High Sensitivity Bioanalysis Using the Xevo TQ-XS Tandem Quadrupole Mass Spectrometer

Importance of the Topic

Bioanalysis plays a crucial role in drug discovery and development by delivering insights into pharmacokinetics and dose response. As therapeutic candidates become more potent and studies adopt smaller sample volumes for ethical reasons, highly sensitive analytical methods are essential. In addition, robust instrument-to-instrument reproducibility is needed for seamless method transfer between laboratories.

Objectives and Study Overview

This study demonstrates a high sensitivity UPLC-MS/MS approach employing the Xevo TQ-XS tandem quadrupole mass spectrometer to quantify midazolam and imipramine in human plasma down to 0.2 pg/mL on column. Reproducibility was evaluated across three independent UPLC-MS/MS systems, and limits of detection, quantification, linearity, and precision were assessed.

Instrumentation

• UPLC system: ACQUITY UPLC I-Class
• Mass spectrometer: Xevo TQ-XS tandem quadrupole
• Column: ACQUITY BEH C18, 2.1 x 50 mm, 1.7 um
• Software: MassLynx v4.2 and TargetLynx XS v4.2

Methodology

• Sample preparation: Protein precipitation by adding acetonitrile (3:1 v/v), centrifugation at 25,000 g, followed by 1:1 dilution with deionized water
• Injection volume: 10 uL
• LC conditions: Mobile phase A 0.1% formic acid with 10 mM ammonium formate in water; mobile phase B 0.1% formic acid in acetonitrile; gradient 25–45% B over 1.9 min; flow rate 0.45 mL/min; column temperature 55 degC; sample temperature 10 degC
• MS conditions: ESI positive mode; MRM transitions Midazolam 326.1->291.0 (cone 60 V, collision 26 V) and Imipramine 281.1->86.0 (cone 30 V, collision 16 V); capillary voltage 0.5 kV

Main Results and Discussion

Both analytes exhibited baseline retention at 1.1 min (midazolam) and 1.6 min (imipramine). Calibration curves were linear from 0.2 to 100 pg/mL with r2 > 0.99 (1/x weighting). The limit of detection was confirmed at 0.2 pg/mL (250 ag on column) across all three systems. Lower limits of quantification were 0.4 pg/mL for imipramine and 1.0 pg/mL for midazolam, with inter-system precision below 8% CV. Measured concentrations at QC levels were within 15% of nominal values.

Benefits and Practical Applications

• Enables trace-level quantification in small-volume plasma samples
• Simple and rapid protein precipitation sample treatment
• Demonstrates consistent performance across multiple instruments for reliable method transfer

Future Trends and Applications

Advances in mass spectrometry sensitivity, microflow UPLC, and automation promise further reductions in sample volume and analysis time. Integration with high-resolution platforms and machine learning–driven data processing will broaden applications to multiplexed assays, real-time monitoring, and personalized medicine. Continued miniaturization and improved ion sources will expand bioanalysis in clinical diagnostics and environmental testing.

Conclusion

The described UPLC-MS/MS method using Xevo TQ-XS achieves sub-pg/mL detection of midazolam and imipramine in human plasma with high reproducibility across instruments, supporting reliable pharmacokinetic and bioequivalence studies.

Reference

Billy J. Molloy and Robert S. Plumb. High Sensitivity Bioanalysis Using the Xevo TQ-XS Tandem Quadrupole Mass Spectrometer. Waters Corporation Application Note, March 2021.