Column switching in capillary liquid chromatography–tandem mass spectrometry for the quantitation of pg/ml concentrations of the free basic drug tolterodine and its active 5-hydroxymethyl metabolite in microliter volumes of plasma

Abstract A capillary column switching system was developed for the determination of low, unbound concentrations of the basic drug tolterodine and its active 5-hydroxymethyl (5-HM) metabolite in human plasma. Free concentrations of tolterodine and 5-HM at p M and n M (pg/ml and ng/ml) levels were obtained by ultrafiltration of 40–400 μl plasma at 37°C. The free fraction (%) was independent of the plasma concentrations of the analytes. Detection of the analytes was performed by sheathless electrospray tandem mass spectrometry in the multiple-reaction monitoring mode. The selectivity of the mass spectrometric detection and the additional clean-up on the pre-column allowed direct injection of the ultrafiltrated plasma samples. Tolterodine and 5-HM were pre-concentrated on a reversed-phase capillary pre-column (1 cm×200 μm) and subsequently backflushed onto the separation column (25 cm×200 μm). The stability of the chromatographic system was good; a large number of ultrafiltrated plasma samples could be injected and the relative standard deviation of the retention times was typically ≤1% (within-day). The accuracy was between 86 and 105% and the precision was between 1 and 7% without the use of an internal standard. Linear calibration curves were obtained between 100 p M and 100 n M .