An improved derivatization method for sensitive determination of fatty acids by high-performance liquid chromatography using 9-(2-hydroxylethyl)-carbazole as derivatization reagent with fluorescence detection |
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Authors: | Jinmao You Weibing Zhang XiangIan Jia Yukui Zhang |
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Institution: | (1) Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 116011, P.R. China;(2) Department of Chemistry, Qufu Normal University, Qufu, 273165 Shandong, P.R. China |
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Abstract: | Summary Tagging techniques with reagents used for fluorescent detection for short and long-chain fatty acids using high-performance
liquid chromatography are evaluated in terms of the tagging reactions, handing, flexibility, stability of the reagents. Emphasis
is given to the applications of the tagging techniques to relatively high molecular mass fatty acids. The fatty acids or carboxylic
compounds were derivatized to their corresponding esters with 9-(2-hydroxy ethyl)-carbazole (HEC) in acetonitrile at 60°C
with N, N′-carbonyldiimidazole (CDI) as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters
of C1−C20 fatty acids was completely separated with 45 min using gradient elution on a reversed-phase C18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (λex 293 nm). Studies on derivatization conditions indicated that fatty acids react rapidly and smoothly with HEC in the presence
of CDI and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method
to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility
for fatty acids derivatives. The relative standard deviations (n=6) for each fatty acid derivative are <5.0%. The detection
limits are at 38–57 fmol levels for C14−C20 fatty acids and lower levels for <C14 fatty acids. |
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Keywords: | Column liquid chromatography Fluorescent detection 9-(2-hydroxyethyl)-Carbazole Fatty acids Plasma samples |
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