Development and validation of an HPLC confirmatory method for the residue analysis of four sulphonamides in cow's milk according to the European union decision 2002/657/EC

In the present study, an HPLC method was developed and validated for the determination of four sulphonamides: sulphadiazine (SDZ), sulphaquinoxaline (SQX), suplhamethazine (SMTH), and sulphadimethoxine (SDM). Two of them, SQX and SDM, were determined in cow's milk. The analytical column, a Kromasil, C(18) 5 mu m, 250 x 4 mm analytical column, was operated at ambient temperature. The mobile phase, a mixture of 0.5% acetic acid as solvent A, CH(3)CN as solvent B, and CH(3)OH was delivered to the analytical column according to a gradient program. PDA detection was performed for the detection and confirmation of separated analytes with monitoring at 260nm. Method validation was performed by means of intra-day (n=5) and inter-day accuracy and precision (n=5), sensitivity, and linearity. Limits of detection (LOD) and limits of quantification (LOQ) were 13 and 40 mu g/kg, respectively. Solid phase extraction was applied to remove all matrix interference from milk samples after deproteinization with 8M HCl. High extraction recoveries (>84%) were achieved using DSC-18 cartridges with CH(3)OH-0.5% CH(3)COOH as eluent. CC alpha values were 111.8 and 117.1 mu g/kg for SDM and SQX, respectively, and CC values were 116.6 and 134.0 mu g/kg, respectively. The method was applied to the analysis of twenty two milk samples from the local market. SQX was identified in seven of these samples.