Quantification of eleven antimycotics in human serum by TurboFlow chromatography coupled to high-resolution accurate-mass Orbitrap mass spectrometry for clinical research

Importance of the topic

The accurate quantification of antifungal drugs in human serum is critical for optimizing treatment of mycoses. These agents exhibit narrow therapeutic ranges, high interindividual variability in absorption, metabolism, and clearance, and potential drug–drug interactions. Rapid and reliable therapeutic drug monitoring supports dose adjustment, enhances efficacy, and reduces the risk of toxicity.

Goals and overview of the study

This work aimed to establish and validate a streamlined analytical method for simultaneous measurement of eleven antifungal compounds in human serum. The approach integrates online TurboFlow sample cleanup with high-resolution accurate-mass Orbitrap mass spectrometry, targeting clinical research applications and routine therapeutic drug monitoring.

Methodology and instrumentation

Sample preparation involves protein precipitation of 50 µL serum with a fortified internal standard mixture, followed by centrifugation and transfer of the supernatant.
Chromatography is performed using a TurboFlow Cyclone MCX-2 column (0.5 × 50 mm) for online cleanup, coupled to a Hypersil GOLD analytical column (50 × 2.1 mm, 1.9 µm) at 30 °C. A multi-segment gradient delivers total runtimes of 4.0 minutes.
Mass spectrometric detection employs a Q Exactive Focus instrument with heated electrospray ionization in positive mode. Full-scan acquisition at 70 000 resolution (FWHM at m/z 200) covers m/z 110–1300. Data processing uses a 5 ppm extraction window in dedicated software.

Instrumentation

• Transcend II TLX-1 system with TurboFlow technology
• TurboFlow Cyclone MCX-2 online cleanup column
• Hypersil GOLD analytical reverse-phase column
• Q Exactive Focus hybrid quadrupole-Orbitrap mass spectrometer
• Heated electrospray ionization source (HESI)

Main results and discussion

The method achieved linear calibration curves for all eleven analytes with correlation coefficients above 0.995. The lower limits of quantification provided signal-to-noise ratios ≥7. Intra- and inter-assay accuracy fell within ±12% (±20% at the LLOQ) and precision did not exceed 17% at the LLOQ and 13% for higher concentrations. Carryover was below 20% of the LLOQ signal for all drugs and below 5% for internal standards. Matrix effects were compensated by isotope-labeled standards, with normalized matrix factors showing variation coefficients ≤9.2%. Stability studies confirmed reliable analyte concentrations after up to five weeks at −20 °C/−80 °C, 24 h in the autosampler at 10 °C, and multiple freeze-thaw cycles, with minor deviations for amphotericin B and reduced benchtop stability for anidulafungin and micafungin beyond 4–6 hours at room temperature.

Benefits and practical applicability

This streamlined method delivers rapid (<5 min) analysis, minimal sample handling, and high throughput. Online TurboFlow cleanup enhances robustness and reduces matrix interferences. High-resolution full-scan acquisition allows flexible panel extension without revalidation of targeted transitions, making the workflow suitable for clinical research laboratories and therapeutic drug monitoring services.

Future trends and possibilities

Advances may include further automation of sample preparation, expansion of the analyte panel to emerging antifungals or metabolites, adaptation to alternative biological matrices (e.g., plasma, cerebrospinal fluid), and integration with laboratory information systems for real-time dose adjustment. The full-scan HRAM platform also supports retrospective data mining for novel biomarkers.

Conclusion

A robust and validated TurboFlow-LC–Orbitrap MS method enables simultaneous quantification of eleven key antifungal agents in human serum with high sensitivity, accuracy, and throughput. The approach addresses critical needs in clinical research and therapeutic drug monitoring, facilitating optimized antifungal therapy.