AccQ-Tag柱前衍生反相高效液相色谱法测定谷物及食糜中氨基酸含量

用AccQ-Tag法测定不同谷物及食糜中氨基酸的含量，并优化原料与酸解剂的比例。采用内标法定量，6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯为柱前衍生剂，用反相高效液相色谱仪，2475荧光检测器（激发波长250nm、发射波长395nm），Waters AccQ-Tag Nova-PakTMC18柱（3.9mm×150mm，4μm），柱温37℃，以醋酸钠（pH4.95）缓冲液为流动相A，纯乙腈为流动相B，水为流动相C进行梯度洗脱，进样量为10μL。结果发现，5mg原料的最优酸解剂为500μL；17种氨基酸线性回归方程在25～500μmol/L范围内线性良好（胱氨酸（Cys）浓度在12.5～250μmol/L之间），相关系数r为0.9996～0.9999，测定的平均回收率为97.56%～103.92%（n=3），相对标准偏差为0.31%～2.75%（n=3）。本法快速简便，能够适用于谷物及食糜中氨基酸含量的测定。

Determination of Amino Acid Contents in Cereals and Ileal Digesta by Reversed-Phase High Performance Liquid Chromatography with AccQ-Tag Pre-column Derivatization Method

The contents of amino acids in different cereals and ileal digesta were determined by reversed-phase high performance liquid chromatography (RP-HPLC) according to the AccQ-Tag method. The ratio of material to acidolysis agent was optimized in this paper. Quantification was performed using an internal standard method after pre-column derivatization with 6-aminoquinolinyl-N-hydroxysuccinimidate (AQC). The analysis procedure was carried out using a Waters 2695 HPLC system fitted with a Waters AccQ-Tag Nova-PakTMC18 column (3.9 mm × 150 mm, 4 μm) and a Waters 2475 fluorescence detector (Waters Corp., USA) at an excitation wavelength of 250 nm and emission wavelength of 395 nm at 37 ℃. Sodium acetate (pH 4.95), acetonitrile and water were used respectively as mobile phases A, B and C for gradient elution and the injection volume was 10 μL. The results showed that the optimum ratio of material to acidolysis agent was 1:100 (mg/μL). The calibration curves of 17 amino acids were linear within the range of 25?500 μmol/L (12.5–250 μmol/L for cystine) with correlation coefficients (r) of 0.999 6?0.999 9. The average recoveries were in the range of 97.56%–103.92% with relative standard deviation (RSD) of 0.31%?2.75% (n = 3). This method is rapid, simple and accurate for the determination of amino acids in cereals and ileal digesta.