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Simple method for isolating free urinary catecholamines by boric acid gel chromatography

This paper describes a field effect transistor (FET) based sugar molecule sensor, using a totally synthetic and chemically stable sugar-detecting moiety phenylboronic acid (PBA). PBA was introduced to the FET sensing surfaces in two different ways. One is in the form of a thin copolymer gel layer directly attached to the FET gate insulator, the other method utilizes self-assembled monolayer (SAM) formed on an extended Au gate surface, a functional group of which was reacted with PBA. Reversible and prompt changes in the FET’s electric characteristics were demonstrated in response to the change in glucose and sialic acid (Neu5Ac) concentrations depending on the pH of the milieu. The developed FET detection scheme could be extended to the construction of stable, readily miniaturizable, and label-free carbohydrate molecule sensing systems due to the ability of PBA moieties to interact with these molecules.

Demonstrated in this study is that without pretreatment and preconcentration nanomolar-level catecholamines in human urine samples can be quantitatively determined with ease by utilizing capillary electrophoresis coupled with amperometric detection. The detector employs a parallel-opposed dual-electrode scheme assembled with an on-capillary electrode and a disk electrode and takes advantage of the redox cycling of analytes between the two working electrodes to improve the limit of detection. The matrix effect of urine samples significantly decreases the detection sensitivity from that obtained in standard solutions. Therefore, calibration curves derived from standard solutions cannot be used in quantitative determination of catecholamines. Methods of standard addition and internal standard have been studied. The results suggest that isoproterenol is a good internal standard to facilitate the measurements of dopamine, epinephrine, and norepinephrine in human urine samples.

High-performance liquid chromatography with electrochemical detection (HPLC-ED) is a popular method for measuring biogenic amines, owing to its simplicity, versatility, sensitivity, and specificity. Recent developments in microbore column HPLC-ED have been facilitated by miniaturization of solvent delivery, column packing, sample injection and micro-flow cell construction. The aim of this paper is to present an overview of recent developments in microbore column HPLC-ED, in terms of advantages and limitations. This paper covers the recent advancements and important factors of HPLC-ED analysis of biogenic amines using microbore columns. Particular emphasis is placed on applying this technique to microdialysis, for which great sensitive is required. Its potential in future biomedical applications is also discussed.

This chapter discusses the use of bioaffinity chromatography (BAC) in the isolation, determination, or removal of biologically active substances. The preparation of specific sorbents utilizing the exceptional properties of biologically active substances to form specific and reversible complexes has enormously facilitated the isolation of a number of antibodies, antigens and haptens, cells and cell organelles, cofactors and vitamins, and glycoproteins and saccharides. The different conditions applied during BAC depend on the nature of the substances to be isolated. In high-performance liquid bioaffinity chromatography (HPLBAC), the biospecificity of BAC is combined with a high-performance (pressure) technology based on the rigid particles of a uniform small size (high-performance liquid chromatography, HPLC). The separation times in HPLBAC are short (minutes) compared to hours for traditional, soft-gel BAC. Moreover, HPLBAC users have at their disposal a wide selection of HPLC equipment, including high-speed pumps, sophisticated injection units, detectors of various kinds, auto-sampling devices, and data-handling capabilities. This enables the users to fine-tune the separation process conveniently and promote higher productivity.

A fully automated reversed-phase high-performance liquid chromatographic method with fluorimetric detection for the determination of urinary free noradrenaline was developed. Urine samples, diluted with buffer were injected into a boric acid gel column (12 μm, TSK: 10 × 4.6 mm I.D.) without prior extraction. Urinary noradrenaline was simultaneously extracted and derivatized with an alkaline mobile phase of pH 11, containing o-phthalaldehyde and 2-mercaptoethanol, in a boric acid gel column. After switching columns, the fluorescent derivatized catecholamines were separated with an ODS-4PW column (TSK) and a mobile phase of pH 2 and the fluorescence was monitored with excitation at 340 and emission at 440 nm. The retention times of noradrenaline and dopamine were 11.0 and 14.2 min, respectively. The detection limits for noadrenaline and dopamine were 0.2 and 20 ng, respectively.

This method has the advantages of not requiring preliminary extraction of urinary catecholamines, high sensitivity and stability of o-phthalaldehyde-derivatized catecholamines.