Quantitative detection of inhaled salmeterol in human urine and relevance to doping control analysis

Salmeterol is a frequently prescribed β₂-agonist used for the treatment of asthma. Due to performance-enhancing effects of some β₂-agonists, salmeterol appears on the prohibited list published by the World Anti-Doping Agency and its therapeutic use is allowed but restricted to inhalation. Because the data on urinary concentrations originating from therapeutic use are limited, no discrimination can be made between use and abuse when a routine sample is found to contain salmeterol. Therefore, the urinary excretion of 100 μg of inhaled salmeterol was investigated. A liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of urine samples. Sample preparation consists of an enzymatic hydrolysis of the urine samples followed by a liquid-liquid extraction at pH 9.5 with diethyl ether/isopropanol (5/1). Analysis was performed using selected reaction monitoring after electrospray ionization. The method was linear in the range of 0.5-50 ng/mL. The limits of quantification were 500 pg/mL. The inaccuracy ranged between 10.4% and -3.7%. Results show that salmeterol could be detected for 48 hours. The maximum urinary concentration detected was 1.27 ng/mL. Cumulative data showed that only 0.27% of the administered dose is excreted as parent drug within the first 12 hours. Analysis of 47 routine doping samples, declared to contain salmeterol during routine analysis, did not exhibit concentrations that could be considered originating from supratherapeutic doses.