Research and Application of Immunoaffinity Column Method for Detection of Aflatoxin M1 in Milk

1. Toxicological Characteristics and Detection Significance of Aflatoxin M1

Aflatoxin M1 (AFM1) is a significantly toxic secondary metabolite from the aflatoxin family, primarily produced by fungi such as Aspergillus flavus and Aspergillus parasiticus under humid conditions, which can easily contaminate crops. When dairy cows consume feed contaminated with aflatoxin B1, AFM1 metabolites are secreted through milk into the dairy supply chain.

From a toxicological perspective, AFM1 has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Its molecular structure contains furan rings and coumarin groups that can form adducts with DNA, leading to genetic mutations. Long-term consumption of dairy products containing AFM1 may induce malignant tumors such as liver cancer and gastric cancer, posing particular risks to infants' growth and development. The World Health Organization recommends that AFM1 levels in dairy products should not exceed 0.5 μg/kg, while EU standards are even stricter at 0.05 μg/kg.

2. Technical Principles and Advantages of Immunoaffinity Column Method

The immunoaffinity column purification technique is based on the principle of specific antigen-antibody binding established for sample pretreatment methods. The Copure® immunoaffinity column is covalently coupled with monoclonal antibodies against AFM1 on its solid-phase carrier; these antibodies selectively capture AFM1 molecules from sample extracts while washing away other interfering matrix components. Compared to traditional solid-phase extraction methods, this approach offers three significant advantages:

First, the specificity of antibody-antigen binding provides high selectivity that effectively eliminates interference from complex matrices like proteins and fats in milk. Second, optimized elution conditions ensure over 90% recovery rates—much higher than liquid-liquid extraction's typical recovery rate of 60-70%. Finally, the entire purification process can be completed within 30 minutes significantly enhancing detection throughput—making it a recommended standard method according to national standard GB5009.24-2016.

3. Experimental Materials and Instrument Configuration

3.1 Main Reagents and Consumables Chromatographic-grade methanol and acetonitrile (from Merck Germany), phosphate-buffered saline (PBS; pH7.4) must be freshly prepared before use; Copure® AFM1 immunoaffinity columns (10ml/column) need to equilibrate at room temperature prior to use; standards purchased from TRC USA (purity ≥98%). All glassware must be treated with a solution containing sodium hypochlorite at concentrations around five percent to avoid cross-contamination. 3.2 Instrument Equipment Parameters The liquid chromatography system consists of Thermo Fisher U3000 binary pump equipped with fluorescence detector (FLD). The chromatographic column selected was Commasil® AQ-C18 (4.6×250mm，5μm), whose polar modified surface effectively retains AFM while improving peak shape stability set at an oven temperature maintained at forty degrees Celsius ensuring retention time consistency—the automatic sampler fitted with one hundred microliter quantitative loop ensures accurate sampling precision.

Four: Optimization Of Sample Pretreatment Process

**4.. Extraction Process Improvement ** nWeighing four grams ± one milligram homogenized milk samples into fifty-milliliter polypropylene centrifuge tubes adding ten milliliters methanol using vortex-ultrasonic combined extraction strategy: first vortexing three minutes denaturing proteins then ultrasonic treatment five minutes promoting toxin dissolution followed by low-temperature centrifugation(8000rpm，ten min）taking eight milliliters supernatant undergoing second round centrifugal purification step eliminating over ninety percent fat globules casein micelles present therein。 n 4.. Details On Purification Using Immunoadhesion Columns: After centrifugation dilute extractive liquids using PBS buffer up until fifty mL maintaining constant flow rate two mL/min through columns particularly monitoring pressure below point five MPa preventing damage caused towards antibody activity during wash stage employing ten mL ultra-purified water(resistivity eighteen-point-two mega ohm cm )to remove non-specific adsorbents utilizing four mL methanol added twice each resting thirty seconds increasing elution efficiency nitrogen blowing concentration controlling bath temperatures remaining between forty-five plus minus two degrees Celsius avoiding thermal decomposition target substances。 n ### Five Chromatography Conditions And Validation Methods : 5.. Optimal Chromatographic Parameters: Flow phase utilizes water-acetonitrile/methanol ratio fifty-fifty establishing seventy-thirty proportion during gradient elution enabling symmetrical peaks forming obtained concerning eight-point-two minute theoretical plate number exceeding five thousand detected limits reaching zero-point-zero-one ng/ml post optimization parameters defined excitation slit width measuring ten nanometers emission slit twenty nanometers photomultiplier tube voltage set six hundred fifty volts confirming limits reached subsequently validate results achieved across various methodologies employed demonstrated linear ranges spanning zero-point-zero-five -five ng/ml correlation coefficient r² equals zero point nine-nine-nine-ninety-five indicating strong dose-response relationships observed among triplicate spiking levels returning recoveries falling eighty-five-hundred-ten percent daily precisions recorded less than six percent RSD demonstrating improved efficiencies compared QuEChERS methodologies utilized previously evaluated overall matrix effects indicate signal suppression ratios lower fifteen percentage values supporting findings conclusively validated testing outcomes confirm reliable reproducibility verified against industry standards consistently applied throughout study conducted . n ### Six Practical Applications Quality Control Recommendations: Experiment protocols necessitate strict implementation involving blank controls including solvent only alongside negative control comprising cow’s milk samples introduced quality assurance measures wherein every batch incorporates reference materials containing half microgram per kilogram amounts verifying acceptable returns existing between eighty-five-to-one-hundred-ten percentages suggesting isotope internal calibration techniques when approaching limit threshold applying isotopic labeling strategies targeting similar compounds anticipated future developments aimed automating purifications reducing processing times achieving less fifteen-minute durations ultimately optimizing designs engineered enhance affinities targeting related analogues potential establishment comprehensive screening processes regarding all types mycotoxins emerging issues requiring attention timely action taken accordingly developing automated solutions capable addressing concerns raised proactively moving forward successfully integrated operations achieved via continuous monitoring systems established effective oversight management structures instituted efficiently managing resources deployed sustainably maintaining highest operational integrity practices adhered continuously implemented .