Quantitation by High Resolution Mass Spectrometry: Using Target Enhancement and Tof-MRM to Achieve Femtogram-level On-column Sensitivity for Quantitation of Drugs in Human Plasma

Significance of the topic

The adoption of high resolution mass spectrometry for quantitative bioanalysis addresses critical needs for sensitivity and selectivity in complex biological matrices. By integrating targeted acquisition modes on time-of-flight platforms, researchers can achieve femtogram-level detection of pharmaceuticals in plasma, enabling more accurate pharmacokinetic, metabolic and safety studies.

Objectives and study overview

This study evaluates three data acquisition approaches on a quadrupole time-of-flight system to quantify four drugs in human plasma. Targets include alprazolam, verapamil, buspirone and clopidogrel. The goal is to compare full-scan MS, MS E and ToF-MRM modes with target enhancement in terms of detection limit, quantification limit, linear dynamic range and data file size.

Methodology

Human plasma samples were protein-precipitated with acetonitrile, diluted 1 1 with water and spiked with analytes over a concentration range from 100 ng/mL to approximately 1 pg/mL. Chromatography was performed on an ACQUITY UPLC I-Class system with a BEH C18 column using a fast gradient. Electrospray ionization in positive mode and a SYNAPT G2-S HDMS QTof mass spectrometer operated at high resolution (>20000 FWHM) were used for detection. Acquisition modes included full-scan MS across 50–1000 m/z, MS E alternating low and high collision energies, and ToF-MRM with quadrupole precursor selection and synchronized ToF pusher for target enhancement.

Instrumentation

• ACQUITY UPLC I-Class System
• SYNAPT G2-S HDMS QTof Mass Spectrometer
• ACQUITY BEH C18 Column 2.1 x 50 mm 1.7 μm
• MassLynx and TargetLynx Software

Results and discussion

ToF-MRM delivered 2 to 8 times greater signal response compared to full-scan and MS E modes at low pg/mL levels. Limits of detection improved 4 to 8 fold and limits of quantification improved 4 to 12 fold, reaching as low as 1.5 pg/mL (7.5 fg on column) for three analytes. Linear dynamic ranges of 3.6 to 3.9 log units were achieved with correlation coefficients above 0.99. Data file size per injection was reduced by over 95 relative to full-scan approaches, simplifying data handling and storage.

Benefits and practical applications

The ToF-MRM mode offers triple-quadrupole-like quantitative performance on an HRMS platform while retaining high-resolution mass accuracy. Key advantages include:

• Exceptional sensitivity in complex matrices
• Wide linear dynamic range suitable for bioanalytical validation
• Significant data storage and processing efficiency
• Combined qualitative and quantitative capability for drug discovery and development

Future trends and potential applications

The integration of targeted acquisition modes on high resolution systems is likely to expand into routine bioanalysis, metabolite profiling, stability studies and PKPD investigations. Continued development of synchronized ToF timing schemes and advanced data processing will further close the sensitivity gap with tandem quadrupole instruments and support comprehensive screening plus quantitation workflows.

Conclusion

Target enhancement with ToF-MRM on a quadrupole time-of-flight platform provides femtogram-level detection, robust linearity and streamlined data management for drug quantitation in human plasma. This approach bridges the gap between qualitative high resolution workflows and routine quantitative assays, offering a versatile solution for pharmaceutical and bioanalytical laboratories.

Reference

1. Waters application note 720004728en Targeted High Resolution Quantification with ToF-MRM and HD-MRM
2. Waters application note 720004762en Effect of MS Scan Speed on UPLC Peak Separation and Metabolite Identification Time-of-Flight HRMS vs Orbitrap