Improving limit of quantification in clinical research by combining PaperSpray with FAIMS technology

Importance of the Topic

Rapid and sensitive quantification of drugs in clinical research samples is essential for pharmacokinetic studies, therapeutic drug monitoring, and biomarker discovery. Traditional LC-MS workflows often require extensive sample cleanup to reduce matrix effects, increasing turnaround time and labor. PaperSpray mass spectrometry offers direct analysis of dried biological samples with minimal preparation, but suffers from high background noise that limits the achievable limit of quantification (LOQ). Integrating Field Asymmetric Ion Mobility Spectrometry (FAIMS) adds an orthogonal gas-phase separation step to selectively filter analyte ions, reducing chemical interferences and enhancing signal-to-noise (S/N).

Study Objectives and Overview

The primary aim of this work was to evaluate the combined performance of PaperSpray and FAIMS for quantifying the antidepressant amitriptyline directly from dried human whole blood. Key goals included:

• Comparing LOQ and S/N for amitriptyline with and without FAIMS
• Assessing calibration linearity and precision across a clinically relevant concentration range
• Demonstrating reduction of background interferences in complex matrices

Methodology and Used Instrumentation

Sample Preparation:

• Amitriptyline was spiked into human blood (0.1–500 ng/mL); amitriptyline-d₃ internal standard at 50 ng/mL
• Ten microliters of each sample were spotted onto VeriSpray sample plates and dried at 45 °C for 30 minutes
• VeriSpray rewet and spray solvent: 95% methanol, 5% water, 0.01% acetic acid; ten successive 10 µL dispenses with timed delays

Instrumentation:

• Thermo Scientific TSQ Altis triple-quadrupole mass spectrometer
• VeriSpray PaperSpray ion source
• FAIMS Pro interface operated in normal resolution mode with optimized compensation voltage (CV –29.5 V)
• Electrospray conditions: ion transfer tube at 350 °C; collision gas at 2 mTorr; spray voltage 4.0 kV (no FAIMS), 3.3 kV (with FAIMS)
• Data acquisition: multiple reaction monitoring of three transitions per analyte; 2-minute chronograms per sample

Main Results and Discussion

Calibration and LOQ:

• Both with and without FAIMS, calibration curves were linear across 0.1–500 ng/mL
• Without FAIMS, LOQ was 2.5 ng/mL (S/N ≥ 4)
• With FAIMS, LOQ improved to 1.0 ng/mL due to >4-fold reduction in background

Signal-to-Noise and Background Reduction:

• Matrix blank signal area decreased over four times when FAIMS was applied
• At 1 ng/mL amitriptyline, S/N increased from 2.7 (no FAIMS) to 4.3 (with FAIMS)

Precision and Accuracy:

• Intra-day precision (%RSD) was below 2% at QC levels (20, 100, 400 ng/mL) for both modes
• Inter-day precision on mid-level QC (100 ng/mL) remained under 1% RSD and within ±12% accuracy for three consecutive days (without FAIMS)

Benefits and Practical Applications

By combining PaperSpray with FAIMS, laboratories can achieve rapid, high-throughput quantification of small molecules in complex biological matrices without conventional sample cleanup. This workflow:

• Delivers improved LOQ and S/N for low-abundance analytes
• Reduces analysis time and solvent usage
• Minimizes matrix interferences, enhancing data reliability in clinical and pharmacokinetic studies

Future Trends and Possibilities of Use

Emerging developments may include:

• Integration of PaperSpray-FAIMS with high-resolution MS for broader untargeted screening
• Automation of sample spotting and data processing for point-of-care testing
• Expansion to other clinical analytes such as metabolites, peptides, and lipids
• Enhanced FAIMS waveform optimization and multi-CV scanning for multiplexed analyses

Conclusion

The combination of PaperSpray ionization with FAIMS technology significantly enhances analytical performance for direct blood analysis. FAIMS reduces chemical noise, allowing up to 2.5-fold lower LOQs and higher S/N ratios without added sample preparation. This streamlined approach supports fast, robust quantification suitable for clinical research and therapeutic monitoring.