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

Significance of the topic

Accurate measurement of antifungal drug levels in human plasma or serum plays a pivotal role in therapeutic drug monitoring and clinical research. Reliable quantification supports individualized dosing, improves patient safety, and enhances the understanding of pharmacokinetics in fungal infection management.

Aims and overview of the study

This work describes the development and evaluation of a liquid chromatography tandem mass spectrometry method capable of quantifying eight commonly used antimycotics in a single analytical run. The assay covers 5-fluorocytosine, fluconazole, hydroxyitraconazole, isavuconazole, itraconazole, ketoconazole, posaconazole, and voriconazole in human plasma or serum. Key objectives included establishing linear response ranges, ensuring high accuracy, and assessing intra- and inter-assay precision.

Methodology and instrumentation

Sample preparation involves offline protein precipitation of 50 µL plasma or serum with internal standards followed by centrifugation. Chromatography is performed on a binary UHPLC system with a 3.7 minute gradient and a 4 µL injection volume at 40 °C. Detection uses a triple quadrupole mass spectrometer with heated electrospray ionization in positive and negative switching mode. Selected reaction monitoring transitions with isotopically labeled internal standards enable specific quantification and confirmation of each analyte.

Main results and discussion

The method showed excellent linearity across calibration ranges for all eight antimycotics. Accuracy bias values ranged from –8.9 % to 1.6 % at two control levels. Intra-assay precision (%CV) remained below 12.7 % for all compounds, while inter-assay precision did not exceed 9.4 %. Representative chromatograms and calibration curves confirmed sensitivity at the lowest calibrator concentrations.

Benefits and practical applications

• Simultaneous quantification of eight antimycotics streamlines workflow in clinical research laboratories
• Rapid 3.7 minute analysis time enhances sample throughput
• Simple offline protein precipitation reduces sample handling and risk of matrix effects
• High accuracy and precision support reliable therapeutic monitoring and pharmacokinetic studies

Future trends and potential applications

Emerging developments may include integration with automated sample preparation platforms, expansion to additional antifungal metabolites, and application to pediatric or special patient populations. High resolution mass spectrometry and microflow chromatography could further improve sensitivity and reduce solvent consumption.

Conclusion

A rapid and robust LC MS MS method for eight key antifungal agents in human plasma or serum has been established. The assay meets performance criteria for linearity accuracy and precision, providing a valuable tool for clinical research and therapeutic drug monitoring.

Reference

Thermo Fisher Scientific Technical Note 65149 2018