Development of Precise Chromatographic Separation, Preparation and Detection Tools for Biomedical Use

For the analysis of small amounts of a component in biological samples, we need to measure only the target molecules in a complicated mixture of various components. To obtain this objective, we developed analytical methods and reagents which have both a highly sensitive fluorescence/chemiluminescence detection and highly selective separation methods such as HPLC. This strategy includes (1) the development of a fluorescence reagent, (2) utilization of a highly selective enzymatic reaction and productaccumulating function of the enzyme, (3) highly selective and sensitive immunoreaction, and (4) development of HPLC systems with fluorescence/chemiluminescnce detection. In this review, various methods and reagents, developed in our laboratory, related to HPLC used in the biomedical or biochemical field are described. 2. Reagents (1,2-DNS and DTAN) for fluorescence detection of aromatic aldehydes and chemiluminescence reagent (mCED) for catechol amines 1,2-Diaminonaphthalene monosulfate (1,2-DNS), an aromatic 1,2-diamino compounds, was first found to react with aromatic aldehydes in dilute sulfuric acid to form 2-substituted naphtho [1,2d ] imidazoles [1, 2] (Figure 1). The fluorescence produced by the aromatic aldehydes in this method is generally higher than those of the fluorescence from aliphatic aldehydes. Since then, various 1,2diamino benzene derivatives have been developed as fluorogenic reagents for aromatic aldehydes. 2,2 ’ -Dithiobis ( 1aminonaphthalene) (DTAN) reacts with aromatic aldehydes in an acidic medium in the presence of tributylphosphine, which serves to reduce DTAN to 1-amino-2-thionaphthol, to produce the 2substituted naphtho [1,2-d ] thiazoles [3, 4] (Figure 1). DTAN has been applied to the sensitive HPLC assay of catechol-O -methyl transferase in rat liver by determination of the 3 -and 4-O -methylated products (vanillin and isovanillin, respecFocusing Review