Fluorometric detection of O2-* is performed based on desulfonylation of 3 to the corresponding fluoresceins 4 through nucleophilic substitution, and this fluorescing process is quite specific toward O2-* over H2O2, t-BuOOH, NaOCl, 1O2, HO*, NO*, and ONOO-. Furthermore, effects of glutathione, cytochrome P450 reductase/NADPH, and diaphorase/NADH are relatively small on the fluorescing process of probe 3 with X = Y = F, which is useful to detect O2-* released from neutrophils stimulated by phorbol myristate acetate with satisfactory sensitivity.

A strategy for designing probes based on protection-deprotection chemistry involving fluoresceins and their benzenesulfonyl (BES) derivatives has led to the development of a much more practical superoxide (O(2) (-.)) probe than the previously reported bis(2,4-dinitro-BES) tetrafluorofluorescein (6 a). Examination of various BES derivatives, developed from the starting point of the prototype probe 6 a, yielded 4,5-dimethoxy-2-nitro-BES tetrafluorofluorescein (BESSo; 7 j) as the optimal reagent. A microtiter plate assay with BESSo showed a tenfold improved detection limit for O(2) (-.) compared with such an assay based on 6 a. BESSo showed markedly better specificity for O(2) (-.) than for GSH or other reactive oxygen species, and this specificity was significantly higher than that of Fe(2+) and some reducing enzymes. These features have resulted in the development of an assay based on BESSo that is capable of providing more unambiguous results for O(2) (-.) release from neutrophils, with or without stimulation by phorbol myristate acetate, as compared with an assay based on 6 a. Intracellular generation of O(2) (-.) in human Jurkat T cells stimulated by butyric acid has been measured by using flow cytometry and fluorescence microscopy utilizing the acetoxymethyl derivative of BESSo.

We have applied a sample pre-treatment method with a cartridge column filled with polyvinylpolypyrrolidone (PVPP) to the effective removal of polyphenols and simple UV spectrophotometry of caffeine in tea. The absorption maximum length (l l max ) for caffeine was close to those for tea catechins in aqueous 1% acetic acid; therefore, the UV spectrum of a non-treated green tea sample had a large absorption wave. In contrast, the absorbance of the green tea sample was gradually reduced by PVPP cartridge treatment using PVPP from 0 to 50 mg, and was nearly constant using a pre-treatment cartridge with more than 100 mg PVPP, because tea catechins were effectively removed and caffeine was mostly recovered from a green tea sample by means of PVPP cartridge treatment. The PVPP pre-treatment cartridge also removed polyphenols successfully from oolong and black tea samples. Comparison with conventional HPLC analysis indicated that the present pre-treatment method with a PVPP cartridge was useful for the simple and selective UV spectrophotometric determination of caffeine in green, oolong and black tea samples.

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