Automatische Flow Injection Analysis-Verfahren für die Lebensmittel-, Umwelt- und klinische Analytik

Abstract

In the doctoral thesis new fluorimetric and fully automated flow injection analysis (FIA) procedures were described. These FIA procedures are based on the derivatization reaction of ammonia and reducing sulphur compounds with o-phthalaldehyde (OPA) and the fluorimetric detection of the generated isoindol derivates. A selective, fast reacting and totally aqueous OPA reagent was developed for the sensitive determination of ammonia/ammonium. The detection limit (DL) was 20 nM. This detection is combined with enzyme catalyzed conversion of ammonia releasing analytes. On this basis FIA procedures were developed for the determination of urea, creatin, creatinin, glutamate, glutamine, and lysine (DL 0.1-1.0 µM). A multichannel FIA set-up was constructed for the sequential determination of ammonia, creatin, creatinin and urea or of glutamate, glutamine and ammonia. The precise determination of ammonia releasing enzyme activities, e.g. of urease was implemented. This opened up a way to determine inhibitors of urease, e.g. Cu(II) and Ag(I) ions. By the in situ preparation of inverse OPA reagents by premixing of OPA and ammonium solutions highly sensitive detection procedures were implemented for the FIA determination of sulphite (DL 50 nM), sulphide (DL 800 nM) and thiols. The fluorimetric detection of thiocholine (DL 80 nM) can be applied for an automatic assay for acetylcholinesterase inhibitors. Acetylcholinesterase was immobilized on magentic microbeads. A FIA set-up with a magnetic separation and incubation cell was constructed and applied for the sensitive determination of carbofuran and paraoxon(DL 13.5 and 17.5 nM). The inverse OPA reagents could also be used for the determination of other polar thiols, e.g. reduced glutathione (DL 60 nM) and cysteine (DL 5 µM). A selective procedures was developed for the determination of reduced glutathione in the presence of dithiotreitol, cysteine, oxidized glutathione, cystine and methionine. Oxidized glutathione was determined after its reduction by dithiotreitol. Fully automated FIA immunoassays (FIIA) were developed on the basis of antibodies immobilized on magnetic microbeads and the magnetic separation. Atrazine, metolachlor, carbofuran and 2,4-dichlorphenoxyacetic acid were determined in the lower mgl-1 range (0.1). By the combination of the FIIA procedure with a solid phase extraction system (SPE) the detection limit of 2,4-dichlorphenoxyacetic acid could be improved 50-fold. SPE can be combined also with the FIA set-up for determination of acetylcholinesterase inhibitors (carbofuran and paraoxon) with the aim to shift the detection limit to lower concentrations about the factor 50 (DL 270 and 350 fM).