Analysis for Voriconazole Using Fully Automated Sample Preparation LC-MS/MS System and Comparison of Measurement Methods

Importance of the Topic

Voriconazole is a deep-seated antifungal agent requiring precise therapeutic drug monitoring (TDM) to maintain effective plasma concentrations (1.0–2.0 µg/mL) and avoid toxicity (>4.0–5.0 µg/mL). Reliable measurements are critical for patient safety and treatment efficacy.

Objectives and Overview of the Study

This study evaluated a fully automated LC-MS/MS workflow incorporating a stable isotope-labeled internal standard for quantifying voriconazole. Results were compared with reference values from the Japanese Society of TDM Quality Control 2020 survey to assess accuracy.

Methodology

The analytical procedure involved:

• Automated addition of sample/calibrator, internal standard (13C2,2H3-voriconazole), and reagents using the CLAM-2030 module
• Protein precipitation and extraction with acetonitrile and isopropanol/water
• Filtration and transfer of processed samples into the LC-MS/MS system
• Quantification by MRM (350.20→281.20 for voriconazole, 355.20→284.20 for internal standard)
• Calibration curve prepared over 0.1–10 µg/mL; QC samples at 0.30, 0.75, and 1.75 µg/mL

Used Instrumentation

• CLAM-2030 fully automated sample preparation module
• Nexera HPLC system with DOSIMMUNE analytical column and mobile phases
• LCMS-8050 triple quadrupole mass spectrometer (ESI+, positive mode)
• Stable isotope-labeled voriconazole ([13C2,2H3]-voriconazole) from Alsachim

Main Results and Discussion

Calibration showed excellent linearity (r=0.9993) across 0.1–10 µg/mL. QC measurements yielded accuracy between 94.1–106.7% and repeatability (%RSD) below 10%. In the TDM control survey, voriconazole results differed from reference values by 0.0%, 2.5%, and 2.9% at low (1.0 µg/mL), middle (4.0 µg/mL), and high (7.0 µg/mL) levels, respectively, confirming high accuracy.

Benefits and Practical Applications

The integrated automated workflow minimizes manual errors, increases throughput, and standardizes sample processing. Use of stable isotope-labeled internal standards effectively compensates for matrix effects, ensuring robust quantification in clinical and laboratory settings.

Future Trends and Potential Uses

Emerging developments may include multiplexed LC-MS/MS assays for simultaneous monitoring of multiple drugs, enhanced automation with real-time data integration, and expansion of stable isotope libraries to support broader TDM panels and personalized medicine approaches.

Conclusion

The combination of fully automated sample preparation and stable isotope-labeled internal standards with LC-MS/MS delivers a reliable, precise, and efficient workflow for voriconazole TDM, aligning with regulatory quality control standards.

Reference

• Japanese Society of TDM Quality Control. Report on the Results of the 2020 TDM Control Survey for Infectious Disease Therapeutics.
• Sano Y. Mass Spectrometry on Drug Metabolism and Pharmacokinetics Studies: Intended for Beginners. J Mass Spectrom Soc Jpn. 2016;64(3):81–85.