A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LCMS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LCMS/MS with conventional C18 and monolithic columns were in the range of 88111% and 0.514%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41108% and 381%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples. Symbiosis Pro 在線固相萃取-液相色譜聯(lián)用系統(tǒng) SYMBIOSIS PICO在線固相萃取SPE-LC聯(lián)用系統(tǒng)