Improving ion-trap GC-MS quantitation limits for therapeutic agents extracted from rat plasma

Insufficient quantitation limits using ion-trap gas-chromatography mass-spectrometry (GC-MS) prevented the assay of some samples during a preliminary screening of preclinical rat plasma samples (50 microliter) containing novel, polar therapeutic agents. Few options were available for improving the lower limit of quantitation. The limited amount of sample available precluded the extraction additional plasma. Lipid-liquid extraction recoveries were greater than 90% throughout the range of the standard curve (500-2000 ng ml-1). Chromatography was optimized and multiple, equivalent sites for analyte fragmentation were precluded, using MS-MS to improve assay sensitivity. Quantitation limits were decreased 10-fold however, by using a larger syringe to increase the injection volume from 5 to 50 microliter, in combination with a universal programmable injector. These large injection volumes required changes in the injector events program and in column plumbing. Additionally, evaluation of injection liner packing material demonstrated a 2-fold improvement in sensitivity, using carbofrit, relative to silanized glass wool. Converting to inert ion-trap electrodes did not appear to affect the detection limit, perhaps due to over-riding peak broadening during gas chromatography. The changes described produced a 20-fold improvement in the lower limit quantitation.