A rapid HPLC method for determination of Sudan dyes and Para Red in red chilli pepper

A rapid high-performance liquid chromatography (HPLC) system consisting of an ultraviolet-visible (UV–VIS) detector was developed for the separation and determination of Sudan dyes (I, II, III, and IV) and Para Red in red chilli peppers. The chromatographic separation was achieved on a reverse phase C₁₈ column with isocratic elution, using a mobile phase of acetonitrile/methanol (80:20, v/v); detector was set at 506 nm. All four Sudan dyes and Para Red were separated in less than 9 min. Among 80 red chilli peppers screened, only one of them contained 0.10, 0.04, and 0.05 mg/kg Sudans I, III, and IV, respectively. No Sudan II and Para Red were detected in any of the red chilli peppers analysed. The method was ‘in-house’ validated using red chilli peppers based on following criteria: limit of detection (LOD), limit of quantification (LOQ), recovery, repeatability, reproducibility, and linearity in red chilli peppers. Depending on the dye involved, LOD and LOQ were in the range of 1.2–5.4 and 4–18 μg/kg in red chilli, respectively. The recovery, repeatability (expressed as coefficient of variation, CV_r), and reproducibility (CV_R) varied from 89 to 98%, from 0.82 to 4.09%, and from 1.33 to 4.65%, respectively. Linearity obtained for all dyes and Para Red were all r² > 0.9999 (in the range of 0.01–5 mg/l). The applicability of the method to the determination of Sudan dyes and Para Red in red chilli peppers was demonstrated. This method has potential to be used for illegal Sudan dyes and Para Red in red chilli peppers and some foodstuffs due to its simple, reliable, rapid, and excellent precision.     

高效萃取体系RP-HPLC测定食品中的对位红和苏丹红

本文研究使用高效萃取体系,建立反相高效液相色谱(RP-HPLC)同时测定食品中对位红、苏丹红Ⅰ～Ⅳ号及苏丹红7B.样品用甲醇-乙腈-丙酮萃取液提取,采用聚硅烷C1s色谱柱(Shiseido C18 150mm×4.6 mm i.d,5μm)分离以水-乙腈为流动相,流速1.0mL/min,等度洗脱;用紫外-可见光二极管阵...     

UPLC-ESI-MS/MS analysis of Sudan dyes and Para Red in food

An analytical method for the simultaneous determination of Sudan dyes (Sudan Red G, Sudan I, Sudan II, Sudan III, Sudan Red 7B and Sudan IV) and Para Red in food by ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) was developed. Samples were extracted with acetonitrile, and water added into the extract. The supernatant was analysed by UPLC-MS/MS after refrigeration and centrifugation. The sample was separated on an Acquity BEH C₁₈ column, and detected by MS/MS with the multiple reaction monitoring mode. Matrix calibration was used for quantitative testing of the method. The linear matrix calibrations of Sudan dyes and Para Red were 2–50 and 10–250 ng g^?1, respectively, and the regression coefficients were >0.9945. The recoveries were 83.4–112.3% with good coefficients of variation of 2.0–10.8%. The limits of detection were between 0.3 and 1.4 ng g^?1 for the six Sudan dyes, and between 3.7 and 6.0 ng g^?1 for Para Red. The limits of quantification were between 0.9 and 4.8 ng g^?1 for the six Sudan dyes, and between 12.2 and 19.8 ng g^?1 for Para Red.     

高效液相色谱法测定食品中可能掺杂的16 种工业染料

建立食品中16 种工业染料的高效液相色谱检测法,包括分散红11、分散黄9、分散橙3、分散橙11、分散红1、分散红9、分散黄54、分散橙37、分散橙61、苏丹红1、分散黄7、分散橙149、苏丹红2、苏丹红3、苏丹红7B、苏丹红4。选择糖果、巧克力、番茄酱与番茄沙司为基质,使用Agilent ZORBAX SB-C18色谱柱进行分离,流速为0.8 mL/min,流动相为乙腈和水,验证过程中确定检出限为0.26～0.88 mg/kg、定量限为0.82～2.52 mg/kg,16 种染料的回收率在81.0%～110.3%之间,相对标准偏差在0.3%～7.5%之间。表明方法稳定可靠,可用于日常的非法添加染料的测定和筛查。     

Rapid Analysis of Multiple Sudan Dyes in Chili Flakes Using Surface-Enhanced Raman Spectroscopy Coupled with Au–Ag Core-Shell Nanospheres

Sudan dyes are often illegally added as colorants into a variety of foodstuffs and have been tied to many food safety issues. In this study, surface-enhanced Raman spectroscopy (SERS) coupled with Au–Ag core-shell nanospheres (Au@Ag) was applied to analyze standard solutions of Sudan I–IV and Sudan dyes in chili flakes. With the use of 90 ± 5 nm Au@Ag (Au seed 20 ± 2 nm) as SERS substrate, the lowest detectible concentrations for Sudan I and II were 0.10 mg/L, for Sudan III was 0.08 mg/L, and for Sudan IV was 0.2 mg/L. The use of principal component analysis (PCA) could successfully classify different Sudan dyes based upon the SERS spectra of their standard solutions. For chili flakes, the use of acetonitrile as extraction solvent led to an overall higher sensitivity for analysis of Sudan dyes with SERS method compared to that of methanol, ethanol, and n-hexane. The lowest detectible concentrations for Sudan I–III in chili flakes were 1 mg/kg and for Sudan IV was 2 mg/kg, which were about ten times as much as that for their standard solutions due to the interference of non-target compounds from sample matrices. Partial least squares (PLS) models developed for quantitative analyses showed relatively high linear correlation between the actual and predicted amounts of Sudan dyes in chili flakes (R ² _cv = 0.869–0.959). The results showed great potential of applying Au@Ag as SERS substrate for qualitative and quantitative analysis of Sudan I–IV with simplified sample preparation method.     

基质固相分散-高效液相色谱法测定川味香肠中的苏丹红染料

建立了川味香肠中苏丹红I～IV的基质固相分散(MSPD)-高效液相色谱(HPLC)分析方法。样品以无水硫酸钠作为分散基体,研磨均匀后与中性氧化铝同时装柱,正己烷淋洗净化,以丙酮-正己烷(5∶95,V/V)溶液洗脱。用InertsilODS-spC18柱(4.6mm×250mm,5μm)进行分离,流动相A为含0.1%甲酸的甲醇,流动相B为0.02mol/L的乙酸铵溶液(A∶B=85∶15,V/V),等度洗脱,柱温40℃。二极管阵列检测器检测,检测波长为490nm,利用保留时间和光谱图定性,外标法定量。4种苏丹红染料在0.10～50.00μg/mL范围内线性关系良好,相关系数均大于0.9999,苏丹红I、II、III、IV的检出限(LOD)分别为0.008～0.011mg/kg(信噪比S/N=3)。在添加浓度为5.0～25.0mg/kg范围内平均回收率达85.54%～94.66%,相对标准偏差(RSD)为0.87%～4.23%(n=6)。     

Discovery and structural elucidation of the illegal azo dye Basic Red 46 in sumac spice

An unknown red dye was discovered in a sumac spice sample during routine analysis for Sudan dyes. LC-DAD and LC-MS/MS did not reveal the identity of the red substance. Nevertheless, using LC-high-resolution MS and isotope ratio comparisons the structure was identified as Basic Red 46. The identity of the dye was further confirmed by comparison with a commercial hair-staining product and two textile dye formulations containing Basic Red 46. Analogous to the Sudan dyes, Basic Red 46 is an azo dye. However, some of the sample clean-up methodology utilised for the analysis of Sudan dyes in food prevents its successful detection. In contrast to the Sudan dyes, Basic Red 46 is a cation. Its cationic properties make it bind strongly to gel permeation columns and silica solid-phase extraction cartridges and prevent elution with standard eluents. This is the first report of Basic Red 46 in food. The structure elucidation of this compound as well as the disadvantages of analytical methods focusing on a narrow group of targeted analytes are discussed.     

Discovery and structural elucidation of the illegal azo dye Basic Red 46 in sumac spice

An unknown red dye was discovered in a sumac spice sample during routine analysis for Sudan dyes. LC-DAD and LC-MS/MS did not reveal the identity of the red substance. Nevertheless, using LC-high-resolution MS and isotope ratio comparisons the structure was identified as Basic Red 46. The identity of the dye was further confirmed by comparison with a commercial hair-staining product and two textile dye formulations containing Basic Red 46. Analogous to the Sudan dyes, Basic Red 46 is an azo dye. However, some of the sample clean-up methodology utilised for the analysis of Sudan dyes in food prevents its successful detection. In contrast to the Sudan dyes, Basic Red 46 is a cation. Its cationic properties make it bind strongly to gel permeation columns and silica solid-phase extraction cartridges and prevent elution with standard eluents. This is the first report of Basic Red 46 in food. The structure elucidation of this compound as well as the disadvantages of analytical methods focusing on a narrow group of targeted analytes are discussed.     

Dye use in fresh meat preparations and meat products: a survey by a validated method based on HPLC-UV-diode array detection as a contribution to risk assessment

The use of dyes in meats has to be monitored, due to food safety concerns. The presence of twelve dyes (Amaranth, Ponceau 4R, Carmine, Ponceau SX, Ponceau 3R, Allura Red AC, Carmoisine, Erythrosine, Sudan I, Sudan II, Sudan III and Sudan IV) in meat was investigated in 130 meat product and fresh meat preparation samples, and the dietary exposure was estimated. Carmine and Ponceau 4R were the identified dyes. Except for one sample of salami, they were regularly indicated on the label, and their concentrations were in the range 1.3–8.1 and 6.2–86.4 mg kg⁻¹ for Ponceau 4R and Carmine, respectively. Concerning fresh meat preparations, Carmine was detected in three samples, confirming that this dye is still used, although it is no longer authorised in these products. Regarding dietary exposure estimate, the daily intake from meat product consumption resulted lower than 1% of the ADI, both for Carmine and Ponceau 4R.     

三种专用固相萃取柱-高效液相色谱法检测食品中苏丹红含量

建立了专用固相萃取柱-高效液相色谱法检测食品中苏丹红Ⅰ、Ⅱ、Ⅲ、Ⅳ的方法。样品经过正己烷提取,通过苏丹红专用固相萃取柱净化和富集,40℃水浴旋蒸至干后用甲基叔丁基醚-甲醇(体积比4:6)溶解定容,借助Waters Symmetry C₁₈色谱柱(150 mm×4.6 mm,5 μm)分离,以乙腈-水为流动相梯度洗脱,二极管阵列检测器检测,外标法定量。结果表明,四种苏丹红在0.16~2.56 μg/mL内线性相关系数(r)均大于0.9999,不同食品的检出限在2.3~9.7 mg/kg之间。不同品牌的苏丹红专用固相萃取柱去除基质干扰和富集目标物的能力不同,可根据食品种类选择合适的固相萃取柱。ProElut SDH SPE柱普遍适用于不同种类食品的前处理;CNW Poly-sery MIP-SDR SPE柱适用于除辣椒粉以外的大部分食品的前处理;Cleanert Sudan SPE柱适用于浅色、低油脂食品的前处理。对六种食品加标2.0 mg/kg,经过ProElut SDH SPE柱处理后,回收率为83.7%~91.1%,相对标准偏差为2.4%~6.2%(n=6)。该方法净化和富集效果理想,与GB/T 19681-2005相比,具有操作简便、重复性好、准确度高、分析时间短、节省溶剂等特点。     

固相分散萃取-高效液相色谱法测定郫县豆瓣中四种苏丹红染料的研究

建立了郫县豆瓣中苏丹红Ⅰ～Ⅳ的固相分散萃取(SPDE)-高效液相色谱(HPLC)分析方法。样品用无水硫酸钠作为分散剂,以乙腈提取样品中的苏丹红,提取液用中性氧化铝层析柱进行净化。用Inertsil ODS-sp C18柱(4.6mm×250mm,5μm))分离,流动相A为乙腈,流动相B为0.1%甲酸水溶液(A∶B为90∶10,V/V),等度洗脱,流速1mL/min,柱温40℃。二极管阵列检测器检测,检测波长为517nm,利用保留时间和光谱图定性,外标法定量。4种苏丹红染料在0.10～20.00μg/mL范围内线性关系良好,相关系数均大于0.9999,苏丹红Ⅰ～Ⅳ的检出限(LOD)分别为0.009～0.013mg/kg(信噪比S/N=3)。在添加浓度为0.5～10.0mg/kg范围内平均回收率达81.67%～93.28%,相对标准偏差(RSD)为1.07%～4.61%(n=6)。     

紫外-可见分光光度法快速检测蛋黄中苏丹红Ⅳ的研究

采用紫外一可见分光光度计对新鲜鸭蛋蛋黄的石油醚一丙酮的提取液进行光谱扫描,建立了纯蛋黄液、标准苏丹红Ⅳ样品以及含有苏丹红Ⅳ的蛋黄液的光谱扫描曲线.通过对光谱扫描曲线的相似度比较及对光谱扫描原始曲线图的数学处理,分别运用波长差和一级微分两种方法,快速检测出新鲜鸭蛋黄中苏丹红Ⅳ.实验结果表明.波长差法的回收率高于一次微分法,而且数值分布相对集中,在对被检测样品中的苏丹红Ⅳ的定量上的精确性和稳定性优于一次微分法,而一次微分法则可直观对苏丹红Ⅳ定性.运用波长差法计算样品RSD(%),得备检样品加标苏丹红Ⅳ 2μg/mL的RSD(%)为14.2,平均回收率为106.01%,加标4μg/mL的RSD(%)为9.4,平均回收率为105.68%;运用一次微分法进行计算,得备检样品加标苏丹红Ⅳ 2μg/mL的RSD(%)为10.41,平均回收率为82.46%,加标4μg/mL的RSD(%)为6.73,平均回收率为94.21%.数据表明样品中加标苏丹红Ⅳ浓度越大.该方法对样品检测的稳定性越好.     

固相萃取-液质联用测定辣椒粉中苏丹红

该试验建立了分子印迹固相萃取-超高效液相色谱-串联质谱法检测辣椒粉中苏丹红Ⅰ、Ⅱ、Ⅲ、Ⅳ的方法。辣椒粉样品经过正己烷提取,氮吹浓缩,苏丹红专用分子印迹固相萃取柱净化和富集,以体积分数为0.2%的甲酸水溶液和乙腈为流动相梯度洗脱,经Agilent ZORBAX Eclipse Plus C₁₈(2.1 mm×50 mm,1.8μm)柱分离,ESI+电喷雾模式扫描,MRM多反应模式监测,外标法定量。结果显示,该方法下4种苏丹红的检出限均为5μg/kg,4种化合物在0~50μg/L质量浓度范围内线性关系良好,相关系数均在0.9990以上;当加标浓度为10,50和100μg/kg时,回收率在86.5%~102.2%之间,相对标准偏差均小于6.0%。该方法可为辣椒粉中4种苏丹红的定性定量分析提供参考。     

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.     

Determination of Trace Para Red Residues in Foods through On-Line Molecularly Imprinted Solid Phase Extraction Coupled with High-Performance Liquid Chromatography

ABSTRACT:  In this article, we prepared a novel imprinted polymer by a room temperature ionic liquid-mediated surface molecular imprinting technique in combination with a sol–gel process. This polymer was characterized by static and kinetic adsorption experiments and exhibited good recognition ability and offered fast kinetics for the adsorption of Para Red. A simple and sensitive analytical method, based on the coupling of molecularly imprinted solid phase extraction with high-performance liquid chromatography (HPLC), had been developed for determination of trace Para Red. With a loading flow rate of 0.42 mL min⁻¹ for 25 mL, an enrichment factor of 1061 was achieved. Under the selected experimental condition, the detection limit (S/N = 3) of Para Red was 6.6 ng L⁻¹, and the peak area precision (RSD) for 5 replicate detections of 0.15 μg L⁻¹ Para Red was 4.1%. The applicability of this method for determination of the blank chili sauce sample, spiked with Para Red at 5 to 25 ng g⁻¹ levels, was demonstrated, with recoveries ranging from 86% to 95%.
Practical Application: In this paper, a simple and sensitive analytical method, based on the coupling of molecularly imprinted solid phase extraction with high performance liquid chromatography, had been developed for determination of trace Para Red. It was applied to the analysis of spiking Para Red in chili sauce sample with satisfactory recovery and repeatability. This proposed method has the potential to be used for monitoring the illegal addition of Para Red in foods in the future due to its simple, reliable, rapid, and excellent precision.     

高效液相色谱-线性离子阱质谱法测定辣椒制品中23种工业染料

目的 建立辣椒酱和辣椒面中23种违禁工业染料的高效液相色谱-线性离子阱质谱(HPLC-LIT-MS)测定方法.方法 样品经乙腈超声提取,目标化合物在梯度洗脱程序下经RP-Amide色谱柱分离后,采用HPLC-LIT-MS进行二级质谱选择反应监测(SRM)模式进行测定.结果 辣椒酱和辣椒面基质中23种工业染料添加水平为0.3、0.6和1.2 mg/kg时,回收率分别为73.9％～133.3％和77.2％ ～128.4％,检出限(LODs)为0.5～ 56.0 μg/kg,定量限(LOQs)为2.0～ 186.0 μg/kg.结论 该方法快速简便,精密度和准确度均满足辣椒酱和辣椒面中23种工业染料的痕量分析要求.对73份辣椒制品进行检测,两份样品检出罗丹明B、碱性橙和苏丹红Ⅳ.     

Exposure assessment to Sudan dyes through consumption of artificially coloured chilli powders in India

In view of detection of Sudan dyes in chillies, the Spices Board of India formulated a mandatory testing programme in all chilli consignments exported from India. However, no surveillance data on use and levels of Sudan dyes in domestic chilli products are available in India. Hence chilli powders were monitored to check the magnitude of artificial coloration and the likely exposure assessment of Sudan dyes. Among 800 non‐branded, loose chilli powder samples, over 66% were found to employ artificial colouration while only 33% were free from Sudan dyes. None of the branded chilli powder samples was found to contain Sudan dyes. The maximum content of Sudan I noted was as high as 11.8 mg g^?1, which at the rough per capita consumption estimates of 0.5–1.0 g chilli powder per day amounts to an intake of 5.8–11.8 mg of Sudan I. This may lead to unwarranted health consequences, hence regular monitoring of the chilli powder samples is advisable to safeguard the health of unsuspecting consumers.     

Method development and survey of Sudan I–IV in palm oil and chilli spices in the Washington,DC, area

Sudan I, II, III and IV dyes are banned for use as food colorants in the United States and European Union because they are toxic and carcinogenic. These dyes have been illegally used as food additives in products such as chilli spices and palm oil to enhance their red colour. From 2003 to 2005, the European Union made a series of decisions requiring chilli spices and palm oil imported to the European Union to contain analytical reports declaring them free of Sudan I–IV. In order for the USFDA to investigate the adulteration of palm oil and chilli spices with unapproved colour additives in the United States, a method was developed for the extraction and analysis of Sudan dyes in palm oil, and previous methods were validated for Sudan dyes in chilli spices. Both LC-DAD and LC-MS/MS methods were examined for their limitations and effectiveness in identifying adulterated samples. Method validation was performed for both chilli spices and palm oil by spiking samples known to be free of Sudan dyes at concentrations close to the limit of detection. Reproducibility, matrix effects, and selectivity of the method were also investigated. Additionally, for the first time a survey of palm oil and chilli spices was performed in the United States, specifically in the Washington, DC, area. Illegal dyes, primarily Sudan IV, were detected in palm oil at concentrations from 150 to 24 000 ng ml^–1. Low concentrations (< 21 µg kg^–1) of Sudan dyes were found in 11 out of 57 spices and are most likely a result of cross-contamination during preparation and storage and not intentional adulteration.     

A new liquid–liquid extraction method for determination of 6 azo-dyes in chilli products by high-performance liquid chromatography

A liquid–liquid extraction method was developed for purifying and enriching the sample of 6 azo-dyes (including Para red, Sudan red G, Sudan I, Sudan II, Sudan III and Sudan IV) by formic acid and chloroform. The results indicated that formic acid can facilitate the solution of fat-soluble azo-dyes, which was helpful for the separation of the fat-soluble interferences from the samples. The extracting conditions of 6 azo-dyes were also optimised. And the proposed extraction method was evaluated by the determination of 6 azo-dyes in chilli products. The mean recoveries of 6 azo-dyes were from 94.1% to 99.2% for chilli powder and from 94.2% to 98.6% for chilli spice. The CCα and CCβ obtained for 6 azo-dyes were in the range of 1.7–2.1 and 2.8–3.4 μg/kg for chilli spice and chilli powder. The inexpensive and simple method was acceptable for routine monitoring 6 azo-dyes in chilli products.     

食品中铝含量测定方法的研究

目的通过对食品中总铝含量测定方法的研究,探讨如何应用现行标准检测方法为含铝食品添加剂的管理提供更好的技术支持。方法应用电感耦合等离子体光谱技术和质谱技术,对不同基质的生物成分标准物质中铝含量进行定量分析,比较不同酸消解体系对总铝含量测定值的影响,并对面制品、水产品等样品进行铝本底值的测定。结果单硝酸体系并不能将样品完全消解,需加入适量氢氟酸消解才能准确测定总铝含量。大量食品中铝存在本底值,且部分贝类、海蜇等水产品中总铝的含量已经超过100 mg/kg的国家限量标准。结论应用铝的国家标准检测方法测定总铝含量,其结果偏低,需采用硝酸-氢氟酸消解体系方可准确测定总铝含量,如果相关规定中铝含量不以"总铝含量计",现行标准检测方法才可得以更好地应用,且不同基质食品有着不同铝的本底值,不同的检测方法也有不同的方法检出限,在管理含铝食品添加剂使用情况时,需综合考虑所使用的检测方法及样品基质本底情况进行科学的判别。