Determination of benzidine in the food colours tartrazine and sunset yellow FCF, by reduction and derivatization followed by high-performance liquid chromatography.

Free and bound benzidine, a non-sulphonated aromatic amine (NSAA), were determined in the food colours tartrazine and sunset yellow FCF. Bound amines were released by reducing with sodium dithionite, then total NSAAs were extracted into chloroform, transferred to aqueous acid solution and diazotized with sodium nitrite before coupling with 2-naphthol-3,6-disulphonic acid, disodium salt (R-salt). Coloured benzidine and aniline derivatives (BZDRS and ANRS) were analysed using reversed-phase ion pair high-performance liquid chromatography (HPLC) and an absorbance detector set at 548 nm. Levels of total benzidine were similar to those reported in studies conducted in the USA, and ranged from < 5 to 270 ng/g. Total aniline was also determined (0.2-188 micrograms/g). Recoveries of benzidine with this method were found to be lower than expected (average ca 46%), but were reproducible. Detection limits were 15-20 ng BZDRS/g (3-4 ng benzidine/g). Mass spectrometry (LC-MS) was evaluated for identifying and determining purity of the standards, but difficulties were encountered when the methodology was applied to commercial samples.     

Determination of total non-sulphonated aromatic amines in soft drinks and hard candies by reduction and derivatization followed by high-performance liquid chromatography.

Utilizing elements of methodology developed previously for food colours, total free and bound non-sulphonated aromatic amines (NSAA) were determined in commercial samples of soft drink beverages and hard candies. Bound amines in the samples were reduced using sodium dithionite, then total NSAA were extracted into chlorofom, transferred to aqueous acid solution and diazotized with sodium nitrite before coupling with 2-naphthol-3,6-disulphonic acid, disodium salt (R-salt). The coloured derivatives were analysed using reversed-phase ion pair high-performance liquid chromatography (HPLC) and an absorbance detector set at 512 nm. Solid phase extraction cartridges were utilized for extraction and clean-up of the food colours present in the sample, and the concentration of each dye was determined quantitatively using HPLC and absorbance detector wavelengths of 426, 516 or 625 nm. Levels of total NSAA were compatible with those observed previously in food colours. Commercial soft drinks were found to contain (expressed in terms of total free plus bound NSAA in the beverage) 0.19-12.6 ng/ml of aniline, 0.83-8.25 ng/ml 1-naphthylamine and 0.62-1.12 ng/ml 2-naphthylamine. Levels of 0.66-9.15 ng/g of aniline and 2.48-10.6 ng/g 1-naphthylamine were found in commercial samples of hard candies. Bound NSAA in hard candies appeared to survive the manufacturing process. Recoveries averaged 96.9% for tartrazine and 89.6-97.2% for the bound amines when hard candies were prepared in the laboratory.     

高效液相色谱法同时测定熟肉制品中日落黄、 胭脂红和诱惑红

目的 建立对熟肉制品中日落黄、胭脂红和诱惑红三种合成色素同时测定的方法。方法 样品采用无水乙醇-氨水-水(7: 2: 1)提取, 加正己烷去除脂肪, 氮吹后定容, 用高效液相色谱法测定。经Symmetry Shield RP18柱分离, 甲醇-0.02 mol/L乙酸铵溶液梯度洗脱, 检测波长为508 nm。结果 本方法的相对标准偏差(n=6)在0.92%～3.2%之间, 平均回收率在86.4%～99.1%之间, 检出限在0.005～0.008 mg/kg之间。结论 本方法灵敏度高, 分离效果好, 结果稳定可靠, 适用于熟肉制品中日落黄、胭脂红和诱惑红的同时测定。     

Stability of sunset yellow FCF and tartrazine in liquid model food systems

Effect of various preservatives, sugar, ascorbic acid and light on the stability of sunset yellow and tartrazine was studied. While sulphur dioxide enhanced the rate of degradation of sunset yellow, it stabilized tartrazine in aqueous model systems. At 30 and 45% sugar levels, the rate of degradation of both sunset yellow and tartrazine was practically same in the presence of benzoic acid and sorbic acid. At 15% sugar level, the rate of degradation of tartrazine was slightly higher in the presence of sorbic acid. Incorporation of ascorbic acid enhanced the degradation of both sunset yellow and tartrazine and its effect was considerably higher in the presence of light. Increase in sugar concentration enhanced the stability of both the dyes, but light enhanced their degradation.     

液相色谱法测定葡萄酒中柠檬黄、苋菜红、胭脂红、日落黄含量结果的不确定度评定

目的评定高效液相色谱法(high performance liquid chromatography,HPLC)测定红酒中人工合成着色剂柠檬黄、苋菜红、胭脂红、日落黄含量的不确定度。方法依据JJF1059-1999《测量不确定度评定与表示》,分析人工合成着色剂含量测定过程中的不确定度来源,通过建立数学模型量化不确定度分量,计算合成不确定度及扩展不确定度。结果本研究测得的红酒中人工合成着色剂的含量分别为柠檬黄(52.6±7.2)mg/kg(k=2)、苋菜红(33.6±7.4)mg/kg(k=2)、胭脂红(40.7±5.6)mg/kg(k=2)、日落黄(50.2±6.8)mg/kg(k=2)。测定过程的不确定度来源主要有试样中人工合成着色剂含量、试样质量、试样定容体积、试样稀释倍数等。结论本方法可反映测量的置信度和准确性,为日常实际检验工作提供参考。     

高效液相色谱法同时测定肉制品中 胭脂红、诱惑红和赤藓红

目的建立高效液相色谱法同时测定肉制品中胭脂红、诱惑红和赤藓红的分析方法。方法样品经石油醚除脂、酸性水洗脱、乙醇氨解吸后,采用Waters Xbridge C_(18)(150 mm×4.6 mm,5μm)色谱柱进行分离,甲醇、0.02 mol/L乙酸铵水溶液为流动相进行梯度洗脱,二极管阵列检测器254 nm波长检测,外标法定量。结果胭脂红、诱惑红、赤藓红可以有效分离,线性范围均为5~50μg/mL,相关系数均大于0.999,加标回收率为92.82%~96.46%,相对标准偏差为0.97%~3.86%。结论此方法操作简单、快速,适用于肉制品中色素的测定。     

Preparation of HPLC test solutions for organic impurities in aluminum lakes of food red no. 40 (allura red AC) and food yellow no. 5 (sunset yellow FCF)

The HPLC determination of organic impurities in Food Red No. 40 aluminum lakes (R-40Als) as directed by Japan's Specifications and Standards for Food Additives, 7th Ed. (JSFA-VII), has problems, such as reproducibility and low recovery. ICP analyses suggested that the problem was caused by the aluminum in the test solution. In the improved method for preparation of the test solution, aluminum was precipitated as a hydroxide gel by boiling with 1% aqueous ammonia. After centrifugation, the supernatant was used for the HPLC analysis of the organic impurities in the lakes. Recoveries of organic impurities were more than 85% from R-40Al spiked at the 0.1 and 1.0% levels of R-40. The proposed method was also adapted for Food Yellow No. 5 aluminum lakes.     

液相色谱法快速检测染色梅鱼中日落黄、柠檬黄的含量

目的建立一种快速、低成本检测染色梅鱼中日落黄、柠檬黄的方法。方法将染色梅鱼剥皮,鱼皮经70%甲醇氨水提取液提取,经EclipseXDB-C_(18)色谱柱分离,以甲醇和乙酸铵水溶液为流动相,进行梯度洗脱,流速为1.0 mL/min,柱温35℃,利用紫外检测器检测日落黄、柠檬黄的含量。结果日落黄、柠檬黄在1.25、2.5、12.5、25 mg/kg添加水平的加标回收率为80%～90%,相对标准偏差小于10%(n=6),柠檬黄检出限为0.05 mg/kg,日落黄检出限为0.025 mg/kg。结论该方法快速、准确、成本低,适合染色梅鱼中日落黄、柠檬黄的快速检测。     

Determination of aldehydes in tequila by high-performance liquid chromatography with 2,4-dinitrophenylhydrazine derivatization

In this work, the determination of aldehydes in different tequila brands was carried out by high-performance liquid chromatography after derivatization with 2,4-dinitrophenylhydrazine. For the comparative purposes, two commercial brandies were also analyzed. The derivatization agent (50 μl of 3.5 mmol l⁻¹ DNPH in HCl, 2 mol l⁻¹) was added directly to the sample (500 μl) and dinitrophenylhydrazones formed were extracted with hexane. After evaporation of the solvent in nitrogen stream, the residues were dissolved in 100 μl of acetonitrile. The calibration standards were prepared from respective dinitrophenylhydrazones, following the same procedure as for beverage samples. Reversed phase chromatographic separation was achieved on Luna C18 column (250 mm×4.6 mm, 5 μm), using gradient elution (acetonitrile:water, from 68 to 80% of acetonitrile in 20 min) and a total flow rate 1 ml min⁻¹. Spectrophotometric detection for furanic aldehydes was at 390 nm (for other aldehydes at 365 nm). The assignation of chromatographic peaks was accomplished by comparison of their relative retention times and UV/Vis spectra with those of external standards. The method of standard addition was also used. The aldehydes identified were 5-hydroxymethylfurfuraldehyde (t _ret=4.1 min), formaldehyde (t _ret=5.1 min), syringaldehyde (t _ret=5.6 min), acetaldehyde (t _ret=6.2 min), 2-furaldehyde (t _ret=7.2 min) and 5-methylfurfuraldehyde (t _ret=8.9 min). At least four chromatographic peaks with retention times higher than 12 min remained unidentified. The quantification results showed drastically higher concentrations of 2-furaldehyde and 5-methylfuraldehyde in tequilas as referred to brandies. Furthermore, 100% tequilas contained higher levels of these two compounds (for four brands analyzed, mean values 18.6 and 5.97 μg ml⁻¹, respectively) as compared to the mixed brands (five brands analyzed, mean values 6.46 and 3.30 μg ml⁻¹). The results obtained confirm that the profile of furanic aldehydes depends on the type of fructans contained in the raw material and also on heating treatment applied or not prior to fermentation. In contrast to other polysaccharides, inulin hydrolyzes at elevated temperature and the contribution of Maillard browning reactions increases the production of furanic compounds. Our results indicate that the levels of 2-furaldehyde and 5-methylfuraldehyde could be used for discrimination between 100% and mixed tequila brands.     

高效液相色谱法测定糕点中的柠檬黄、日落黄和亮蓝

建立了高效液相色谱法测定糕点中的柠檬黄、日落黄和亮蓝的方法。采用C18柱分离,以甲醇及0.02mol/L乙酸铵为流动相梯度洗脱,测定波长为410nm,检出限柠檬黄为0.0002mg/kg,亮蓝、日落黄为0.0005mg/kg,回收率在92%～105%之间,相对标准偏差RSD%<2。改进了样品的前处理方法,简单易行,回收率及重复性较好。     

Spectroscopic and molecular docking studies of the interactions of sunset yellow and allura red with human serum albumin

Binding interactions of human serum albumin (HSA) with sunset yellow (SY) and allura red (AR), two food colorants, were investigated at the molecular level through fluorescence and UV absorption as well as molecular docking. The collective results of the study under the simulated physiological conditions proposed a static type of binding occurring between the two dyes and HSA. When compared with AR (293 K: Ksv = (4.21 ± 0.36) × 10⁴ L·mol⁻¹; K_b = (0.30 ± 0.23) × 10⁶ L·mol⁻¹), SY (293 K: Ksv = (6.80 ± 0.10) × 10⁴ L·mol⁻¹; K_b = (3.11 ± 2.01) × 10⁶ L·mol⁻¹) had stronger quenching ability and higher affinity for HSA due to less steric hindrance. It can be deduced that the energy transfer from HSA to the two dyes occurred with high probability based on the Förster resonance energy transfer theory (r < 7 nm, 0.5 R₀ < r < 2.0 R₀). The spectral analysis suggested that the formation of the dye-HSA complex resulted in the change in microenvironment around Tyr and Trp residues and in the secondary structure of the protein. According to molecular docking simulation, the two structural analogs almost bound to the same site of HSA, near Sudlow's Site I, but significant difference existed in the number and location of hydrogen bond (H-bond) formed between the dyes and HSA. From the molecular docking along with the thermodynamic parameters (AR: ΔH^o = −(58.79 ± 15.24) kJ·mol⁻¹, ΔS^o = −(115.1 ± 31.10) J·mol⁻¹·K⁻¹; SY: ΔH^o = −(52.24 ± 3.15) kJ·mol⁻¹, ΔS^o = −(50.07 ± 11.14) J·mol⁻¹·K⁻¹), it could be inferred that H-bond and van der Waals forces were the major binding forces involved in formation of the dye-HSA complexes.     

果汁中胭脂红、柠檬黄和日落黄含量检测能力验证研究

目的通过分析、评定实验室的检测结果,了解国内实验室对果汁中胭脂红、柠檬黄和日落黄的检测能力。方法采用果汁添加标准品的方式制备样品,通过F检验、Ss≤0.3?和|x-y|≤0.3?法对所制备的样品进行均匀性和稳定性检验。以稳健分析算法A得出的稳健平均值和稳健标准差,作为本次能力验证计划的指定值和能力评定标准差,利用z比分数进行参加实验室的能力评定。结果参加实验室检测胭脂红、柠檬黄和日落黄能力评定的满意率分别为79%、87%和77%。结论大部分参加实验室对胭脂红、柠檬黄和日落黄具备较好的检测能力。     

甲基绿吸收光谱法同时测定饮料中的酒石黄和日落黄

研究建立了一种简便、快速、能同时测定饮料中共存着色剂——酒石黄和日落黄的吸收光谱新方法.在pH 9.43 Tris-HCl溶液中,甲基绿与酒石黄和日落黄发生显色反应,生成离子缔合物,在400～750 nm范围内,酒石黄体系在574 nm波长处产生1个能进行定量分析的特征负吸收峰,共存色素日落黄不干扰测定,酒石黄的质量浓...     

钛胶反相高效液相色谱法测定饮料中的柠檬黄

建立了基于钛胶柱对饮料中合成色素柠檬黄的高效液相色谱快速测定方法.色谱条件:分离柱为Titania Sachtopore-RP柱(250 mm×4.6 mm i.d.,5μm),流动相为甲醇:水=45∶55(体积比)、流动相中含乙酸铵1.5 mmol/L,流速0.8 mL/min,柱温35℃,检测波长426 nm.线性范围为5 mg/L～80 mg/L,最低检出限为4μg/L,回收率为91.7％～93.7％,相对标准偏差(n=8)小于0.7％.准确度和精密度均可满足饮料中柠檬黄的测定要求.     

柠檬黄、日落黄在含乳饮料中褪色分析及改进

目的分析人工偶氮类色素柠檬黄、日落黄在含乳饮料中的褪色原因。方法通过测定Lab值,比色法计算其ΔE值,对比柠檬黄和日落黄与β-胡萝卜素在普通水体系以及含乳体系中的褪色速率,同时设计试验对比以乳清分离蛋白和酪朊酸钠作为蛋白质来源体系中复配柠檬黄日落黄色素的褪色速度,并使用Ellman’s法测定两者体系中巯基的数量。通过测定不同浓度半胱氨酸体系中复配柠檬黄日落黄色素的褪色情况,验证巯基数量对合成偶氮类色素褪色的影响。结果在普通水体系中复配柠檬黄日落黄稳定性较好,褪色速度较慢,而在含乳体系中复配柠檬黄日落黄色素的褪色速度快于天然色素。Ellman’s法测定乳清分离蛋白的巯基数量远远大于酪朊酸钠,同时乳清蛋白体系中复配柠檬黄日落黄色素褪色速度快于酪朊酸钠体系。半胱氨酸浓度梯度实验显示,半胱氨酸浓度越高复配柠檬黄日落黄色素褪色越快。结论通过不同实验法案能够确定,含乳饮料中的巯基是导致合成偶氮类色素快速褪色的原因之一。     

Determination of carotenoids in foods by high-performance liquid chromatography.

The mean values of alpha and beta-carotene of 10 fruits and vegetables from supermarkets in Cairo and Alexandria have been determined using HPLC method, and the average vitamin A activities (in retinol equivalents) calculated. Carrots, spinach and sweet potatoes were the best sources of provitamin A activity. Beta-carotene was the more prevalent carotenoid compared with alpha-carotene. An analysis of variance including all foods investigated demonstrated that there were no significant differences among either locations or times of analysis. There was no difference between the analysed values of vitamin A activity and the USDA Handbook No. 8 values.     

First-derivative spectrophotometric determination of ponceau 4R,sunset yellow and tartrazine in confectionery products

A first-derivative spectrophotometric method was developed for quantitative determination of synthetic organic dyes, Ponceau 4R (E124), Sunset Yellow (E110) Tartrazine (E102), which are used in food products under governmental regulations all over the world because of their toxicity and carcinogenity. In this study, Ponceau 4R with Sunset Yellow, and Tartrazine with Sunset Yellow, were simultaneously determined in their binary mixtures by first-derivative spectrophotometry. The method was applied to different sugar confectionery products. Recoveries were 93.8–101.2%, 92.1–107.9% and 94.9–99.2% for Ponceau 4R, Sunset Yellow and Tartrazine, respectively. The results were also compared by an independent method, thin layer chromatography (TLC). A t-test indicates that the differences obtained via the present methods and TLC were insignificant at the 95% confidence level. In addition, the present method is very rapid, economical and accurate.     

原位衍生化-分散液液微萃取-气相色谱法测定环境水样中酚类物质的含量

本研究建立了原位衍生化-分散液液微萃取-气相色谱测定水样中酚类化合物含量的方法,并考察了衍生化反应过程以及萃取过程的相关因素对实验结果的影响。实验结果表明最佳条件为:在5 m L分析水样中（p H=7.00）,加入800μL衍生剂乙酸酐,置于50℃水浴中加热30 min,猝冷至室温后,以200μL二氯甲烷为萃取剂,以10μL甲醇为分散剂,密封后振荡后离心5 min,取1.00μL萃取液进行分析。本法线性范围为0.02⁴.00μg/m L,相关系数R2≥0.9951,方法检出限为1.98μg/L。实际水样测定的相对标准偏差≤6.90%,回收率为59.6%¹19.2%。      

高效液相色谱法测定烧烤制品中5种杂环胺的含量

目的 建立高效液相色谱法检测烧烤制品中5种杂环胺(heterocyclic aromatic amines, HAAs)含量的方法。方法 样品经乙酸乙酯、三乙胺、氨水提取, 丙基磺酸(propanesulfonic acid, PRS)和C18固相萃取小柱净化, 采用TSK-gel ODS-80TM色谱柱分析, 以乙腈和0.05 mol/L醋酸-醋酸铵缓冲液(pH=3.4)为流动相进行梯度洗脱, 紫外-荧光检测器的串联方式检测杂环胺含量。结果 2-氨基-1-甲基-6-苯基-咪唑并[4,5-b]吡啶(2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, PhIP)、9H-吡啶并[4,3-b]吲哚(9H-pyrido[4,3-b]indole, Norharman)、1-甲基-9H-吡啶并[4,3-b]吲哚(1-methyl-9H-pyrido[4,3-b]indol, Harman)、2-氨基-9H-吡啶并[2,3-b]吲哚(2-amine-9H-pyrido[2,3-b]indol, AaC)在0.1~500 μg/L范围呈良好的线性关系, 2-氨基-3-甲基咪唑并[4,5-f]喹啉(2-amino-3-methylimidazo(4,5-f)quinoline, IQ)在0.1~1000 μg/L范围呈良好的线性关系, 相关系数r2均大于99.3%, 加标回收率为74.06%~93.77% (n=6), 相对标准偏差(relative standard deviation, RSD)为3.41%~5.93% (n=6)。结论 该方法简便快速、结果准确、灵敏度高, 适合于肉制品中的杂环胺含量的检测。通过检测结果可知不同原料中5种杂环胺含量不同, 相同原料烧烤条件不同, 产生的杂环胺含量也不相同。     

超高效液相色谱紫外-柱前衍生法测定肉制品中的9种生物胺

摘 要：目的 建立超高效液相色谱紫外-柱前衍生法测定肉制品中的9种生物胺。方法 实验选取西式火腿肠为样品,以三氯乙酸/高氯酸混合溶液对样品中生物胺进行提取,调节提取液于pH 10碱性环境下,使用1 mL的10 mg/mL丹磺酰氯溶液进行衍生化处理,60℃水浴衍生15 min,100 μL氢氧化钠中止衍生,乙酸乙酯进行最终萃取。使用超高效液相色谱仪紫外配二极管阵列检测器进行测定,采用流动相：1%乙酸乙腈-10 mmol/L乙酸铵溶液;波长：254 nm;柱温：35℃;进样量：3 μL;流速：0.3 mL/min;ACQUITY BEH C18(2.1 mm*100 mm, 1.7 μm)色谱柱进行分析。结果 在线性范围5~250mg/kg内9种生物胺的相关性均＞0.999,检出限范围为0.2~0.5 mg/kg,定量限范围为0.5~1.5 mg/kg,9种生物胺的日内精密度相对标准偏差RSD (relative standard deviation)范围为1.58%~ 5.21%,回收率均≥85%。结论 本研究所建方法具有良好的准确性、精密度、灵敏度,可为我国肉制品中生物胺的检测提供参考依据。