Assessment of meat authenticity using bioinformatics,targeted peptide biomarkers and high-resolution mass spectrometry

In recent years a significant increase of food fraud has been observed, ranging from false label claims to the use of additives and fillers to increase profitability. Recently in 2013 horse and pig DNAs were detected in beef products sold from several retailers. Mass spectrometry (MS) has become the workhorse in protein research, and the detection of marker proteins could serve for both animal species and tissue authentication. Meat species authenticity is performed in this paper using a well-defined proteogenomic annotation, carefully chosen surrogate tryptic peptides and analysis using a hybrid quadrupole-Orbitrap MS. Selected mammalian meat samples were homogenised and proteins were extracted and digested with trypsin. The samples were analysed using a high-resolution MS. Chromatography was achieved using a 30-min linear gradient along with a BioBasic C8 100 × 1 mm column at a flow rate of 75 µl min^–1. The MS was operated in full-scan high resolution and accurate mass. MS/MS spectra were collected for selected proteotypic peptides. Muscular proteins were methodically analysed in silico in order to generate tryptic peptide mass lists and theoretical MS/MS spectra. Following a comprehensive bottom-up proteomic analysis, we detected and identified a proteotypic myoglobin tryptic peptide (120–134) for each species with observed m/z below 1.3 ppm compared with theoretical values. Moreover, proteotypic peptides from myosin-1, myosin-2 and β-haemoglobin were also identified. This targeted method allowed comprehensive meat speciation down to 1% (w/w) of undesired product.     

液相色谱/元素分析仪-同位素质谱法鉴定蜂蜜 掺假的方法改进

目的改进液相色谱/元素分析仪-同位素质谱法鉴别蜂蜜掺假的方法。方法对现有欧盟标准方法优化液相色谱条件,结合元素分析仪-同位素质谱法,将二糖分离为麦芽糖、蔗糖,提出一个新的参数—麦芽糖、蔗糖δ~(13)C值之差δ~(13)C_(M-S)。结果根据本研究检测113个国内外不同来源纯正蜂蜜样本的数据,提出纯正蜂蜜δ~(13)C值新要求:蜂蜜蛋白质与蜂蜜同位素差值δ~(13)C_(P-H)大于-0.97‰;果糖、葡萄糖δ~(13)C值之差δ~(13)C_(F-G)在-0.60‰至0.56‰范围内;麦芽糖、蔗糖δ~(13)C值之差δ~(13)C_(M-S)在-0.73‰至0.98‰范围内;各个组分δ~(13)C最大差值δ~(13)C_(max)小于2.05‰;根据上述4个参数来确认蜂蜜是否掺假。在日常检测和市场销售的160个样品中,原方法阳性检出率为16.2%,而新方法阳性检出率达21.9%。结论本研究提升了蜂蜜掺假检测能力,此方法的建立更好、更精确打击掺假的同时,也维护消费者权益。     

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration by digital PCR

The alarming problem of meat adulteration emphasises the demand for accessible analytical approaches for food regulatory agencies to detect and, specially, to measure altered meat fractions. This study proposes a novel cross-species triplex droplet digital polymerase chain reaction (ddPCR) assay to simultaneously identify and quantify the ratios of pork/beef meat fractions from a total DNA content, including processed and autoclaved meat, without requiring a standard, achieving high sensitivity with a limit of quantification estimated at 0.1% (w/w) and a limit of detection down to 0.01% (w/w). A single copy nuclear gene, β-actin, was employed as a target, accompanied with myostatin gene as a cross-species target to quantify the meat background. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and time.     

A simple sample preparation for simultaneous determination of chloramphenicol and its succinate esters in food products using high-performance liquid chromatography/high-resolution mass spectrometry

ABSTRACT

A simple method is described for the determination of chloramphenicol and its succinate esters in food products. Examination of food products using high-performance liquid chromatography/high-resolution mass spectrometry showed the presence not only of chloramphenicol but also of its succinate forms. A scheme is proposed for determining chloramphenicol and its succinate esters (calculated as chloramphenicol) in meat (beef, pork, poultry), milk, liver, kidney, eggs, fish and honey. Analytes are extracted from a 1.0 g sample with 5 ml acetonitrile. It was found that using the method of standard addition and diluting the extract with water leads to the elimination of matrix effects and also eliminates errors associated with peak splitting due to the separate elution of the differing forms of the analyte. Validation results were satisfactory, with recoveries from 85% to 111% (meat, milk, liver, kidney, eggs, fish and honey) and a relative standard deviation (RSD) lower than 13% for spiked levels of 0.3, 1.0 and 5 µg kg^–1. The limits of detection and quantification (calculated as chloramphenicol for all forms) were 0.1 and 0.3 µg kg^–1, respectively. The RSD of the results of the analysis was < 10%. The duration of the analysis was less than 1 h.     

液相色谱质谱技术在食品掺假中的应用

食品欺诈是食品安全风险防控领域中的重要方面, 其中食品掺假作为食品欺诈的一种类型, 因其掺假物的种类性质、方式等不同往往可能带来或者引入食品安全问题。针对全球性食品掺假行为, 本文综述了近年来液相色谱-质谱联用技术用于解决肉制品、牛奶、食用植物油、蜂蜜4类食品掺假问题, 总结了该4类食品的主要掺假行为, 其中肉制品掺假分为地理来源改变、肉种类替代、添加剂非法使用、过敏原引入、病死畜禽肉; 乳制品掺假主要有奶源产地改变、不同动物奶、添加富氮化合物、添加剂违规; 植物油掺假主要有油种类混合、掺入不可食用油、植物油产地改变、添加工业染料; 蜂蜜常见的掺假方式有蜂蜜产地、植物源、添加糖浆。对液相色谱-质谱鉴别食品真伪的前处理方法(液液萃取、固相萃取)也进行了总结, 以期为液相色谱-质谱联用技术在食品真实性技术鉴别应用提供有益参考。     

超高效液相色谱-串联质谱法检测生肉中的阿托品和山莨菪碱

目的建立超高效液相色谱串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)同时检测生肉中的阿托品和山莨菪碱的残留量。方法生肉样品加水均质后经乙腈超声提取,采用正己烷除油,旋干后定容上机检测。实验中采用了Agilent Eclipse Plus C_18色谱柱,以0.1%甲酸水溶液-甲醇为流动相,进行梯度洗脱,流速为0.3 m L/min,柱温40℃。结果阿托品和山莨菪碱在0.5~20 ng/m L的质量浓度范围内线性关系良好,相关系数均在0.999以上。分别对其进行1.0、5.0、10 ng/m L不同浓度的加标实验,回收率介于70.77%~89.09%。阿托品和山莨菪碱的方法检出限分别为0.056、0.025μg/kg,定量限分别为0.072、0.030μg/kg。结论该实验方法简单、快速,回收率较好,方法检出限较低,适用于快速检测生肉样品中阿托品和山莨菪碱的残留量。     

液相色谱—串联质谱同时检测肉类中11种鼠药残留

目的:建立同时测定肉类中11种鼠药残留的液相色谱—串联质谱法.方法:样品经乙腈提取,QuEChERs萃取盐包净化,采用液相色谱—串联质谱仪对肉类中11种鼠药残留进行定性定量分析.流动相为甲醇-0.05 mol/L乙酸铵水溶液,采用电喷雾电离,正离子扫描及多反应监测(MRM)模式,采用外标法进行定量分析.结果:标准溶液在...     

气相色谱-质谱法测定腌制肉类食品中的多种脂肪酸

建立了腌制肉类食品中多种脂肪酸的气相色谱-质谱检测方法。样品采用ASE快速溶剂萃取仪提取、1%硫酸-甲醇溶液甲酯化,采用气相色谱质谱测定。样品添加回收率为80.2%⁹6.2%,RSD为2.4%⁶.8%;32种脂肪酸甲酯在0.010⁰.10mg/mL质量浓度范围内良好的线性关系,相关系数为0.9915⁰.9990;检出限为0.02⁰.14μg/kg,定量限为0.06⁰.46μg/kg;方法具有快速、准确、灵敏度高的特点,适用于腌制肉类食品中多种脂肪酸的检测分析。      

超高效液相色谱-高分辨质谱组学技术鉴别咖啡掺假

目的 采用超高效液相色谱-高分辨质谱法(ultra performance liquid chromatography-high resolution mass spectrometry,UPLC-HRMS)对咖啡及其掺假物建立高分辨质谱数据库,建立一种基于组学技术对咖啡掺假进行鉴别的方法。方法 咖啡及其掺假物经甲醇-水(7:3,V:V)超声提取后,经BEH C18色谱柱(2.1 mm×100 mm,1.7μm)分离,电喷雾电离(electrospray ionization,ESI)离子源,在高分辨质谱Full MS/dd MS2扫描模式下采集数据,建立咖啡及掺假物黑玉米、黑豆、大麦的高分辨质谱数据。采用Compound Discoverer 3.3 (CD 3.3)组学软件对UPLC-HRMS数据进行主成分分析(principal component analysis,PCA)和火山图差异分析,根据咖啡及掺假物中差异化合物的精准分子质量数、母离子同位素组成及碎片离子信息质量信息检索mzCloud和Chem Spider在线数据库,对差异成分进行匹配鉴定。结果 PCA结果表明咖啡与掺...     

Analysis and comparison of key proteins in Maiwa yak and bovine milk using high-performance liquid chromatography mass spectrometry

Yak milk is an essential and predominant food resource for Tibetan people for subsistence purposes and to combat altitude-induced challenges. Due to its unique qualities, yak milk has recently been gaining broader attention from consumers across China as well in other parts of the world. One of the key characteristics of yak milk is the protein content, which is about 40 to 60% higher than that of native bovine milk. In this work, a sensitive and reproducible high-throughput analytical method was developed employing both ultra high-performance liquid chromatography Orbitrap (Thermo Fisher Scientific) high-resolution accurate mass spectroscopy (UHPLC-HRAM-MS) and UHPLC coupled with triple quadrupole tandem MS (UHPLC-QqQ-MS) to simultaneously analyze 8 milk proteins. A total of 15 Maiwa yak milk samples and 15 bovine milk samples were qualitatively and quantitatively analyzed using targeted proteomics and compared for α-lactalbumin, β-lactoglobulin, α_S1-casein, α_S2-casein, β-casein, κ-casein, lactoferrin, and osteopontin. Peptides of β-lactoglobulin were used to specifically distinguish yak and bovine milk. The results showed that this novel detection method could quantitatively detect these major and minor milk proteins with >0.99 linear correlation coefficient and a recovery rate between 90 and 120%, with relative standard deviations typically less than 10%. The data revealed that yak milk not only had higher overall milk protein content than bovine milk but higher lactoferrin and osteopontin contents as well. The lactoferrin content of yak milk was about 30% higher than that of bovine milk, and the osteopontin content of yak milk was nearly twice that of bovine milk. The application of this method demonstrates that UHPLC-HRAM-MS and UHPLC-QqQ-MS are suitable for high-throughput qualitative and quantitative analysis of major and minor proteins of yak and bovine milk.     

元素分析/液相色谱-碳同位素比值质谱法在纯正 苹果汁掺假鉴别中的应用

目的建立一种鉴别检测苹果汁掺假情况的元素分析/液相色谱-同位素比值质谱法(elemental analysis/liquid chromatography-isotope ratio mass spectrometry,EA/LC-IRMS)。方法通过对不同产区不同种类118个纯正苹果汁的糖、有机酸、果糖、葡萄糖、二糖、寡糖碳同位素比值(δ~(13)C值)的测定,建立纯正苹果汁同位素数据库进而提出纯正苹果汁应满足的δ~(13)C值要求。结果有机酸和糖差值Δδ~(13)CO-S在-1.38‰至1.09‰范围内,果糖和葡萄糖差值Δδ~(13)CF-G在-0.70‰至0.57‰范围内,而各组分最大差值Δδ~(13)Cmax4.36‰。对市售105个苹果汁进行检测,采用本方法检出32个阳性样品,而采用本实验室的糖浆标志物法仅检出12个阳性样品。结论本方法大大提高了苹果汁的掺假鉴别,有很大的实际应用潜力。     

超高效液相色谱-串联质谱法检测预包装畜禽肉制品中多种兽药残留

目的 建立预包装畜禽肉制品中54种兽药残留的超高效液相色谱-串联质谱检测方法。方法 样品经甲酸乙腈提取,Oasis PRIME HLB固相萃取柱净化,经Waters Acquity UPLC BEH C18色谱柱分离,以甲醇和0.1％甲酸水溶液为流动相进行梯度洗脱,电喷雾正离子(ESI+)模式电离,MRM-IDA-EPI扫描模式检测。结果 经方法学验证,54种化合物在0.5ng/mL～50 ng/mL范围内均呈现良好的线性关系,相关系数均?0.990,检出限0.5 μg/kg ～10 μg/kg,回收率为76.3%～92.4%,相对标准偏差为3.1%～15.2%。结论 本方法操作简便,回收率高。适用于预包装畜禽肉制品中54种兽药残留的同时定性、定量分析及确证。     

Determination of phospholipid molecular species in pork meat by high performance liquid chromatography-tandem mass spectrometry and evaporative light scattering detection

Normal phase high performance liquid chromatography has been optimized for both evaporative light scattering detection and tandem mass spectrometry in order to characterize the natural phospholipids (PL) (classes and molecular species) of raw and cooked pork meat. The PL fraction included phosphatidylcholine (PC) (42.9%±4.5 for raw vs 42.6%±8.0 for cooked meat), plasmalogen-phosphatidylethanolamine (pPE) and phosphatidylethanolamine (PE) (26.7%±3.1 vs 28.5%±2.3), cardiolipin (CL) (8.3%±2.9 vs 6.3%±0.7), sphingomyelin (Sph) (7.5%±0.9 vs 8.3%±2.1), phosphatidylinositol (PI) (6.8%±0.7 vs 6.5%±2.1) phosphatidylserine (PS) (4.9%±0.5 vs 4.6%±1.4) and lysophosphatidylcholine (LPC) (2.9%±1.3 vs 3.3%±2.6). Arachidonic acid (absent in Sph) was mainly present in pPE and PI and formed molecular species with a saturated fatty acid, such as stearic (as in PI, PS, PE and PC) or palmitic acid (as in PE and PC), or the respective vinyl ethers in pPE (p18:0 and p16:0); however, in PC, arachidonic acid also formed combinations with oleic and linoleic acid. Palmitic acid formed the most abundant molecular species in PC, but not in CL, PE, PI and PS. Unexpectedly, the cooked pork meat showed an increased content of the molecular species of PI and LPC with more unsaturated fatty acids (18:0/20:4 and 18:2, respectively) with respect to raw meat.     

QuEChERS-气相色谱-串联质谱法测定熟肉制品中9种N-亚硝胺类化合物

目的建立同时测定熟肉制品中9种N-亚硝胺类化合物的气相色谱-串联质谱分析方法。方法样品采用快速基质分散净化(QuEChERS)法处理后,用HP-INNOWax石英毛细管色谱柱(30 m×0.25 mm,0.25μm)分离,经气相色谱-串联质谱多反应监测技术进行定性及定量分析。结果 9种N-亚硝胺类化合物在0.20～50.00μg/kg范围内线性关系良好,相关系数均0.997,方法检出限为0.02～0.1μg/kg。3个加标水平的平均回收率为78%～120%,相对标准偏差为3.42%～15.24%。结论本方法准确、快速、灵敏度高,可用于熟肉制品中9种N-亚硝胺类化合物的检测。     

高效液相色谱-串联质谱法测定食品、保健食品中一种新型他达拉非衍生物

目的 建立高效液相色谱－串联质谱法测定食品、保健食品中一种新型的非法添加物3-羟基丙基去甲他达拉非的分析方法。方法 样品采用甲醇超声提取,以Agilent Eclipse Plμs C18(2.1×100 mm, 1.8 μm) 色谱柱分离待测物,以0.1%甲酸溶液-甲醇为流动相,梯度洗脱,流速为0.4 mL/min;在电喷雾正离子化模式下,采用多反应监测(MRM)模式检测,标准曲线进行外标法定量。结果 3-羟基丙基去甲他达拉非在2-50 ug/mL 范围内线性良好,相关系数R2≥0.999,方法检出限和定量限分别为50 ug/kg和100 ug/kg, 回收率为74.58%~117.97%, RSD≤5.0%。结论 该方法简单、准确、高效、专属性强,适用于食品和保健食品中非法添加3-羟基丙基去甲他达拉非的定性和定量检测。     

超高效液相色谱-三重四极杆质谱法测定保健食品中28种降糖类非法添加化合物

目的建立超高效液相色谱-三重四极杆质谱法测定保健食品中28种降糖类非法添加化合物的分析方法。方法采用Merck SeQuant ZIC-HILIC色谱柱(100 mm×2.1 mm,3.5μm),以乙腈-含10 mmol/L甲酸铵的0.1%甲酸水溶液为流动相梯度洗脱,分离二甲双胍等3种化合物;采用Waters CORTECS T3色谱柱(100 mm×2.1 mm,2.7μm),以乙腈-0.1%甲酸水溶液为流动相梯度洗脱,分离苯乙双胍等25种化合物,在电喷雾离子化正离子或负离子模式下,以多反应监测方式(multiple reaction monitoring,MRM)检测。结果二甲双胍等3种化合物在进样浓度为50~1000μg/L范围内线性关系良好,妥拉磺脲等5种化合物在25~500μg/L范围内线性关系良好,其余20种化合物在5~100μg/L范围内线性关系良好,相关系数(r)均大于0.99。各化合物在定量限(limit of quantity,LOQ)1倍、2倍、10倍添加水平下的平均加标回收率为48.6%~134.4%,相对标准偏差(relative standard deviation,RSD)为0.1%~8.4%(n=6)。二甲双胍等3种化合物的定量限为500μg/kg,妥拉磺脲等5种化合物的定量限为250μg/kg,苯乙双胍等20种化合物的定量限为50μg/kg。结论本方法灵敏、高效,可用于测定保健食品中降糖类非法添加化合物含量。     

色谱质谱技术在食品安全分析检测中的应用

食品安全是关系国计民生的重要议题。准确、深入、高效的食品安全分析检测技术是防控食品安全事件发生及处理相关贸易纠纷、立法追责的基础和保障。色谱质谱技术作为高效的分离和检测手段是目前食品安全分析领域最重要、最主流的技术手段。近年来,色谱质谱领域的新发展也推动了食品安全分析检测向更快速,更有效,更可靠,更安全的目标迈进,本综述就其中最重要并有望代表未来食品安全分析发展趋势的色谱质谱技术做了总结和点评。如用于食品样品预处理的固相微萃取技术;气相色谱及气相色谱质谱联用技术中的快速气相色谱方法及其与质谱联用方法,二维气相色谱方法及其与质谱联用方法;液相色谱及液相色谱质谱联用技术中的超高效液相色谱及其与质谱联用方法,毛细管液相色谱和纳流液相色谱及其与质谱联用方法,超临界色谱及其与质谱联用方法,二维液相色谱及其与质谱联用方法;质谱分析技术中的超高分辨率质谱方法,常压敞开式离子源质谱技术等。     

元素分析-同位素质谱法鉴定麦卢卡蜂蜜中碳-4植物糖掺假

目的建立元素分析-同位素质谱法(elementanalyzerstableisotopeproportionalmassspectrometry,EA-IRMS)鉴定麦卢卡蜂蜜中碳-4植物糖掺假的新方法。方法样品经前处理后,利用元素分析-同位素质谱仪测定蜂蜜的碳同位素比值δ~(13)C_H、蜂蜜中总蛋白质的碳同位素比值δ~(13)C_P、蜂蜜中花粉的碳同位素比值δ~(13)C_F。用麦卢卡蜂蜜中δ~(13)C值更为稳定的δ~(13)C_F值为标准,δ~(13)C_H值与其进行比较,参照GB/T 18932.1-2002《蜂蜜中碳-4植物糖含量测定方法稳定碳同位素比率法》计算方式,来建立新的计算麦卢卡蜂蜜中碳-4植物糖含量X(%)的计算方式。结果对纯正麦卢卡蜂蜜提出:0X(%)7,δ~(13)C_Fδ~(13)C_Pδ~(13)C_H。新方法利用测定麦卢卡蜂蜜中δ~(13)C值更为稳定的δ~(13)C_F值,代替了原有的比较不稳定的δ~(13)C_P为标准,解决了原方法对于麦卢卡蜂蜜中碳-4植物糖检测的假阳性问题。结论本方法显示出较好的准确度,大大提高了掺假检测的能力。     

网销市场中驴肉真伪及掺假分析

目的为了保障食品安全和维护消费者合法权益,对网销市场中驴肉(鲜肉和制品)真伪及掺假情况进行分析。方法本研究根据现行行业标准SN/T 3730.4-2013和SN/T 3730.5-2013,分析市场中鲜驴肉(9份)和加工驴肉制品(18份)中的驴源性及马源性。结果 9份鲜肉样品中有4份为驴肉,剩余5份为马肉;加工肉制品中,7份含有驴源性成分,5份含有马源性成分,剩余6份既含有驴源性成分又含有马源性成分。结论市场中存在利用马肉冒充驴肉的欺诈现象,相关部门应加强监管,避免欧洲"马肉风波"在中国上演。     

超高效液相色谱-串联质谱法测定猪肉中巴氯芬的残留

目的建立超高效液相色谱-串联质谱法测定猪肉中巴氯芬残留量的方法。方法样品采用0.1moL/L盐酸-甲醇溶液(20:80,V:V)提取,混合型强阳离子交换柱(MCX)净化,经C_(18)反相色谱柱分离后,正离子扫描,多反应监测模式检测巴氯芬,基质标曲定量。结果该方法在1～500 ng/mL浓度范围内线性良好, r~2=1,检出限为1μg/kg,定量限为5μg/kg,回收率为75.28%～77.86%,精密度RSD10%。结论该方法灵敏度高、特异性强,适用于猪肉中巴氯芬残留量的分析。