Analysis of Drugs in Whole Blood by PaperSpray-FAIMS-MS/MS

Significance of the Topic

In clinical and research settings, precise measurement of immunosuppressant drugs in whole blood is critical for therapeutic monitoring and pharmacokinetic studies. PaperSpray mass spectrometry offers rapid analysis with minimal sample preparation, but matrix-related background limits sensitivity and quantitation. Integrating Field Asymmetric Ion Mobility Spectrometry (FAIMS) reduces interferences, enhancing signal-to-noise and lowering detection limits, thus addressing key challenges in high-throughput drug monitoring.

Study Objectives and Overview

The study evaluated the Thermo Scientific FAIMS Pro interface’s impact on PaperSpray-MS/MS performance for quantifying Everolimus, Sirolimus, Tacrolimus, and Cyclosporin A in whole blood. Comparative metrics included background reduction, sensitivity, linearity, accuracy, and precision between methods with and without FAIMS.

Methodology

EDTA blood samples were spiked with target analytes and isotopically labeled internal standards, dried on VeriSpray paper substrates, and analyzed by two approaches:

• PaperSpray-MS/MS: Direct ionization from dried spots without mobility separation.
• PaperSpray-FAIMS-MS/MS: Workflow augmented by FAIMS Pro using optimized dispersion and compensation voltages to transmit target ions selectively and neutralize interferences.

Calibration curves comprised nine concentration levels (0–80 ng/mL) for Everolimus, Sirolimus, Tacrolimus, and six levels (0–1600 ng/mL) for Cyclosporin A. Thermo Scientific TraceFinder integrated analyte signals; weighted least squares regression fitted response factors.

Instrumental Setup

• VeriSpray PaperSpray source on TSQ Altis mass spectrometer.
• FAIMS Pro parameters: dispersion voltage –5000 V, electrode temperature 100 °C, optimized compensation voltages per compound.
• Selected reaction monitoring transitions customized for each analyte and internal standard.

Main Results and Discussion

• Background Reduction: FAIMS Pro decreased background by 67–100%, boosting signal-to-blank ratios (up to 480× improvement for Cyclosporin A at high concentrations).
• Sensitivity Gains: Limits of detection improved for all analytes (e.g., Everolimus LOD from 11.7 to 4.6 ng/mL; Tacrolimus LOD from 4.3 to 0.8 ng/mL).
• Linearity: r² values increased for Everolimus (0.965→0.995) and Sirolimus (0.986→0.996); Tacrolimus and Cyclosporin A maintained excellent linearity.
• Accuracy and Precision: Absolute percent differences at low levels improved for Everolimus, Sirolimus, and Tacrolimus; precision remained within acceptable limits (<20% CV for most levels).
• Quantitation Limits: LOQs were lowered by factors of 4–20 across the drug panel with FAIMS integration.

Practical Benefits

• Streamlined sample preparation and higher throughput compared to conventional LC/MS workflows.
• Enhanced selectivity and reliability for therapeutic drug monitoring.
• Broader dynamic range and robust quantitation in complex biological matrices.

Future Trends and Applications

Further integration of ion mobility with ambient ionization is expected to drive additional sensitivity and selectivity gains. Potential developments include:

• Real-time therapeutic monitoring and point-of-care platforms.
• Automated sample processing combined with FAIMS separation.
• Extension to other low-abundance biomarkers and small molecules in complex matrices.

Conclusion

The FAIMS Pro interface significantly enhances PaperSpray-MS/MS analysis of immunosuppressant drugs in whole blood, delivering superior detection limits, linearity, and accuracy. This method supports high-throughput clinical and research applications with minimal sample preparation.

Reference

1. Manicke NE, Belford M. Separation of opiate isomers using electrospray ionization and paper spray coupled to high-field asymmetric waveform ion mobility spectrometry. J Am Soc Mass Spectrom. 2015;26(5):701–705.
2. Wang H, et al. Paper spray for direct analysis of complex mixtures using mass spectrometry. Angew Chem Int Ed. 2010;49(5):877–880.
3. CLSI. Evaluation of detection capability for clinical laboratory measurement procedures; approved guideline. 2012.