A U.S. population dietary exposure assessment for 4-methylimidazole (4-MEI) from foods containing caramel colour and from formation of 4-MEI through the thermal treatment of food

ABSTRACT

4-Methylimidazole (4-MEI) is formed in caramel colours produced using ammonium compounds (Class III and Class IV caramel colours). 4-MEI can also form in food through Maillard reactions between reducing sugars and amino acids during cooking, roasting or dry-heating. The USFDA has analysed over 700 food and beverage samples collected from 2013 to 2015 for the presence of 4-MEI. These samples include foods containing added caramel colour and foods that are not labelled as containing added caramel colour, but which may contain 4-MEI resulting from thermal treatment. The 4-MEI levels in all food samples were quantified using LC-MS/MS. These data were used to develop a comprehensive dietary exposure assessment for 4-MEI for the U.S. population aged 2 years or more and several sub-populations, using two non-consecutive days of food consumption data from the combined 2009–2012 National Health and Nutrition Examination Survey and 10–14-day food consumption survey data for 2009–2012 from the NPD Group, Inc. National Eating Trends–Nutrient Intake Database. Dietary exposure estimates were prepared for each category of foods labelled as containing added caramel colour and of foods not labelled as containing added caramel colour, but which may contain 4-MEI from thermal treatment. Exposure to 4-MEI from consumption of foods containing added caramel colour was higher than that from foods that contain 4-MEI from thermal treatment for all population groups. Cola-type carbonated beverages were the highest contributors for most populations from foods containing added caramel colour. Coffee was the highest contributor for most populations from foods in which 4-MEI could be formed from thermal treatment. An overall combined exposure to 4-MEI was also estimated that included all foods identified as containing added caramel colour and foods in which 4-MEI could be formed by thermal treatment.     

高效液相色谱-四级杆/静电场轨道肼高分辨率质谱检测蜂蜜中的4-甲基咪唑和2-甲基咪唑

目的建立高效液相色谱-四级杆/静电场轨道肼高分辨率质谱法直接测定蜂蜜中4-甲基咪唑和2-甲基咪唑的方法。方法用水溶解蜂蜜样品,经0.22μm粒径的微孔膜过滤后,采用Agilent HILIC Plus(2.1 mm×100 mm,3.5μm)色谱柱进行分离,以体积比为8:2的乙腈与5 mmol/L乙酸铵水溶液为流动相进行等度洗脱。质谱采用正离子模式,在质荷比(m/z)50~400范围内通过高分辨质谱全扫描模式提取目标化合物的精确质量数。结果在本文建立的色谱条件下,4-甲基咪唑(4-MI)和2-甲基咪唑(2-MI)能够得到较好的分离,高分辨率质谱提取的质量准确度小于5×10-6(5 ppm)。该方法对4-MI的检测限可达50μg/kg,对2-MI的检测限可达25μg/kg。两种待测物质采用外标法定量,线性良好,相关系数均大于0.99。通过加标验证,在50、100和150μg/kg三个加标水平下,蜂蜜中4-MI和2-MI的平均回收率为73.2%~107.9%,相对标准偏差(RSD)小于9.7%。结论该方法样品处理过程简单,不使用有机溶剂,灵敏度高,适用于任何蜜种的测定,可以作为蜂蜜中是否掺入焦糖色素的检测方法。利用该方法对国内外各地区12个蜜种的30个蜂蜜样品进行检测,均未检出4-MI和2-MI。     

Determination of 4(5)-methylimidazole in carbonated beverages by isotope-dilution liquid chromatography-tandem mass spectrometry

The purpose of this study was to develop a method to quantify 4(5)-methylimidazole (4-MEI), a suspected carcinogen, in carbonated beverages by simple sample dilution and isotope-dilution reverse-phase LC-MS/MS. Isotope dilution using hexa-deuterated methylimidazole (d₆-4-MEI) was used to quantify native 4-MEI and to assess matrix effects quantitatively. The accuracy of the method was assessed by intentionally fortifying a negative control sample at three doses: low, medium and high (replicates of n = 5 each) with a known amount of 4-MEI. The respective absolute error in each case was 18.7 ± 0.7%, 14.6 ± 2.8% and 21.1 ± 9.7%. Within-day (intra-) and day-to-day (inter-) repeatability, determined as the relative standard deviation by fortifying a negative control sample (n = 5), were 9.5% and 15.4%, respectively. Average ion suppression of d₆-4-MEI in beer was 63.9 ± 3.2%, while no suppression or enhancement was seen in non-alcoholic samples. The instrument and method limit of detection were calculated as 0.6 and 5.8 ng ml^–1, respectively. 4(5)-Methylimidazole was quantified in a variety of store-bought consumer beverages and it was found that in many of the samples tested consuming a single can of beer would result in intake levels of 4-MEI that exceed the no significant risk guideline of 29 µg day^–1. Conversely, 4-MEI in the samples was orders of magnitude smaller than the European Food Safety Authority acceptable daily intake threshold value of 100 mg kg^–1 bw day^–1.     

Detection and Quantification of 4(5)‐Methylimidazole in Cooked Meat

4(5)‐Methylimidazole (4‐MeI) is a nitrogen‐containing heterocyclic compound found in class III and IV ammoniated caramel colors, a group of additives widely used in the food industry. A suspected carcinogen and neurotoxin and efforts are underway to limit its presence in foods. Several methods have been developed to detect and quantitate 4‐MeI in different food matrices, including roasted coffee, beer, soft drinks, and soy sauce; however, no methods are available to measure 4‐MeI in cooked meat and meat products containing lipids and high levels of interfering nitrogen compounds, such as amino acids and peptides. A rapid method using 0.1 M sodium acetate buffer (pH 5) as an extraction solvent followed by derivatization with isobutylchloroformate and gas chromatograph mass‐spectrometry was developed to quantify 4‐MeI in cooked meat products with added caramel colors containing 4‐MeI. Selected ion monitoring mode was used to monitor 4‐MeI ions fragments. In the 8 commercial meat products tested, 4‐MeI levels ranged from 0.041 to 1.015 mg/kg, with recovery of 94.76% to 103.94%. In addition, a matrix‐matched calibration performed by analyzing a spiked cooked meat sample indicated no significant difference (P > 0.05), which means the meat matrix had no effect on the developed method. This method proved useful in analyzing 4‐MeI in meat products with added caramel color containing 4‐MeI.     

Effect of Various Food Additives on the Levels of 4(5)‐Methylimidazole in a Soy Sauce Model System

In this study, the effect of food additives such as iron sulfate, magnesium sulfate, zinc sulfate, citric acid, gallic acid, and ascorbic acid on the reduction of 4(5)‐methylimidazole (4(5)‐MI) was investigated using a soy sauce model system. The concentration of 4(5)‐MI in the soy sauce model system with 5% (v/v) caramel colorant III was 1404.13 μg/L. The reduction rate of 4(5)‐MI level with the addition of 0.1M additives followed in order: iron sulfate (81%) > zinc sulfate (61%) > citric acid (40%) > gallic acid (38%) > ascorbic acid (24%) > magnesium sulfate (13%). Correlations between 4(5)‐MI levels and the physicochemical properties of soy sauce, including the amount of caramel colorant, pH value, and color differences, were determined. The highest correlations were found between 4(5)‐MI levels and the amount of caramel colorant and pH values (r² = 0.9712, r² = 0.9378). The concentration of caramel colorants in 8 commercial soy sauces were estimated, and ranged from 0.01 to 1.34% (v/v).     

超高效液相色谱-串联质谱法同时测定客家娘酒中的2-甲基咪唑、4-甲基咪唑及5-羟甲基糠醛

目的 建立超高效液相色谱-串联质谱法同时测定客家娘酒中的2-甲基咪唑(2-methylimidazole, 2-MEI)、4-甲基咪唑(4-methylimidazole, 4-MEI)和5-羟甲基糠醛(5-hydroxymethylfurfural, 5-HMF)的分析方法。方法 样品经过直接稀释法和标准加入法分别进行前处理, 使用ACQUITY UPLC BEH HILIC (100 mm× 2.1 mm, 1.7 μm)色谱柱进行样品分离, 以甲醇-5 mmol/L氨水为流动相进行梯度洗脱, 采用电喷雾离子源正离子模式和多反应监测模式进行检测。结果 在优化条件下, 2-MEI和4-MEI在1~500 ng/mL范围内线性关系良好, 相关系数(r2)为0.9998和0.9998, 5-HMF在50~8000 ng/mL线性范围内r2为0.9970。2-MEI、4-MEI和5-HMF在低、中、高3个浓度添加水平下的回收率范围分别为88.7%~108%、77.5%~102%和95.7%~98.4%, 相对标准偏差分别为4.16%~10.50%、2.04%~5.49%和3.10%~6.62%。2-MEI、4-MEI和5-HMF的检出限分别为3、3和17 ng/mL, 定量限分别为10、10和50 ng/mL。结论 本方法操作简便、线性关系良好、回收率高, 适用于客家娘酒中2-MEI、4-MEI和5-HMF的检测。     

Formation of 4(5)‐Methylimidazole in Aqueous d‐Glucose‐Amino Acids Model System

The International Agency for Research on Cancer (IRAC) has classified 4(5)‐methylimidazole (4‐MeI) as a group 2B possible human carcinogen. Thus, how 4‐MeI forms in a d ‐glucose (Glu) amino acids (AA) model system is important, as it is how browning is affected. An aqueous solution of Glu was mixed individually in equimolar amounts at 3 concentrations (0.05, 0.1, and 0.15 M) with aqueous solutions of l ‐Alanine (Ala), l ‐Arginine (Arg), Glycine (Gly), l ‐Lysine (Lys), and l ‐Serine (Ser). The Glu‐AA mixtures were reacted at 60, 120, and 160 °C for 1 h. The 4‐MeI levels were measured by gas chromatography‐mass spectrometry after derivatization with isobutylchloroformate. No 4‐MeI was formed at 60 °C for any treatment combination; however, at 120 °C and 0.05 M, Glu‐Arg and Glu‐Lys produced 0.13 and 0.14 mg/kg of 4‐MeI. At 160 °C and 0.05 M all treatment combinations formed 4‐MeI. At 160 °C and 0.15 M, the observed levels of Glu‐Ala, Glu‐Arg, Glu‐Gly, Glu‐Lys, and Glu‐Ser were 0.21, 1.00, 0.15, 0.22, and 0.16 mg/kg. The AA type, reactant concentrations, and temperature significantly affected (P < 0.001) formation of 4‐MeI as well as browning. Glu‐Lys treatment in all combinations produced the most browning, but Glu‐Arg produced the most 4‐MeI. This method showed that foods processed using low temperatures may have reduced levels of 4‐MeI.     

UPLC-MS-MS检测饮料中的4-甲基咪唑及其同分异构体2-甲基咪唑

建立超高效液相色谱-串联质谱（ultra performance liquid chromatography -tandem mass spectrometry,UPLCMS- MS）法检测饮料中的4-甲基咪唑（4-(5-)-methylimidazole,4-Mel）及2-甲基咪唑（2-methylimidazole,2-Mel） 的方法。以水溶解样品,OASIS®MCX固相萃取柱富集净化后,进入UPLC-MS-MS检测,内标法定量。在优化的条 件下,以ACQUITY UPLC® HILIC色谱柱（2.1 mm×100 mm,1.7 mm）为分析柱,乙腈和5 mmol/L甲酸铵溶液为 流动相,在电喷雾正离子多反应监测模式进行检测。结果表明,4-Mel和2-Mel在9～500 ng/mL范围内线性关系良 好。方法的定量限为3.0 mg/kg。在4 个加标水平下,3 种不同基质饮料中4-Mel和2-Mel的平均回收率分别为97.3% 和93.7%,相对标准偏差分别为3.8%和3.4%,该方法适用于饮料中4-Mel和2-Mel的检测。利用该方法对市售饮料中 4-Mel和2-Mel的含量进行检测,在软饮料、焦糖色饮料及咖啡饮料中检出含有4-Mel或2-Mel;而功能性饮料中未检 出4-Mel和2-Mel。     

UPLC-MS/MS法同时测定焙烤食品中的丙烯酰胺、4-甲基咪唑与5-羟甲基糠醛

建立了同时测定焙烤食品中丙烯酰胺、4-甲基咪唑和5-羟甲基糠醛的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法。样品中加入同位素内标后,以水和乙腈超声提取,QuEChERS净化,正己烷脱脂,UPLC-MS/MS分离检测,丙烯酰胺和4-甲基咪唑以同位素内标定量。在优化条件下,3种待测物的方法检出限在1.5μg/kg~7.0μg/kg之间;在2.0μg/L~500μg/L浓度范围内线性关系良好,相关系数均大于0.9990;在面包、蛋糕和饼干样品中进行3个水平的添加实验,平均回收率(n=6)为87.8~115.7%,日内精密度为3.2~9.6%;日间精密度(n=5)小于11.5%。本方法净化效果好,灵敏度高、准确性好,适用于焙烤食品中丙烯酰胺、4-甲基咪唑和5-羟甲基糠醛的同时测定。     

Intake and risk assessment of nitrate and nitrite from New Zealand foods and drinking water

Exposure to excess nitrite is a potential health risk for humans. One hundred meat and processed foods and 100 vegetable samples purchased from New Zealand retail outlets were prepared as for consumption and analysed for nitrite and nitrate concentration using a standard, validated methodology. Nitrate concentrations ranged from less than the limit of detection (LOD = 5 mg kg^-1) in cheddar cheese and cream cheese-based dips to 3420 mg kg^-1 in lettuce. Nitrite was detected in half the processed foods and meats analysed (levels up to 119 mg kg^-1), but detected in only one vegetable sample above the LOD (broccoli at 27 mg kg^-1 nitrite). Concentration data were combined with 24 h dietary recall information to generate 4398 individual adult daily exposure scenarios for exogenous nitrite and nitrate including a contribution from water assessed from 1021 drinking water samples. The mean adult daily intake of exogenous nitrate and nitrite from food and water combined was 16 and 13% of the Acceptable Daily Intake (ADI), respectively, and therefore should not pose a health risk for the average consumer. A maximally exposed New Zealand adult is estimated to have an intake of up to seven times the ADI for nitrate. When the endogenous conversion of nitrate to nitrite is taken into account, approximately 10% of people with an average rate of conversion and half of all people with a high rate of conversion are estimated to exceed the ADI. Either the ADI is inappropriate and needs to be re-evaluated, or those individuals who have a high rate of conversion of nitrate to nitrite are at risk to adverse effects of nitrite exposure.     

固体硫酸铁催化合成不饱和5（4H）-噁唑酮反应的研究

首次采用固体硫酸铁作催化剂，用饱和噁唑酮与醛酮反应，合成了10种不饱和噁唑酮。并通过m．p测定，IR测定，IR测定等，对产物进行了表征。     

毛细管气相色谱法测定果汁饮料中对羟基苯甲酸乙酯、对羟基苯甲酸丙酯的含量

建立了果汁饮料中对羟基苯甲酸乙酯、对羟基苯甲酸丙酯含量的毛细管气相色谱测定方法。该法采用WBI进样口、毛细管色谱柱J＆WDBl701（中等极性30mxO．53minx1μm、FID检测器进行检测。进样口温度230℃,载气流速10ml／mim．柱室温度1800℃恒温。检测器温度250℃,样品用盐酸溶液酸化,乙酸乙酯提取后直接进样测定。方法简单、快速、重现性好,平均回收率大于90％,RSD小于3．0％,线性范围为10-200μg／ml。     

Candida etchellsii产2(5)-乙基-4-羟基-5(2)-甲基-3(2H)-呋喃酮的影响因素

在埃切假丝酵母(Candida etchellsii)发酵过程中,通过盐度调控和氨基酸添加,强化目标产物HEMF(2(5)-乙基-4-羟基-5(2)-甲基-3(2H)-呋喃酮)的合成效率。分阶段调控发酵体系的盐度(初始阶段控制CaCl浓度为200 g/L,发酵40 h后提升至220 g/L),结合氨基酸添加(向发酵体系中添加丙氨酸、精氨酸和甘氨酸各1.67 g/L)。摇瓶结果表明:酵母C.etchellsii合成HEMF,其产量为110.74 mg/L。7 L发酵罐上罐验证,HEMF产量达到121.51 mg/L,相比空白(200 g/L CaCl浓度下且没有添加氨基酸)提高了21.2倍。分阶段盐度调控结合氨基酸添加策略显著强化了C.etchellsii对HEMF的合成。     

5-(4-(2-甲氧二氧)苯烯)-2-硫代噻唑-4-酮对雪莲果中酪氨酸酶的抑制作用

通过分光光度法探究5-(4-(2-甲氧二氧)苯烯)-2-硫代噻唑-4-酮(MBTO)对雪莲果中酪氨酸酶的抑制作用。结果表明：MBTO对雪莲果中酪氨酸酶有良好的抑制作用。单因素试验最佳抑制剂浓度为30.0μmol/L、最佳作用时间为210s、最佳作用温度为30℃。通过三因素三水平正交试验,分析得最佳反应条件为：pH7.5、抑制剂MBTO浓度30.0μmol/L、作用时间330s、作用温度30℃,可达到最佳抑制效果,在此条件下MBTO对雪莲果中酪氨酸酶的抑制率达84.24%。     

高糖食品中2-（4-甲氧基苯氧基）丙酸钠的风险评估

为探讨化学合成的芳氧羧酸类化合物2-（4-甲氧基苯氧基）丙酸钠在食品生产中作为食品添加剂的安全性,本文采用动物实验对化学合成的2-（4-甲氧基苯氧基）丙酸钠进行毒理学评价,采用高效液相色谱法对食品中2-（4-甲氧基苯氧基）丙酸钠的使用率进行研究。毒理学实验结果表明,化学合成的2-（4-甲氧基苯氧基）丙酸钠的半数致死量LD₅₀为6577 mg/kg bw,哺乳动物微粒体酶实验及小鼠精子畸形实验分别显示其未见明显的致突变性及遗传毒性。色谱检测结果表明,月饼、糖果及糕点等3类高糖食品的2-（4-甲氧基苯氧基）丙酸钠使用率较高,其中平均使用量分别为149.48±1.26、52.30±1.87、71.31±1.39 mg/kg;中位值使用量分别为118.05±2.11、72.58±0.94、（65.77±1.02）mg/kg;95%位值使用量分别为439.97±2.06、340.77±1.45、137.63±1.62 mg/kg。综合毒理学评价结果及2-（4-甲氧基苯氧基）丙酸钠的使用情况进行风险评估,结果表明添加量为95%位值使用量时,化学合成的2-（4-甲氧基苯氧基）丙酸钠暴露量已超出其ADI值,可能会对高糖食品的安全性造成影响。      

Reactive dicarbonyl compounds and 5-(hydroxymethyl)-2-furfural in carbonated beverages containing high fructose corn syrup

α-Dicarbonyl compounds, namely glyoxal (GO), methylglyoxal (MGO) and 3-deoxyglucosone (3-DOG), as well as 5-(hydroxymethyl)-2-furfural (5-HMF) were found and measured in carbonated soft drinks (CSD). It was realized that high fructose corn syrup (HFCS) in regular CSDs was the major source of α-dicarbonyl compounds in beverages after comparison of levels in regular and diet CSDs. In two most commonly used HFCS formulas, 42% and 55% HFCS, the highest level of dicarbonyl found was 3-DOG, followed by MGO, and then GO. The stability of dicarbonyls in CSDs containing HFCS and (−)-epigallocatechin gallate (EGCG) which were incubated at 35 and 45 °C was investigated. It was found that EGCG decreased from 1 mg/mL to 0.5 and 0.3 mg/mL for 35 and 45 °C, respectively, in 16 days storage. Moreover, the reactions of EGCG with MGO, 3-DOG and 5-HMF were observed from the comparison of storage CSDs with and without EGCG under acidic conditions.     

Survey and risk assessment of trace elements in foods from Taiwan containing red mould rice (Monascus) by ICP-MS

The concentrations of seven trace elements (As, Cd, Cr, Pb, Se, Cu and Zn) in 93 red mould rice (Monascus) food samples in Taipei, Taiwan, were determined by inductively coupled plasma-mass spectrometry (ICP-MS) after wet digestion. The results, calculated in mg?kg^?1 (wet weight) for each sample, revealed the general scenario of food safety in Taiwan: As (0.005–12.04), Cd (<0.0005–2.22), Cr (0.014–6.95), Cu (0.012–8.70), Pb (0.001–0.64), Se (<0.001–1.29) and Zn (0.020–67.02). Three food samples were identified with As concentrations higher than regulatory limits: a dietary supplement sample and a seaweed sample with As concentrations that exceeded the limit of Taiwan's health food standard of 2?mg?kg^?1, and a canned eel sample with an As concentration that exceeded the limit of Canada's fish standard of 3.5?mg?kg^?1. This study suggests that the estimated intakes of these seven trace elements from the consumption of foods containing Monascus pose little risk, as the trace element contents in the majority of samples were lower than the permissible/tolerable intakes per week according to the guidelines recommended by the Food and Agricultural Organization/World Health Organization (FAO/WHO). Moreover, their concentrations in foods containing Monascus differ widely for different food varieties, suggesting that external contaminants and raw materials are the main sources of trace elements. This study shows that ICP-MS is a simple method proposed for the determination of As, Cd, Cr, Pb, Se, Cu, and Zn in foods containing Monascus.     

3-苯基-4-氨基-5-硫酮-1,2,4-三唑对牛蒡中酪氨酸酶的抑制作用

以3-苯基-4-氨基-5-硫酮-1,2,4-三唑(APTT)为效应物,通过SPSS设计正交试验,研究不同APTT浓度、时间以及温度对牛蒡酪氨酸酶提取液的护色效果的影响,并探讨pH值对抑制剂及反应体系作用效果的影响,数据用SPSS软件处理,在25℃条件下,牛蒡与APTT比为0.5:3(g/mL)时,500μmol/L抑制剂对底物作用420s为最佳抑制反应条件,其ΔA为0.0082:pH7.0时,抑制效果最佳。APTT对牛蒡中酪氨酸酶有良好的抑制作用。     

以三聚氯化氰为脱水剂合成饱和5（4H）—恶唑酮反应的研究

从N酰基氨基酸出发，以三聚氯为氰为脱水剂，合成出8种2（取代苯基）5恶唑酮。通过熔点测定，红外光谱测定，核磁测定对产品进行了表征。