Quantification of eight antimycotics in human plasma using an Orbitrap Exploris 120 high-resolution accurate mass mass spectrometer for clinical research

Significance of the topic

Antifungal agents are critical for treating and preventing invasive fungal infections, particularly in immunocompromised patients. Precise quantification of these drugs in plasma supports therapeutic drug monitoring (TDM) and helps avoid subtherapeutic exposure or toxicity. High-resolution accurate mass (HRAM) spectrometry offers enhanced selectivity and sensitivity compared to traditional HPLC-UV methods, reducing sample preparation time and improving throughput.

Study objectives and overview

This work aimed to develop and validate a rapid, robust UHPLC-HRAM MS method to quantify eight clinically relevant antimycotics in human plasma within a 3.5-minute run time. The analytes included fluconazole, itraconazole (and its OH-metabolite), voriconazole, posaconazole, isavuconazole, ketoconazole, and 5-fluorocytosine. Method performance was assessed for linearity, accuracy, precision, carryover, and lower limit of quantification (LLOQ) using commercially available calibrators and quality controls.

Methodology and instrumentation

• Sample preparation: 50 µL plasma precipitated with 100 µL of internal-standard–spiked protein-precipitation solution, vortexed, centrifuged, diluted 1:10, and transferred to vials.
• Liquid chromatography: Thermo Scientific Vanquish Flex Binary UHPLC, 40 °C column, 5 µL injection, gradient elution (0–98 % B over 2.2 min), total runtime 3.5 min at 0.6 mL/min.
• Mass spectrometry: Thermo Scientific Orbitrap Exploris 120 with heated electrospray ionization (H-ESI) in positive mode; Full Scan 60 000 resolution (m/z 70–1400) for quantification and data-dependent MS² at 15 000 resolution for confirmation; HCD collision energy 30 %; EASY-IC mass calibration.

Main results and discussion

The method exhibited linear responses with 1/x weighting across relevant clinical ranges. Calibration curves showed biases within ±10 %. No significant carryover was observed. Accuracy (bias) for quality controls ranged from –4.3 % to 6.0 %. Intra-assay precision (%CV) was below 9.3 % and inter-assay precision below 7.0 % for all analytes. LLOQs ranged between 0.0233 mg/L (posaconazole) and 0.490 mg/L (5-fluorocytosine), meeting clinical research requirements.

Benefits and practical applications

• High throughput: 3.5-minute analysis per sample.
• Reduced sample preparation complexity via simple protein precipitation.
• Enhanced selectivity and sensitivity with HRAM detection.
• Fit-for-purpose for clinical pharmacokinetic studies and TDM in research settings.

Future trends and applications

Further developments may include expanding the analyte panel to additional antifungals or other drug classes, automation of sample processing, integration with laboratory information management systems (LIMS), and application in multicenter studies. Advances in software for real-time data processing and AI-driven quality control could further streamline workflows.

Conclusion

A fast, accurate UHPLC-HRAM MS method on the Orbitrap Exploris 120 was successfully validated for quantifying eight antimycotics in human plasma. The approach offers high throughput, robust performance, and is well suited for clinical research and TDM applications.

References

Renoulin G, De Nardi C. Quantification of eight antimycotics in human plasma using an Orbitrap Exploris 120 high-resolution accurate mass mass spectrometer for clinical research. Technical Note TN66064-EN, Thermo Fisher Scientific, 2021.