A liquid chromatography-tandem mass spectrometry method for the quantitative determination of diastereomers of a phosphoramidate nucleotide prodrug (PSI-7851) in human plasma

A rapid and stereospecific method using high performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) for the separation and determination of PSI‐7851 diastereomers in human K₂EDTA plasma has been developed. The analytical method involves direct protein precipitation with acetonitrile, followed by separation of the diastereomers on a Luna C₁₈ column, positive mode electrospray ionization and selected reaction monitoring mode mass spectrometry detection. The mobile phase composition and pH were investigated for the resolution of the two diastereomers of PSI‐7851. The optimized method showed good resolution (R_s = 4.8) within short analysis time (approximately 8 min). The assay range was 5–2500 ng/mL for both diastereomers using a 1/x² weighted linear regression analysis for standard curve fitting. Replicate sample analysis indicated that intra‐ and inter‐day accuracy and precision were within ±15.0%. The recovery of diastereomers from human plasma was greater than 85% and no significant matrix effect was observed. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies. Copyright © 2011 John Wiley & Sons, Ltd.