反相高效液相色谱法测定白肋烟烟叶中的游离氨基酸

Determination of Free Amino Acids in Burley Tobacco by High Performance Liquid Chromatography

建立了一种用于烟草中游离氨基酸测定的反相高效液相色谱法．实验采用超声波水解、邻苯二甲醛／3-巯基丙酸作为衍生剂进行柱前衍生．色谱柱为依利特C18柱（4．6mmi．d．×250mm，5μm），流动相A为18mmol／L的醋酸钠溶液（pH7．2）含体积分数为0．002％的三乙胺和0．3％的四氢呋喃，流动相B组成为：100mmol／L的醋酸钠溶液（pH7．2）-乙腈-甲醇（体积比为1：2：2），流速为1．0mL／min，柱温为40℃．荧光检测器，激发波长350nm，发射波长450nm．方法的回收率为95．3％～100．7％，RSD为2．32％～9．24％（n=6）．该方法简便、准确、重现性好．测定了不同肥料配比生产的白肋烟烟叶中17种游离氨基酸的含量．结果表明，不论有机肥与无机肥怎样配比，天冬氨酸的含量与各氨基酸相比都是最高的；随着饼肥的加入，大部分氨基酸的含量是先增加后逐渐降低的趋势，15％饼肥＋85％无机肥与30％饼肥＋70％无机肥配比时，大部分游离氨基酸的含量接近，30％饼肥＋70％无机肥配比时的游离氨基酸总量最高，综合考虑，30％饼肥＋70％无机肥配比时的烟叶质量最好．

A reversed-phase HPLC method was developed for determining free amino acids in burley tobacco. The test was done with phthalaldehyde （OPA）/3-mercaptopropionic acid（3-MPA） as the precolumn derivative reagent. Chromatographic column was Elitte Cls column （4.6 mm i.d.×250 mm, 5μm）. Mobile phase A was 18 mmol/L NaAc （ pH 7.2） including 0. 002 % （V/V） triethylamine and 0.3 % （V/V） furanidine. Mobile phase B was 100 mmol/L NaAc （ pH 7.2） -acetonitrile-methanol （ V/ V = 1 ： 2： 2）. The column temperature was 40℃ and the flow rate was 1.0 mL/min. The fluorescence detector was used with 350 nm excitation wave length and 450 nm emission wave length. The average recoveries of the method ranged from 95.3 % to 100.7 % with the relative standard deviation of 2.32 % -9.24 %. The method is simple, accurate with good repeatability. Seventeen kinds of free amino acids in burley leaves which were produced by way of different ratio of cake fertilizer and inorganic fertilizer were determined. The results show that aspartie acid is of the highest content however ratio of cake fertilizer and inorganic fertilizer. The contents of most of free amino acids are increased and then gradually decreased with the increase of organic manure. The contents of most free amino acids are very close at 15 % ： 85 % ratio and 30 % ： 70 % ratio of cake fertilizer to inorganic fertilizer. The total amount of free amino acids is the highest at 30 % ： 70 % ratio of cake fertilizer to inorganic fertilizer. Considering comprehensively, the aualitv of burlev leaves is the best at 30 % ： 70 % ratio of cake fertilizer to inorganic fertilizer.