Determination of Sudan Red in Food

Sudan red detection methods include high performance liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, thin layer chromatography and so on
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1.
Principle

After the sample was purified by solvent extraction and solid phase extraction, it was analyzed by reversed-phase high performance liquid chromatography with ultraviolet-visible light detector and quantified by external standard method
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2.
Reagents

(1) Acetonitrile : chromatographically pure
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(2) Acetone : chromatographically pure and analytically pure
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(3) Formic acid : analytically pure
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(4) Ether : analytically pure
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(5) n-Hexane : analytically pure
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(6) Anhydrous sodium sulfate : analytically pure
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(7) Chromatography column tube: 1cm (inner diameter)×5cm (height) syringe tube
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(8) Alumina for chromatography (neutral 100-200 mesh): dry at 105°C for 2 hours, cool to room temperature in a desiccator, add 2 mL of water per 100 g to deactivate, mix well, seal, and place for 12 hours before use
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(9) Alumina chromatography column: insert a thin layer of absorbent cotton into the bottom of the chromatography column, dryly load the treated alumina to a height of 3 cm, lightly compact and add a thin layer of absorbent cotton, pre-prepared with 10 mL of n-hexane After washing and washing the impurities in the column, set aside
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(10) 5% acetone in n-hexane solution: draw 50 mL of acetone and dilute to 1L with n-hexane
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(11) Standard materials: Sudan Red I, Sudan Red II, Sudan Red III, Sudan Red IV; purity ≥95%
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(12) Standard stock solution: Weigh 10.
0 mg each of Sudan Red I, Sudan Red II, Sudan Red III and Sudan Red IV (calculated based on actual content), dissolve in ether and dilute to 250 mL with n-hexane
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3.
Apparatus

(1) High performance liquid chromatograph (equipped with UV-visible light detector)
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(2) Analytical balance: Sensitivity 0.
1mg
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(3) Rotary evaporator
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(4) Homogenizer
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(5) Centrifuge
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(6) 0.
45um organic filter membrane
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4.
Operation steps (taking powdered samples such as red pepper powder as an example)

(1) Pre-treatment Weigh 1～5g (accurate to 0.
001g) sample in a Erlenmeyer flask, add 10mL to 30mL n-hexane, sonicate for 5min, filter, wash the residue with 10mL n-hexane several times until the eluate is colorless , Combine the n-hexane solution, use a rotary evaporator to concentrate to less than 5mL, slowly pour it into the alumina chromatography column, in order to ensure the chromatographic effect, keep the n-hexane liquid level in the column at about 2mm and load the sample.
During the analysis process, the column should not be dried out.
Rinse the concentration bottle with n-hexane several times and inject it into the column at the same time
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(2) Determination

①Recommended chromatographic conditions:

Chromatographic column: ZotbaxSB-C ₁₈ 3.
5um 4.
6mm×150mm (or equivalent chromatographic column); mobile phase: solvent A 0.
1% formic acid aqueous solution: acetonitrile=85:15, solvent B 0.
1% formic acid in acetonitrile solution: acetone= 80:20; gradient elution conditions: see Table 6-8; column temperature: 30°C; detection wavelength: Sudan Red 1478nm; Sudan Red II, Sudan Red III, Sudan Red IV 520nm; switch after Sudan Red I peaks
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Table 6-8 Gradient elution conditions

②Standard curve: draw standard stock solution 0, 0.

5.

In the formula, X——Sudan content in the sample, mg/kg

p——The concentration of Sudan red in the sample solution derived from the standard curve, ug/mL

V——Constant volume of sample solution, mL

m——sample mass, g