Ultrafast Method for Simultaneous Measurement of Triazole Antifungals in Human Serum Using Online SPE/MS/MS

Significance of the topic

Therapeutic drug monitoring of triazole antifungals is critical in clinical settings to ensure effective dosing, avoid toxicity, and manage resistance. Traditional HPLC and LC/MS/MS methods can be time-consuming and resource-intensive, limiting throughput when rapid results are needed for patient management.

Study objectives and overview

This study presents an ultrafast online solid-phase extraction coupled with tandem mass spectrometry (SPE/MS/MS) method to quantify five triazole antifungal agents—voriconazole, ketoconazole, fluconazole, itraconazole, and posaconazole—in human serum. The goal was to achieve high accuracy and precision at dramatically reduced analysis times compared to conventional techniques.

Methodology and instrumentation

Sample preparation uses simple protein precipitation followed by a tenfold dilution in ammonium hydroxide solution and direct injection into the SPE/MS/MS system. Key components include:

• Agilent RapidFire 365 high-throughput SPE module
• Agilent 6460 Triple Quadrupole mass spectrometer
• Reversed-phase C18 SPE cartridges
• MassHunter software for acquisition and quantitative analysis

System parameters were optimized for 14-second cycle times per sample, with separate aqueous and organic washes to minimize interferences.

Results and discussion

Calibration curves for each antifungal demonstrated excellent linearity (R2 > 0.995) over a 0.2–25 µg/mL range. Intra- and interday accuracy values remained within ±10%, and precision (%CV) stayed below 10% across quality control levels. The method achieved over 240 injections per hour, with carryover below 5% for all analytes. A marathon test of more than 2,000 sequential injections showed stable instrument response (CV 2.3–6.9%), confirming robustness and cartridge longevity.

Practical benefits and applications

This ultrafast SPE/MS/MS workflow delivers more than tenfold reductions in analysis time and solvent usage compared to typical HPLC approaches. It supports high-throughput clinical research and quality control environments requiring rapid turnaround and reliable quantitation of multiple antifungal agents.

Future trends and potential uses

Advancements may include expansion to broader drug panels and biomarkers, integration with automated sample handling, and real-time data interpretation via machine learning. Such developments could further streamline laboratory operations and support personalized dosing strategies in critical care.

Conclusion

An online SPE/MS/MS method was developed that quantifies five key triazole antifungals in human serum with high accuracy, precision, and throughput. By leveraging rapid sample cleanup and direct injection, the approach significantly enhances laboratory efficiency without compromising analytical performance.

Reference

1. Verdier MC et al. Liquid chromatography-tandem mass spectrometry method for simultaneous quantification of four triazole antifungal agents in human plasma. Clin Chem Lab Med. 2010;48(10):1515-22.
2. B Czasopismo et al. Rapid HPLC–MS/MS method for simultaneous quantitation of four routinely administered triazole antifungals in human plasma. Clin Chim Acta. 2012;413:240-245.