Application of LC-SIM-MS. Determination of a neurokinin receptor antagonist in human plasma by column-switching HPLC and automated sample processing

A sensitive and selective HPLC column-switching procedure with single-quadrupole massspectrometric detection has been developed for the determination of a neurokinin receptor antagonist in human plasma. The analyte and its deuterated internal standard were isolated from plasma by precipitation of protein with acetonitrile and the deproteinated sample was injected on to the trapping column of a column-switching system (2.1 mm×10mm, XTerra MS C8, 5μm). After flushing, the analytes were back-flushed on to the analytical column (2.1 mm×30 mm, XTerra MS C18, 3.5μm) and separated by elution with a gradient prepared from acetonitrile and 5 mM ammonium formate. The effluent from the analytical column (300 μL min−1) was passed to a turbo ion-spray interface. Selected ion monitoring (SIM) in the positive-ion mode was used for mass-spectrometric detection. The limit of quantification was 0.5 ng mL−1. The mean precision of the procedure was 6.3%, the mean accuracy 99%. The short chromatographic cycle time (4.5 min) enabled increased overnight sample throughput, which was realized by automating the sample work-up procedure, using 96-well plate technology and a Tecan Genesis robotic sample processor. The use of a study-oriented LIMS with an Oracle data-base reduced pre-and post-run data handling and data storage activity and thus facilitated management of the large amount of analytical data generated. The robotic procedure was accurate, precise, and robust. It could be successfully applied to the analysis of several thousand samples from clinical trials.