High Sensitivity Bioanalysis using the Waters Xevo TQ Absolute Tandem Quandrupole Mass Spectrometer

Significance of the Topic

Bioanalysis of potent drug candidates at ultra-low concentrations is critical for pharmacokinetic studies and ethical research designs requiring minimal sample volumes. Advances in UPLC-MS/MS sensitivity support reliable detection at the femtogram level and facilitate seamless method transfer between laboratories.

Objectives and Study Overview

This study aimed to demonstrate a robust, high-sensitivity UPLC-MS/MS workflow for quantifying midazolam and imipramine in rat plasma. Key goals included establishing detection limits, assessing linearity across a wide concentration range, and verifying reproducibility using a simple sample preparation protocol.

Methodology

Sample preparation involved protein precipitation of rat plasma (100 µL) with acetonitrile (300 µL), centrifugation, dilution of the supernatant, and injection of 10 µL into the UPLC-MS/MS system.

Used Instrumentation

• UPLC: ACQUITY UPLC I-Class PLUS with BEH C18 2.1 × 50 mm, 1.7 µm column; column temp 55 °C; flow rate 0.45 mL/min; gradient time 1.9 min; mobile phases 0.1% formic acid in water with 10 mM ammonium formate (A) and 0.1% formic acid in acetonitrile (B).
• MS: Xevo TQ Absolute Tandem Quadrupole; ESI+; MRM transitions midazolam 326.1>291.0 (CV=60 V, CE=26 eV), imipramine 281.1>86.0 (CV=30 V, CE=16 eV); capillary voltage 0.5 kV.
• Data management: MassLynx v4.2, TargetLynx XS v4.2.

Main Results and Discussion

Both analytes were baseline resolved with retention times of 1.1 min (midazolam) and 1.5 min (imipramine). Calibration was linear from 0.2 to 100 pg/mL (r2 > 0.99). The limit of detection and quantification for both compounds was 0.2 pg/mL, with precision (%CV) under 11% at the lowest levels, demonstrating exceptional sensitivity and reproducibility.

Benefits and Practical Applications

• Robust, reproducible quantification at sub-picogram levels.
• Minimal sample preparation and rapid analysis.
• Applicable to low-dose pharmacokinetic and toxicokinetic studies.
• Facilitates inter-laboratory method transfer and high-throughput workflows.

Future Trends and Applications

Opportunities include extending the approach to additional trace-level analytes, integrating automated and miniaturized sample preparations, expanding to clinical microdosing and biomarker analysis, and combining with multiplexed detection strategies for comprehensive pharmacological profiling.

Conclusion

This application note validates a highly sensitive, fast, and reproducible UPLC-MS/MS method for detecting midazolam and imipramine in plasma at femtogram levels, underscoring its value in modern bioanalytical research.