异硫氰酸苯酯柱前衍生RP-HPLC法同时测定莲雾中15种游离氨基酸的含量

Simultaneous Determination of Free Amino Acids in Wax Apple by Reversed Phase High Performance Liquid Chromatography with Phenyl Isothiocyanate Precolumn Derivatization

采用异硫氰酸苯酯(PITC)为柱前衍生化试剂,结合反相高效液相色谱法,建立了同时测定莲雾果实中15种游离氨基酸含量的方法。采用Atlantis T3柱为固定相,以0.1 mol/L乙酸钠缓冲液(pH6.5,含0.5%三乙胺)和乙腈-水(4∶1)为流动相进行梯度洗脱,流速为1.0 mL/min,柱温35℃,紫外检测器波长254 nm。各氨基酸组分在2.0~500 mg/L范围内具有良好的线性关系,相关系数r均大于0.99,检出限为0.08~0.35 mg/L,定量限为0.35~1.25 mg/L,加标回收率为85.61%~106.68%,相对标准偏差RSD均小于5%。该方法使得15种氨基酸很好的分离,且准确度和精密度好,灵敏度和回收率高,适用于莲雾果实游离氨基酸含量的分析。

A high performance liquid chromatography method for simultaneous determination of 15 free amino acids in wax apple fruit by pre-column derivatization with phenyl isothiocyanate(PITC)was established.The separation was achieved using a Atlantis T3 column under the gradient elution with the mobile phases composed of 0.1 mol/L sodium acetate buffer(pH=6.5)and acetonitrile-water(4:1)at the flow rate of 1.0 mL/min,column temperature of 35℃and UV detector wavelength at 254 nm.The calibration curves of amino acid component had good linearity over the range from 2.0 to 500 mg/L with correlation coefficient all above 0.99.The detection limits were in the range of 0.08~0.35 mg/L and the quantitation limit were in the range of 0.35~1.25 mg/L.The spiked recoveries were between 85.61%and 106.68%and the RSDs were less than 5%.This method could separate the 15 amino acids with high accuracy,high sensitivity and recovery.The method was successfully used to analyze the free amino acids in wax apple fruit.