HYPHENATION OF COUPLED-COLUMN LIQUID-CHROMATOGRAPHY AND THERMOSPRAY TANDEM MASS-SPECTROMETRY FOR THE RAPID-DETERMINATION OF BETA(2)-AGONISTRESIDUES IN BOVINE URINE USING DIRECT LARGE-VOLUME SAMPLE INJECTION, SET-UP OF SINGLE-RESIDUE METHODS FOR CLENBUTEROL AND SALBUTAMOL

In a previous work, coupled-column LC (LC/LC) with direct large-volume sample injection for the analysis of beta(2)-agonists in bovine urine was investigated, in view of its combination with thermospray (TSP) t andem mass spectrometric (MS/MS) detection. In this work, the potentia l of both conventional LC and LC/LC coupled to TSP-MS/MS for the rapid determination of beta(2)-agonists in bovine urine was evaluated. It w as first established that, in order to avoid disturbances in TSP-MS/MS detection due to changes in pressure and flow rate of the mobile phas e, LC/LC is preferable to conventional LC. Then, selecting clenbuterol and salbutamol as model compounds for the class of beta(2)-agonists, two single-residue LC/LC/TSP-MS/MS methods were developed, permitting the determination of the analytes in bovine urine samples with a limit of quantification of at least 0.1 ng, ml(-1), with a sample throughpu t of 4 h(-1). The methods were validated by analysing both spiked urin e samples and samples containing clenbuterol or salbutamol residues. M ean recoveries from urine samples spiked with clenbuterol at levels of 5 and 0.22 ng ml(-1) were 107% (n = 9, RSD = 7.4%) and 102% (n = 6, R SD = 14%), respectively. A bovine urine sample containing 1.28 ng ml(- 1) clenbuterol residue (previously quantified by GC/MS) was re-analyse d by LC/LC/TSP-MS/MS on four different days (n = 3, each day) and yiel ded a mean concentration of 1.31 ng ml(-1) with a reproducibility of 8 .4%. Bovine urine samples spiked with salbutamol at levels of 10.8 and 0.55 ng ml(-1) yielded mean recoveries of 101% (n = 3, RSD = 1%) and 89% (n = 3, RSD = 13%), respectively, In bovine urine samples containi ng salbutamol residues, the concentrations of unconjugated salbutamol were found to be in the range 8-57 ng ml(-1).