title: A liquid chromatography-tandem mass spectrometry method for the quantitative determination of diastereomers of a phosphoramidate nucleotide prodrug (PSI-7851) in human plasma.
abstract: 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.
authors.affiliation: Pharmasset, Inc., 303A College Road East, Princeton, NJ 08540, USA. dbao@pharmasset.com
authors.forename: Donghui
authors.initials: D
authors.lastname: Bao
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Ross
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sofia
mesh_terms: D000704:Analysis of Variance; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: C553297:2'-deoxy-2'-fluoro-2'-C-methyluridine 5'-monophosphate; D014542:Uridine Monophosphate