A High Sensitivity LC/MS/MS Method for Quantitative Analysis of Eight Antifungal Drugs in Human Serum

Importance of the topic

Invasive fungal infections carry significant morbidity and mortality risks, especially in immunocompromised patients. Triazoles and echinocandins are frontline antifungal therapies, yet their pharmacokinetics vary widely due to individual bioavailability and drug–drug interactions. Precise dosing through therapeutic drug monitoring (TDM) is therefore critical to maximize efficacy and minimize toxicity.

Objectives and Study Overview

This study aimed to establish a rapid, sensitive and reliable LC/MS/MS method for simultaneous quantitation of eight antifungal drugs in human serum. The target analytes include five triazoles (fluconazole, posaconazole, voriconazole, itraconazole and hydroxy-itraconazole) and three echinocandins (anidulafungin, caspofungin and micafungin). Method performance was assessed using spiked serum samples as a precursor to full clinical validation.

Methodology and Instrumentation

Sample preparation involved protein precipitation by adding a 1:1 mixture of acetonitrile and methanol to human serum at a 3:1 solvent-to-sample ratio. After vortexing, centrifugation and filtration, the clear supernatant was injected into the UHPLC–MS/MS system. Chromatographic separation was achieved on a C18 column under a 9-minute gradient (5–90% B mobile phase) at 0.4 mL/min. The mass spectrometer operated in multiple reaction monitoring (MRM) mode, predominantly in positive electrospray ionization, with optimized transitions for each drug.

Used instrumentation

• UHPLC: Shimadzu Nexera
• Column: Kinetex C18, 100 × 2.1 mm, 1.7 µm, 40 °C
• Mass spectrometer: Shimadzu LCMS-8060 triple quadrupole with heated ESI interface
• Mobile phases: Water/0.1% formic acid (A), Acetonitrile/0.1% formic acid (B)

Main Results and Discussion

• Run time of nine minutes enabled separation of all eight analytes.
• Linearity was excellent (R² 0.9968–0.9998) over ranges: 5–5000 ppt for fluconazole and voriconazole; 20–5000 ppt for posaconazole, itraconazole, hydroxy-itraconazole and caspofungin; 200–5000 ppt for anidulafungin and micafungin.
• Limits of detection (LOD) ranged from 0.4 to 43 ppt; limits of quantitation (LOQ) from 1.3 to 131 ppt.
• Repeatability at mid (200 ppt) and high (1000 ppt) levels showed RSD ≤15% for most compounds; echinocandins exhibited slightly higher variability.
• Recoveries varied from 69% to 135%, and matrix effects ranged 39–119%, reflecting some impact of protein precipitation and serum components on analyte response.

Benefits and Practical Applications of the Method

This streamlined LC/MS/MS approach offers:

• High sensitivity for low-level detection essential to TDM.
• Fast throughput suitable for routine clinical laboratory workflows.
• Broad analyte panel covering key triazoles and echinocandins for comprehensive monitoring.
• Simple sample preparation without solid-phase extraction.

Future Trends and Potential Applications

Future developments may include microflow LC to reduce solvent use, automation of sample preparation for higher throughput, adaptation to dried blood spot analysis for remote monitoring, and extension of the panel to emerging antifungal agents. Integration with clinical decision support tools could further individualize patient therapy.

Conclusion

A robust, nine-minute LC/MS/MS method has been developed for quantifying eight major antifungal drugs in human serum with high sensitivity and reproducibility. The technique is well suited for therapeutic drug monitoring and holds promise for routine clinical implementation.

References

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3. Farowski F. et al., Antimicrob. Agents Chemother. 54(5):1815–1819 (2010)