Liquid chromatographic determination of multiple sulfonamide residues in bovine milk: collaborative study

A collaborative study involving 8 laboratories was conducted on the determination of 8 sulfonamide residues in raw bovine milk using a liquid chromatographic (LC) method. The sulfonamides are extracted with chloroform-acetone, the organic phase is evaporated, the residues are dissolved in an aqueous potassium phosphate solution, and the fatty residues are removed by washing with hexane. The aqueous layer is collected, filtered, and injected onto an LC system, and the analyte is detected by ultraviolet (UV) absorption at 265 nm. To quantitate all 8 sulfonamides isocratically, 2 chromatographic conditions are required: 12% methanol in the mobile phase for 5 sulfonamides, and 30% methanol in the mobile phase for 4 sulfonamides. Sulfamethazine (SMZ), the most widely used sulfonamide, is detected by both systems. Collaborators were instructed to analyze 3 replicates each of control milk and control milk fortified at 3 levels. They were also provided with 20 blind incurred samples (10 samples in duplicate) to analyze. For 10 ppb fortified milk, the average interlaboratory recovery for the 8 sulfonamides ranged from 56.2% for sulfaquinoxaline (SQX) to 82.7% for SMZ in the 12% methanol mobile phase (SMZ12). Also at this level, Sr ranged from 3.2 for SQX to 8.9 for SMZ12, and SR ranged from 6.9 for sulfadimethoxine to 17.2 for SMZ in the 30% methanol system (SMZ30). At 10 ppb, RSDr and RSDR ranged from 5.7% for SQX to 10.8% for SMZ12, and 10.1% for sulfamerazine to 20.9% for SMZ30, respectively. These results demonstrate that the method is suitable for the determination of the 8 sulfonamide residues in milk at 10 ppb. However, the identification of positives by this procedure needs additional confirmation by procedures comparable to the specificity achievable by liquid or gas chromatography combined with mass spectrometry.