Determination of biogenic amines by RPHPLC with fluorescent detection after derivatization with 2-(9-carbazole)ethyl chloroformate (CEOC) |
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Authors: | Jinmao You Yukui Zhang |
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Affiliation: | (1) Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 116011 Dalian, P.R. China;(2) Department of Chemistry, Qufu Normal University, 273165 Qufu Shandong, P.R. China |
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Abstract: | Summary The use of 2-(9-carbazole)ethyl chloroformate (CEOC) for pre-column derivatization of biogenic amines (BA) has been tested for the first time. The reagent reacts completely with BA within 3 min at ambient temperature in acetonitrile solution to form stable derivatives that are readily analyzed by reversed-phase HPLC. Study of the derivatization conditions revealed derivatization yields to be excellent in borate buffer over the pH range 9.0–10.0. Maximum yields were obtained by use of a three- to fourfold molar excess of reagent. The reaction is extremely tolerant of common buffer salts, no decrease in reaction yield is discernible in well-buffered samples. The emission maximum for the CEOC-derivatives is 360 nm (λ ex = 293 nm). All the derivatives fluoresced strongly and direct injection of the reaction mixture was possible, with no significant disturbance from the major fluorescent reagent degradation by-products, 2-(9-carbazole)ethanol (CEOC-OH) and bis-(2-(9-carbazole)ethyl) carbonate (CEOC)2. Separation of the derivatized BA by high-performance liquid chromatography with gradient elution was tested on a Hypersil BDS C18 column. Excellent response linearity was observed over the concentration range from 0.25 to 94.6 μmol L−1 for the labeled BA. Detection limits were 117–840 fmol at a signal-to-noise ratio of 3∶1. Analysis of BA in a shrimp sauce extract was conducted to demonstrate the applicability of the technique to real sample matrixes; results were satisfactory. |
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Keywords: | Column liquid chromatography Fluorescent detection 2-(9-Carbazole)ethyl chloroformate Biogenic amines |
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