高效液相色谱法测定雨水中溶解态氨基酸的含量

A High Performance Liquid Chromatography Method for Measuring Dissolved Amino Acids in Rain

本研究采用邻苯二甲醛/9-芴甲基氯甲酸酯-高效液相色谱-荧光检测器(OPA/FMOC-HPLC-FLD)联用技术,测定雨水中主要游离态氨基酸(DFAA)和结合态氨基酸(DCAA)的含量。雨水中的DFAA经阳离子交换树脂柱富集纯化后,可直接上机测定;类似地,经浓缩、酸解、再浓缩后,直接上机测定雨水中的DCAA。不同氨基酸的保留时间和峰面积的平均相对标准偏差分别为0.053%(0.014%～0.254%)和1.219%(0.223%～3.846%)。除赖氨酸外,其余氨基酸的检出限和定量限都较低,分别为0.90μmol/L(0.19～1.42μmol/L)和2.95μmol/L(0.39～4.74μmol/L)。样品加标回收率为59.87%～125.72%,均值为89.58%,空白回收率为57.47%～118.74%,均值为90.60%,保证了样品测定的准确性。利用本方法测得贵阳地区雨水中结合态氨基酸的浓度为27.25～493.87 nmol/L(均值为182.44 nmol/L),其中含量最多的是蛋氨酸,占总DCAA的24.61%;游离氨基酸的浓度为3.94～126.45 nmol/L(均值24.59 nmol/L),其中丝氨酸含量最高,占总DFAA的30.25%。本研究确立了最优的雨水溶解态氨基酸的提取和测定条件,并保证了雨水氨基酸在低浓度范围内定量的准确可靠性。

The dissolved free amino acids(DFAA) and the dissolved combined amino acids(DCAA) in rainwater were determined by the OPA/FMOC-HPLC-FLD method(phthalaldehyde/9-fluorene methyl chloroformate-high performance liquid chromatography-fluorescence detector). Rainwater samples were acidified and then experienced following processes of adding internal standard, enriching by cation exchange resin column, eluting, lyophilizing and redissolving, then DFAA could be measured on HPLC;similarly, after concentrated, digested, lyophilized, and redissolved, DCAA could be measured on HPLC. The average relative standard deviations of retention times and peak areas for different amino acids were 0.053%(0.014%-0.254%) and 1.219%(0.223%-3.846%), respectively. Except lysine,the limit of detection(LOD) and the limit of quantitation(LOQ) of other amino acids were 0.90 μmol/L(0.19-1.42 μmol/L) and 2.95 μmol/L(0.39-4.74 μmol/L), respectively. The recoveries of spiked samples were 59.87%-125.72% with an average of 89.58%. The blank recovery rates were 57.47%-118.74% with an average of 90.60%, which ensured the accuracy of sample determination. The concentration of DCAA in rainwater of Guiyang measured by this method was 182.44 nmol/L(27.25-493.87 nmol/L), of which methionine accounted for 24.61%. The average concentration of DFAA in rainwater was 24.59 nmol/L(3.94-126.45 nmol/L), the minimum content was tyrosine and the maximum content was serine. Our extraction and analysis methods can ensure the accurate and reliable quantification of amino acids in low concentration ranges and can be used for determining concentrations of amino acids in rainwater.