DETERMINATION OF SULFAMETHAZINE AND ITS MAJOR METABOLITES IN EGG ALBUMIN AND EGG YOLK BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Abstract

A quantitative liquid chromatographic method for the determination of sulfamethazine (SMZ), N4-acetyl sulfa-methazine (N4-acetyl-SMZ), and desamino sulfamethazine (desamino-SMZ) in egg albumin and egg yolk is described. Egg albumin or yolk was homogenized in acetonitrile and centrifuged. The supernatant was evaporated to dryness and residue reconstituted in the mobile phase. Albumin extract was directly analyzed by high performance liquid chromatography (HPLC). Hexane was added to the yolk sample, vortex mixed, and centrifuged to separate the layers. The top hexane layer was removed and a small amount of salt was added to break the emulsions. The lower aqueous layer was analyzed by HPLC. The HPLC system included a reversed phase column, a gradient mobile phase of 5–15% acetonitrile and 0.01M phosphate buffer, and a UV detector set at 268 nm. The recovery of SMZ and N4-acetyl-SMZ, both fortified at 1 ppm levels, from egg albumin was 101 and 88% and from egg yolk was 79 and 91%, respectively. The recovery of desamino-SMZ at 2 ppm fortification level from egg albumin and egg yolk was 84 and 63%, respectively. The method was applied to detect the presence of SMZ and its potential metabolites in eggs collected after dosing hens with SMZ. Parent drug, SMZ, was the major compound transferred to both egg albumin and yolk. Small concentration of N4-acetyl-SMZ metabolite were also detected in some eggs; however, no desamino-SMZ or other metabolites were detected.