High-Sensitivity Analysis of Drugs in Ultra-Small Volumes Plasma Samples Using Micro-Flow LCMS/ MS

Importance of the Topic

Accurate quantification of drugs and metabolites in minute plasma volumes is vital for discovery-stage pharmacokinetic studies, especially when sample availability is limited by micro-sampling in small animal models. High-sensitivity analytical methods reduce animal usage, lower costs, and improve data quality for safety and efficacy assessments.

Objectives and Study Overview

This study demonstrates a proof-of-concept micro-flow LC–MS/MS method for quantifying the antiarrhythmic drug verapamil and its metabolite nor-verapamil in ultra-small plasma samples. The goals were to:

• Develop a trap-and-elute micro-flow configuration to maximize sensitivity.
  
• Evaluate signal enhancement versus semi-micro flow.
  
• Assess recovery, accuracy, and precision using 2 µL plasma inputs.
  

Methodology and Instrumentation

The workflow combined micro-sampling and isotope-dilution MRM analysis:

1. Sample Preparation:
   – 2 µL plasma spike with verapamil, nor-verapamil, and deuterated internal standard.
   – Dilution with acetonitrile/formic acid, centrifugation, and 5 µL injection.
   
2. Chromatography:
   – Shimadzu Nexera MIKROS system with trap (Shim-pack MCT LC8) and analytical column (0.2×100 mm, 2.7 µm biphenyl).
   – Mobile phases water/formic acid and acetonitrile/formic acid.
   – Flow at 4 µL/min, 11 min total run time.
   
3. Detection:
   – LCMS-8060 triple quadrupole with micro-ESI source.
   – MRM transitions for verapamil (455.0 > 150.25) and nor-verapamil (440.95 > 165).
   – Linear calibration 0.5–185 µg/L, 1/x weighting.
   

Key Results and Discussion

Signal intensity improved markedly compared to semi-micro flow:

• Verapamil: >4.5-fold increase in peak area, >10-fold improvement in signal-to-noise.
  
• Nor-verapamil: >3.5-fold increase in signal.
  

Recovery and matrix effects were evaluated at low QC (0.5 µg/L):

• Overall recoveries >97% for both analytes.
  
• Calibration accuracy between 85–115% across seven levels.
  

Benefits and Practical Applications

The micro-flow trap-and-elute approach offers:

• High sensitivity without increasing injection volume.
  
• Compatibility with ultra-small plasma samples (2 µL).
  
• Potential integration with automated micro-sampling devices to adhere to 3R principles.
  

Future Trends and Applications

Emerging directions include applying micro-flow LC–MS/MS to:

• High-throughput drug screening in early drug discovery.
  
• Clinical microsampling for pediatric and neonatal studies.
  
• Integration with novel sampling devices for minimally invasive monitoring.
  

Conclusion

The presented micro-flow LC–MS/MS method dramatically enhances sensitivity for verapamil and nor-verapamil quantification in ultra-small plasma volumes, enabling reduced sample requirements and supporting ethical micro-sampling strategies in pharmacokinetic research.

Reference

[1] Rapid Communications in Mass Spectrometry 2014, 28, 1293–1302