Improving Sensitivity for Trastuzumab Emtansine Quantification using Trap-Elute MicroLC-MS with Large Volume Sample Loading

Importance of Topic

Quantification of antibody–drug conjugates (ADCs) such as trastuzumab emtansine at low concentrations in biological matrices is critical for preclinical pharmacokinetic studies. Combining immunoaffinity enrichment with a trap-elute microLC-MS/MS workflow enhances sensitivity while preserving throughput.

Study Objectives and Overview

This work aimed to develop an ultra-sensitive assay for trastuzumab emtansine in mouse plasma by leveraging large-volume injection on a trap-elute microLC system. The key goals were to improve the lower limit of quantitation (LLOQ), maintain high throughput, and ensure robust quantification performance.

Methodology and Instrumentation

The workflow consisted of:

• Immunocapture: Streptavidin magnetic beads were coated with biotinylated anti-human IgG for selective enrichment of trastuzumab emtansine from 25 µL plasma.
• Proteolysis: Enriched eluent was neutralized and subjected to trypsin digestion in a deep-well thermo-shaker.
• Trap-elute microLC: M5 MicroLC system operated at 1 µL/min for analytical separation and 50 µL/min for sample loading onto a C18 trap column.
• Mass spectrometry: SCIEX QTRAP 6500+ with OptiFlow™ Turbo V source and SteadySpray™ probe; MRM transitions were optimized for signature peptides.

Main Results and Discussion

Injection-volume studies demonstrated that increasing injection volume to 50 µL improved peak areas without compromising chromatographic performance. The optimized assay achieved:

• LLOQ of 0.5 ng/mL and LOD of 0.25 ng/mL in mouse plasma.
• Linear dynamic range spanning four orders of magnitude (0.5–5000 ng/mL) with r = 0.992 (1/x² weighting).
• Accuracy between 85 % and 120 % and intra- and inter-assay CVs below 15 %.

Benefits and Practical Applications

This microLC-MS/MS immunoaffinity method delivers:

• Enhanced sensitivity for small-volume preclinical samples.
• High throughput comparable to conventional analytical-flow assays.
• Reduced matrix interference and simplified sample preparation.
• Minimal source optimization for routine implementation in bioanalysis labs.

Future Trends and Opportunities

Potential developments include:

• Automation of immunoaffinity capture and digestion steps to increase sample throughput.
• Expansion to multiplexed quantification of multiple ADCs or mAbs in a single run.
• Integration of nano- or low-flow LC to further boost sensitivity for ultra-low abundance targets.
• Application in translational studies bridging preclinical and clinical bioanalysis.

Conclusion

A trap-elute microLC-MS/MS assay with large-volume injection on the SCIEX QTRAP 6500+ platform enables reliable quantification of trastuzumab emtansine at sub-nanogram levels in mouse plasma, combining sensitivity, dynamic range, and throughput for biotherapeutic research.

Reference

1. Lei Xiong and Remco van Soest. Ultra-Sensitive Quantification of Trastuzumab Emtansine in Mouse Plasma using Trap-Elute MicroLC-MS Method. SCIEX technical note RUO-MKT-02-8288-A, 2018.