Indirect detection of anti-acetylcholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase.

The inhibiting compounds were separated by micro-column liquid chromatography in the mobile phase containing the natural substrate acetylcholine. A home-made packed bed microbioreactor system containing immobilized enzyme acetylcholinesterase (ACHE) in human red blood cell membrane and choline oxidase (CHO) from alcaligenes was used for the post-column conversion of acetylcholine to hydrogen peroxide which was detected by an electrochemical detector. The inhibition effect of the solutes caused a decrease in the acetylcholinesterase activity, a decrease in the formation of hydrogen peroxide and also a decrease in the response corresponding to the concentration of the solutes. The rate of the enzyme regeneration was also recorded. The micro-system was compared with a conventional LC system comprising commercially prepared enzyme reactor. The stability of the enzymes is at least 3 weeks at ambient temperature. The limit of detection depends on biological activity of inhibition and for galanthamine was 1 pmol.

Cation-exchange liquid chromatography of choline and acetylcholine on free shielded silanols of silica-based reversed-phase stationary phases.

Free anionic functions present on the surface of reversed-phase packing materials were used for the selective cation-exchange preconcentration and separation of the neurotransmitters choline and acetylcholine from a biological matrix. The cation-exchange behaviour of different reversed-phase packing materials in the neat aqueous mobile phase, the properties of an end-capped column, the dependence of capacity factors and peak shape on the concentration of counter ions, ionic strength, pH and the addition of acetonitrile and optimum conditions for enzymatic conversion of solutes to hydrogen peroxide were studied. The studied reversed-phase columns exhibit better pH stability and longer lifetimes than normal silica-based cation exchangers. Acetylcholine is an effective and sensitive test sample for the measurement of adsorption on silica support. A large sample volume was injected onto a precolumn inserted instead of an injection valve and after injection the solutes were focused and separated on an analytical column with a mobile phase containing tetramethylammonium perchlorate as the counter ion.

Identification of products formed during UV irradiation of tamoxifen and their use for fluorescence detection in high-performance liquid chromatography.

During the UV irradiation of tamoxifen, isomerization of the trans to the cis isomer takes place and consequently corresponding highly fluorescent phenanthrene derivatives are formed. Their formation can be used for the sensitive and selective detection of tamoxifen in high-performance liquid chromatography (HPLC). The structure of photoproducts was identified by 1H NMR spectroscopy, HPLC, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Owing to the variety of products formed and the higher selectivity and fluorescence response, on-line postcolumn photocyclization is preferred to the precolumn mode. A chromatographic system for the separation of isomers and photoproducts is suggested.

Column liquid chromatography of methotrexate and its metabolites using a post-column photochemical reactor and fluorescence detection.

On irradiation with short-wavelength UV light in the presence of hydrogen peroxide, methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid are cleaved into highly fluorescent products. This reaction can be used for the sensitive and selective detection of the compounds in biological fluids, following reversed-phase high-performance liquid chromatographic separation. Study of the effect of the mobile phase composition and irradiation time on fluorescence signal intensity showed that a residence time of ca. 3 s in the on-line photochemical reactor was best. The detection limit for methotrexate was 0.4 ng, for 7-hydroxymethotrexate 1.0 ng and for 2,4-diamino-N10-methylpteroic acid 0.6 ng. The addition of dimethylformamide to the mobile phase enhanced the selectivity of separation.

Determination of methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid in biological fluids by liquid chromatography with fluorimetric detection.

A high-performance liquid chromatographic method involving post-column cleavage and fluorimetric detection has been developed for the determination of methotrexate and its metabolites in biological fluids. The cleavage is based on photooxidative reaction of methotrexate and its metabolites to highly fluorescent products. The photoreaction occurs during the flow of eluate containing a certain amount of hydrogen peroxide through a PTFE capillary irradiated by UV light. The method allows the determination of methotrexate, 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid in plasma, urine and ascitic fluid samples at concentrations as low as 2 X 10(-8)M.