Highly sensitive assay for tiotropium, a quaternary ammonium, in human plasma by high-performance liquid chromatography/tandem mass spectrometry

Tiotropium bromide, a long-acting inhaled bronchodilator analogous to ipratropium bromide, is currently undergoing development for the treatment of chronic obstructive pulmonary disease. To evaluate its systemic absorption in humans, we have developed a rapid and sensitive method for its determination in human plasma based on high-performance liquid chromatography with tandem mass spectrometric detection (HPLC/MS/MS). Reversed-phase chromatography of tiotropium and the internal standard clenbuterol was carried out using acetonitrile/10 mM ammonium acetate (1% formic acid) 40:60 as mobile phase in a run time of 3.0 min. The sample preparation involved deproteination with acetonitrile, extraction into dichloromethane and back-extraction into hydrochloric acid. The assay was linear over the concentration range 0.500-50.0 pg/mL with intra- and inter-day precision (as relative standard deviation) both