Direct simultaneous determination of drug discovery compounds in monkey plasma using mixed-function column liquid chromatography/tandem mass spectrometry

A direct injection method based on a single column and high-performance liq uid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was develop ed for the simultaneous determination of two drug candidates in monkey plas ma samples in support of pharmacodynamic studies. Each diluted monkey plasm a sample containing internal standard was directly injected into a mixed-fu nction column for sample cleanup, enrichment and chromatographic separation . The proteins and macromolecules first passed through the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column with an aqueous liquid mobile phase were then chemi cally eluted by switching to a strong organic mobile phase at a constant fl ow rate of 1.0 ml/min. The column effluent was also diverted from waste to mass spectrometer for analyte detection. Samples from two different analyte studies were assayed in one analytical procedure and the calibration curve s were prepared using both analytes. The calibration curves were linear ove r the range of 5-2500 ng/ml for both analytes. The retention times for anal ytes and the internal standard were found to be consistent and no column de terioration was observed after 200 injections. The apparent on-column recov eries for the test compounds in monkey plasma samples were greater than 90% with 6% CV (N = 5). The total analysis time was less than 5 min per sample . (C) 2002 Elsevier Science B.V. All rights reserved.