Quantification of nine antimycotic drugs in human plasma or serum by LC-HRAM(MS) for clinical research

Importance of the topic

Accurate measurement of antifungal drugs in patient plasma or serum is essential for optimizing therapy, avoiding toxicity, and ensuring effective treatment of invasive fungal infections. High-resolution mass spectrometry offers superior selectivity and speed compared with traditional HPLC–UV methods, making it attractive for clinical research and therapeutic drug monitoring.

Objectives and Study Overview

• Develop a robust LC–HRAM(MS) method for quantifying nine common antifungal agents in human plasma or serum.
• Employ a simple offline sample preparation with protein precipitation and internal standard addition.
• Evaluate method performance in terms of sensitivity, linearity, accuracy, precision, limit of quantification, and carryover.

Methodology and Used Instrumentation

• Sample preparation: 50 µL plasma or serum processed by protein precipitation with 100 µL reagent containing stable isotope–labeled internal standards; supernatant transferred for analysis.
• Chromatography: Thermo Scientific Vanquish™ Duo UHPLC in direct injection mode; 10 µL injection; 3.5 min run; 40 °C column temperature; water and acetonitrile gradient with 0.1% formic acid.
• Mass spectrometry: Thermo Scientific Q Exactive™ Plus hybrid quadrupole-Orbitrap with HESI-II source in positive ion mode; FullMS-ddMS2 acquisition; resolution 70 000 (FullMS) and 17 500 (ddMS2); scan range 120–1 150 m/z; stepped NCE 15, 25, 35.

Main Results and Discussion

• Linearity: All analytes showed linear response (quadratic for ketoconazole) across calibration ranges and down to LLOQs between 0.013 and 0.461 ng/mL.
• Accuracy: Bias within ±10% (±15% at lowest level) for calibrators; control sample bias between –2.0% and 4.2%.
• Precision: Intra-assay CVs ≤ 8.2%; inter-assay CVs ≤ 6.4% across three days and two controls.
• Carryover: No detectable carryover after highest calibrator.
• Trueness: External quality controls for itraconazole and OH-itraconazole showed bias between –5.4% and 1.4%.

Benefits and Practical Applications

• High selectivity and resolution of Orbitrap technology reduce interference and improve confidence in quantification.
• Short run time (3.5 min) and simple protein precipitation streamline workflow and increase throughput.
• Wide calibration range and low LLOQs support monitoring of both prophylactic and therapeutic antifungal concentrations.
• Method meets research-level requirements for sensitivity, accuracy, and precision, facilitating pharmacokinetic and therapeutic drug monitoring studies.

Future Trends and Opportunities

Integration of online sample cleanup and automation could further reduce hands-on time. Expanding panels to include additional emerging antifungal agents or metabolites will support broader clinical studies. Combining HRAM–MS data with advanced data analytics and machine learning may improve individualized dosing algorithms.

Conclusion

The described LC–HRAM(MS) method using a Vanquish Duo UHPLC coupled to a Q Exactive Plus Orbitrap provides fast, accurate, and precise quantification of nine antifungal drugs in plasma or serum. It offers significant advantages over traditional HPLC–UV approaches and is well suited for clinical research and therapeutic drug monitoring.