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A sensitive and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for determination of tulobuterol in rat plasma for the first time. Plasma samples were extracted by liquid–liquid extraction method with methyl tert -butyl ether and the analyte and clenbuterol (IS) were separated on a Venusil MP C 18 column (100 mm × 2.1 mm, 3 μm) using 0.1% formic acid–water–methanol as mobile phase, with a runtime of 5 min. The analyte was detected in multiple reaction monitoring (MRM) mode with positive electrospray ionization. Transitions of m / z 228.2 → 154.0 for tulobuterol and m / z 277.1 → 203.0 for the clenbuterol were monitored. The linear range was 0.5–100 ng/ml ( r = 0.9967) for tulobuterol with the lower limit of quantitation of 0.5 ng/ml. The intra-day and inter-day precisions were less than 10.3% for the analyte and the accuracy was less than −8.6%. The RSD of matrix effect and recovery yield were within ±15% of nominal concentrations and tulobuterol was stable during stability studies. The validated method has been successfully applied to a pharmacokinetic study of three doses of tulobuterol patch in rats for the first time. [ABSTRACT FROM AUTHOR]