Determination of aflatoxin M₁ in milk and dairy products using high performance liquid chromatography-fluorescence...

The determination of aflatoxin M₁ in milk using high performance liquid chromatography with photochemical post-column derivatization and fluorescence detection is described. The samples were first extracted and clean-up using the immunoaffinity AFLATEST column originally targeted for aflatoxins B₁, B₂, G₁ and G₂. The separation of aflatoxin M₁ were performed using C18 Hypersil gold (150mm×4.6mm, 5μm) column at 40°C under isocratic elution. Fluorescence detector (FLD) was set at 360nm and 440nm as excitation and emission, respectively. The use of methanol to replace acetonitrile as the mobile phase resulted in ∼67% peak area enhancement of AFM₁. The limit of detection (LOD) and quantification (LOQ) of the analytical method after post-column derivatization without evaporation/reconstitution with mobile phase was 0.0085μgL^-1 and 0.025μgL^-1 respectively. However, LOD and LOQ improved to 0.002 and 0.004μgL^-1 respectively with the addition of evaporation/reconstitution step. The method was statistically validated, showing linear response (R²>0.999), good recoveries (85.2-107.0%) and relative standard deviations (RSD) were found to be ≤7%. The proposed method was applied to determine AFM₁ contamination in various types of milk and milk products. Only 2 samples were contaminated with aflatoxin M₁ (10% incidence). However, the contamination level is below the Malaysian and European legislation limits.