Novel High-Throughput Quantitation of Potent hERG Blocker Dofetilide in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry: Application in a Phase 1 ECG Biomarker Validation Study.

The authors developed a novel, sensitive high-throughput ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of dofetilide in human plasma. To compensate for the matrix effect, a deuterated internal standard was used. The method employed a very low sample volume (50 mu L) of plasma for sample processing by using simple protein precipitation extraction in a 96-well plate. The extracted samples were chromatographed on an Acquity BEH C-18 column (2.1 x 100 mm, 1.7 mu m) and eluted in a gradient manner at a flow rate of 0.5 mL/min for 2 min using 5 mM ammonium formate (0.1% formic acid) and methanol. The calibration curve was linear from 25 to 2,500 pg/mL with a correlation coefficient (r(2)) >= 0.99 (0.9969-0.9980; n = 3). The developed method was validated as per the current United States Food and Drug Administration's guidance for industry on 'Bioanalytical Method Validation'. The multiple reaction-monitoring mode was employed for quantitation of dofetilide with m/z 442.2/198.2 and dofetilide d(4) with m/z 446.2/198.2. The validated method was used for evaluation of dofetilide concentration in the Comprehensive in vitro Proarrhythmia Assay phase 1 electrocardiogramic biomarker validation study.