Comparison of Tandem and High Resolution Mass Spectrometry for the Quantification of the Monoclonal Antibody, Trastuzumab in Plasma

Significance of the topic

Demand for precise bioanalytical quantification of therapeutic proteins continues to grow as monoclonal antibodies become more prevalent in drug development. High selectivity, sensitivity and broad dynamic range are critical for accurate pharmacokinetic and toxicokinetic studies.

Objectives and study overview

This work compares the quantitative performance of a tandem quadrupole mass spectrometer (Xevo TQ-XS) and a high-resolution QTof instrument (Xevo G2-XS QTof) for measuring trastuzumab in plasma. Unique tryptic peptides (FTISADTSK and DTYIHWVR) serve as surrogates for antibody quantification.

Methodology and instrumentation

Sample preparation involved immunoaffinity purification from plasma using a 96-well protein A plate, followed by enzymatic digestion and solid-phase extraction clean-up. Chromatographic separation used ACQUITY UPLC H-Class with a Peptide BEH C18 column and an eight-minute gradient. Quantitative analysis on the Xevo TQ-XS employed multiple reaction monitoring (MRM) for targeted peptide transitions. The Xevo G2-XS QTof was operated in three modes: Tof-MRM (product ion and precursor ion targeting) and full-scan MS.

Main results and discussion

• Xevo TQ-XS MRM achieved lower limits of quantification (LLOQs) of 10–25 ng/mL and a linear dynamic range spanning ≥4.3 orders of magnitude for both peptides.
• Xevo G2-XS QTof Tof-MRM delivered comparable performance with LLOQs of 25–50 ng/mL and linearity ≥4.0 orders, while alternative HRMS modes showed reduced sensitivity and narrower dynamic range.
• Quality-control samples on both platforms met accuracy (±15%) and precision criteria (%CV ≤15%).
• Chromatographic profiles demonstrated high specificity and signal-to-noise advantages for HRMS with narrow mass extraction windows.

Benefits and practical applications

The study confirms that high-resolution QTof instrumentation can match tandem quadrupole sensitivity for mAb quantification while offering added qualitative insights in a single run. This dual capability streamlines workflows in bioanalysis, supporting robust pharmacokinetic studies and QA/QC in pharmaceutical research.

Future trends and applications

• Broader adoption of HRMS-based MRM techniques in regulated bioanalysis.
• Integration of data-independent acquisition for simultaneous multi-attribute monitoring.
• Advanced software for enhanced mass accuracy and automated data processing.
• Expansion of high-throughput immunoaffinity-LC-MS workflows for complex biologics.

Conclusion

The Xevo G2-XS QTof system, operated in Tof-MRM mode, delivers sensitive, accurate and robust quantification of trastuzumab in plasma, with performance comparable to a state-of-the-art tandem quadrupole platform. This validates HRMS as a versatile tool for protein therapeutic analysis.

References

No external literature references were cited in the original text.