Evaluation of an automated and integrated flow-through immunoanalysis system for the rapid determination of cephalexin in raw milk

Abstract

In the present work, an evaluation of an automated flow-through amperometric immunoanalysis system for the quantitation of the antibiotic drug cephalexin (Ce) in milk is described. The detection limit of the method was calculated as 1 μg/l ( S/ N=3), while the quantitation limit being 3 μg/l, well below the EU regulation mandated limit of 0.1 mg/kg for cephalexin in milk. Spiked milk samples with increased concentrations of cephalexin were investigated as blind coded duplicate samples. Total 20 milk samples spiked with cephalexin between 3 and 30 μg/l were analyzed leading to a good correlation referred to the spiked values. The samples could be detected with a mean recovery of 97±23%, indicating a good agreement with the spiked concentration. The precision and repeatability was determined using spiked samples with four different concentrations which were investigated on four different days. A mean within-day variation of 6.9% and a mean between-day variation of 10.9% were obtained. Correct classifications were achieved in false negative and false positive studies when the cut-off values were set at 2.0 and 3.7 μg/l, respectively, further proving the detection and quantification capabilities of the method. The method was specific for cephalexin and free of interferences from other cephalosporins and penicillins at concentrations up to at least 1000 μg/l. Milk matrix properties in terms of bacteriological quality, somatic cell content, and pH have no significant effects on the determination. The effect of the milk fat was eliminated by a simple-defatting step. For the investigation of samples with concentrations higher than 30 μg/l, a dilution step for the sample would be required. In addition, 17 natural contaminated milk samples of cephalexin-treated cows were also analyzed and the results obtained were confirmed by an enzyme-linked immunosorbent assay (ELISA) method. This intra-laboratory study demonstrated that the proposed method is suitable for the rapid and reliable determination of cephalexin in raw milk samples.