Automated 96-well SPE and LC-MS-MS for determination of protease inhibitors in plasma and cartilage tissues

Bioanalytical methods based on automated solid-phase extraction (SPE) and h igh-performance liquid chromatography with electrospray tandem mass spectro metry (LC-MS-MS) have been developed and utilized for the determination of MMP inhibitors in plasma and cartilage tissues. The SPE methods were automa ted using a 96-wed extraction plate and a 96-channel programmable liquid-ha ndling workstation. The LC-MS-MS methods were developed using a rapid gradi ent LC separation, followed by sample introduction through an ionspray inte rface in the positive ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized SPE methods, crude plasma o r ground cartilage supernatant samples were loaded onto an SPE plate to rem ove proteins and other interfering components in the matrixes to render rel atively dean extracts for LC-MS-MS analysis. Compared to the simple plasma protein precipitation method, the automated SPE method afforded significant time-saving in sample preparation and improved sensitivity in MS detection , The methods were validated and successfully applied to the analysis of pr otease inhibitors in plasma and cartilage tissues.