This review summarizes the synthesis, features and application of benzofurazan (i.e., 2,1,3−benzoxadiazole)−bearing fluorescence labeling reagents for the determination of biologically important molecules, such as biogenic amines, amino acids, carboxylic acids, thiols and drugs. Ammonium 4−fluoro−2,1,3−benzoxadiazole−7−sulfonate (SBD−F), 4−fluoro−7−aminosulfonyl−2,1,3−benzoxadiazole (ABD−F) and 4−fluoro−7−(N,N−dimethylaminosulfonyl)−2,1,3−benzoxadiazole (DBD−F) were synthesized from the investigation of various substituted groups at the 7−position of the 4−fluoro−2,1,3−benzoxadiazole. The fluorescence property of these derivatives was almost the same, but the reactivity and solubility were different for each reagent. The synthesized reagents were applied to the sensitive determination of biological thiols and amines, such as cysteine, homocysteine and cysteinylglycine in human plasma, glutathione in human blood cells, α−lipoic acid in animal tissues, and histamine and polyamines in hair. Furthermore, the chiral derivatization reagents, e.g., 4−(3−aminopyrrolidin−1−yl)−7−(N,N−dimethylaminosulfonyl)−2,1,3−benzoxadiazole (DBD−APy), 4−(3−aminopyrrolidin−1−yl)−7−aminosulfonyl−2,1,3−benzoxadiazole (ABD−APy), 4−(3−aminopyrrolidin−1−yl)−7−nitro−2,1,3−benzoxadiazole (NBD−APy), 4−(3−isothiocyanatopyrrolidin−1−yl)−7−(N,N−dimethylaminosulfonyl)−2,1,3−benzoxadiazole (DBD−PyNCS), 4−(3−isothiocyanatopyrrolidin−1−yl)−7−nitro−2,1,3−benzoxadiazole (NBD−PyNCS), 4−(2−chloroformylpyrrolidin−1−yl)−7−(N,N−dimethylaminosulfonyl)−2,1,3−benzoxadiazole (DBD−Pro−COCl), and 4−(2−chloroformylpyrrolidin−1−yl)−7−nitro−2,1,3−benzoxadiazole (NBD−Pro−COCl), were developed for the resolution of various chiral molecules, in terms of reactivity, separatability, handling easiness, sensitivity and selectivity. The chiral separation of various racemates was efficiently performed by reversed−phase chromatography after labeling with the chiral reagents. Some applications utilizing these reagents for the analyses of bioactive chiral compounds and drugs are also described in this paper.

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Fluorogenic derivatization (FD)-HPLC is a powerful tool for analyzing biological samples due to its high sensitivity and reproducibility. In recent years, we have developed FD-LC-MS/MS, a novel proteomics method that consists of HPLC separation of fluorogenic derivatized biological macromolecules with DAABD-Cl and identification of the separated derivatives. To develop the analytical system for differential analysis, high-resolution HPLC conditions were optimized. The analytical system was then developed to clarify the flow dynamics of signaling in cells. The affinity column for extracting albumin in plasma was also evaluated. FD-LC-MS/MS method is superior to other proteomics methods in sensitivity, quantitativity and reproducibility, and enables demonstrating flow dynamics in cells.

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Microchip electrophoresis (MCE) is one of the versatile separation techniques in microfluidic systems since MCE has remarkable advantages such as high resolution, rapid analysis, small consumptions of samples and reagents, and high integratability to other chemical operations on planer substrate. However, there still remains a significant problem about a poor sensitivity due to a short optical pathlength and extremely small volume of analytes introduced into the separation channel. To overcome this drawback, a lot of on-line sample preconcentration methods have been developed and applied to MCE for improving the sensitivity. In this review, the author highlights recent progresses on combinations of chemical operations integrated onto microfluidic devices for enhancing a detector signal, e.g., solid phase extraction (SPE), affinity capturing, electrokinetic filtrating/trapping, and polymerase chain reaction (PCR) with MCE mainly from 2009. The other integrated techniques and methods which are expected to be combined with MCE are also introduced briefly.

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