Simultaneous in vivo Quantification and Metabolite Identification of Plasma Samples Using High Resolution QTof and Routine MSE Data Analysis

Importance of the Topic

High resolution mass spectrometry enables simultaneous accurate quantification of a parent drug and comprehensive metabolite identification in a single analytical run. This dual capability streamlines drug metabolism and pharmacokinetic workflows and enhances the depth of in vivo bioanalytical studies.

Objectives and Study Overview

This study compared a high resolution QTof platform operated in MS E mode with a conventional triple quadrupole mass spectrometer in MRM mode. Propranolol was used as a model compound in a rat pharmacokinetic study to evaluate quantitative performance, consistency of pharmacokinetic parameters, and the added value of concurrent metabolite profiling.

Methodology and Instrumentation

Sample collection involved Sprague-Dawley rats dosed orally with propranolol at 75 mg/kg and plasma drawn from predose to 24 hours. Extraction used protein precipitation with acetonitrile containing 0.1% formic acid and an internal standard, followed by dilution. Chromatography employed a ballistic gradient on an ACQUITY UPLC BEH C18 1.7 µm, 2×50 mm column with a two-minute gradient. Quantitative acquisition was performed on a Thermo Quantum Ultra in MRM mode and a Waters Xevo G2 QTof in MS E mode. Data processing and noncompartmental pharmacokinetic analysis used MassLynx, TargetLynx, UNIFI Scientific Information System, and Phoenix WinNonLin.

Main Results and Discussion

Both platforms produced calibration curves with r2 values above 0.99 over 2.5 to 10000 ng/mL and met accuracy and precision acceptance criteria. Quality control sample results were comparable, with deviations within ±20%. Overlay of pharmacokinetic profiles showed nearly identical concentration-time curves and area under the curve values. Key pharmacokinetic parameters such as half-life and exposure metrics differed by less than 7% between instruments. The high resolution dataset additionally revealed multiple phase I and phase II propranolol metabolites, demonstrating the advantages of full scan MS for pathway elucidation.

Benefits and Practical Applications

• Combines quantitative analysis and metabolite identification in one run
• Eliminates extensive MRM method development for each metabolite
• Delivers high mass accuracy and rich fragmentation information
• Maintains alignment with established DMPK workflows and regulatory expectations

Future Trends and Potential Applications

Future developments will likely see broader adoption of high resolution platforms in routine bioanalysis, enhanced informatics for automated data interpretation, integration with machine learning for predictive metabolism studies, and expansion into simultaneous pharmacodynamic marker profiling.

Conclusion

High resolution QTof mass spectrometry demonstrates robustness and reliability equivalent to traditional tandem quadrupole systems for in vivo pharmacokinetic analysis while offering the added value of comprehensive metabolite profiling. This integrated quan/qual approach represents a powerful tool for modern DMPK research.

Reference

• Wrona M, Rainville P, Langlois E, Ewing N, Laterreur J. Simultaneous in vivo Quantification and Metabolite Identification of Plasma Samples Using High Resolution QTof and Routine MS E Data Analysis. Waters Application Note, May 2013.