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附件二：检测方法 ( 英文 )
European Commission
HEALTH & CONSUMER PROTECTION DIRECTORATE-GENERAL
Directorate D – Food safety: production and distribution chain
D3 – chemical and physical risks – surveillance
NEWS notification: 03/99
Subject: Corrected method for the detection of Sudan
The previous method under NEWS 3/92 is hereby corrected (the mistake was in point 4.12.1: the correct figure is 100ml instead of 50ml).
The commission has received the following notification from France:
Analysis and dosage of the colorants Sudan and Bixin in chilli powder and pepper-based products
O. Vérétout, L. Demesse [1] , L. Szymanski [2]
1. Field of Application
The method describes the detection of Sudan 1, Sudan II, Sudan III, Sudan IV, Sudan Orange B, Sudan Red 7B and Bixin in pepper-based products .
2. Definition
Sudan colorants are non-animal synthetic colorants used in the chemical industry to colour oils, waxes, floor waxes, soaps….they are generally insoluble in water but soluble in organic solvents。
Bixin is a food colorant not permitted for use in chilli powder and spices .
3. Principal
The colorants are extracted by acetonitrile, after filtration, the extract is chromatographed in HPLC in reverse phase (RP). A variable UV/VIS detector is used for identification and quantification.
4. Reagents and products.
Unless indicated to the contrary, use only reagents of analytic quality and distilled water, demineralised water or water of equivalent quality .
4.1 Acetonitrile, of HPLC quality.
4.2 Water, of HPLC quality.
4.3 Ice cold acetic acid
4.4 Chloroform
4.5 Sudan 1, Aldrich Chemical Company
4.6 Sudan II, Acros organics
4.7 Sudan III, Acros organics
4.8 Sudan IV, Acros organics
4.9 Sudan Orange B Acros organics
4.10 Sudan Red 7B, Acros organics
4.11 Bixin, Extra synthetic
4.12 Working solutions
4.12.1 Stock solutions
Weigh 50.0 mg of pure colorant (taking account of the stated purity of the product) and transfer it into a 100ml phial in the following manner:
Colorants
Transfer and putting into solution
QS 100ml
Sudan 1
Acetonitrile
Acetonitrile
Sudan II
Acetonitrile
Acetonitrile
Sudan III
Chloroform
Acetonitrile
Sudan IV
Chloroform
Acetonitrile
Sudan Orange G?
Acetonitrile
Acetonitrile
Sudan Red 7B
Acetonitrile
Acetonitrile
Bixin
Chloroform
Chloroform
4.12.2 Working reference solutions
Take 5 ml of the stock solutions in a single phial of 50 ml, bring up to reference level with acetonitrile.
From this solution set the calibration scale.
Transfer by pipette 0.5ml, 1ml, 2.5ml, 4ml, 5ml of this solution to a series of 50ml phials and bring up to reference level with acetonitrile.
These solutions contain respectively 0.5μg/ml, 1 μg/ml, 2.5 μg/ml, 4 μg/ml, and 5 μg/ml in colorants.
5. Equipment
5.1 Analytical scales, accurate to the nearest 1mg and capable of reading to 0.1mg.
5.2 Stoppered Erlenmeyer flasks, 250 ml
5.3 Funnels about 10cm diameter
5.4 Measuring phials 50ml capacity
5.5 Pipettes of 0.5ml, 1 ml, 2.5ml, 4ml and 5ml capacity
5.6 Test tubes, 100ml
5.7 Disposable syringes 5ml capacity
5.8 Micro filters with membranes, (diameter of the pores, 0.45 μm)
5.9 Paper filters, 185mm diameter
5.10 Laboratory bladed grinder
5.11 Ultra Turrax homogeniser, armed with a dispersing tool 25mm in diameter
5.12 Liquid chromatograph, with a UV/VIS detector allowing for spectrum analysis and a pump allowing for an elution gradient to be obtained
5.13 Analytical column, LiChroCART 250-4 HPLC Cartbridge Supersher 100 RP 18.
5.14 Flasks for automatic injection
6. Preparation of the sample for testing
Carefully mix the sample (powder) in a sealed receptacle of sufficient capacity.
For solid samples grind up a sufficient quantity using the laboratory grinder.
7. Modus Operandi
7.1 Preparation of the sample for testing
7.1.1 Peppers, chilli powder
Weigh about 1g within 10mg in a ground Erlenmeyer flask and add 100ml acetonitrile using a graded test tube.
Agitate for 1 hour and filter on filter paper in a ground Erlenmeyer flask.
7.1.2 Spices in powdered form
Weigh about 5g within 10mg in a ground Erlenmeyer flask and add 100ml acetonitrile using a graded test tube.
Agitate for 1 hour and filter on filter paper in a ground Erlenmeyer flask.
7.1.3 Sauce, pepper paste, peppered oil, harissa
Weigh about 20g to within 10mg in a ground Erlenmeyer flask and add 100ml acetonitrile using a graded test tube.
Agitate for 1 hour and filter on filter paper in a ground Erlenmeyer flask .
7.1.4 Merguez sausuage, chorizo, meat
Weigh about 20g within 10mg in a ground Erlenmeyer flask and add 100ml acetonitrile using a graded test tube.
After adding acetonitrile mix for a few minutes in the Ultra Turrax to disperse the product thoroughly.
Agitate for 1 hour and filter on filter paper in a ground Erlenmeyer flask.
NB: The test samples and dilutions are adapted to the results obtained by HPLC and the quantity of pepper within the product .
7.2 Determination by HPLC
7.2.1 Elution solvents
Solvent A: Acidified water (165ml acetic acid plus 1000 ml water)
Solvent B: Acetonitrile
7.2.2 Elution gradient
Duration in minutes
Solvent A%
Solvent B%
Curve of gradient
Initial
30
70
Linear
20.0
5
95
Linear
30.0
0
100
Linear
42.0
0
100
Drip rate 0.7 ml min -1
The time from the setting up of the column to start time is determined by the level of control over the stability of the base line.
The setting up time for the column between each injection is 10 minutes.
7.2.3 Injection volume 10 μl.
7.2.4 Detector
Do a sweep with a wavelength of between 300nm and 600nm.
Three fixed wavelengths (432nm, 478nm and 520nm) are used for quantification.
7.2.5 Calibration curves
Calibration curves are produced by using 5 standard working solutions.
They are produced at a wavelength of 432nm for Sudan Orange G, 478 nm for Sudan 1, Sudan II and Bixin and 520nm for Sudan III, Sudan IV and Sudan Red B.
7.2.6 Determination.
The results obtained at 7.1 are filtered on a membrane of 0.45μm in flasks designed for use with an automatic injector.
The calibration curves are validated at each analytical series with the help of one of the working solutions.
7.3 Calculations
The level of colorant is calculated with the aid of the following equation:
R = C x V x D all over M mg/kg
Where
C is the concentration in μg/ml of the test solution read on the calibration curve.
M mass in g of the test material
V volume in ml of the test solution
D dilution of the test solution
8. Sudan I
8.1 First approach at determination of the performance parameters of the method
In line with Plan A of the Norm NF V03 110 we obtain:
The model is linear
Detection limit at 478nm:0.013μg/ml
Quantification limit at 478nm: 0.106 μg/ml.
The recuperation rates for the added powdered chilli samples is higher than 90%
This type of study is being developed for other matrices and all colorants measured out in this way.
In Annex A: Control Chromatograms and absorption spectra.
8.2 Confirmation of the presence of Sudan I by LC/MS.
In the case of a complex matrix or the setting up of a new matrix, confirmation of the presence of the molecule Sudan I is necessary.
However if the spectral analysis is not satisfactory (presence of the analyte in weak concentration or possible presence of an artefact) this technique would also be applied.
8.3 Equipment
8.3.1 Liquid chromatography, linked to an Electrospray mass spectrometer
8.3.2 Analytical column, PHENOMENEX LUNA C18 3μm 150X2 nm
8.3.3 Column Oven, regulated at 30 °C
8.4 Determination by HPLC
8.4.1 Elution solvents
Solvent A: 20% acidified water (0.1% acetic acid)
Solvent B: 80% acetonitrile
Drip rate 0.2 ml min -1
8.4.2 Injection volume: 10μl.
8.4.3 Detector
Electrospray positive
Typical settings: Power of spray: 5300V
Nozzle: 120V
Skimmer: 9,50V
Temp. of auxiliary gas: 300°C
8.4.4 Determination
The extracts obtained at 7.12 are diluted by a factor of 10 then 100 then filtered on a membrane of 0.45μm in flasks designed for automatic injection.
8.5 Results
The presence of Sudan I is confirmed by the appearance of a peak at the same retention time as the control to an accuracy of 5% variation and by obtaining a mass spectrum at 249,1022 +/- 0.01 u.
In Annex B: Chromatogram and spectrum of the control for Sudan I at 30 ng/ml:
ANNEXES A AND B: GRAPHS
[Annex A: Chromatogram at 432 nm and absorption spectra for Sudan Orange G, Sudan I, Bixin and Sudan II, Sudan III, Sudan Red 7B and Sudan IV]
[Annex B: Chromatogram and control spectrum for Sudan I at 30 ng/ml]
检测方法 ( 中文 )
欧洲委员会
健康与消费者保护综合委员会
第四分委员会―生产与流通过环节的食品安全
第三部―物理化学危害物监督
新方法声明： 03/99
主题：苏丹红检测方法的更正
以前的 3/92 新方法声明在此更正（原方法 4.12.1 存在错误：正确的数字是 100ml 而不是 50ml ）
代办处已收到来自法国的方法声明内容如下：
辣椒粉及以辣椒为主要成分的产品中苏丹红和胭脂树橙的含量分析
O.Veretout,L.Demesse,L.Szymanski
1 应用范围
本方法涉及以辣椒为主要成分的产品中苏丹红 1 号、苏丹红 2 号、苏丹红 3 号、苏丹红 4 号、苏丹橙 B 、苏丹红 7B 和胭脂树橙的检测。
2 定义
苏丹红是应用于诸如油彩、蜡、地板蜡和香皂等化工产品中的一种非生物合成着色剂，一般不溶于水易溶于有机溶剂，胭脂树橙是一种食品着色剂但不允许在辣椒粉和调味品中使用。
3 方法要点
上述着色剂经乙腈提取后，过滤，滤液用反相高效液相色谱仪进行色谱分析。以波长可变的紫外―可见检测器定性与定量。
4 试剂与标准品
除有特殊指明外，本方法中涉及试剂均为分析纯，实验用水均为蒸馏水、去离子水或相同质量的分析用水。
4.1 乙腈 色谱纯
4.2 水 色谱级
4.3 冰醋酸
4.4 氯仿
4.5 苏丹红 1 号 (Aldrich Chemical Company)
4.6 苏丹红 2 号（ Acros organics 化工合成有机物）
4.7 苏丹红 3 号（ Acros organics 化工合成有机物）
4.8 苏丹红 4 号（ Acros organics 化工合成有机物）
4.9 苏丹橙 B （ Acros organics 化工合成有机物）
4.10 苏丹红 7B （ Acros organics 化工合成有机物）
4.11 胭脂树橙 ( 特殊合成产品 )
4.12 标准溶液
4.12.1 标准贮备液
称取 50.0mg 着色剂（按产品标明的纯度折算成纯着色剂）并按以下方式移入 100ml 容量瓶定容。
着色剂
溶解和转移溶剂
定容溶剂
苏丹红 1 号
乙腈
乙腈
苏丹红 2 号
乙腈
乙腈
苏丹红 3 号
氯仿
乙腈
苏丹红 4 号
氯仿
乙腈
苏丹橙 B
乙腈
乙腈
苏丹红 7B
乙腈
乙腈
胭脂树橙
氯仿
氯仿
4.12.1 标准工作液
取上述标准贮备液各 5ml 移入 50ml 容量瓶中以乙腈定容，再分别从以上容量瓶中吸取 0.5ml 、 1ml 、 2.5ml 、 4ml 和 5ml 溶液移入 50ml 容量瓶中以乙腈定容，此时溶液中各种着色剂的浓度分别为 0.5,1,2.5,4 和 5 μ g/ml 。
5 仪器与设备
5.1 万分之一天平
5.2 250ml 具塞三角瓶
5.3 直径 10cm 的漏斗
5.4 50ml 容量瓶
5.5 5ml 移液枪
5.6 100ml 量筒
5.7 5ml 一次性注射器
5.8 0.45 μ m滤膜
5 . 9 185mm滤纸
5.10 打浆机
5.11 Ultra Turrax 均质机
5.12 配有紫外 - 可见检测器的高效液相色谱仪
5.13 色谱柱 LiChroCART250-4HPLC Cartbridge Supersher 100RP18
5.14 进样瓶
6 样品制备
将采集样品放入一个容量较大的密闭容器中混合均匀。对于固体样品要用打浆机或粉碎机磨细。
7 操作方法
7.1 样品处理
7.1.1 甜椒或红辣椒粉
称取 10g （准确至 0.01g ）样品于三角瓶中，用量筒加入 100ml 乙腈。
7.1.2 粉状调味品
称取 5g （准确至 0.01g ）样品于三角瓶中，用量筒加入 100ml 乙腈。
7.1.3 调味辣椒酱、原味辣椒酱、辣椒油等
称取 20g （准确至 0.01g ）样品于三角瓶中，用量筒加入 100ml 乙腈。
7.1.4 Merguez 香肠、西班牙加调料的口利左香肠和肉制品
称取 20g （准确至 0.01g ）样品于三角瓶中，用量筒加入 100ml 乙腈。
之后在 Ultra Turrax 中充分混合数分钟，振荡 1 小时后过滤于三角瓶中。检测样品取样量和其稀释液浓度要视产品中的辣椒含量而定，以符合液相色谱检测限的要求。
7.2 高效液相色谱测定
7.2.1 流动相
溶剂 A ：酸性水溶液（ 165ml 乙酸溶于 1000ml 水中）
溶剂 B ：乙腈
7.2.2 梯度洗脱
时间
溶剂 A ％
溶剂 B ％
梯度曲线
0
30
70
线性
20.0
5
95
线性
30.0
0
100
线性
42.0
0
100
流速 :0.7ml/min
基线稳定后开始进样
两次进样间隙时间为 10 分钟
7.2.3 进样量 :10 μ l
7.2.4检测波长
在 300nm 到 600nm 波长范围进行扫描 , 确定三个测定波长（ 432nm,478nm 和 520nm ）
7.2.5 标准曲线
用 5 个标准工作溶液的测定值绘制标准曲线 , 各种色素的标准曲线分别在最大吸收波长处由 5 点回归计算（苏丹橙 B 在 432nm 波长有最大吸收，苏丹红 1 号 , 苏丹红 2 号 和胭脂树橙在 478nm波长有最大吸收和 苏丹红 3 号，苏丹红 4 号和苏丹红 B 在 520nm 波长有最大吸收）。
将 7.1 制好的样过 0.45 μ m 的膜装入自动进样器的小瓶后进行液相色谱测定得到结果。
标准回归曲线经过每次实验配制的系列标准溶液测定结果的验证。
7.3 计算
着色剂含量按以下公式计算：
R=C×V×D/M 单位： mg/kg
C- 样品中待测组分的浓度 , 单位： μ g/ml
M- 检测样品取样量（ g ）
V- 样品溶液体积（ ml ）
D- 样品溶液的稀释倍数
8 苏丹红 1 号
8.1 第一步是确定方法的操作条件
应用 Norm NF V03 110 获得以下操作条件：
标准曲线模型是线性的
478nm 下的检测限是 0.013 μ g/ml
478nm 下定量的最低浓度为： 0.106 μ g/ml
在辣椒粉样品中的添加回收率高于 90%
对于其它食品基质中的色素方法也进行了研究并可应用类似方法对所有着色剂进行检测。
8.2 应用 LC/MS 确证苏丹红 1 号
对于复杂食品基质本底或一种新的基质本底，确证苏丹红 1 号分子的存在是非常必要的。
如果光谱分析结果不令人满意（如待分析物浓度较低或可能存在结构类似物时）也可以应用这种技术进行确证。
8.3 设备
8.3.1 液相色谱与电喷雾离子化质谱仪联用
8.3.2 色谱柱： PHENOMENEX LUNA C18 3 μ m 150×2nm
8.3.3 柱温箱温度调至30℃
8.4 HPLC测定
8.4.1 流动相
溶剂 A:20% 酸性水溶液 (0.1% 乙酸溶液 )
溶剂 B:80% 乙腈
流速 :0.2ml/min
8.4.2 进样量 :10 μ l
8.4.3 检测器
正电喷雾
设定条件 : 喷雾电压 :5300V
喷雾口电压 :120V
周边电压 :9.50V
辅助气体温度 :300 ℃
8.4.4 测定
步骤7.12 取得的提取物先稀释 10 倍后再稀释 100 倍后经 0.45 μ膜过滤于自动进样瓶中 .
8.5 结果
样品中苏丹红 1号组分与样准品出峰时间基本一致相对误差为5%,并经质谱检测由m/Z为249和1022的离子定性,误差为0.01u.
[1] Laboratoire de Paris-Massy, 25, Av de la republique, 91744 Massy Cedex, CLHP UVIVIS section
[2] Laboratoire de Paris-Massy, 25, Av de la republique, 91744 Massy Cedex, LCIMS section
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附件二：检测方法 ( 英文 )
European Commission
HEALTH & CONSUMER PROTECTION DIRECTORATE-GENERAL
Directorate D – Food safety: production and distribution chain
D3 – chemical and physical risks – surveillance
NEWS notification: 03/99
Subject: Corrected method for the detection of Sudan
The previous method under NEWS 3/92 is hereby corrected (the mistake was in point 4.12.1: the correct figure is 100ml instead of 50ml).
The commission has received the following notification from France:
Analysis and dosage of the colorants Sudan and Bixin in chilli powder and pepper-based products
O. Vérétout, L. Demesse [1] , L. Szymanski [2]
1. Field of Application
The method describes the detection of Sudan 1, Sudan II, Sudan III, Sudan IV, Sudan Orange B, Sudan Red 7B and Bixin in pepper-based products .
2. Definition
Sudan colorants are non-animal synthetic colorants used in the chemical industry to colour oils, waxes, floor waxes, soaps….they are generally insoluble in water but soluble in organic solvents。
Bixin is a food colorant not permitted for use in chilli powder and spices .
3. Principal
The colorants are extracted by acetonitrile, after filtration, the extract is chromatographed in HPLC in reverse phase (RP). A variable UV/VIS detector is used for identification and quantification.
4. Reagents and products.
Unless indicated to the contrary, use only reagents of analytic quality and distilled water, demineralised water or water of equivalent quality .
4.1 Acetonitrile, of HPLC quality.
4.2 Water, of HPLC quality.
4.3 Ice cold acetic acid
4.4 Chloroform
4.5 Sudan 1, Aldrich Chemical Company
4.6 Sudan II, Acros organics
4.7 Sudan III, Acros organics
4.8 Sudan IV, Acros organics
4.9 Sudan Orange B Acros organics
4.10 Sudan Red 7B, Acros organics
4.11 Bixin, Extra synthetic
4.12 Working solutions
4.12.1 Stock solutions
Weigh 50.0 mg of pure colorant (taking account of the stated purity of the product) and transfer it into a 100ml phial in the following manner:
Colorants |
Transfer and putting into solution |
QS 100ml |
Sudan 1 |
Acetonitrile |
Acetonitrile |
Sudan II |
Acetonitrile |
Acetonitrile |
Sudan III |
Chloroform |
Acetonitrile |
Sudan IV |
Chloroform |
Acetonitrile |
Sudan Orange G? |
Acetonitrile |
Acetonitrile |
Sudan Red 7B |
Acetonitrile |
Acetonitrile |
Bixin |
Chloroform |
Chloroform |
4.12.2 Working reference solutions
Take 5 ml of the stock solutions in a single phial of 50 ml, bring up to reference level with acetonitrile.
From this solution set the calibration scale.
Transfer by pipette 0.5ml, 1ml, 2.5ml, 4ml, 5ml of this solution to a series of 50ml phials and bring up to reference level with acetonitrile.
These solutions contain respectively 0.5μg/ml, 1 μg/ml, 2.5 μg/ml, 4 μg/ml, and 5 μg/ml in colorants.
5. Equipment
5.1 Analytical scales, accurate to the nearest 1mg and capable of reading to 0.1mg.
5.2 Stoppered Erlenmeyer flasks, 250 ml
5.3 Funnels about 10cm diameter
5.4 Measuring phials 50ml capacity
5.5 Pipettes of 0.5ml, 1 ml, 2.5ml, 4ml and 5ml capacity
5.6 Test tubes, 100ml
5.7 Disposable syringes 5ml capacity
5.8 Micro filters with membranes, (diameter of the pores, 0.45 μm)
5.9 Paper filters, 185mm diameter
5.10 Laboratory bladed grinder
5.11 Ultra Turrax homogeniser, armed with a dispersing tool 25mm in diameter
5.12 Liquid chromatograph, with a UV/VIS detector allowing for spectrum analysis and a pump allowing for an elution gradient to be obtained
5.13 Analytical column, LiChroCART 250-4 HPLC Cartbridge Supersher 100 RP 18.
5.14 Flasks for automatic injection
6. Preparation of the sample for testing
Carefully mix the sample (powder) in a sealed receptacle of sufficient capacity.
For solid samples grind up a sufficient quantity using the laboratory grinder.
7. Modus Operandi
7.1 Preparation of the sample for testing
7.1.1 Peppers, chilli powder
Weigh about 1g within 10mg in a ground Erlenmeyer flask and add 100ml acetonitrile using a graded test tube.
Agitate for 1 hour and filter on filter paper in a ground Erlenmeyer flask.
7.1.2 Spices in powdered form
Weigh about 5g within 10mg in a ground Erlenmeyer flask and add 100ml acetonitrile using a graded test tube.
Agitate for 1 hour and filter on filter paper in a ground Erlenmeyer flask.
7.1.3 Sauce, pepper paste, peppered oil, harissa
Weigh about 20g to within 10mg in a ground Erlenmeyer flask and add 100ml acetonitrile using a graded test tube.
Agitate for 1 hour and filter on filter paper in a ground Erlenmeyer flask .
7.1.4 Merguez sausuage, chorizo, meat
Weigh about 20g within 10mg in a ground Erlenmeyer flask and add 100ml acetonitrile using a graded test tube.
After adding acetonitrile mix for a few minutes in the Ultra Turrax to disperse the product thoroughly.
Agitate for 1 hour and filter on filter paper in a ground Erlenmeyer flask.
NB: The test samples and dilutions are adapted to the results obtained by HPLC and the quantity of pepper within the product .
7.2 Determination by HPLC
7.2.1 Elution solvents
Solvent A: Acidified water (165ml acetic acid plus 1000 ml water)
Solvent B: Acetonitrile
7.2.2 Elution gradient
Duration in minutes |
Solvent A% |
Solvent B% |
Curve of gradient |
Initial |
30 |
70 |
Linear |
20.0 |
5 |
95 |
Linear |
30.0 |
0 |
100 |
Linear |
42.0 |
0 |
100 |
Drip rate 0.7 ml min -1
The time from the setting up of the column to start time is determined by the level of control over the stability of the base line.
The setting up time for the column between each injection is 10 minutes.
7.2.3 Injection volume 10 μl.
7.2.4 Detector
Do a sweep with a wavelength of between 300nm and 600nm.
Three fixed wavelengths (432nm, 478nm and 520nm) are used for quantification.
7.2.5 Calibration curves
Calibration curves are produced by using 5 standard working solutions.
They are produced at a wavelength of 432nm for Sudan Orange G, 478 nm for Sudan 1, Sudan II and Bixin and 520nm for Sudan III, Sudan IV and Sudan Red B.
7.2.6 Determination.
The results obtained at 7.1 are filtered on a membrane of 0.45μm in flasks designed for use with an automatic injector.
The calibration curves are validated at each analytical series with the help of one of the working solutions.
7.3 Calculations
The level of colorant is calculated with the aid of the following equation:
R = C x V x D all over M mg/kg
Where
C is the concentration in μg/ml of the test solution read on the calibration curve.
M mass in g of the test material
V volume in ml of the test solution
D dilution of the test solution
8. Sudan I
8.1 First approach at determination of the performance parameters of the method
In line with Plan A of the Norm NF V03 110 we obtain:
The model is linear
Detection limit at 478nm:0.013μg/ml
Quantification limit at 478nm: 0.106 μg/ml.
The recuperation rates for the added powdered chilli samples is higher than 90%
This type of study is being developed for other matrices and all colorants measured out in this way.
In Annex A: Control Chromatograms and absorption spectra.
8.2 Confirmation of the presence of Sudan I by LC/MS.
In the case of a complex matrix or the setting up of a new matrix, confirmation of the presence of the molecule Sudan I is necessary.
However if the spectral analysis is not satisfactory (presence of the analyte in weak concentration or possible presence of an artefact) this technique would also be applied.
8.3 Equipment
8.3.1 Liquid chromatography, linked to an Electrospray mass spectrometer
8.3.2 Analytical column, PHENOMENEX LUNA C18 3μm 150X2 nm
8.3.3 Column Oven, regulated at 30 °C
8.4 Determination by HPLC
8.4.1 Elution solvents
Solvent A: 20% acidified water (0.1% acetic acid)
Solvent B: 80% acetonitrile
Drip rate 0.2 ml min -1
8.4.2 Injection volume: 10μl.
8.4.3 Detector
Electrospray positive
Typical settings: Power of spray: 5300V
Nozzle: 120V
Skimmer: 9,50V
Temp. of auxiliary gas: 300°C
8.4.4 Determination
The extracts obtained at 7.12 are diluted by a factor of 10 then 100 then filtered on a membrane of 0.45μm in flasks designed for automatic injection.
8.5 Results
The presence of Sudan I is confirmed by the appearance of a peak at the same retention time as the control to an accuracy of 5% variation and by obtaining a mass spectrum at 249,1022 +/- 0.01 u.
In Annex B: Chromatogram and spectrum of the control for Sudan I at 30 ng/ml:
ANNEXES A AND B: GRAPHS
[Annex A: Chromatogram at 432 nm and absorption spectra for Sudan Orange G, Sudan I, Bixin and Sudan II, Sudan III, Sudan Red 7B and Sudan IV]
[Annex B: Chromatogram and control spectrum for Sudan I at 30 ng/ml]
检测方法 ( 中文 )
欧洲委员会
健康与消费者保护综合委员会
第四分委员会―生产与流通过环节的食品安全
第三部―物理化学危害物监督
新方法声明： 03/99
主题：苏丹红检测方法的更正
以前的 3/92 新方法声明在此更正（原方法 4.12.1 存在错误：正确的数字是 100ml 而不是 50ml ）
代办处已收到来自法国的方法声明内容如下：
辣椒粉及以辣椒为主要成分的产品中苏丹红和胭脂树橙的含量分析
O.Veretout,L.Demesse,L.Szymanski
1 应用范围
本方法涉及以辣椒为主要成分的产品中苏丹红 1 号、苏丹红 2 号、苏丹红 3 号、苏丹红 4 号、苏丹橙 B 、苏丹红 7B 和胭脂树橙的检测。
2 定义
苏丹红是应用于诸如油彩、蜡、地板蜡和香皂等化工产品中的一种非生物合成着色剂，一般不溶于水易溶于有机溶剂，胭脂树橙是一种食品着色剂但不允许在辣椒粉和调味品中使用。
3 方法要点
上述着色剂经乙腈提取后，过滤，滤液用反相高效液相色谱仪进行色谱分析。以波长可变的紫外―可见检测器定性与定量。
4 试剂与标准品
除有特殊指明外，本方法中涉及试剂均为分析纯，实验用水均为蒸馏水、去离子水或相同质量的分析用水。
4.1 乙腈 色谱纯
4.2 水 色谱级
4.3 冰醋酸
4.4 氯仿
4.5 苏丹红 1 号 (Aldrich Chemical Company)
4.6 苏丹红 2 号（ Acros organics 化工合成有机物）
4.7 苏丹红 3 号（ Acros organics 化工合成有机物）
4.8 苏丹红 4 号（ Acros organics 化工合成有机物）
4.9 苏丹橙 B （ Acros organics 化工合成有机物）
4.10 苏丹红 7B （ Acros organics 化工合成有机物）
4.11 胭脂树橙 ( 特殊合成产品 )
4.12 标准溶液
4.12.1 标准贮备液
称取 50.0mg 着色剂（按产品标明的纯度折算成纯着色剂）并按以下方式移入 100ml 容量瓶定容。
着色剂 |
溶解和转移溶剂 |
定容溶剂 |
苏丹红 1 号 |
乙腈 |
乙腈 |
苏丹红 2 号 |
乙腈 |
乙腈 |
苏丹红 3 号 |
氯仿 |
乙腈 |
苏丹红 4 号 |
氯仿 |
乙腈 |
苏丹橙 B |
乙腈 |
乙腈 |
苏丹红 7B |
乙腈 |
乙腈 |
胭脂树橙 |
氯仿 |
氯仿 |
4.12.1 标准工作液
取上述标准贮备液各 5ml 移入 50ml 容量瓶中以乙腈定容，再分别从以上容量瓶中吸取 0.5ml 、 1ml 、 2.5ml 、 4ml 和 5ml 溶液移入 50ml 容量瓶中以乙腈定容，此时溶液中各种着色剂的浓度分别为 0.5,1,2.5,4 和 5 μ g/ml 。
5 仪器与设备
5.1 万分之一天平
5.2 250ml 具塞三角瓶
5.3 直径 10cm 的漏斗
5.4 50ml 容量瓶
5.5 5ml 移液枪
5.6 100ml 量筒
5.7 5ml 一次性注射器
5.8 0.45 μ m滤膜
5 . 9 185mm滤纸
5.10 打浆机
5.11 Ultra Turrax 均质机
5.12 配有紫外 - 可见检测器的高效液相色谱仪
5.13 色谱柱 LiChroCART250-4HPLC Cartbridge Supersher 100RP18
5.14 进样瓶
6 样品制备
将采集样品放入一个容量较大的密闭容器中混合均匀。对于固体样品要用打浆机或粉碎机磨细。
7 操作方法
7.1 样品处理
7.1.1 甜椒或红辣椒粉
称取 10g （准确至 0.01g ）样品于三角瓶中，用量筒加入 100ml 乙腈。
7.1.2 粉状调味品
称取 5g （准确至 0.01g ）样品于三角瓶中，用量筒加入 100ml 乙腈。
7.1.3 调味辣椒酱、原味辣椒酱、辣椒油等
称取 20g （准确至 0.01g ）样品于三角瓶中，用量筒加入 100ml 乙腈。
7.1.4 Merguez 香肠、西班牙加调料的口利左香肠和肉制品
称取 20g （准确至 0.01g ）样品于三角瓶中，用量筒加入 100ml 乙腈。
之后在 Ultra Turrax 中充分混合数分钟，振荡 1 小时后过滤于三角瓶中。检测样品取样量和其稀释液浓度要视产品中的辣椒含量而定，以符合液相色谱检测限的要求。
7.2 高效液相色谱测定
7.2.1 流动相
溶剂 A ：酸性水溶液（ 165ml 乙酸溶于 1000ml 水中）
溶剂 B ：乙腈
7.2.2 梯度洗脱
时间 |
溶剂 A ％ |
溶剂 B ％ |
梯度曲线 |
0 |
30 |
70 |
线性 |
20.0 |
5 |
95 |
线性 |
30.0 |
0 |
100 |
线性 |
42.0 |
0 |
100 |
流速 :0.7ml/min
基线稳定后开始进样
两次进样间隙时间为 10 分钟
7.2.3 进样量 :10 μ l
7.2.4检测波长
在 300nm 到 600nm 波长范围进行扫描 , 确定三个测定波长（ 432nm,478nm 和 520nm ）
7.2.5 标准曲线
用 5 个标准工作溶液的测定值绘制标准曲线 , 各种色素的标准曲线分别在最大吸收波长处由 5 点回归计算（苏丹橙 B 在 432nm 波长有最大吸收，苏丹红 1 号 , 苏丹红 2 号 和胭脂树橙在 478nm波长有最大吸收和 苏丹红 3 号，苏丹红 4 号和苏丹红 B 在 520nm 波长有最大吸收）。
将 7.1 制好的样过 0.45 μ m 的膜装入自动进样器的小瓶后进行液相色谱测定得到结果。
标准回归曲线经过每次实验配制的系列标准溶液测定结果的验证。
7.3 计算
着色剂含量按以下公式计算：
R=C×V×D/M 单位： mg/kg
C- 样品中待测组分的浓度 , 单位： μ g/ml
M- 检测样品取样量（ g ）
V- 样品溶液体积（ ml ）
D- 样品溶液的稀释倍数
8 苏丹红 1 号
8.1 第一步是确定方法的操作条件
应用 Norm NF V03 110 获得以下操作条件：
标准曲线模型是线性的
478nm 下的检测限是 0.013 μ g/ml
478nm 下定量的最低浓度为： 0.106 μ g/ml
在辣椒粉样品中的添加回收率高于 90%
对于其它食品基质中的色素方法也进行了研究并可应用类似方法对所有着色剂进行检测。
8.2 应用 LC/MS 确证苏丹红 1 号
对于复杂食品基质本底或一种新的基质本底，确证苏丹红 1 号分子的存在是非常必要的。
如果光谱分析结果不令人满意（如待分析物浓度较低或可能存在结构类似物时）也可以应用这种技术进行确证。
8.3 设备
8.3.1 液相色谱与电喷雾离子化质谱仪联用
8.3.2 色谱柱： PHENOMENEX LUNA C18 3 μ m 150×2nm
8.3.3 柱温箱温度调至30℃
8.4 HPLC测定
8.4.1 流动相
溶剂 A:20% 酸性水溶液 (0.1% 乙酸溶液 )
溶剂 B:80% 乙腈
流速 :0.2ml/min
8.4.2 进样量 :10 μ l
8.4.3 检测器
正电喷雾
设定条件 : 喷雾电压 :5300V
喷雾口电压 :120V
周边电压 :9.50V
辅助气体温度 :300 ℃
8.4.4 测定
步骤7.12 取得的提取物先稀释 10 倍后再稀释 100 倍后经 0.45 μ膜过滤于自动进样瓶中 .
8.5 结果
样品中苏丹红 1号组分与样准品出峰时间基本一致相对误差为5%,并经质谱检测由m/Z为249和1022的离子定性,误差为0.01u.
[1] Laboratoire de Paris-Massy, 25, Av de la republique, 91744 Massy Cedex, CLHP UVIVIS section
[2] Laboratoire de Paris-Massy, 25, Av de la republique, 91744 Massy Cedex, LCIMS section
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