Bosman J., Franke J.P., Koole A., De Zeeuw R.A.

Department of Analytical Chemistry and Toxicology, University Centre for Pharmacy, Groningen Institute of Drug Studies, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands

In our search for methods to detect the use of ß-agonists as illegal growth promoters in cattle we evaluated HPLC with electrochemical detection (ECD). For the separation of eight ß-agonists, namely brombuterol, cimaterol, clenbuterol, fenoterol, mabuterol, mapenterol, salbutamol and terbutaline, HPLC systems were developed on Lichrosorb RP select B with two isocratic mobile phases. Due to great differences in the polarity of the ß-agonists separation of these substances within one isocratic run within a reasonable time was not possible. A gradient run gave difficulties in the stability of the ECD signal. The voltammetric behaviour of the eight ß-agonists were tested, based on which an optimal potential for the ECD was selected.
For the extraction of the ß-agonists an SPE procedure was developed using mixed-mode SPE columns (TSC 300 mg, Merck). At the 50 ng/mL level, brombuterol, clenbuterol, mabuterol and mapenterol gave clean extracts from 5 mL human urine and acceptable recoveries (>70%). The extraction of the more polar ß-agonists (salbutamol, terbutaline, cimaterol and fenoterol) gave high recoveries (>80%) when extracted from water. Urine samples, however, introduced endogenous peaks especially interfering with the peaks of terbutaline and salbutamol, so that no reliable recovery data for these substances could be determined.
To increase the selectivity and the sensitivity of the procedure, immunoaffinity chromatography (IAC) with antibodies raised against salbutamol is being evaluated as an extra clean-up step prior to the SPE procedure.