OPA-FMOC在线柱前衍生化HPLC法测定甘露聚糖肽中氨基酸组成及含量

Determination of amino acids in mannatide by online pre-column OPA-FMOC derivatization HPLC

建立了OPA-FMOC在线柱前衍生化高效液相色谱法测定甘露聚糖肽中氨基酸组成及含量的方法,采用色谱柱为Agilent ZORBAX Eclipse XDB-C18柱（250 mm×4.6 mm,5μm）,流动相A为40 mmol/L Na_2HPO_4缓冲溶液（pH 7.8）;流动相B为甲醇：乙腈：水（45∶45∶10,V/V/V）;梯度洗脱;流速：1.5 m L/min;柱温：40℃;检测波长：338,262 nm。20种氨基酸在53 min内得到较好地分离,各氨基酸在5～500μg/m L范围内线性关系良好,相关系数在0.9941～0.9999之间;平均加标回收率在85.6%～102.6%之间;检出限在0.56～8.33 ng（进样量1μL）范围内。甘露聚糖肽样品中含有16种氨基酸,氨基酸总量平均为3.56%（n=3）。

To establish a high performance liquid chromatography（ HPLC） method for the determination of composition and content of amino acids in mannatide by online pre-column OPA-FMOC derivatization. The sample was analyzed on a ZORBAX Eclipse XDB-C18column（ 250 mm × 4. 6 mm,5 μm） with gradient elution of 40 mmol / L Na_2HPO_4 buffer solution（ pH 7. 8）（ A） and methanol-acetonitrile-water（ 45： 45： 10,V / V / V）（ B） at the flow rate of 1. 5 m L / min. The column temperature was 40 ℃,and the detection wavelength was 338 nm and 262 nm. 20 kinds of amino acids got a good separation in 53 minutes,and the calibration curves were linear in the range of 5 ～ 500 μg / m L with the correlation coefficient of 0. 9941 ～ 0. 9999; the average recovery ranged in 85. 6% ～ 102. 6% and the limit of detection ranged in 0. 56 ～ 8. 33 ng（ Injection volume was 1 μL）. Totally 16 kinds of amino acids were determined in mannatide and total content of amino acids was3. 56 %（ n = 3）. This method was simple, rapid, accurate and reproducible, which was suitable for determination of composition and content of amino acids in mannatide.