An UHPLC-MS/MS method was developed and validated to quantify BMS-927711, a drug candidate to treat migraine, in rat dried blood spots (DBS). The DBS samples were extracted using an improved liquid-liquid extraction (LLE) strategy involving in the sonication of DBS punches in 20% MeOH aqueous solution containing the internal standard, [C-13(2), D-4]-BMS-927711, and then with a 100 mM NH4OAc buffer solution, followed by an automated LLE with EtOAc-hexane (70:30, v/v). The presence of 20% MeOH as an organic modifier in the elution solution significantly improved the analyte elution efficiency and assay performance. A novel inter-well volume replacement dilution workflow was introduced for DBS sample dilution before LLE step. This was a simple two-step process, firstly a small portion of the DBS blank solution was discarded, and then the same volume of a concentrated DBS sample solution was spiked into the leftover blank solution to achieve a desired dilution. Chromatographic separation was achieved on an Acuity UPLC (R) BEH C-18 column (2.1 mm x 50 mm, 1.7 mu m) and the analyte was detected by selected reaction monitoring (SRM) with positive electrospray ionization on an AB Sciex Triple Quad 5500 mass spectrometer. The standard curve was linear from 5.00 to 5000 ng/mL with assay precision <= 4.9% CV, and assay accuracy within +/- 3.1%Dev of the nominal values. Accurate sample dilution was achieved by using inter-well volume replacement with a precision of <= 4.2% CV and an accuracy of +/- 3.3% for dilution QC at 50,000 ng/mL with 100-fold dilution (n = 18). This robust UHPLC-MS/MS assay has been successfully applied to the non-clinical studies in rats. By using inter-well volume replacement workflow, accurate dilution was demonstrated using only one DBS blank sample for a typical dilution of <50-fold, and using only two blank DBS samples for a dilution of up to 625-fold. Moreover, this new workflow makes it easier to automate DBS sample dilution. (C) 2013 Elsevier B.V. All rights reserved.
