柱前衍生-高效液相色谱测定牛皮蓝湿革中18种氨基酸

Determination of eighteen amino acids in wet-blue leather by pre-column derivatization combined with high performance liquid chromatography

建立了一种柱前衍生-高效液相色谱测定牛皮蓝湿革水解液中18种氨基酸含量的方法。以2,4-二硝基氟苯(DNFB)为衍生试剂,常规C18色谱柱(4.6 mm×250 mm,5μm)分离。通过优化DNFB用量、衍生反应时间、流动相种类和梯度洗脱程序,40 min内可实现18种氨基酸的完全基线分离。在优化的衍生反应和色谱分离条件下,18种氨基酸在5~100μmol/L范围内线性关系良好,线性相关系数(R2)>0.9956,检出限和定量限分别为0.02~0.47μmol/L和0.08~1.57μmol/L,日内和日间相对标准偏差(RSD)为1.1%~3.7%和1.9%~5.7%。将该方法用于牛皮蓝湿革经微波消解所得水解液的分析,氨基酸的含量组成符合蓝湿革的特征;加标回收率为91.7%~112.5%,RSD为0.36%~5.3%,灵敏度和准确度好。

A method of pre-column derivatization combined with high performance liquid chromatography(HPLC)was proposed to determine eighteen kinds of amino acids in wet-blue leather.2,4-dinitrofluorobenzene(DNFB)and conventional C18 column(4.6 mm×250 mm,5μm)were used as derivatization reagent and separation column.Complete baseline separation of eighteen kinds of amino acids could be achieved within 40 min by optimizing the amount of DNFB,derivative reaction time,mobile phase type and gradient elution program.Under the optimized conditions,good linear relationships in the range of 5-100μmol/L were obtained for eighteen kinds of amino acids,and the corresponding correlation coefficients(R2)were greater than 0.9956.The limits of detection(LODs)and limits of quantitation(LOQs)were in the ranges of 0.02-0.47μmol/L and 0.08-1.57μmol/L,respectively,along with the intra-day and inter-day relative standard deviations(RSDs)of 1.1%-3.7%and 1.9%-5.7%,respectively.Moreover,the established pre-column derivatization-HPLC method was applied to analyze wet-blue leather hydrolysate digested by microwave,revealing the content and composition of amino acids conformed to the characteristics of wet-blue leather.The spiked recoveries of eighteen kinds of amino acids were attained ranging from 91.7%to 112.5%,together with the RSDs of 0.36%-5.3%,showing excellent sensitivity and accuracy.