基于UPLC-QqQ-MS/MS同步检测热加工食品中典型晚期糖基化终末产物

建立超高效液相色谱-三重四极杆串联质谱法测定热加工食品中羧甲基赖氨酸（Nε-carboxymethyllysine,CML）和羧乙基赖氨酸（Nε-carboxyethyllysine,CEL）含量的同步检测方法。首先脱脂样品经硼氢化钠还原8 h,沉淀蛋白,酸水解后,经Oasis MCX固相萃取小柱净化和富集,以乙腈和含0.1%甲酸的1 mmol/L乙酸铵溶液为流动相梯度洗脱,采用ACQUITY UPLC HSS T3-C18色谱柱分离,多反应监测模式进行定性定量分析。CML和CEL在0.25～500 ng/mL范围内线性良好,相关系数高于0.999,方法检出限分别为8 ng/g和10 ng/g,定量限分别为36 ng/g和40 ng/g。平均回收率分别为96%～103%和94%～107%,相对标准偏差分别为1.48%～2.43%和1.23%～1.84%。利用该方法检测13 种市售热加工食品发现,婴儿肉松和婴儿饼干中CML和CEL含量显著高于烘焙食品和油炸食品（P＜0.05）。结果表明该方法快速、准确、高效,适用于热加工食品中CML和CEL的同步快速检测。     

基于蛋白沉淀法超高效液相色谱-串联质谱法检测面包中两种蛋白结合型晚期糖基化终末产物

目的 建立面包中两种典型晚期糖基化终末产物（AGEs）的超高效液相色谱-串联质谱检测方法。方法以结合型羧甲基赖氨酸（CML）和羧乙基赖氨酸（CEL）为检测对象，面包样品经还原孵育、蛋白沉淀、加同位素内标、酸水解、氮吹、九氟戊酸水溶液复溶后，多反应监测定性及定量分析。结果 方法的检出限CML为4.5 ng/g,CEL为0.5 ng/g；定量限CML为20 ng/g,CEL为2 ng/g;CML加标回收率为89.62%～95.65%,CEL的加标回收率为86.38%～97.17%；标准曲线线性范围为CML2.5～800 ng/mL和CEL0.25～80 ng/mL，决定系数均大于0.999。结论 该方法准确且灵敏，能够满足面包中AGEs的检测需求。     

UPLC-MS/MS分析食物煎炸过程中2种晚期糖基化终末产物的研究

建立了一种分析食用油煎炸食物中羧甲基赖氨酸(CML)和羧乙基赖氨酸(CEL)含量的超高效液相色谱-串联质谱法检测方法,并研究了煎炸温度、时间以及不同食用油对CML和CEL含量的影响。样品经硼氢化钠还原4 h后,采用HLB固相萃取小柱进行净化和富集,加标回收率达到86.0%～92.9%。CML和CEL在0.01～1.00μg/mL浓度范围内线性良好,相关系数R~2为0.999 4～0.999 5,检出限均为15.0μg/kg,定量限均为45.0μg/kg。在煎炸温度100～200℃,两者生成量随温度的升高而增加;在煎炸时间10～60 min,两者生成量随煎炸时间的延长呈现先增加后不变的趋势,在40 min时达到最大值;在比较不同食用油煎炸食物中发现,橄榄油煎炸的食物中CML和CEL含量最低。     

不同产地进口三文鱼挥发风味物质组成特征研究

目的初步探索不同产地三文鱼中挥发性风味物质种类和含量组成特征,区分三文鱼的产地。方法采用固相微萃取-气相色谱质谱检测出不同产地进口三文鱼中的挥发性风味成分,通过组成特征分析各成分在三文鱼产地差异分析中的作用。结果检出法罗群岛、挪威、澳大利亚、智利、英格兰、加拿大各含有56、51、53、53、43、43种挥发性成分。醛类化合物和碳氢类化合物是三文鱼挥发性风味成分的主要组成。智利三文鱼含有醛类相对百分含量最高,挪威三文鱼含有醛类相对百分含量最低。反式-2-癸烯醛、2,4-辛二烯醛仅在智利的三文鱼中检出。2,4-十二碳二烯醛和十七醛仅在澳大利亚三文鱼中有检出。2,3-辛二酮仅法罗群岛三文鱼中有检出。1-羟基-2-甲基-1-苯基-3-戊酮仅在澳大利亚三文鱼中检出。2,6-双(1,1-二甲基乙基)-2,5-环己二烯-1,4-二酮和5-十二烷基二氢-2(3H)-呋喃酮仅在加拿大三文鱼中检出。丁酸1-丙基戊酯仅在英格兰三文鱼中检出。2,2-二甲基癸烷仅在挪威三文鱼中被检出。正二十烷仅在挪威三文鱼中检出28.13%(匹配度93%),3-十二炔仅在法罗群岛检出5.64%(86%)。结论特征性的挥发性风味成分可作为特征指标对三文鱼产地进行区分。     

高效液相色谱-串联质谱法测定油条中羧甲基赖氨酸

建立了利用高效液相色谱-串联质谱法(HILIC-HPLC-MS/MS)测定油条中羧甲基赖氨酸(CML)含量的检测方法。样品经硼氢化钠还原、盐酸水解,过混合型阳离子交换小柱萃取净化,样品溶液在HILIC色谱柱中以甲醇-水(均含2mmol/L乙酸铵和0.1%甲酸)为流动相进行梯度洗脱,CML被保留并与样品基质分离。选用电喷雾正离子(ESI+)模式电离,多反应监测模式(MRM)进行串联质谱分析。以内标法定量,D4-CML为内标物,分析时间为12min。本方法添加回收率范围89.5%~99.1%,相对标准偏差范围2.1%~2.6%,仪器检出限为1μg/L,定量限为3μg/L。应用该方法测得15种不同来源的油条中CML含量为69.3±8.2~182.2±3.1mg/(kg蛋白质),5.3±0.5~15.4±0.3mg/(kg样品)。本方法操作简便、快速、灵敏度高、精密度好,为进一步研究食品中CML的产生机理提供了有效的检测方法。     

冻藏对原料肉加工前后羧甲基赖氨酸和羧乙基赖氨酸含量的影响

目的探究冻藏对原料肉加工前后羧甲基赖氨酸(N~ε-carboxymethyl-lysine,CML)和羧乙基赖氨酸(N~ε-carboxyethyllysine,CEL)含量的影响。方法以鸡胸肉为研究对象,使用反复冻融的方式模拟原料肉的冻藏过程,通过TBARs值与脂肪酸含量的变化评价原料肉脂肪氧化程度,同时考察原料肉与烤制后其中CML和CEL的含量。结果反复冻融会造成原料肉的汁液损失,游离氨基酸含量增加。在冻融前期(0～2 cycles)脂肪氧化程度较低, TBARs值为0.25 mg/kg,后期(4～6 cycles)程度加大,TBARs值为0.53 mg/kg。未冻融的新鲜原料肉中的CML及CEL含量均为最低,经1次冻融后CEL含量增加一倍达到(165.01±2.33)(μg/g干基),CML含量增加不显著(P0.05)。热加工后肉饼中CEL含量随原料肉冻融次数逐渐升高,于第6次冻融后达到最高值133.26±7.16(μg/g肉饼),与鲜肉对比含量增加15%,同时CML的含量增加44%。结论冻藏过程中,原料肉的反复冻融会导致脂肪氧化, CML和CEL的累积,同时会影响热加工后肉制品的品质。     

糖醋排骨中羧甲基赖氨酸分析方法的建立及其烹饪过程中动态变化

采用高效液相色谱-三重四极杆质谱联用技术建立了一种操作简单、稳定性好的检测中式菜肴糖醋排骨中羧甲基赖氨酸（Nε-(carboxymethyl)lysine,CML）的方法。样品用正己烷去脂肪,硼氢化钠还原,盐酸水解蛋白,Oasis MCX固相萃取小柱净化。以5 mmol/L的乙酸铵溶液和甲醇-水（80∶20,V/V）溶液作为流动相,采用Phenomenex Synergi 4 μ Hydro-RP 80A色谱柱,在正离子模式下采用多重反应监测分析,同位素内标法定量。该方法检出限为1.40 μg/g,加标回收率为90.94%～105.26%,相对标准偏差为5.80%～15.46%。采用该分析方法对中式菜肴糖醋排骨加工过程中CML的含量变化进行分析,结果表明,加工温度和加热时间以及糖和油脂的添加均可以显著影响糖醋排骨中CML的含量。     

超高效液相色谱-稳定性同位素稀释质谱法测定预包装食品中不同阶段美拉德反应产物含量

建立基于稳定同位素内标稀释的超高效液相色谱-串联三重四极杆质谱同步检测预包装食品中羧甲基赖氨酸(N^ε-(carboxymethyl)-lysine,CML)、羧乙基赖氨酸(N^ε-(carboxyethyl)-lysine,CEL)、甲基乙二醛-羟基咪唑酮异构体(methyglyoxal-derived hydroimidazolones,MG-H1、MG-H2、MG-H3)、乙二醛-氢咪唑酮(glyoxal-derived hydroimidazolone,G-H1)、糠氨酸7种不同阶段美拉德反应产物(Maillard reaction products,MRPs)的含量。采用Kinetex C₁₈色谱柱进行分离，多反应监测模式进行定量分析。该方法的加标回收率为88.4%～111.3%，精密度相对标准偏差小于9%，检出限和定量限分别为2.1～14.2 ng/mL和7.4～41.2 ng/mL。采用该方法对134种常见预包装食品中的7种MRPs进行定量，并对得到的数据进行主成分分析和相关性分析。结果表明，CML、MG-H1...     

山奈、白芷和香叶对猪肉加热过程中晚期糖化终末产物含量的影响

采用超高效液相色谱—串联质谱联用仪结合羧甲基赖氨酸(Nε-carboxymethyllysine,CML)和羧乙基赖氨酸(Nε-carboxyethyllysine,CEL)的同位素内标物作为AGEs的分析方法,研究了山奈、白芷和香叶3种食用香料的添加以及加热(100℃,5～120 min)对猪肉中结合态CML和CEL含量的影响。研究表明:加热对猪肉中AGEs的产生有促进作用,且AGEs的含量随着加热时间的延长而增加,经120min加热后的猪肉中CML和CEL的含量约为生肉的5～8倍,分别达到(35.60±2.20)～(40.68±5.66)mg/kg(CML)和(13.32±1.49)～(16.85±2.44)mg/kg(CEL)。在0～40min的加热时间过程中,加入或未加入香料的猪肉中CML[R~2=0.971～0.996;k=0.355～0.590mg/(kg·min)]和CEL[R~2=0.942～0.997;k=0.150～0.368mg/(kg·min)]的形成均符合零级反应动力学方程。香料的添加对于猪肉在加热过程中AGEs形成的影响因香料的种类、加热时间、肉的批次的不同而不同,总体来说,香料对猪肉加热过程中CML的形成影响较小,但是对CEL的形成有一定的促进作用,尤其是加热5 min处理的肉样,其CEL含量增加13%～61%。     

HPLC法测定婴幼儿配方乳粉中羧甲基赖氨酸

乳由于热损伤而发生美拉德反应,产生羧甲基赖氨酸(CML)。研究显示,CML与糖尿病、动脉粥样硬化、人体细胞老化等均呈正相关。本文建立了HPLC方法对婴幼儿乳粉中羧甲基赖氨酸的测定方法。样品经预处理,采用高效液相色谱仪(HPLC)进行测定,色谱柱为C18柱,柱温为35℃,流动相为甲醇和醋酸钠缓冲液(p H 6.50,0.048 mol/L)(96∶4,体积比),流速为0.8 m L/min,荧光检测器,激发波长340 nm,发射波长455 nm,外标法定量。结果显示,CML在0.2μg/m L~35.0μg/m L范围内呈良好线性关系,相关系数(r)为0.999 1。最低检出浓度为0.16μg/m L。选择市售2种品牌奶粉进行加标回收试验,回收率分别为92%和107%。     

Simultaneous determination of N ε-(carboxymethyl) lysine and N ε-(carboxyethyl) lysine in cereal foods by LC–MS/MS

N ^ε-(carboxymethyl) lysine (CML) and N ^ε-(carboxyethyl) lysine (CEL) are two advanced glycation end products. Few studies have focused on the simultaneous determination of CML and CEL content in foods, especially in Eastern foods. In this study, a stable isotope dilution LC–MS/MS method was developed for the simultaneous determination of CML and CEL in foods. The CML and CEL contents in three cereal foods consumed in China were determined by the developed method. Sample preparation consisted of lyophilization, defatting, grinding, reduction, protein precipitation, acid hydrolysis, and solid-phase extraction. The limit of quantification for CML and CEL was 4 and 3 ng/g, respectively. CML and CEL content in fried dough stick was determined for the first time. CML and CEL contents in fried dough sticks were 28.06–66.69 and 10.67–30.22 μg/g of fried dough sticks, respectively. The highest CML and CEL contents in biscuits were 117.53 and 46.09 μg/g of biscuits, respectively. CML and CEL contents in bread crusts were higher than those in bread crumbs.     

Simultaneous Analysis of Nε‐(Carboxymethyl)Lysine and Nε‐(Carboxyethyl)Lysine in Foods by Ultra‐Performance Liquid Chromatography‐Mass Spectrometry with Derivatization by 9‐Fluorenylmethyl Chloroformate

Isotope dilution ultra‐performance liquid chromatography–electrospray tandem mass spectrometry with derivatization by 9‐fluorenylmethyl chloroformate was successfully applied to quantify N^ε‐(carboxymethyl)lysine (CML) and N^ε‐(carboxyethyl)lysine (CEL) in processed foods. We demonstrate that this analytical method is well validated for the determination of CML and CEL contents in processed foods. Relative standard deviations (RSD) indicate repeatability (RSD < 6% for CML and CEL) and reproducibility (RSD < 6% for CML and < 7% for CEL) in this method. Percent recovery is also good. We obtain recoveries of 102% to 112% for CML and 86% to 114% for CEL. CML levels detected in the samples vary from 2.29 to 480 mg/kg food, whereas CEL is detected in significantly lower concentrations ranging from 0.56 to 107 mg/kg food. These data could help consumers make better food choices by monitoring intake of advanced glycation end‐products, which may pose a risk to human health.     

Automated determination of oxytetracycline residues in muscle, liver, milk and egg by on-line dialysis and post-column reaction detection HPLC.

An automated method for control of oxytetracycline (OTC) residues in chicken and bovine muscle, salmon liver, bovine milk and hen egg has been developed. Tissue homogenate, decreamed milk or whole egg solution was dialysed and the dialysate enriched on a small polystyrene column on-line to HPLC. OTC and the internal standard (tetracycline) were separated on a polystyrene column by ion-pair chromatography. The column effluent was mixed with sodium hydroxide and irradiated at 366 nm. Monitoring the resulting derivatives with a fluorescence detector (excitation: 358 nm, emission: 460 nm), OTC could be detected at 1 ng/ml in milk, 1 ng/g in egg, 3-4 ng/g in muscle and 8 ng/g in liver. Relative standard deviations at 50 and 200 ng/g (milk: 20 and 100 ng/ml) ranged from 1.6 to 3.1%.     

不同产地三文鱼的稳定同位素指纹特征及原产地溯源

本研究从深圳口岸获取我国三文鱼主要进口国（法罗群岛、挪威、智利、加拿大以及澳大利亚）的三文鱼样品共16?份,并采集中国产三文鱼（虹鳟）样品2?份,分析三文鱼肌肉、表皮、鳞片以及骨骼中的碳、氮、氢、氧、硫稳定同位素比值,比较不同产地以及不同部位同位素分布差异,采用判别分析对不同产地进行判别。三文鱼的碳、硫以及氢、氧稳定同位素具有显著的产地差异。不同组织之间同位素呈现明显的同位素分馏效应。鳞片以及表皮同位素比值对产地的指示效果优于肌肉和骨骼,结果表明采用稳定同位素能完全将以上产地的样品区分开,且还能将中国的虹鳟与进口的三文鱼进行区分。对市场随机购买的6?份三文鱼样品的产地鉴别结果表明,稳定同位素技术能有效鉴别市场中三文鱼的产地造假行为,可用于对市场上三文鱼的产地追溯,以期为我国食品安全提供技术保障。     

金属有机骨架基质固相分散高效液相色谱法检测食用油中苯并[a]芘

目的建立基于金属有机骨架材料(metal-organic frameworks, MOFs) MIL-101(Cr)的基质固相分散进行食用油样品前处理,并以高效液相色谱串联荧光检测器(high performance liquid chromatography-fluorescence detector, HPLC-FLD)对苯并[a]芘进行定量检测。方法吸附后将体系装填于6 mL固相萃取空柱,以丙酮洗脱,氮气吹干定容。色谱流动相为乙腈-水(80:20, V:V),流速0.3 mL/min,激发波长369 nm,发射波长404 nm,外标法定量。结果苯并[a]芘在1～50ng/g范围内呈良好线性,相关系数r~2为0.9997,加标回收率为96.0%～99.9%。方法检测限及定量限分别为0.33ng/g和1.09ng/g,实际样品检测的相对标准偏差为2.8%～8.9%。结论该方法操作简单,测定结果准确,同时克服了常规固相萃取法易堵塞、渗漏、过载及负压过高等问题,可用于食用油中苯并[a]芘的检测。     

Effects of oxidised linoleic acid on the formation of Nε‐carboxymethyl‐lysine and Nε‐carboxyethyl‐lysine in Maillard reaction system

This study investigated the effects of oxidised linoleic acid (18:2) on N^ε‐carboxymethyl‐lysine (CML) and N^ε‐carboxyethyl‐lysine (CEL) formation in Maillard reaction systems. Model systems of lysine/glucose (L/G), lysine/18:2 (L/18:2), lysine/18:2/glucose (L/18:2/G), myofibrillar protein/glucose (MFP/G), MFP/18:2 and MFP/18:2/G were maintained at 37 °C for 6 weeks. The results showed that CML/CEL contents in L/G (6.99 and 0.96 mmol mol^?1 lysine, respectively) were significantly higher than those in L/18:2/G (1.43 and 0.41 mmol mol^?1 lysine, respectively), and there is a small amount of CML/CEL generation in L/18:2. However, the CML/CEL levels in MFP/G (197.2 and 83.8 ng mg^?1 protein, respectively) were markedly lower than those in MFP/18:2/G (283.2 and 118.5 ng mg^?1 protein, respectively). 18:2 favours the formation of CML/CEL in MFP/18:2/G, not in L/18:2/G. All these findings indicated that the role of 18:2 on CML/CEL formation in Maillard reaction system was complex, and depended on CML/CEL formation rate and substrate types (lysine or lysine residue in protein).     

超高效液相色谱-串联质谱法测定牛乳中6种兽药残留

目的:建立高效液相色谱-串联质谱法（high performance liquid chromatography-tandem mass spectrometry,HPLC-MS/MS）测定牛奶样品中6种兽药残留（硝碘酚腈、碘醚柳胺、氯氰碘柳胺、托曲珠利、三氯苯达唑、水杨酸钠）的检测方法。方法:样品经过乙腈溶液提取,以MAX固相萃取柱净化,采用Waters X Bridge BEH-C₁₈色谱柱分离,流动相以乙腈和0.1%甲酸水溶液梯度洗脱,并使用电喷雾离子源,正负离子切换扫描模式进行检测,外标法定量。结果:6种兽药在0~10 ng/mL范围内呈现良好的线性关系,决定系数（R2）均大于0.995。6种兽药方法的检出限为0.06~0.18 μg/kg,方法的定量限为0.5~2.0 μg/kg。在添加量为0.5~8.0 μg/kg的加标回收实验下,6种兽药加标回收率为67.1%~105.5%,相对标准偏差均小于10%。结论:该方法前处理操作简便,分析速度快,灵敏度高,可用于牛奶样品中的兽药残留测定。