高效液相色谱法测定脂肪酸组成柱前衍生条件的优化研究

目的:制备脂肪酸苯酰肼对照品,通过该对照品计算衍生率,探究脂肪酸最优的高效液相色谱（HPLC）柱前衍生条件。方法:选取正十四碳酸（myristic acid,MA）为代表,以吡啶为催化剂,1-乙基-（3-二甲基氨基丙基）碳酰二亚胺盐酸盐（1-EDC·HCl）为偶联剂,MA与2-硝基苯肼盐酸盐（2-NPH·HCl）反应,合成正十四碳酸苯酰肼（myristic benzhydrazide,MBH）;采用硅胶柱层析和重结晶对衍生产物MBH进行分离纯化,MS和1H-NMR进行结构鉴定,TLC和HPLC进行纯度检测;以衍生率为指标,对影响MA衍生为MBH的HPLC的柱前衍生条件进行研究。结果:成功制备了纯度为99.81%的MBH对照品,可用于脂肪酸衍生条件优化研究中衍生率的测定;最优的衍生条件为:2-NPH·HCl浓度为0.04 mol/L,1-EDC·HCl浓度为0.45 mol/L,60℃水浴加热15 min,此时MA的衍生率达到99.85%。在此衍生条件下,衍生同摩尔浓度的亚油酸（linoleic acid,LA）,经HPLC分析发现,LA衍生产物的峰面积与MA衍生产物的峰面积的相对差值仅为1.3%。结论:获得了脂肪酸最优的HPLC柱前衍生条件,该衍生条件能最大化地衍生脂质中各种脂肪酸。 

Optimization of pre- column derivatization condition of fatty acid by high performance liquid chromatography

Objective: Fatty acid benzhydrazide was produced which was used as a reference substance for determination of derivatization yield.The best conditions for the pre- column derivatization of fatty acids using high performance liquid chromatography were studied.Methods: Myristic acid( MA) was selected to represent all of fatty acids( FAs) in the experiment.MA reacted with 2- nitrophenylhydrazine hydrochloride( 2- NPH·HCl) in the case of1- ethyl-3-( 3- dimethylaminopropyl) carbodiimide hydrochloride( 1- EDC·HCl) as coupling agent and pyridine as catalyst,and Myristic benzhydrazide( MBH) was produced. The MBH was separated and purified by silica gel column chromatography and recrystallization.Its structure was identified by MS and1 HNMR.Its purity was detected by TLC and HPLC. Derivatization yield as a indicator,the pre- column derivatization conditions of MA using high performance liquid chromatography were studied.Results: MBH was obtained and it purity was 99.81% which could be used as a reference substance for determination of derivatization yield in the study of optimization of derivatization condition of fatty acid. The best conditions for the pre- column derivatization were as follows: the concentration of 2- NPH·HCl was 0.04 mol / L,the concentration of 1- EDC·HCl was 0.45 mol / L,the temperature of the derivatization reaction was 60 ℃,the time of the derivatization reaction was 15 min.The derivatization yield was99.85% under above the derivatization condition. Linoleic acid derivative was prepared using same derivatization condition.The difference between the peak area of LA derivative and MA derivative was only 1.3%.Conclusion: The optimal pre- column derivatization condition of fatty acid by HPLC was obtained and various fatty acids could be maximum derivatized under above the derivatization condition.