Quantification of eight antimycotics in human plasma by liquid chromatography-tandem mass spectrometry for clinical research

Significance of the Topic

Therapeutic drug monitoring of antifungals is essential due to their narrow therapeutic ranges and variable pharmacokinetics. Reliable quantification in plasma ensures optimal dosing, improves patient outcomes, and reduces toxicity risks.

Objectives and Study Overview

The study aimed to develop and validate a rapid, sensitive LC-MS/MS method for simultaneous quantification of eight antimycotics in human plasma using a 3.6-minute chromatographic run. The method supports clinical research and therapeutic drug monitoring by assessing key performance metrics.

Methodology and Instrumentation

Sample preparation involved simple protein precipitation of 50 µL plasma with 100 µL acetonitrile containing deuterated internal standards. After vortexing, centrifugation, and dilution to 500 µL, 2 µL was injected. Chromatography employed a Vanquish Flex Binary UHPLC system with a Hypersil GOLD 50×2.1 mm, 1.9 µm column at 40 °C, using water and acetonitrile (both 0.1 % formic acid) in a 3.6-minute gradient. Detection used a TSQ Quantis triple quadrupole mass spectrometer with heated electrospray ionization in positive mode and SRM acquisition. Data processing utilized TraceFinder 5.1 software.

Key Results and Discussion

The method achieved linear calibration (1/x weighting) across the therapeutic ranges for all eight compounds. Lower limits of quantification ranged from 0.0138 mg/L (voriconazole) to 0.245 mg/L (5-fluorocytosine). Accuracy bias was within ±6 %, and intra-assay precision (CV) remained below 12.4 %, with inter-assay CV under 10.8 %. No carryover was observed.

Benefits and Practical Applications

• High throughput and short run time facilitate large-scale studies.
• Minimal sample preparation reduces labor and potential errors.
• Broad analyte coverage supports comprehensive antifungal monitoring.
• Robust sensitivity meets clinical research requirements.

Future Trends and Potential Applications

Ongoing trends include extending multiplex assays to additional antifungals and metabolites, integrating automation for sample preparation, and adapting methods for point-of-care platforms. Data analytics and real-time monitoring may further enhance individualized dosing.

Conclusion

The developed LC-MS/MS method provides a fast, reliable, and sensitive approach for quantifying eight key antifungal agents in human plasma. Its performance characteristics meet clinical research demands for therapeutic drug monitoring.

References

Renoulin G, De Nardi C. Quantification of eight antimycotics in human plasma by LC-MS/MS for clinical research. Thermo Fisher Scientific Technical Note 66059; 2021.