Synthesis and Application of a New Fluorescent Reagent 2-(2-Naphthoxy)ethyl 2-(piperidino) ethanesulfonate for Liquid Chromatography

• Main Authors: Chi-Yu Lu, 呂濟宇
• Other Authors: Hsin-Lung Wu, Ph. D.
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/89071386339181844742

博士 === 高雄醫學大學 === 藥學研究所 === 90 === Summary A new fluorescent derivatization reagent, 2-(2-naphthoxy)ethyl 2-(piperidino) ethanesulfonate (NOEPES), was synthesized for high performance liquid chromatography. The reagent has a favorable auxochrome (substituted alkoxy) attached to the chromophoric (fluorophoric) naphthalene moiety, giving good spectrophotometric property for a derivative. The main results of this work were summarized as follows : 1. In the synthesis of NOEPES, 2-(2-naphthoxy)ethanol reacted with 2-chloroethanesulfonyl chloride in dichloromethane using triethylamine as a catalyst at 0 oC for 2 h to give an intermediate. The intermediate was not isolated and further reacted with piperidine in dichloromethane at 0 oC for 1 h to give NOEPES. 2. The chemical reactivity of NOEPES was studied briefly on several organic and inorganic analytes by TLC. The results indicate that NOEPES is reactive to various organic functional groups (acid, mercaptan, imide, phenol, thiouracil and sulfonamide) and inorganic analytes (S2-, I-, Br-, SCN-, CN— and NO2-). 3. The new reagent NOEPES was used for the simultaneous determination of biologically important very-long-chain fatty acids (docosanoic, tetracosanoic and hexacosanoic acids) and long-chain fatty acids (dodecanoic, tetradecanoic, hexadecanoic, octadecanoic, hexadecenoic, octadecenoic and octadecadienoic acids) as fluorogenic derivatives. The method is based on the derivatization of the fatty acids with NOEPES in toluene in the presence of potassium carbonate and 18-crown-6. The resulting naphthoxyethyl derivatives were separated by isocratic HPLC and monitored with a fluorometric detector. The linear range for the determination of very-long-chain fatty acids was 0.028 - 1.4 M with a detection limit of about 56 fmol (S / N = 3, per 10 L injection). The linear range for the determination of long-chain fatty acids was 0.05 - 2.5 M with a detection limit of about 0.15 pmol (S / N = 3, per 20 L injection). 4. The reagent contains a tertiary amino moiety and it can be protonated by a simple acid treatment to form a water soluble species for the removal of excess NOEPES, resulting in a simpler and cleaner chromatogram. 5. Application of NOEPES to the analysis of biologically important free very-long-chain and long-chain fatty acids in plasma and milk products was performed. Only a very small amount of human plasma (100 L and 10 L) was used to analyze free very-long-chain and long-chain fatty acids. Application of the new reagent to the analysis of diagnostic important free fatty acids in human plasma and milk products proved simple and feasible.