THE DETERMINATION OF HYDROXYLATED NATURAL PRODUCTS

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7-[(Chlorocarbonyl)methoxy]-4-methylcoumarin (CMMC) is a fluorescent reagent for precolumn derivatization of hydroxy compounds in liquid chromatography. The synthesis of the acyl chloride involves the use of thionyl chloride, which has been subsequently shown to unexceptably introduce highly fluorescent impurities due to chlorination in addition to formation of the acyl chloride. An improved derivatization reagent utilizes a reactive acyl imidazole, instead of the acyl chloride, offerring a purer product while maintaining desirable reagent characteristics such as ease of handling, mild derivatization conditions, etc. Reaction of 7-[(imidazolemethanoyl)methoxy]-4-methylcoumarin (IMMC) with various hydroxy compounds produces esters which are readily separable under reversed-phase HPLC conditions compatible with detection by thermospray mass spectrometry. When observed by positive ion thermospray mass spectrometry, these compounds give [M+H]+ ions as the base peak of their mass spectra with no discernible fragmentation. In the negative ion thermospray mass spectra of these compounds, the proton- abstracted anion of 7-hydroxy-4-methylcoumarin (appearing at m/z 175) is, without exception, the base peak.