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。     

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。     

煎炸过程对棕榈油品质及南美白对虾中 AGEs含量的影响

以煎炸油和煎炸南美白对虾为研究对象,探究煎炸时间及煎炸物料对煎炸油品质的影响;采用液相色谱-质谱联用及荧光技术测定煎炸对虾中晚期糖基化终末产物（AGEs）的含量,探究煎炸过程对煎炸对虾中AGEs生成的影响。结果表明,高温煎炸可加速棕榈油氧化,使其酸值（AV）、p-茴香胺值（p-AnV）、羰基值（CV）显著增加,24 h时达到最大值。煎炸后,对虾水分含量显著降低,脂肪、丙二醛和荧光AGEs含量显著升高,煎炸批次对其无显著影响。煎炸对虾外层丙二醛、荧光AGEs、非荧光AGEs（羧甲基赖氨酸（CML）、羧乙基赖氨酸（CEL）和甲基乙二醛氢咪唑酮（MG-H1））含量显著高于内部,与未煎炸生虾相比除内部CEL含量有所降低外其余均高于未煎炸生虾。随着煎炸批次的增加,煎炸对虾外层CML、CEL和MG-H1含量均逐渐降低,在煎炸第5批时趋于稳定,煎炸第1批时取得最大值,分别为25.37、335.68 mg/kg和468.20 mg/kg,分别为未煎炸生虾的7、4.5倍和16.5倍;煎炸对虾内部CML、CEL和MG-H1含量均先升高后降低,在煎炸第5批时趋于稳定,煎炸第3批时取得最大值,分别为12.56、85.27 mg/kg和204.20 mg/kg,分别为未煎炸生虾的3.5、1倍和7倍。     

超高效液相色谱-串联质谱法测定动物源性食品中硝基咪唑类药物及其代谢物的残留量的研究

目的建立超高效液相色谱-串联质谱法测定动物源性食品中硝基咪唑类药物及其代谢物的分析方法。方法试样经三氯乙酸溶液提取, MCX固相萃取小柱净化, 5%的甲醇水(V/V)溶液复溶。电喷雾正离子模式电离,多反应监测模式检测,基质匹配内标法定量。结果硝基咪唑及其代谢物在0.25～50 ng/mL范围内线性关系良好,相关系数均大于0.9915,加标回收率为73.5%～120.4%;相对标准偏差在2.4%～13.4%之间,定量限为0.1～0.5μg/kg。结论本方法简便、灵敏,适用于动物源性食品中硝基咪唑类药物及其代谢物残留的定性、定量分析。     

固相萃取UPLC-MS/MS测定黄酒中2-甲基咪唑和4-甲基咪唑

建立同位素内标超高效液相色谱串联质谱法同时测定黄酒中2-甲基咪唑和4-甲基咪唑的定量分析方法。样品加入同位素内标后,以质量分数1%的三氯乙酸提取,经MCX固相萃取柱净化,超高效液相色谱串联质谱分离并检测,内标法定量。在优化条件下,2-甲基咪唑和4-甲基咪唑的检出限分别为0.5μg/kg和0.05μg/kg,在0.1 ng/mL～1 000 ng/mL和1 ng/mL～1 000 ng/mL浓度范围内线性关系良好,相关系数大于0.999。     

超高效液相色谱-串联质谱法检测保健食品中4-甲基咪唑含量

目的建立超高效液相色谱-串联质谱法测定保健食品中4-甲基咪唑含量的分析方法。方法样品采用90%乙腈水溶液超声提取后,提取液经过Waters BEH Hilic色谱柱,根据亲水作用色谱原理分离待测物,以0.1%甲酸乙腈-0.1%甲酸水溶液作为流动相洗脱,正离子电喷雾离子化,多反应监测模式检测,超高效液相色谱-串联质谱法(ultra performance liquid chromatography tandem mass spectrometry, UPLC-MS/MS)对样品进行分析。结果该方法用于4-甲基咪唑检测时,专属性、精密度和样品稳定性良好,线性范围在0.11～1.7ng/mL之间,相关系数r为0.9998;加标回收率在94.16%～116.1%之间,方法准确性良好;检出限为1 ng/g,定量限为3 ng/g,方法灵敏度较好。样品中检出4-甲基咪唑,含量在12.4～14.9 ng/g之间。结论该方法专属性强,准确性好,灵敏度高,适用于保健品中4-甲基咪唑的筛查和测定。     

超高效液相色谱-串联质谱法同时测定小麦粉中硫脲、曲酸、噻苯咪唑、噻二唑、四环素

建立超高效液相色谱-串联质谱法同时测定小麦粉中硫脲、曲酸、噻苯咪唑、噻二唑、四环素5 种非法添加物的快速定量检测方法。样品经80%乙醇溶液-乙腈（1∶1,V/V）提取、离心、氮吹浓缩后,采用多反应监测模式进行定性、定量分析。结果表明：本方法中硫脲、曲酸、四环素在100～5 000 ng/mL、噻苯咪唑在1～50 ng/mL、噻二唑在1～50 μg/mL范围内线性关系良好,相关系数大于0.999。硫脲、曲酸、四环素检出限为25 μg/kg,定量限为50 μg/kg;噻苯咪唑检出限为0.5 μg/kg,定量限为1 μg/kg;噻二唑检出限为0.5 mg/kg,定量限为1 mg/kg。基质添加回收率范围在88.6%～102.2%之间,相对标准偏差在0.7%～5.6%之间（n=6）。该方法前处理简单、快速,可用于小麦粉中硫脲、曲酸、噻苯咪唑、噻二唑、四环素5 种非法添加物的快速测定。     

超高效液相色谱-串联质谱法检测牛组织中常山酮残留量

为了建立牛组织中常山酮残留量的超高效液相色谱-串联质谱检测方法,将牛组织中的常山酮经蛋白酶酶解后,采用乙酸乙酯提取,HLB固相萃取柱净化,超高效液相色谱-串联质谱法进行检测。在0.5 ng/mL～50 ng/mL线性范围内,常山酮的回归方程呈良好的线性关系,R2＞0.998,牛组织样品在添加水平为0.5、5 μg/kg时,回收率为73.2%～93.60%,相对标准偏差（relative standard deviation,RSD）为 5.91%～7.96%,方法检出限为 0.25 μg/kg,定量限为 0.5 μg/kg。本试验适用于牛组织中常山酮残留量的检测。     

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.     

添加糖与酱油对油炸鲟鱼晚期糖化终末产物形成的影响研究

本论文首次分析糖和酱油添加对油炸鲟鱼晚期糖化终末产物（AGEs）形成的影响。以鲟鱼肉为原料，添加绵白糖（0%、1%、2%、3%）、酱油（0%、1%、2%）并制备油炸鲟鱼饼（170 ℃/9 min），分析鲟鱼饼AGEs以及其前体物质糠氨酸、二羰基化合物的变化规律。结果表明，仅添加绵白糖时，油炸鲟鱼饼AGEs以及其前体物质糠氨酸、二羰基化合物含量随绵白糖添加水平增加而逐渐增加；仅添加酱油时，油炸鲟鱼饼糠氨酸、荧光AGEs、羧乙基赖氨酸（CEL）含量随着酱油添加水平增加而逐渐增加，而羧甲基赖氨酸（CML）、甲基乙二醛氢咪唑酮（MG-H1）含量随着酱油添加水平的增加而显著减少（P<0.05），添加酱油对AGEs抑制作用与酱油成分对二羰基化合物消耗有关。当绵白糖和酱油同时添加时，进一步促进糠氨酸、荧光AGEs和MG-H1的形成，两者对油炸鲟鱼饼AGEs形成具有显著交互作用（P<0.05）。因此，添加绵白糖会促进AGEs的形成，而酱油由于其复杂成分导致其添加对不同AGEs标志物形成影响各异。这些工作将为油炸水产品AGEs形成与控制提供理论依据与指导。     

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.     

Validation and measurement uncertainty evaluation of the GC-MS method for the direct determination of hexamethylenetetramine from foods

A simple GC-MS method has been developed and validated for the direct determination of hexamethylenetetramine (HMT). The separation of HMT was performed using a MXT-1 column. The calibration curve was linear over the concentration range 0.1-25 μg/mL, with a good correlation coefficient (r =0.9996). The recoveries of HMT from foods spiked at 1, 5, and 10 μg/g ranged from 91.7% to 115.2%. Intra-day (n=5) and inter-day (n=5) precision were less than 7%. The limit of detection and the limit of quantification of the method were 0.05 and 0.15 μg/mL, respectively. The uncertainties associated with food matrix and calibration contributed most to the overall expanded uncertainty. The method validation data indicated that quantitative method could be applied to the direct determination of non-hydrolyzed HMT in foods.     

Forty years of furosine - forty years of using Maillard reaction products as indicators of the nutritional quality of foods

The Maillard reaction products (MRPs) most widely used as markers of the nutritional quality of foods are furosine, N(epsilon)-carboxymethyllysine (CML), hydroxymethylfurfural, pyrraline, pentosidine and pronyl-lysine. One of the MRPs identified first was furosine, which was quantified in foods 40 years ago as a chemical indicator of the Amadori compound N(epsilon)-fructoselysine. Since then, furosine has gained broad attention by food chemists and biomedical researchers, as its formation upon heat treatment is well characterised. Moreover, it represents the Amadori products from early Maillard reactions in which amino acids react with reducing carbohydrates, resulting in a loss of their availability. This is of importance for the essential amino acid lysine, which is also the limiting amino acid in many proteins. In order to evaluate the nutritional quality of a protein, the concomitant analysis of free - and nutritionally available - lysine and the amount of lysine reacted to form the respective MRP is essential, even for mildly processed foods. The other chemical markers of heat treatment such as CML, pyrraline, pentosidine or pronyl-lysine seem to be useful markers of the advanced stages of Maillard reactions. Compared to the conditions in which furosine is formed, these compounds are generated under more severe conditions of heat treatment. However, the concentrations analysed are significantly lower than those of furosine. Therefore, the nutritional evaluation of a food protein should include not only furosine, but also other chemical markers of heat treatment such as, for example, CML, pyrraline and pentosidine.     

Interference-free simultaneous determination of Sudan dyes in chili foods using solid phase extraction coupled with HPLC–DAD

A simple, rapid, inexpensive and robust solid phase extraction (SPE) cleanup method coupled with high performance liquid chromatography–diode array detector (HPLC–DAD) for the determination of four Sudan dyes in chili products was developed. The interference substances were removed completely by normal SPE-Alumina N, resulting in a clean and interference-free baseline. Without the use of expensive LCMS/MS instrument the low levels of Sudan dyes were identified and quantified in chili foods with good precision and recovery. Depending on the Sudan dyes involved, the limit of determination (LOD) and limit of quantification (LOQ) were in the range of 4.1–5.8 and 13.2–19.1 μg/kg, respectively. The recovery, repeatability and reproducibility varied from 93.2% to 103%, from 0.3% to 4.4%, and from 0.6% to 4.9%, respectively. This method is suitable for the routine analysis of Sudan dyes for the common factory laboratory due to its sensitivity, simplicity, convenience and low-cost.