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 , L. Szymanski
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
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]
