CN112630319A - Lc-ms/ms内标法测定肉制品中猪肉含量的方法 - Google Patents
Lc-ms/ms内标法测定肉制品中猪肉含量的方法 Download PDFInfo
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Abstract
本发明属于食品检测技术领域,具体涉及一种LC‑MS/MS内标法测定肉制品中猪肉含量的方法,以特定的猪肉特征多肽作为检测的分析对象,以同位素标记的所述特征多肽为内标物,采用内标标准曲线法定量。本发明基于高效液相色谱‑串联质谱技术,结合筛选出可用于准确定量分析的猪肉多肽,制备相应的同位素内标多肽,建立了测定肉制品中猪肉含量的内标定量方法。该方法通过绘制内标法猪肉标准曲线,可准确计算其中所含有的猪肉含量,是一种灵敏、稳定、特异、高通量、操作性强、定量准确的分析方法。
Description
技术领域
本发明属于食品检测技术领域,具体涉及一种LC-MS/MS内标法测定肉制品中猪肉含量的方法。
背景技术
肉制品是我国居民饮食结构中的重要组成部分,深受消费者的喜爱。由于我国居民消费以猪肉为主,当前国内猪肉制品所占的市场份额最大,占比超60%。近几年,受非洲猪瘟疫情的影响,生猪跨省调运,生猪交易市场被关闭,猪肉价格显著增长,进而导致猪肉制品生产企业成本明显增高。
一些不法商家为了降低成本,使用大量植物蛋白替代畜禽肉成分,或者用鸡、鸭肉(低价肉)替代或部分替代猪肉,但在配料表中却不按事实标注,虽然蛋白总量符合要求,但动物蛋白较低,使得产品价格低于市场价格,致合规性肉类制品企业的产品在市场上受阻,竞争力不足,长此以往影响企业的生存及发展。因此建立肉制品中猪肉含量的测定方法既顺应市场需求,也可为规范行业标识、维护市场公平提供强有力的技术支持。
发明内容
本发明的目的是克服现有技术的不足,提供一种LC-MS/MS内标法测定肉制品中猪肉含量的方法。本发明选择定量较好的猪肉多肽作为分析对象,通过前处理方法提取待测肉制品中猪肉特异性多肽,并加入合成的同位素内标多肽,用于校正仪器状态、基质效应以及实验前处理过程中的损失,然后进行LC-MS/MS分析,并建立猪肉对应多肽及内标的标准曲线,以猪肉含量作为横坐标,定量多肽与对应内标多肽的峰面积比值作为纵坐标,通过内标法计算得到待测肉制品中猪肉含量。
具体来说,本发明提供了如下技术方案:
一种LC-MS/MS内标法测定肉制品中猪肉含量的方法,包括以下步骤:对含猪肉的待测肉制品进行预处理,得到待测肉制品的多肽样品溶液;采用液相色谱串联质谱对所述多肽样品溶液进行检测,以猪肉的特征多肽作为检测的分析对象,以同位素标记的所述特征多肽为内标物,采用内标法对待测肉制品中猪肉的含量进行定量检测;
其中,所述特征多肽选自Pep_1至Pep_3中的一种或两种以上:
Pep_1的序列如下:EPITVSSDQMAK(SEQ ID No.1);Pep_1的序列如下:GGPLTAAYR(SEQ ID No.2);Pep_1的序列如下:HDPSLLPWTASYDPGSAK(SEQ ID No.3)
本发明的发明人利用高分辨质谱对纯猪肉及猪肉制品进行数据采集,并使用蛋白质组学数据库和软件进行数据分析,筛选出猪肉的专属性多肽链;将得到的多肽链信息转化为离子对信息导入至LC-MS/MS,通过优化仪器条件,对样品信息进行数据采集及处理,筛选响应值高、稳定性好、定量准确的多肽链及离子信息;制备同位素内标多肽,通过标准曲线数据拟合及样品分析,进一步筛选出如上表所示的使用内标法定量的多肽信息。图1为猪肉的总离子流图和3种特征多肽的提取离子流图。
优选的,上述LC-MS/MS内标法测定肉制品中猪肉含量的方法中,所述同位素标记的特征多肽的信息如如ISTD_1至ISTD_3所示,其中,所述ISTD_1为同位素标记Pep_1后的多肽,所述ISTD_2为同位素标记Pep_2后的多肽,所述ISTD_3为同位素标记Pep_3后的多肽:
优选的,上述LC-MS/MS内标法测定肉制品中猪肉含量的方法中,所述特征多肽为Pep_2。
优选的,上述LC-MS/MS内标法测定肉制品中猪肉含量的方法中,所述内标法的步骤包括:
配制含不同浓度梯度标准猪肉多肽样品与相同浓度内标物的混合标准品工作溶液,以标准猪肉多肽样品对应的猪肉质量浓度为横坐标,以特征多肽与内标物的色谱峰面积比为纵坐标,建立标准曲线;
对加入有相同浓度内标物的待测肉制品的多肽样品溶液进行检测,根据所述标准曲线计算待测肉制品中猪肉的含量。
优选的,上述LC-MS/MS内标法测定肉制品中猪肉含量的方法中,所述预处理包括以下步骤:
(1)提取样品高丰度蛋白:向待测肉制品中加入提取液,在液氮条件下进行冷冻研磨,然后离心取上清液;
(2)向步骤(1)取得的上清液中加入NH4HCO3和胰蛋白酶进行酶解,加入三氟乙酸终止反应,经过HLB固相萃取柱净化,供上机检测。
优选的,上述LC-MS/MS内标法测定肉制品中猪肉含量的方法中,所述提取液为0.04~0.06mol/L Tris-HCl、6~8mol/L尿素、1~3mol/L硫脲。
本发明基于高效液相色谱-串联质谱技术,结合筛选出的可用于准确定量分析的猪肉特征多肽,制备相应的同位素内标多肽,建立了测定肉制品中猪肉含量的内标定量方法。该方法通过绘制内标法猪肉标准曲线,可准确计算其中所含有的猪肉含量,是一种灵敏、稳定、特异、高通量、操作性强、定量准确的分析方法。
附图说明
图1为猪肉的总离子流图和3种特征多肽的提取离子流图。
具体实施方式
以下结合具体实施例对本发明作进一步详细说明,但不用来限制本发明的范围。
以下实施例中所使用的实验方法如无特殊说明,均为常规方法。以下实施例中所用的实验原料和相关设备等,如无特殊说明,均可从商业途径得到。
实施例1
实施例1提供一种LC-MS/MS内标法测定肉制品中猪肉含量的方法,以猪肉的特征多肽作为检测的分析对象,以同位素标记的所述特征多肽为内标物,采用内标标准曲线法定量。所述特征多肽和同位素内标多肽的信息如表1所示。
表1定量多肽离子信息
1、样品前处理
将5组模拟肉丸(成分含量见表2)作为待测肉制品,用制样机研磨均匀,称取0.5g,加入5mL提取液,液氮冷冻研磨30s,15000r/min离心10min,取300μL上清液,加入30μL内标物溶液(200μg/mL),加入50mmol/L二硫苏糖醇溶液10μL,56℃振荡反应1h,放置室温,加入100mmol/L碘乙酰胺溶液20μL,暗处反应30min,再加入50mmol/L二硫苏糖醇溶液15μL,暗处反应15min;加入25mmol/L Tris-HCl缓冲溶液750μL,然后加入20μg胰蛋白酶,37℃反应过夜;向上述酶解液中加入0.4%TFA终止反应,并加入1mL水,全部过HLB固相萃取柱(乙腈、0.1%乙酸水活化,上样),1mL 60%乙腈+0.5%乙酸洗脱并过0.22μm滤膜,即得添加内标物的待测肉制品的多肽样品溶液,准备上机。
表2 5组模拟肉丸的成分含量
2、猪肉标准曲线的建立
取猪肉(冷冻猪肉,需化冻)称取0.5g,加入5mL提取液,液氮冷冻研磨30s,15000r/min离心10min,分别取上清液20μL、50μL、100μL、200μL、300μL至5个管并加入适量的提取液分别定容为300μL,后续处理步骤同步骤1,即得含不同浓度梯度标准猪肉多肽样品与相同浓度内标物的混合标准品工作溶液。
采用液相色谱串联质谱检测上述混合标准品工作溶液,以标准猪肉多肽样品对应的猪肉质量浓度为横坐标,以猪肉多肽峰面积/内标多肽峰面积为纵坐标,绘制标准工作曲线。
其中,以所述取上清液20μL最终得到的标准猪肉多肽样品为例,其对应的猪肉质量浓度是指(0.5g/5mL)×(20μL/1mL),即(猪肉质量/提取液体积)×(取的上清液体积/混合标准品工作溶液的上机体积)。
3、液相色谱串联质谱的检测条件
色谱条件:色谱柱Hypersil GOLD C18,规格2.1mm×100mm,1.9μm;流速0.2mL/min;柱温40℃,进样量20μL;流动相:A相0.1%FA/H2O,B相0.1%FA/ACN,色谱梯度为:0~0.2min,3%~10%B;0.2~16min,10%~40%B;16~17min,40%~80%B;17.5~18.5min,80%~3%B;
质谱条件:喷雾电压3500V;鞘气38Arb;辅气15Arb;离子传输管温度275℃;离子源雾化温度380℃;传输线温度275℃;离子源温度380℃;采集周期为0.3s;碰撞气为1.5mTorr;Q1和Q3分辨率均为0.7,正模式。
4、标准工作曲线数据分析
在0.5-15mg/mL的猪肉质量浓度范围内,用ISTD方法评估校准曲线的线性。5点校准曲线的线性良好,R2均大于0.99,结果显示如表3所示。
表3 5点校准曲线的线性回归方程
5、结果计算及分析
定性测定
选择了猪的3条特异多肽,1个母离子,3个子离子,在相同实验条件下,样品中待测多肽与标准样品保留时间比相对偏差在±2.5%;且样品谱图各组分定性离子的相对丰度与标准肉样对应的定性离子的相对丰度进行比较,偏差不超过表4规定的范围,则可判定为存在对应的肉样组分。
表4定性时相对离子丰度的最大允许偏差
相对离子丰度/% | >50 | >20~50 | >10~20 | ≤10 |
允许的相对偏差/% | ±20 | ±25 | ±30 | ±50 |
定量测定
采用液相色谱串联质谱检测步骤1添加相同浓度内标物的待测肉制品的多肽样品溶液,然后将得到的特征多肽和内标多肽的峰面积比代入标准曲线的线性回归方程中计算得到待测肉制品多肽样品对应的猪肉质量浓度C,根据公式:
其中,C-由标准曲线得到的待测肉制品多肽样品对应的猪肉质量浓度,mg/mL;
V-待测肉制品多肽样品溶液的上机体积,mL;本实施例中为1mL;
m-待测肉制品的称样量,g;本实施例中为0.5g;
计算得到待测肉制品中猪肉的含量X,%,结果如表5所示。
表5待测肉制品的猪肉含量计算结果
对比例1
与实施例1相比,区别在于:不加入内标物,采用外标法对待测肉制品进行定量测定,以标准猪肉多肽样品对应的猪肉质量浓度为横坐标,以特征多肽的色谱峰面积为纵坐标,建立标准曲线,其线性方程如表6所示,测定结果如表7所示。
表6 5点校准曲线的线性回归方程
表7待测肉制品的猪肉含量计算结果
对比例2
与实施例1相比,区别在于:以猪肉的其他特征多肽Pep_4至Pep_7作为检测的分析对象进行肉制品定量分析。
Pep_4的序列如下:DTLVSQLSR(SEQ ID No.4);Pep_5的序列如下:TLAFLFAER(SEQID No.5);Pep_6的序列如下:YLEFISEAIIQVLQSK(SEQ ID No.6);Pep_7的序列如下:TVLGNFAAFVQK(SEQ ID No.7)。所述特征多肽Pep_4至Pep_7和其同位素内标多肽的定量离子信息如8所示。采用内标法对待测肉制品进行定量测定,其线性方程如表9所示,测定结果如表10所示。
表8其他特征多肽的定量离子信息
表9 5点校准曲线的线性回归方程
表10待测肉制品的猪肉含量计算结果
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对其作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
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Claims (6)
3.根据权利要求1或2所述的LC-MS/MS内标法测定肉制品中猪肉含量的方法,其特征在于,所述特征多肽为Pep_2。
4.根据权利要求1-3任一项所述的LC-MS/MS内标法测定肉制品中猪肉含量的方法,其特征在于,所述内标法的步骤包括:
配制含不同浓度梯度标准猪肉多肽样品与相同浓度内标物的混合标准品工作溶液,以标准猪肉多肽样品对应的猪肉质量浓度为横坐标,以特征多肽与内标物的色谱峰面积比为纵坐标,建立标准曲线;
对加入有相同浓度内标物的待测肉制品的多肽样品溶液进行检测,根据所述标准曲线计算待测肉制品中猪肉的含量。
5.根据权利要求1-4任一项所述的LC-MS/MS内标法测定肉制品中猪肉含量的方法,其特征在于,所述预处理包括以下步骤:
(1)提取样品高丰度蛋白:向待测肉制品中加入提取液,在液氮条件下进行冷冻研磨,然后离心取上清液;
(2)向步骤(1)取得的上清液中加入NH4HCO3和胰蛋白酶进行酶解,加入三氟乙酸终止反应,经过HLB固相萃取柱净化,供上机检测。
6.根据权利要求5所述的LC-MS/MS内标法测定肉制品中猪肉含量的方法,其特征在于,所述提取液为0.04~0.06mol/L Tris-HCl、6~8mol/L尿素、1~3mol/L硫脲。
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CN110007019A (zh) * | 2019-03-29 | 2019-07-12 | 绿城农科检测技术有限公司 | 一种同时定量检测母乳中三种抗体含量的方法 |
CN110736802A (zh) * | 2019-12-20 | 2020-01-31 | 中国农业科学院蜜蜂研究所 | 一种液相色谱串联质谱定量检测意蜂蜜中α-葡萄糖苷酶的方法 |
CN111122756A (zh) * | 2019-12-04 | 2020-05-08 | 青岛海关技术中心 | 检测可食用肉的特征多肽及方法 |
CN111595971A (zh) * | 2020-05-22 | 2020-08-28 | 浙江大学 | 一种灰尘中尘螨过敏原蛋白Der f1和Der p1及其硝化产物的检测方法 |
-
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- 2020-11-27 CN CN202310118201.0A patent/CN116539739A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110007019A (zh) * | 2019-03-29 | 2019-07-12 | 绿城农科检测技术有限公司 | 一种同时定量检测母乳中三种抗体含量的方法 |
CN111122756A (zh) * | 2019-12-04 | 2020-05-08 | 青岛海关技术中心 | 检测可食用肉的特征多肽及方法 |
CN110736802A (zh) * | 2019-12-20 | 2020-01-31 | 中国农业科学院蜜蜂研究所 | 一种液相色谱串联质谱定量检测意蜂蜜中α-葡萄糖苷酶的方法 |
CN111595971A (zh) * | 2020-05-22 | 2020-08-28 | 浙江大学 | 一种灰尘中尘螨过敏原蛋白Der f1和Der p1及其硝化产物的检测方法 |
Non-Patent Citations (3)
Title |
---|
YINGYING LI,ET AL.: "Simultaneous determination of heat stable peptides for eight animal and plant species in meat products using UPLC-MS/MS method", 《FOOD CHEMISTRY》 * |
古淑青等: "基于液相色谱-串联质谱法的肉类特征肽段鉴别及掺假测定", 《色谱》 * |
张颖颖等: "测定肉串源性成分的液相色谱-串联质谱与聚合酶链式反应方法对比研究", 《肉类研究》 * |
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