Cookson-type reagents, 4-substituted 1,2,4-triazoline-3,5-diones, having a chromophore, fluorophore or electrophore at the 4-position, have been synthesized for high-performance liquid chromatographic measurements of conjugated dienes. The reactivity of these reagents and the properties of their adducts were examined by using 7-dehydrocholesterol (7-DHC) as a model compound. 4-(1-Anthryl)- and 4-(1-pyrenyl)-1,2,4-triazoline-3,5-dione quantitatively produced adducts, in 5 min under ice-cooling, that could be detected using an ultraviolet detector but not a fluorescence detector. The adducts with 4-[2-(1-pyrenyl)ethyl]- and 4-amino-1,2,4-triazoline-3,5-dione were also easily formed and could be easily detected using fluorescence [detection limit: signal-to-noise ratio (S/N) = 5, 0.025 pmol] and electrochemical (S/N = 5, 0.029 pmol) detectors, respectively. The chromatographic behaviour of the adducts with 7-DHC and vitamin D3, and the application of the method to the detection of 7-DHC in human skin, are also reported.
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