Immuno Precolumns for Selective On-Line Sample Pretreatment of Aflatoxins in Milk Prior to Column Liquid Chromatography

Abstract

A column liquid chromatographic system for on-line sample pretreatment and determination of aflatoxin Ml in milk is described. The system consists of an immuno precolumn packed with immobilized monoclonal or polyclonal antibodies, a second precolumn packed with C-18 bonded silica and a C-18 analytical column. Defatted milk is directly loaded on the immuno precolumn. Desorption takes place by eluting the immuno precolumn with methanol-water (70:30, v/v). The eluate is diluted on-line with water with subsequent reconcentration of the analytes on the C-18 bonded silica precolumn and separation on the analytical column followed by fluorescence detection.

The applicability of automated immunoaffinity sample preparation with both a polyclonal and a monoclonal immuno precolumn is discussed and aspects such as the interference of milk proteins and the use of an external standard are investigated. Both types of immuno precolumns can be used for at least 20 analyses of milk samples, with relative standard deviations of 5–10% and detection limits of 20 ng/l for aflatoxin M1 in milk. The linear dynamic range is 20–400 ng/1 of aflatoxin M1 in milk if 2.4-ml milk samples are used.

Key Words:

• Immunoaffinity precolumns
• column switching
• column liquid chromatography
• aflatoxins
• milk