柱前在线衍生-反相高效液相色谱内标法快速测定饲料中氨基酸

Rapid Determination of Amino Acids in Feedstuffs by High Performance Liquid Chromatography Using Internal Standard Method with Online Pre-column Derivatization

采用邻苯二甲醛和氯甲酸芴甲酯联合柱前在线衍生，用反相C18短柱梯度洗脱，二极管阵列检测器（用于检测胱氨酸和赖氨酸）和荧光检测器（用于检测其它氨基酸）联合检测，建立了一种高效液相色谱内标法快速测定饲料中氨基酸含量的方法。此方法对传统样品水解液制备的方法进行了简化，在不须预先除去干扰物质的情况下，提高了氨基酸测定的准确度，18种氨基酸除胱氨酸和色氨酸未检出（完全被破坏）和蛋氨酸为87．32％（部分被氧化破坏）外，其它氨基酸的回收率为94．14％-102．25％，相对标准偏差为2．11％-4．52％（n=5）；线性相关性好（相关系数为0．9900—0．9995）；线性范围为4．5—1000μmol／L；方法检出限低，灵敏度高，分离时间短，适合饲料中氨基酸的快速测定。

A high performance liquid chromatographic method for rapid determination of amino acids in feedstuffs, was performed automatically by the autosampler, based on the online derivatization with o-phthaldialdehyde and 9-fluorenylmethyl chloroformate and the detection was carried out with diode array detector （for cystine and lysine ） and fluorescence detector （for other amino acids except cystine lysine） combined a C18 short column using gradient elution with norvaline as internal standard, adding to the samples before hydrolysis. The steps of sample preparation were simplified. The accuracy of determination of amino acids was improved without separating the interferents before analysis. The recoveries of standard spiking of 18 amino acids were in the range of 94.1% - 102.3%, except cystine, tryptophan （destroyed completely） and methionine （87.3%）. The relative standard deviations （ RSD, n = 5 ） were in the range of 2.1% - 4.5 %. The ratio of peak area of amino acids versus their concentration had a good linearity （ correlation coefficients were 0.9900 - 0. 9995 ） in the range of 4.5 -1000 μmol/L. The limits of detection of the most amino acids were low, and sensitivities of detection were high. This method was suitable for rapid determination of amino acids in feedstuffs.