Analyze how aflatoxin exist in milk powder

Posted on  November 11, 2019, Edited by Samantha, Category  

Aflatoxin, as a mycotoxin, is a metabolite of fungi produced when the grain or fresh forage is not dried and stored in time, and widely exists. Because of its strong toxicity and carcinogenicity, strict regulations of it have been set in the national food standards and feed standards. Aflatoxin M1 belongs to mycotoxin and is the hydroxylation metabolite of aflatoxin B1 in animals. It is highly toxic and carcinogenic, should be prevented from being added to food. Because it is harmful to liver tissue and may induce milk allergic and liver cancer, it was listed as a carcinogen by WHO Cancer Research Institute as early as 1993.

Because of the low content and similar structure of aflatoxin, it requires high sensitivity and specificity of the detection method, which integrates separation and detection. In this paper, high-performance liquid chromatography (HPLC) was used to analyze aflatoxin. The problem of low content of aflatoxin was overcome, and a high sensitivity result was obtained.

Sample pretreatment

The milk powder from milk markets is taken 5g and placed in a 100mL measuring bottle. The milk powder is dissolved by adding 20 mL warm water at 50 degrees Celsius to make it oscillate for the 30S. The sodium chloride is added to make it dissolve by oscillating for 2g. The 50 mL acetonitrile is added to oscillate for the 30S. After mixing, the acetonitrile is added to the bottle to meet the scale. The scroll oscillation makes it homogeneous. Filtration, 40 mL of filtrate, 40 C nitrogen flow down to the near dry. The residue was dissolved by adding 20 mL 10% acetonitrile. The affinity column was washed with 10 mL water after the sample passed through the affinity column completely.

At last, 2 mL acetonitrile was used for elution. 1.5 ml of eluent was collected and dried with nitrogen. Adding 200 uL hexane and 200 uL trifluoroacetic acid, shaking and mixing, reacting at 40 C for 10 minutes, taking out, drying under nitrogen flow, adding 10% acetonitrile 0.5 ml, shaking for 10 seconds.

experimental condition

Instrument type: Dionex Ultimate 3000 Series
Pump: LPG-3400SD
Automatic Sampler: WPS-3000SL
Column Temperature Box: TCC-3000RS
Detector: FLD
Chromatography Software: Chromeleon Chromatography Data System
Detector type, working parameters, and S/N number:
Fluorescence detector, FLD-3400
Response time = 1

FLD FlowCell Temperature Nominal = 35.00 [C]
FLD FlowCell ReadyTempDelta = 1.00 [C]
BaselineBehavior = Append
Data Collection Rate Nominal = 10.00 [Hz]
Emission 1.ExWavelength = 365.0 [nm]
Emission 1. Em Wavelength = 435.0 [nm]
Emission 1. Sensitivity = 6
Emission 1.PMT = Auto
Emission 1. FilterWheel = Auto
Chromatographic conditions:

Chromatographic conditions
Chromatographic conditions

Chromatographic Column:
Thermo Scientific Hypersil GOLD C18, 5 um, 100 x 2.1 mm No. 25005-102130
Mobile phase:
Sampling volume: 20 mL
Flow velocity: 800 mL/min
Column temperature: 25 C

Fig. 1 Sample determination chromatogram (1 ppb plus standard chromatogram; 2 blank sample chromatogram)


In this paper, Aflatoxin B1, B2, G1, G2, M1 in sesame were analyzed by using Thermo Scientific Hypersil GOLD C18, 5 microns, 100 *2.1 mm column, pre-column derivatization combined with fluorescence detector. Good peaks, separation and high sensitivity detection results were obtained.

For more related mycotoxin detection kits, please click here.

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