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Determination of five positive control drugs in hERG external solution (buffer) by LC-MS/MS to support in vitro hERG
Tamara I King1, Amruta Indapurkar1, Isra Tariq1
1Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
Insights
Accurate drug concentration verification is crucial for assessing cardiac safety, particularly hERG channel block. This study developed and validated LC-MS/MS methods to quantify five common hERG blockers in external solutions, ensuring reliable proarrhythmic risk assessment.
Area of Science:
- Pharmacology and Toxicology
- Analytical Chemistry
- Regulatory Science
Background:
- The hERG channel is critical for cardiac repolarization; its blockade can lead to QT prolongation and Torsade de Pointes (TdP).
- Accurate drug concentration in electrophysiology assays is essential for reliable proarrhythmic risk assessment, as per ICH S7B guidelines.
- Existing analytical methods lack quantification and stability data for key hERG blocker positive controls in specialized assay media.
Purpose of the Study:
- To develop and validate robust analytical methods for quantifying five common hERG channel blockers (dofetilide, cisapride, terfenadine, sotalol, E-4031) in hERG external solution.
- To address the need for verified drug concentrations in patch clamp recordings for accurate proarrhythmic risk assessment.
- To provide stability data for these critical positive control compounds in a relevant assay matrix.
Main Methods:
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for quantification.
- Method validation was performed according to FDA bioanalytical method validation guidance.
- Stability studies were conducted on the five target compounds in the hERG external solution.
Main Results:
- Validated LC-MS/MS methods were successfully developed for the accurate quantification of the five hERG blockers.
- Stability of the compounds in the hERG external solution was assessed.
- The validated methods provide a means to verify drug concentrations, reducing uncertainty in hERG assay results.
Conclusions:
- The developed and validated LC-MS/MS methods are suitable for quantifying critical hERG positive control compounds in external solutions.
- These methods support regulatory compliance and enhance the reliability of drug-induced proarrhythmic risk assessments.
- This work fills a critical gap in analytical methodology for cardiac safety pharmacology studies.
Abstract:
ICH S7B recommends screening for hERG channel block using patch clamp recordings to assess a drug's proarrhythmic risk. Block of the hERG channel has been associated with clinical QTC prolongation as well as the rare, but potentially fatal ventricular tachyarrhythmia Torsade de Pointes (TdP). During recording, drug concentrations perfused to the cells can deviate from nominal concentrations due to molecule-specific properties (such as non-specific binding), thereby introducing error when assessing drug potency. To account for this potential source of error, both the original ICH S7B and the newly released ICH E14/S7B Q&As guidelines call for verifying drug solutions' concentrations. Dofetilide, cisapride, terfenadine, sotalol and E-4031 are hERG blockers commonly used as positive controls to illustrate hERG assay sensitivity. The first four compounds are also clinical drugs associated with high TdP risk; therefore, their safety margins may be useful comparators to better understand an investigational product's TdP risk. Having analytical methods to quantify these five compounds in the hERG external solution that will be used for patch clamp recordings is important from a regulatory science research perspective. However, a literature search revealed no analytical methods or stability information for these molecules in the high salt, serum-free matrix that constitutes the hERG external solution. This study was conducted to develop and validate LC-MS/MS methods to quantify these 5 molecules in hERG external solution. The bioanalytical methods for these positive controls were validated as per the FDA's bioanalytical method validation guidance along with various stabilities.
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