Determination of Eighteen Kinds of Free Amino Acids in Fruits by Ultra Performance Liquid Chromatography-Quadrupole-Exactive Orbitrap Mass Spectrometry
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摘要: 本文采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱（UPLC-Q-Exactive Orbitrap）建立了水果中18种游离氨基酸的快速测定方法。水果样品经粉碎、超声提取、离心、过滤后，使用SHISEIDO CAPCELL PAK BB-H C18色谱柱（2.1 mm×150 mm，3 μm）分离，以0.2%甲酸水溶液和甲醇溶液为流动相进行梯度洗脱，结合保留时间、一级分子离子精确质荷比和二级特征碎片离子对目标化合物进行快速筛查，并准确定量。结果表明，甘氨酸、丙氨酸的线性范围为1~50 nmol/mL，其余16种氨基酸线性范围为0.5~50 nmol/mL，均线性良好（R2≥0.99），检出限为0.5~1 pmol/g，在1倍、2倍、10倍定量限浓度水平进行加标回收实验，回收率范围为79.1%~118.2%，相对标准偏差（relative standard deviations，RSD）为0.5%~5.6%。该方法简便、快捷、准确，适用于多种水果中18种游离氨基酸成分的测定。Abstract: An ultra performance liquid chromatography-quadrupole-exactive orbitrap mass spectrometry(UPLC-Q-Exactive Orbitrap)was developed for the determination of eighteen kinds of free amino acids in fruits.The crushed samples were extracted by microwave ultrasonic instrument,then centrifugation and filtration,finally,separated on a SHISEIDO CAPCELL PAK BB-H C18(2.1 mm×150 mm,3 μm)column using a binary solvent system composed of 0.2% formic acid aqueous solution and methanol solution by gradient elution. Under this conditions,the analytes were rapidly screened and precisely detected with the precision precursor mass,retention time and two-stage ion mass spectra.The linear range of glycine and alanine was 1~50 nmol/mL,and the linear range of other 16 amino acids was 0.5~50 nmol/mL.There were good linear relationships for the 18 analytes with correlation coefficients(R2)larger than 0.99.The method showed the limit of detection(LOD)was 0.5~1 pmol/g. The recoveries for the 18 compounds at different spiked levels mainly ranged from 79.1% to 118.2%,with relative standard deviations(RSD)of 0.5%~5.6%.The method was convenient,rapid and accurate,and was quite effective for the determination of the 18 free amino acids in fruits,which was beneficial for the investigation on nutritional ingredient in fruits.
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