A sensitive high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) assay is established for the determination of tiotropium in human plasma using benzyltriethylammonium chloride as the internal standard (IS). After being treated with C(18) cartridges, plasma samples are separated by HPLC on a reversed-phase C(18) column with a mobile phase of 40mM ammonium acetate buffer-methanol (56:44, v/v). Tiotropium is determined in a single-quadrupole MS. HPLC-ESI-MS is performed in the selected ion monitoring mode using target ions at m/z 392.0 for tiotropium and m/z 192.3 for the IS. The calibration curve is linear over the range 1.5-30 pg/mL. The intra- and inter-assay variability values are less than 10.1% and 13.6%, respectively. The mean plasma extraction recovery of tiotropium is 92.3 +/- 5.0%. The method has been successfully applied to studying the pharmacokinetics of tiotropium in healthy Chinese volunteers.
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