Quantitative determination of a nonpeptide antithrombotic in dog plasma by microbore high-performance liquid chromatography-tandem mass spectrometry utilizing pneumatically assisted electrospray ionization

A method has been developed and is described for the quantitative determination of a nonpeptide antithrombotic in dog plasma. The assay employs reversed phase microbore high-performance liquid chromatography in conjunction with tandem mass spectrometry utilizing pneumatically assisted electrospray ionization. The analyte and internal standard are isolated from the plasma matrix by solid-phase extraction. The mass spectrometer is operated in the positive ion multiple reaction monitoring mode and is set to detect the presence of a precursor-product ion pair for both the analyte and internal standard to generate product ion chromatograms for both species. The analyte is quantified by using weighted least-squares regression of the peak height ratio of drug:internal standard. The method provides linear response for plasma concentrations ranging from 5 ng/mL (25 pg on-column) to 2500 ng/mL. Statistical evaluation and examples of authentic sample assays are also presented.