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。     

三文鱼中晚期糖基化终末产物含量测定及产地特征因子分析

建立超高效液相色谱-串联三重四极杆质谱同时检测三文鱼中羧甲基赖氨酸（N-carboxymethyllysine,CML）和羧乙基赖氨酸（N-carboxyethyllysine,CEL）的分析方法。色谱柱为ACQUITY UPLC HSS T3柱（2.1?mm×150?mm,1.8?μm）,流动相A为100%乙腈、B为0.1%甲酸,电喷雾离子源正离子模式扫描,多反应监测模式定性分析,同位素内标稀释法定量;样品经脱脂、还原、水解和Oasis?MCX固相萃取柱净化等步骤后上机分析;方法平均回收率为90%～95%,精密度相对标准偏差小于5%,CEL检出限为0.032?7?ng/g,定量限为0.142?2?ng/g。采用该法对来自挪威、智利和法罗群岛的21?份三文鱼样品进行含量测定,并对所获数据进行主成分分析。结果表明,即使是相同品种的三文鱼在不同国家和不同个体之间CML和CEL含量存在很大的生物差异性,并且发现CEL有望成为鉴别挪威三文鱼和智利、法罗群岛三文鱼的产地特征因子。     

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...     

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。     

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。     

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%。     

市售鱼加工食品中3 种晚期糖基化终末产物分析及与组分的相关性

分析市售烘烤类、油炸类、干制类、罐头类等鱼加工食品中羧甲基赖氨酸（Nε-carboxymethyllysine,CML）、羧乙基赖氨酸（Nε-carboxyethyllysine,CEL）、甲基乙二醛氢咪唑酮（Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine,MG-H1）3 种晚期糖基化终末产物（advanced glycation end products,AGEs）含量及与组分的相关性。使用ACQUITY UPLC BEH Amide色谱柱建立超高效液相色谱-串联质谱检测AGEs的方法,应用该方法分析65 种市售鱼加工食品AGEs含量。结果表明,鱼加工食品中MG-H1含量高于CEL和CML含量,MG-H1含量为CEL含量的2～11 倍、CML含量的2～20 倍;罐头类鱼加工食品中AGEs含量呈较高水平;油炸类鱼加工食品中乙二醛、甲基乙二醛（methylglyoxal,MGO）含量相对较高。通过主成分分析与相关性分析发现,鱼加工食品中3 种AGEs含量间呈显著正相关（r≥0.54,P＜0.001）;其AGEs含量与脂肪含量呈显著正相关,与蛋白含量呈显著负相关;烘烤类、干制类鱼加工食品中MG-H1含量与MGO含量呈正相关。     

基于实时质谱快速监控牦牛肉水煮过程中羧甲基赖氨酸和羧乙基赖氨酸的生成

建立了基于实时直接分析质谱(direct analysis in real time mass spectrometry,DART-MS)快速测定牦牛肉中羧甲基赖氨酸[Nε-(carboxymethyl)lysine,CML]、羧乙基赖氨酸[Nε-(carboxyethyl)lysine,CEL]含量的方法,并探究了苦...     

紫苏叶提取物对美拉德体系晚期糖基化终末产物生成的抑制作用研究

目的 探究紫苏叶提取物（perilla leaf extract, PLE）对美拉德体系晚期糖基化终末产物（Advanced glycation end-products, AGEs）的抑制作用。方法 本研究通过构建葡萄糖-赖氨酸模型体系，利用高效液相色谱-串联质谱法探究其在加热过程中PLE对Nε-羧甲基赖氨酸（Nε-(1-Carboxymethyl)-L-lysine, CML）、Nε-羧乙基赖氨酸（Nε-(1-Carboxyethyl)-L-lysine, CEL）和吡咯素（Pyrraline, Pyr）三种AGEs的抑制作用，同时通过评价PLE的抗氧化性、测定PLE对α-二羰基化合物抑制率，以探讨PLE 对AGEs的可能抑制途径。结果 PLE对AGEs的生成具有显著的抑制作用，其抑制率与PLE的添加浓度成正比，当添加量为2.0 mg/mL时，对CML、CEL和Pyr的抑制率分别为47.23%、29.59%和37.77%。HPLC显示紫苏叶提取物中的主要活性物质为迷迭香酸、芹菜素-7-O-二葡萄糖苷和野黄芩苷等，推测其抑制途径可能是由于PLE的抗氧化性和清除二羰基化合物的共同作用。结论 PLE对AGEs有抑制作用，对食品安全和人体健康具有重要意义。     

酱油生产过程中游离态羧甲基赖氨酸和5-羟甲基糠醛的同步检测

为快速检测酱油生产过程中2种危害物羧甲基赖氨酸(CML)和5-羟甲基糠醛(5-HMF)的含量变化,采用高效液相色谱质谱联用(HPLC-MS/MS)技术建立了一种同步检测游离态CML和5-HMF的方法。通过质谱参数、色谱条件和前处理的优化,CML和5-HMF的回收率可达98%以上,样品中CML和5-HMF的检测限(LOD)分别为0.3 ng/mL和6.0 ng/mL,定量限(LOQ)分别为1.0 ng/mL和20.0 ng/mL。对酱油生产过程中各工艺工段的样品进行检测,发现发酵原料大曲中CML含量较高,而豆粕的蒸煮过程和老抽酱油的调配是使5-HMF含量增高的关键生产环节。     

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.     

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).     

色氨酸抑制体外模型中晚期糖基化终末产物形成机理

食源性晚期糖基化终末产物(advanced glycation end products,AGEs)与多种慢性疾病密切相关,尤其是糖尿病和肾脏疾病.为减少AGEs对机体的健康风险,降低食品中AGEs含量,本研究从13种氨基酸中筛选出具有良好AGEs抑制效果的色氨酸(tryptophan,Trp),利用牛血清白蛋白(bo...     

单糖的类型对模拟体系中晚期糖基化终末产物生成的影响

为了研究单糖-赖氨酸模拟体系中单糖类型对晚期糖基化终末产物(AGEs)生成的影响。探讨了葡萄糖、半乳糖、果糖、山梨糖在120 ℃加热0~120 min期间pH、反应颜色、底物消耗、AGEs中间体包括3种二羰基化合物乙二醛(GO)、丙酮醛(MGO)和三脱氧葡萄糖醛酮(3-DG)以及果糖基赖氨酸(FL),以及最终三种AGEs包括羧甲基赖氨酸(CML)、羧乙基赖氨酸(CEL)、吡咯素(Pyr)的变化。结果显示随着反应进行,醛糖体系的pH下降程度以及颜色增加程度高于酮糖体系。反应结束时,葡萄糖-赖氨酸(Glu-Lys)、半乳糖-赖氨酸(Gla-Lys)、果糖-赖氨酸(Fru-Lys)和山梨糖-赖氨酸(Glu-Lys)体系中赖氨酸的损失分别为26.65%、28.95%、11.47%、14.90%;而所有体系中单糖在反应结束时的损失均达到了75.00%以上,要远高于赖氨酸。模拟体系中单糖会出现醛酮转换,但比例低于10%。所有体系中GO和3-DG的含量随着时间的延长而下降,这表明体系中GO和3-DG在反应前20 min内就已大量生成。α-二羰基化合物的生成量远高于FL的生成量。反应结束时,酮糖体系的CML和CEL含量要高于醛糖体系。所有体系中,三种AGEs生成量均为CEL > CML > Pyr。本文表明将来研究食品中AGEs的生成和抑制途径,需重点从中间体途径着手,尤其是α-二羰基化合物。     

Simultaneous analysis of Nε-(carboxymethyl)lysine,reducing sugars,and lysine during the dairy thermal process

A new analytical method allowing the simultaneous quantification of Nε-(carboxymethyl)lysine (CML), lysine, and reducing sugars (glucose, lactose, and galactose) is described. It is based on high performance anion-exchange chromatography with pulsed amperometric electrochemical detection. This method demonstrated a low limit of quantification (0.385 to 0.866 mg/L), excellent linear correlation (R² > 0.997), and desired calibration range (3.125 to 25 mg/L). In addition, lactose-lysine solutions containing sulfite (4 to 400 mmol/L) were heated at 110°C for 2 h. The results showed that sulfite inhibited the formation of CML and promoted the consumption of reducing sugars and lysine in the Maillard reaction model. The method proved to be useful for simultaneous analysis of CML, lysine, and reducing sugars (glucose, galactose, and lactose) in the Maillard reaction system. Moreover, sulfite was an effective inhibitor of CML formation.     

发酵食品中多种防腐剂检测方法的建立

该实验建立了高效液相色谱法同时测定苯乳酸、苯甲酸、山梨酸的方法。在以C18柱为分离柱,流动相为甲醇-0.020 mol/L乙酸铵（20∶80,V/V）,色谱柱温为25 ℃,流速为1.0 mL/min,检测波长为220 nm条件下,12 min内可实现苯乳酸、苯甲酸、山梨酸的分离。该方法在0.25～50.00 mg/L范围内具有良好的线性关系（相关系数R2＞0.998）,检出限（LOD）＜50.00 ng/mL,回收率为96.93%～118.42%,精密度试验结果相对标准偏差（RSD）为0.39%～6.21%。该方法操作简便、结果准确度高,可用于发酵食品中苯乳酸、苯甲酸和山梨酸的同时分析检测。