically significant (P less than 0.05). After concentration of the low-molecular-weight cadmium-binding protein(s) ( CdBP ) by ultrafiltration and preparative isoelectric focusing in a granulated gel, the cadmium appeared in one single band with pI values of 5.8 (lung and liver) and 6.0 (kidney), respectively. It is therefore concluded that human lung exposed to cadmium, in this case via cigarette smoke, contains a CdBP , which binds cadmium. The relative degree of binding is less in lung than in liver or kidney, implicating that the metal could be more toxic to the lung than to liver or kidney, as the protein probably serves a role in detoxifying cadmium.

Characterization of bacterial L-(-)-tyrosine decarboxylase by isoelectric focusing and gel chromatography.

The purification of L-(-)-tyrosine apodecarboxylase (TAD) (E.C. 4.1.1.25), obtained from extracts of cells of Streptococcus faecalis, has been investigated by means of preparative isoelectric focusing, molecular sieve chromatography and hydrophobic interaction chromatography. Isoelectric focusing demonstrated two separate fractions possessing enzyme activity that had pI values of 4.5 and ca. 3.2. In the chromatographic methods, however, the activity was obtained in a single peak. It was found that hydrophobic interaction chromatography on phenyl-Sepharose was particularly suitable for purification purposes. The enzyme is very firmly bound to octyl-Sepharose CL-4B but retains most of its activity even in the bound state.

Enantioselective microbial degradation of some racemates studied by chiral liquid chromatography

Abstract Kinetic resolutions of some racemates via the preferential degradation of one of the enantiomers by a microorganism have been studied by analytical chiral liquid chromatography involving a column containing silica-bound bovine serum albumin as the stationary phase (Resolvosil ® -BSA). Resting cells of Nocardia restrictus rapidly degrades the l -forms of N- acetyl- dl -tryptophan and N- benzoyl- dl -alanine . Arthrobacter oxydans produces N- acetyl- d -tryptophan by d -selective hydrolysis of N- acetyl- dl -tryptophan ethyl ester while an opposed enantioselectivity was found on using cells of Nocardia corallina. The technique described is very useful for kinetic investigations of stereoselective reactions promoted by microorganisms.

A new method for the determination of estrogen-2-hydroxylase activity

A new method is described for determining the activity of a monooxygenase, acting by specific hydroxylation at the 2- position of the aromatic ring of estrogenic compounds with the formation of catechol estrogens. The procedure is based on separation of the catechol estrogen product from the substrate by high performance reversed-phase chromatography with amperometric detection in a thin-layer flow-cell by electrooxidation at a graphite-paste anode. Due to the very high detector sensitivity for the enzymatically generated product and an uncomplicated overall performance, the method offers great advantages over the radioenzymatic assay described earlier.