Determination of aflatoxin M1 in milk and dairy products using high performance liquid chromatography-fluorescence with post column photochemical derivatization

Abstract

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.