| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1994: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Various biogenic carboxylic acids such as prostaglandins and unsaturated fatty acids occur in extremely small amounts in biological samples, and play very important roles. In addition many drugs with carboxylic acids, which have strong efficiency against diseases and are administered at extremely small doses, have been developed. Therefore, a sensitive and selective high-performance liquid chromatography (HPLC) method is required for the determination of the biogenic carboxylic acids and drugs.
4- (1-Methylphenanthro [9,10-d] imidazol-2-yl) benzohydrazide has been developed as a highly sensitive and selective fluorescence derivatization reagent for carboxylic acids in HPLC.The reaction conditions were optimized with C16-C20 linear, saturated fatty acids. The derivatization reaction proceeded in aqueous solution in the presence of pyridine and 1-ethyl-3- (3-dimethlaminopropyl) carbodiimide at mild temperatures. The resulting fluorescent derivatives wrere separated by reversed-phase (C18) liquid chromatography with aqueous methanol and were detected with conventional fluorescence detection at 460 nm with excitation at 325 nm. The detection limits (signal-to-noise ratio=3) for the acids are 2-12 fmol for an injeciton volume of 10 mul. The fluorescent derivatives display an excitation maximum at 325 nm, which coincides closely with the light emission of the helium-cadmium laser. Hence, HPLC with the reagent was combined with helium-cadmium laser-induced fluorescence detection. Using this system, attomole detection limits (70-100 amol on-column) were achieved for various carboxylic acis.
Furthermore, the reagent was also successfully applied to the HPLC determination of aldehydes.
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