High-speed liquid chromatography/tandem mass spectrometry using a monolithic column for high-throughput bioanalysis

Area of Science:

• Analytical Chemistry
• Chromatography
• Mass Spectrometry

Background:

• Increasing workload in drug screening necessitates faster, accurate bioanalytical methods.
• Conventional particulate High-Performance Liquid Chromatography (HPLC) columns present limitations in throughput.
• Demand for high-throughput analytical methodologies in drug discovery and development is growing.

Purpose of the Study:

• To evaluate monolithic columns as an alternative to particulate HPLC columns for rapid bioanalysis.
• To demonstrate the feasibility of high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) for drug discovery support.
• To assess the performance and robustness of monolithic columns at high flow rates.

Main Methods:

• Utilized monolithic columns for chromatographic separations instead of conventional particulate HPLC columns.
• Employed high flow rates (6 mL/min) due to the lower pressure drop of monolithic columns.
• Analyzed plasma extracts using LC/MS/MS, measuring separation efficiency, signal/noise ratios, and reproducibility.

Main Results:

• Achieved baseline separation of multiple drug compounds in plasma extracts within one minute.
• Observed consistent separation efficiency and signal/noise ratios across varying flow rates (1-6 mL/min).
• Demonstrated high reproducibility in retention time, separation quality, peak response, and sensitivity over an overnight run.

Conclusions:

• Monolithic columns facilitate significantly reduced runtime in bioanalysis without compromising separation power.
• The developed high-speed LC/MS/MS method using monolithic columns is robust and reproducible.
• This approach effectively supports high-throughput drug discovery programs by enabling rapid and reliable bioanalysis.