液相色谱-串联质谱法检测辐照蛋白类功能食品中酪氨酸同分异构体

Determination of Tyrosine Isomers in Irradiated Protein-containing Functional Food by Liquid Chromatography-Tandem Mass Spectrometry

建立了液相色谱-串联质谱联用法（LC-MS/MS）同时检测辐照蛋白类功能食品中邻酪氨酸（o-Tyrosine）和间酪氨酸（m-Tyrosine）的分析方法.样品在氮气环境下经6 mol/L盐酸酸解过夜,酸解液再经氮气吹干,1 mL水复溶后,用Phenomenex Kinetex PFP色谱柱分离,以5 mmol/L乙酸铵溶液和甲醇为流动相进行梯度洗脱.质谱采用电喷雾负离子（ESI-）模式电离,多反应监测（MRM）模式检测,基质匹配标准溶液外标法定量.酪氨酸同分异构体与基质干扰峰得到良好的分离,在0.005 ～ 1.0 mg/L浓度范围内,其峰面积与浓度呈良好的线性关系,线性系数（r2）不低于0.999.在乳清粉和乳清蛋白粉中添加3种浓度水平的酪氨酸同分异构体,平均加标回收率为81.8％ ～98.2％,相对标准偏差（n=6）低于8.0％,方法定量下限（LOQ）和检出限（LOD）分别为5.0,8.0μg/kg和1.5,2.4μg/kg.将该方法应用于实际样品分析,表明辐照剂量与辐照蛋白类功能食品中o-Tyrosine和m-Tyrosine含量存在相关性,符合拟合多项式（r2 ＞0.98）.该方法可对蛋白类功能食品是否经过辐照给予定性鉴定,从而为辐照蛋白类功能食品中辐照标志物的检测方法研究提供理论依据.

A liquid chromatography-tandem mass spectrometric （LC-MS/MS） method was developed for the determination of tyrosine isomers （o-tyrosine and m-tyrosine） in protein-containing functional food.The sample was dissolved with 6 mol/L hydrochloric acid,staying overnight under 110℃ acid solution after full of nitrogen,then evaporated with a stream of nitrogen to near dryness and redissolved with water.The LC separation was performed on a Phenomenex Kinetex PFP column with 5 mmol/L ammonium acetate solution and methanol as mobile phases by gradient elution.The tyrosine isomers were determined by MS/MS in negative electrospray ionization（ESI） mode,and quantified by the matrix-matched （MRM） external standard method.Good separations were obtained between the tyrosine isomers and other impurity peaks.In 0.005-1.0 mg/L concentration range,the peak areas of tyrosine isomers against concentration showed good linear relationships with correlation coefficient （r2） more than 0.999.The average recoveries of tyrosine isomers in protein-containing function food at three spiked concentration levels ranged from 81.8％ to 98.2％,with relative standard deviations（RSDs,n =6） less than 8.0％.The limit of quantitation （LOQs） were 5.0 μg/kg and 8.0 μg/kg,and the limits of detection（LODs） were 1.5 μg/kg and 2.4 μg/kg,respectively.This method was applied in real samples,and the result indicated that the radiation dose could be quantitatively evaluated by the linear correlation with coefficients of determination （r2） more than 0.98in individual irradiated protein-containing function food samples.The method could be applied in qualitative identification of tyrosine isomers in protein-containing functional food.