Liquid–liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry

Abstract

BMS-299897 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid–liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized 13C 6-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm × 50 mm, 3 μm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25–100 ng/ml for human CSF. Both standard curves were fitted to a 1/ x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within ±7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.