Ultra-Sensitive Quantification of Trastuzumab Emtansine in Mouse Plasma using Trap-Elute MicroLC MS Method

Importance of the Topic

The precise measurement of monoclonal antibodies and antibody–drug conjugates in small animal plasma is essential for pharmacokinetic and preclinical studies. Traditional ligand-binding assays can be limited by specificity and dynamic range, so LC-MS/MS approaches offer an orthogonal solution with improved selectivity and quantitation across a wide concentration range.

Objectives and Study Overview

This study presents a hybrid immunoaffinity trap-elute microflow LC-MS/MS workflow for ultra-sensitive quantification of trastuzumab emtansine in mouse plasma. The aim was to achieve a lower limit of quantitation of 1 ng/mL using only 25 µL of plasma, while maintaining high accuracy, precision, and throughput.

Methods

Mouse plasma samples (25 µL) spiked with trastuzumab emtansine and an internal standard (SILuMab) underwent immunocapture on streptavidin magnetic beads conjugated with biotinylated anti-human IgG. After washing, the bound ADC was eluted with 0.1% TFA and neutralized. Proteins were denatured at 95 °C, digested with trypsin at 50 °C for 1 hour, and the resulting peptides were analyzed by trap-elute microflow LC and MRM detection. The trap phase enabled on-line desalting and concentration, while an analytical microLC column separated signature peptides.

Instrument Used

• SCIEX QTRAP 6500+ mass spectrometer with OptiFlow Turbo V Source and SteadySpray probe (no source adjustment required)
• M5 MicroLC system configured in trap-elute mode, capable of flows down to 1 µL/min
• Phenomenex Luna C18 trap column (20 × 0.3 mm, 5 µm) and Kinetex XB-C18 analytical column (50 × 0.3 mm, 2.6 µm)

Main Results and Discussion

The optimized microflow assay achieved a lower limit of quantitation of 1 ng/mL with accuracy between 87% and 109% and coefficients of variation below 15% across all calibration levels (1–20 000 ng/mL, r² = 0.996, 1/x² weighting). Compared with a conventional analytical-flow method, the microflow setup delivered over five-fold higher peak area and a three-fold improvement in signal-to-noise at low concentrations, enabling reliable quantitation from minimal sample volumes.

Benefits and Practical Applications

• High sensitivity with minimal plasma volume (25 µL) suitable for small animal studies
• Wide dynamic range (4.5 orders of magnitude) for early and late time-point samples
• Robust performance with minimal source or probe adjustments
• Reduced matrix interference via immunoaffinity enrichment
• High throughput through fast trap-elute cycles

Future Trends and Opportunities

Further developments may include automation of immunoaffinity sample preparation, multiplexed quantitation of multiple ADCs or mAbs in a single run, integration with microfluidic platforms for on-chip processing, and extension of the workflow to clinical pharmacokinetic monitoring. Advances in high-resolution MS and novel enrichment reagents will continue to push sensitivity and throughput boundaries.

Conclusion

This hybrid immunoaffinity-microflow LC-MS/MS approach on the SCIEX QTRAP 6500+ with M5 MicroLC delivers ultra-sensitive quantification of trastuzumab emtansine in mouse plasma, combining low sample requirements, broad dynamic range, and high reproducibility. It provides a powerful tool for biotherapeutic development and preclinical pharmacokinetic studies.

References

• Zhang F., Li Y., et al. Quantification of Trastuzumab in Rat Plasma using an Improved Immunoaffinity-LC-MS/MS Method, SCIEX Technical Note.
• Universal Solution for Monoclonal Antibody Quantification in Biological Fluids Using Trap-Elute MicroLC-MS Method, SCIEX Technical Note RUO-MKT-02-8279-A.