Quantification of terbutaline in urine by enzyme-linked immunosorbent assay and capillary electrophoresis after oral and inhaled administrations

Abstract

The International Olympic Committee and World AntiDoping Agency restricts the use of β 2-agonists and only the inhaled administration of terbutaline, salbutamol, formoterol and salmeterol is permitted for therapeutic reasons. The aim of this study was to develop a test for the quantitation of terbutaline in urine and evaluate different parameters to distinguish between oral and inhaled administration of the drug. Urine samples were collected from asthmatic and non-asthmatic recreational swimmers who had received repeated doses of oral (3×2.5 mg plus 1×5 mg during 24 h) and inhaled (12×0.5 mg in 24 h with half of it being in the last 4 h) racemic terbutaline, and single oral (5 mg) or single inhaled doses (1 mg). Total terbutaline concentrations (free+conjugated) were determined by enzyme-linked immunosorbent assay. Results showed that after oral administrations urinary terbutaline concentrations were higher than those detected after inhalation. For confirmation purposes, a chiral capillary electrophoretic procedure was established and validated. A solid-phase extraction with Bond-Elut Certify cartridges was undertaken, separation performed using a 50 m M phosphate buffer (pH 2.5) containing 10 m M of (2-hydroxypropyl)-β-cyclodextrin as running buffer and diode-array UV detection set at 204 nm. The proposed procedure is rapid, selective and sensitive allowing quantitation of free terbutaline enantiomers in urine. No statistical differences were found between total free terbutaline concentrations [ S-(+)+ R-(−)] in urine collected after oral and inhaled administrations of the drug. After oral doses enantiomeric [ S-(+)]/[ R-(−)] ratios lower than those obtained after inhalation were observed probably due to an enantioselective metabolism that take place in the intestine, but differences between both routes of administration were not statistically significant. Although different trends were observed after oral and inhaled doses in total terbutaline, total free terbutaline concentrations and in ratios between its enantiomers, differences observed were not sufficiently significant to establish cut-off values to clearly distinguish between both routes of administration.