SIMPLE CONTINUOUS AND SIMULTANEOUS DETERMINATION OF MULTIPLE SULFONAMIDE RESIDUES

A new continuous separation method was developed for the determination of nine different sulfonamides (sulfaguanidine, sulfamethazine, sulfa pyridine, sulfadiazine, sulfathiazole, sulfamethizole, sulfamethoxazol e, sulfadimethoxine, and sulfaquinoxaline). Bioassay on minimum medium seeded with Bacillus subtilis ATCC 6633 was carried out for detection . An extract taken from an agar block of the clear inhibition zone on minimum medium produced by a mixture of sulfonamides was then subjecte d to high-performance liquid chromatography. For identification, high- performance liquid chromatography analyses were performed using two di fferent columns and analytical conditions. Using a mu-Bondapak C18 col umn, the sulfonamides were separated at room temperature using a mobil e phase of methanol: 0.1 M KH2PO4 (30:70, vol/vol) at a flow rate of 1 .0 ml/min. A variable wavelength detector set at 265 nm and recorder s et at 4 mm/min were used for the detection. The entire mixture was res olved as eight peaks from 4.68 to 50.78 min. When an Asahipak GS-320 c olumn was employed, nine peaks were separated with retention times ran ging from 12.62 to 54.43 min using a mobile phase of acetonitrile: 1% acetic acid (25:75, vol/vol) at a flow rate of 2.0 ml/min. Correlation coefficients of standard curves for individual sulfonamides were line ar (>0.99) with recoveries ranging from 25.2 +/- 8.6% to 64.1 +/- 8.6% for a concentration range of 1.0-25 mug/ml.