超高效液相色谱-串联质谱法检测食品中腰果过敏原

Detection of Cashew Allergens in Foods by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry

基于超高效液相色谱-串联质谱系统建立食品中腰果过敏原的定量检测方法。腰果经提取、胰蛋白酶酶解、净化后,经Easy-nLC 1000纳升液相色谱分离,随后进入四极杆-静电场轨道离子阱高分辨质谱以全扫描模式进行数据采集,使用ProteinPilot软件结合Uniprot蛋白数据库对样品扫描结果进行分析,并基于基本局部比对搜索工具(BLAST)验证肽段特异性,最终筛选出6条腰果特异性肽段,利用超高效液相色谱-三重四极杆质谱系统的多反应监测模式对实际样品进行检测。结果表明,该方法在0.005~10 mg/mL范围内线性关系良好,R^(2)均大于0.99,固体基质中定量限为2.5~5 mg/kg,饮料基质中定量限为0.005~0.01 mg/mL,平均回收率在82.5%~109.9%之间,相对标准偏差不大于8.9%。该方法灵敏度高、特异性好,可用于饼干、面包、蛋糕、桃酥、巧克力和饮料6种食品基质中的腰果过敏原检测。

A quantitative method for the detection of cashew allergens in foods was established based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).Cashew samples were extracted,trypsinized,purified,and separated using an Easy nLC 1000 nano-liquid chromatograph,and then data were collected in the full-scan acquisition mode by quadrupole/orbitrap high-resolution mass spectrometry.The scanning results were analyzed by ProteinPilot software search against the UniProt protein database,and the peptide specificity was verified using the basic local alignment search tool (BLAST).Finally,six cashew specific peptides were selected,and actual samples were detected by UPLC-quadrupole/orbitrap MS in the multi-reaction monitoring (MRM) mode.The results showed that the proposed method had good linearity in the range of 0.005–10 mg/mL with correlation coefficients (R^(2)) greater than 0.99.The quantitation limit was 2.5–5 mg/kg and 0.005–0.01 mg/mL for solid-state and beverage matrices,respectively.The average recoveries were 82.5%–109.9%,and the relative standard deviation (RSD) was not more than 8.9%.This method has high sensitivity and specificity,and it can be used for the detection of cashew allergens in six food substrates including biscuit,bread,cake,walnut cookie,chocolate and beverage.