Immunoaffinity column HPLC determination of aflatoxin M(1) in milk

Aflatoxin M(1) was selectively bound to a monoclonal antibody immobilized to an affinity column. After washing the column free of the milk matrix with 2 volumes of 10% MeOH in H2O, the aflatoxin was eluted with 1 ml of MeOH. The eluate was subjected to high-pressure liquid chromatography (HPLC) using a reverse-phase column and fluorimetric detection. The detection limit was 50-100 ng l(-1) of M(1) in milk. The method was tested with two different batches of affinity columns at a spiking level of 300 ng l(-1) in milk, and showed recoveries of 98.7 and 79.3% and repeatabilities of 8.8 and 5.8% respectively. Howeverr, the method was found to be more reproducible when columns from the same batch were used (spiking levels 50 and 100 ng l(-1)). The detection limit could be further lowered to 10 ng l(-1) by concentrating the eluate from the column. The results at low spiking levels (less than or equal to 100 ng l(-1)) showed that although the recoveries were low, the repeatability was satisfactory enough to allow the implementation of the method with results corrected for recovery. The method compared well with chemical procedures and was fast, sensitive and more selective, with no interfering peaks appearing in the chromatograms.