Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry

A sensitive, efficient, high throughput, direct injection bioanalytical met hod based on a single column and high-performance liquid chromatography (HP LC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for samp le cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica, This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the parking w hile retaining the drug molecules on the bonded hydrophobic phase. The anal ytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. T he column effluent was diverted from waste to the mass spectrometer for ana lyte detection. Within 200 plasma sample injections the response ratio (ana lyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were g reater than 90%. The total analysis time was less than or equal to 5 min pe r sample. Copyright (C) 2000 John Wiley & Sons, Ltd.