Development of a low volume plasma sample precipitation procedure for liquid chromatography/tandem mass spectrometry assays used for drug discovery applications

The demand for high sensitivity bioanalytical methods has dramatically increased in the drug discovery stage; in addition, there has been a growing trend of reducing the sample volume that is required for these assays. A sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedure has been developed and tested to meet these needs. The assay requires only a low plasma sample volume (10 microL) and employs a protein precipitation procedure using a 1:6 plasma/acetonitrile ratio. The supernatant is injected directly into the LC/MS/MS system using the selected reaction monitoring (SRM) procedure for detection. A generic HPLC gradient based on a methanol/water mobile phase with a flow rate set to 0.8 mL/min was used. The test method showed very good linearity between 0.1-1000 ng/mL (R2 = 0.9737), precision (%RSD = 6-9), accuracy (%RE = -2) and reproducibility (%RSD = 11). A drug discovery IV/PO study was assayed using both the new low volume method and our standard volume (50 microL) method. The correlation of the two sets of data from the two methods was excellent (R2 = 0.9287). This new assay procedure has been successfully used in our laboratory for over 100 different rat or mouse discovery PK studies.