Robustness characterization of a quantitative whole blood assay using PaperSpray technology for clinical research

Importance of the Topic

PaperSpray mass spectrometry is a rapidly emerging technique for direct analysis of complex biological samples. Its minimal sample preparation, combined with high throughput and robust quantitation, addresses critical needs in clinical research and therapeutic drug monitoring. This study focuses on validating a quantitative whole blood assay for the opioid metabolite EDDP without traditional sample cleanup.

Study Objectives and Overview

The primary goal was to demonstrate the robustness and reproducibility of a PaperSpray assay for EDDP in human whole blood over 480 injections. Key objectives included:

• Establishing a calibration curve spanning 1.75 to 500 ng/mL
• Verifying lower limit of quantification and precision across multiple runs
• Evaluating assay stability during unattended operation of two fully loaded VeriSpray magazines

Methodology

Human whole blood samples were spiked with EDDP and deuterated EDDP-d3 internal standard. Twenty VeriSpray plates (24 paper strips each) were loaded with 480 samples. Each strip received a 8 μL spot, oven dried, rewetted with methanol/water (95/5) containing 0.01% acetic acid, and ionized under 3.8 kV. A one-minute acquisition per sample captured SRM transitions. Calibration and QC samples were interspersed at the beginning and end of each 240-sample run.

Used Instrumentation

• Thermo Scientific VeriSpray PaperSpray ion source with automated plate loader and racks for 240 samples per magazine
• Thermo Scientific TSQ Quantis triple quadrupole mass spectrometer
• TraceFinder software version 4.1 for data acquisition and processing

Main Results and Discussion

The assay achieved a lower limit of quantification of 3.5 ng/mL, with accuracy within ±4.1% and precision below 4% across combined calibration curves. QC samples at 50, 100, and 200 ng/mL showed accuracy between 1.2% and 5.8% and precision under 2%. Performance remained stable throughout 480 injections; external wiping of the ion transfer tube between magazine loads prevented signal drift without disassembly. These findings confirm high reproducibility and minimal maintenance requirements.

Benefits and Practical Applications

• Walk-away automation allows analysis of up to 480 whole blood samples in 16 hours without manual cleanup
• Sensitivity and precision meet clinical research standards for drug metabolite quantitation
• Reduced consumables and labor lower operational costs in high-throughput laboratories

Future Trends and Potential Applications

Advances in PaperSpray ion source design and mass spectrometer sensitivity will expand applications to other biomarkers and drugs of abuse. Integration with laboratory information management systems could streamline data workflows. Emerging disposable plate formats may further reduce carryover and expand multiplexed assays in clinical toxicology and therapeutic drug monitoring.

Conclusion

The validated PaperSpray assay provides a robust, sensitive, and high-throughput solution for quantitative analysis of EDDP in whole blood. Its minimal maintenance and automation capabilities make it well suited for clinical research environments requiring consistent performance over hundreds of injections.

References

1. Wang H, Liu J, Cooks RG, Ouyang Z. Paper Spray for Direct Analysis of Complex Mixtures Using Mass Spectrometry. Angew Chem Int Ed. 2010;49(5):877–880.