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

Abstract

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 μL) 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 C18 column (2.1 × 100 mm, 1.7 μm) 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 (r2) ≥ 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 d4 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.

Recent Progress in Fast Sample Preparation Techniques

“…The format of a 96-well plate is commonly used in parallel sample pretreating in high-throughput bioanalysis . A particular preparation procedure for batch of samples was reported to execute simultaneously, including SPE, μ-SPE, SPME, − LLE, , membrane separation, and protein precipitation . For example, Carasek’s group developed a high-throughput sample preparation approach based on thin-film SPME and a 96-well plate. , By using cork coating on thin-films as a sorbent of SPME, they extracted contaminants from 96 water samples within 200 min (180 min for extraction and 20 min for desorption) .…”