2.3 Blood Sample Collection; Method of Measurement Blood samples were collected into tubes containing K2-EDTA prior to and 0.33, 0.67, 1, 1.33, 1.67, 2, 2.5, H 89 nmr 3, 3.5, 4, 5, 6, 8, 12, 16, 24, 36, 48 and 60 h after drug administration. This sampling was planned in order to provide a reliable estimate of the

extent of absorption, as well as the terminal elimination half-life, and to ensure that the area under the plasma concentration–time curve (AUC) from time zero to time t (AUC t ) was at least 80 % of the AUC from time zero extrapolated to infinity (AUC ∞ ). Samples were processed and stored under conditions (frozen) that have been shown not to cause significant degradation of the analyte. The experimental samples

were assayed for doxylamine, using a validated bioanalytical ultra-high-performance liquid chromatography method with tandem mass spectrometry detection (UPLC/MS/MS method, Xevo TQ MS, Waters Corp., Milford MA), which involved the solid-phase extraction of doxylamine and the deuterium-labeled internal standard (Doxylamine-d5) from plasma samples (150 μL). The calibration curve ranged from 1.0 to 300.0 ng/mL and the limit of quantification was 1.0 ng/mL. A gradient elution with 0.1 % formic acid in acetonitrile and 0.1 % formic acid in water was used for the mobile phase. A volume of 10 μL was injected into an Acquity UPLC Doramapimod BEH C18 column (1.7 μm learn more particle size, 2.1 mm id × 50 mm length) and the transitions (m/z) for both doxylamine (271.22/167.02) and internal Phospholipase D1 standard (276.24/171.28) were monitored using MRM ion mode ESI+. The parameters evaluated during the validation were linearity and range, selectivity including hemolysed and hyperlipidemic plasma, specificity in the presence of common OTC, intra- and inter-run precision and accuracy, limit of quantification, dilution integrity,

carryover, recovery, matrix effect, stock solution stability, autosampler stability, short-term stability in human plasma at room temperature, freeze-thaw and long-term stability in human plasma. All the evaluated parameters met the acceptance criteria of the current guidelines. For past analytical batches run during the validation, the precision expressed as %CV of calibration standards ranged from 0.8 to 3.7 %, and the % mean accuracy of the back-calculated value of the calibration standards ranged from 94.2 to 103.4 %. The mean correlation coefficient for these analytical batches was 0.9992. The intra-run precision expressed as %CV for all concentration levels of quality control samples ranged from 0.9 to 12.7 %, and the inter-run precision ranged from 1.1 to 7.9 %. The intra-run accuracy expressed as % nominal for all concentration levels of quality control samples ranged from 96.4 to 113.7 %, and the inter-run accuracy ranged from 102.8 to 108.8 %.