Production and characterization of polyclonal antibodies to sulfamethazineand their potential use in immunoaffinity chromatography for urine sample pre-treatment

An immunoaffinity chromatographic (IAC) method for isolating sulfamethazine (SMZ) from incurred urine samples was developed. This was achieved by (i) generating polyclonal antibodies that recognize equally well SMZ and its ma jor urinary metabolites, (ii) evaluating in an ELISA procedure the influenc e of methanol, salt and pH on the antigen-antibody interaction in order to determine the optimum conditions for IAC and (iii) covalent coupling of the IgG fractions of anti-SMZ to CNBr activated Sepharose for the preparation of re-usable immunoaffinity columns, having a high capacity for SMZ (1900 n g SMZ mL(-1) gel). For desorbing SMZ from the immunoaffinity column, differ ent elution modes were evaluated, with 40% MeOH-0.1 mol L-1 HOAc-0.5 mol L- 1 NaCl being the most efficient combination. Using the IAC column for proce ssing SMZ spiked urine samples resulted in high recoveries, ranging from 92 to 100%. Because of the high cross-reactivity with the major metabolites o f SMZ present in urine of treated animals, the antibodies show excellent pr operties for use in both IAC and ELISA. For the isolation and concentration of the parent drug and its major metabolites, the urine could be applied d irectly to the IAC column, without the time-consuming step of deconjugation . Moreover, the use of IAC prior to ELISA for the analysis of incurred urin e samples showed good efficiency for the elimination of matrix interference s. Owing to the urine-tissue relationship, the urine concentrations can be used to predict the presence of the parent drug in tissues and so possible violations of the maximum residue limit (MRL) can be controlled.