Optimization and Quantification of Antibiotics in Dried Plasma Spots Utilizing Paper Spray Mass Spectrometry

Importance of the Topic

The accurate measurement of antibiotic concentrations in biological fluids is critical for effective therapeutic drug monitoring (TDM) and dose optimization. Suboptimal dosing of beta-lactam and other anti-microbial agents can lead to therapeutic failure, resistance development, and adverse outcomes. Paper spray mass spectrometry (PS-MS) offers a rapid, minimally invasive approach well suited to point-of-care applications.

Objectives and Overview of the Study

This work aimed to develop and optimize a user-friendly PS-MS assay for the simultaneous quantitation of four antibiotics—linezolid, meropenem, ampicillin, and piperacillin—in plasma. Key goals included establishing detection limits compatible with clinical TDM, minimizing blank signals from the paper substrate, and identifying optimal solvent-substrate combinations.

Methodology and Instrumentation

• Sample Preparation: Plasma samples were spiked with stable isotope-labeled internal standards, spotted onto various chromatography papers (including carbon-sputtered and untreated substrates), and air-dried.
• Instrument Configuration: Analyses were performed on a Thermo TSQ Altis triple quadrupole mass spectrometer equipped with a Verispray autosampler.
• Optimization Parameters: A factorial design explored eight factors—paper pore size, sample volume, solvent composition and volume, spray mount, paper cut method (laser vs. razor), solvent location, and washing procedures.
• Spray Solvent: A mixture of acetonitrile, tetrahydrofuran, water and 0.1% formic acid was varied; the 60:30:10 ACN:THF:H₂O (0.1% FA) blend gave the most stable signal.

Main Results and Discussion

• Blank Signal Reduction: Razor-cut paper tips produced significantly lower blank background than laser-cut tips, which released pyrolysis products.
• Substrate Treatment: Carbon-sputtered paper with ethyl acetate and untreated paper with THF yielded the highest signal-to-background ratios.
• Critical Factors: Pore size, sample volume, solvent volume, and spray mount were statistically significant for maximizing area under the curve (AUC) and signal-to-noise.
• Detection Limits: Limits of detection ranged from 0.01 to 0.09 µg/mL and quantitation limits from 0.03 to 0.28 µg/mL across analytes, meeting clinically relevant thresholds.
• Surface Treatments: Acid and solvent washes of laser-cut papers reduced blank signals, approaching those of razor-cut controls. Baking alone increased background.

Benefits and Practical Applications

The optimized PS-MS method enables rapid (<5 minutes per sample) antibiotic quantitation with minimal sample preparation. It facilitates real-time TDM in clinical settings, supports individualized dosing strategies, and reduces turnaround time compared to conventional LC-MS workflows.

Future Trends and Potential Applications

Further improvements may include silanized or other chemically modified papers to reduce analyte binding, integration of automated washing protocols, and extension to a broader range of drugs. Portable mass spectrometers coupled with machine-learning algorithms for pattern recognition could enable bedside TDM and decentralized clinical testing.

Conclusion

This study demonstrates a robust PS-MS workflow for four key antibiotics in plasma. By optimizing paper substrate, solvent composition, and operational parameters, clinically relevant detection limits were achieved. Ongoing refinements and validation studies will support translation to routine therapeutic monitoring.

Reference

• Espy RD, Manicke NE, Ouyang Z, Cooks RG. Rapid analysis of whole blood by paper spray mass spectrometry for point-of-care therapeutic drug monitoring. Analyst. 2012;137:2344-9.
• Manicke NE, Abu-Rabie P, Spooner N, Ouyang Z, Cooks RG. Quantitative analysis of therapeutic drugs in dried blood spot samples by paper spray mass spectrometry. J Am Soc Mass Spectrom. 2011;22:1501-7.
• Manicke NE, Yang Q, Wang H, Oradu S, Ouyang Z, Cooks RG. Assessment of paper spray ionization for quantitation of pharmaceuticals in blood spots. Int J Mass Spectrom. 2011;300:123-9.
• Wang H, Liu J, Cooks R, Ouyang Z. Paper spray for direct analysis of complex mixtures using mass spectrometry. Angew Chem Int Ed. 2010;49:889-892.
• Wang H, Ren Y, McLuckey MN, Manicke NE, Park J, Zheng LX, Shi RY, Cooks RG, Ouyang Z. Direct quantitative analysis of nicotine alkaloids from biofluid samples using paper spray mass spectrometry. Anal Chem. 2013;85:11540-4.