A new continuous separation method was developed for the determination of nine different sulfonamides (sulfaguanidine, sulfamethazine, sulfapyridine, sulfadiazine, sulfathiazole, sulfamethizole, sulfamethoxazole, 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 subjected to high-performance liquid chromatography.

For identification, high-performance liquid chromatography analyses were performed using two different columns and analytical conditions. Using a μ-Bondapak C18 column, the sulfonamides were separated at room temperature using a mobile 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 set at 4 mm/min were used for the detection. The entire mixture was resolved as eight peaks from 4.68 to 50.78 min. When an Asahipak GS-320 column was employed, nine peaks were separated with retention times ranging 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 linear (>0.99) with recoveries ranging from 25.2 ± 8.6% to 64.1 ± 8.6% for a concentration range of 1.0–25 μg/ml.

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