Quantification of eight antimycotics in human plasma by LC-HRAM-MS for clinical research

Significance of Topic

The precise measurement of antifungal agents in human plasma is essential for optimizing therapeutic outcomes, preventing toxicity, and managing drug resistance in patients with serious fungal infections or immunocompromised status. Rapid and reliable quantification supports timely dose adjustments and improves patient care in clinical research.

Study Objectives and Overview

This work presents the development and validation of a high-throughput liquid chromatography–high resolution accurate mass spectrometry (LC-HRAM-MS) method to quantify eight commonly used antimycotics in human plasma. The analytical workflow is designed to complete chromatographic separation and detection within a 3.6-minute run time, enabling efficient processing of clinical samples.

Methodology and Instrumentation

Sample Preparation

• Protein precipitation of 50 µL plasma with 100 µL acetonitrile containing stable isotope-labeled internal standards.
• Centrifugation followed by transfer of 50 µL supernatant, diluted to 500 µL with water.

Chromatography

• Vanquish™ Flex Binary UHPLC system with Hypersil GOLD™ 50 × 2.1 mm, 1.9 µm column at 40 °C.
• Gradient elution from 5 % to 100 % acetonitrile (both mobile phases containing 0.1 % formic acid) over 3.6 minutes; flow rate 0.5 mL/min; injection volume 2 µL.

Mass Spectrometry

• Orbitrap Exploris™ 120 equipped with heated electrospray ionization in positive mode.
• Full-scan MS at 60 000 resolution (m/z 100–750) with data-dependent MS2 at 15 000 resolution.
• Instrument settings: spray voltage 3 750 V, sheath gas 55, auxiliary gas 10, vaporizer temp 450 °C.

Main Results and Discussion

Calibration and Sensitivity

• Linear response over therapeutic concentration ranges for all eight antifungals; three compounds required 1/x weighting.
• Bias between nominal and back-calculated calibrator concentrations within ±15 %.

Carryover and Selectivity

• No detectable carryover in blank injections following the highest calibrator.

Accuracy and Precision

• Quality control samples showed biases between –7.6 % and 7.2 %.
• Intra-assay precision (n=5) CVs ≤ 8.4 %; inter-assay precision (n=15) CVs ≤ 5.8 %.

Practical Benefits and Applications

• Rapid turnaround: 3.6-minute analysis time supports high sample throughput.
• Simplified sample preparation reduces handling errors and labor.
• Simultaneous quantification of eight antifungals enhances efficiency in therapeutic drug monitoring and pharmacokinetic studies.

Future Trends and Potential Applications

Advances in HRAM-MS technology and automated sample handling are poised to further reduce analysis time and expand multiplexing capabilities. Integration with clinical decision support systems may enable real-time dose adjustment. Method adaptation could encompass additional antifungal agents and metabolites for comprehensive therapeutic monitoring.

Conclusions

A robust LC-HRAM-MS method on the Orbitrap Exploris 120 platform was successfully developed for eight antimycotics in human plasma. The workflow delivers high selectivity, rapid turnaround, and excellent accuracy and precision, meeting the needs of clinical research laboratories.

Reference

Thermo Fisher Scientific application note TN66046-EN 0621S