Rapid and Sensitive Analysis of Antifungal Agents in Human Serum Using the Agilent 6475 LC/TQ

Importance of the Topic

Invasive fungal infections pose a growing threat to patient health and the emergence of antifungal resistance concerns clinicians. Therapeutic drug monitoring (TDM) of antifungal agents in serum is vital to optimize dosing, minimize toxicity, and improve clinical outcomes.

Objectives and Study Overview

This study aimed to develop and validate a rapid 3.5-minute quantitative LC/TQ method for eight key antifungal agents in human serum. Using the Agilent 6475 triple quadrupole LC/MS system combined with a commercial RECIPE ClinMass TDM kit, the work evaluates sensitivity, linearity, precision, and robustness across multiple days.

Methodology and Instrumentation

Sample preparation involved protein precipitation followed by dilution and direct injection. Briefly, serum calibrators or QC samples were mixed with internal standard and precipitant, agitated, incubated, centrifuged, then the supernatant diluted twentyfold prior to LC injection.

Instrumentation

• Liquid chromatograph Agilent 1290 Infinity II with high-speed pump, multisampler, and multicolumn thermostat
• Analytical column and prefilter supplied in the RECIPE ClinMass TDM kit
• Mass spectrometer Agilent 6475 triple quadrupole with Jet Stream ion source

Chromatographic conditions employed RECIPE mobile phases A and B at 0.6 mL per minute under a 3.5-minute gradient. MRM transitions were optimized via Auto MRM Optimizer in MassHunter software. Data were processed using MassHunter Qualitative and Quantitative Analysis versions B.12.

Main Results and Discussion

Chromatographic separation was achieved for six of eight compounds; posaconazole and hydroxy-itraconazole exhibited minor retention overlap but maintained selectivity via MRM. Calibration curves showed excellent linearity (R2 >= 0.995) over five days. Limits of quantitation ranged from 2.5 to 89 µg per liter for the eight agents. Interday accuracy in QC samples was between 94.1 and 106.2% with relative standard deviations below 5.6%. No significant carryover was observed.

Benefits and Practical Applications

The validated assay allows throughput of up to 350 serum samples per day with minimal manual steps. Its speed, sensitivity, and robustness make it suitable for routine TDM in clinical laboratories, supporting dose adjustment and resistance management.

Future Trends and Possibilities

Further developments may include expansion to other biological matrices, integration with automated sample preparation, multiplexing with additional antifungal or antibacterial compounds, and application of advanced data analysis algorithms for real-time clinical decision support.

Conclusion

A streamlined LC/TQ method on the Agilent 6475 LC/MS with the RECIPE ClinMass kit enables rapid, precise measurement of eight antifungal agents in human serum. The approach meets clinical demands for efficient therapeutic monitoring and can be readily adopted in high-throughput laboratory settings.

References

1. Ashbee HR Barnes RA Johnson EM Richardson MD Gorton R Hope WW Therapeutic Drug Monitoring of Antifungal Agents Guidelines from the British Society for Medical Mycology J Antimicrob Chemother 2014 69(5):1162–1176