Evaluation of three different microbial inhibition tests for the detection of sulphamethazine residues in the edible tissues of rabbit

Abstract

The aim of the present study was to evaluate three microbial inhibition tests (MIT) based on inhibition of growth of the test organisms: (a) four plate test (FPT) containing Bacillus subtilis BGA, (b) screening test for antibiotic residues (STAR) containing Bacillus stearothermophilus var. calidolactis_ATCC 10149 and (c) the Premi®Test containing Bacillus stearothermophilus var. calidolactis. The tests were used to determine sulphamethazine (SMZ) residues in edible tissues of rabbit after oral administration up to day 15 of the withdrawal period (WP). A solvent extraction procedure was used to enhance the capability of the tests to detect SMZ residues at or below the maximum residue limit (MRL). Para-aminobenzoic acid (PABA) was employed to previously identify SMZ residues in the first stage of the residue screening. The presence of SMZ residues in the samples was confirmed and quantified by a validated HPLC method. The Premi®Test detected SMZ residues in the muscle and heart tissue up to day 9 of the WP, and in the liver, lungs and kidneys up to day 10 of the WP. The STAR detected SMZ residues in the edible organs of rabbits up to day 8 of the WP. The kidneys were positive up to day 5 of the WP, the liver until day 4 of the WP and the lungs until day 3 of the WP. No SMZ residues were detected in the muscle and heart. By using the solvent extraction procedure, SMZ residues were detected in the muscle extract up to day 10 of the WP and the muscle was positive until day 6 of the WP. No detection sensitivity was observed using the FPT. After solvent extraction, SMZ residues were detected in the muscle extract until day 8 of the WP and the muscle was positive until day 3 of the WP. No positive results were detected after the addition of PABA into/onto the agar medium. PABA at a concentration of 10 µg ml⁻¹ completely reversed the inhibitory activity of SMZ and enabled reliable identification of SMZ in the examined samples. Using HPLC, SMZ was detected in the muscle samples until day 10 of WP (0.02 mg kg⁻¹) and in the liver until day 12 of the WP (0.09 mg kg⁻¹). The results obtained by the HPLC method and the limit of detection (LOD) of screening tests for SMZ (FPT 0.4 µg ml⁻¹, STAR 0.2 µg ml⁻¹, Premi® Test 0.05 µg ml⁻¹) allowed us to state that the most suitable screening tests for the detection of SMZ residues in the edible tissues of rabbits at level corresponding to the MRL of 0.1 mg kg⁻¹, established for sulphonamides, are the Premi®Test and STAR in conjunction with the solvent-extraction procedure.

Keywords:

• sulphamethazine
• residues
• detection
• rabbit tissues
• microbial inhibition tests
• HPLC

Acknowledgements

This study was supported by VEGA grant No 1/3491/06.