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Updated: May 28, 2026
Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses
Published on: August 30, 2018
Rapid HPLC-MS/MS method for simultaneous quantitation of four routinely administered triazole antifungals in human
Kerstin Yvonne Beste1, Olaf Burkhardt, Volkhard Kaever
1Institute of Pharmacology, Hannover Medical School, Hannover, Germany.
Background:
Fluconazole, itraconazole, posaconazole and voriconazole are four triazole antifungal drugs administered for the prevention and in the treatment of invasive fungal infections. Therapeutic drug monitoring of these antifungals in human plasma is advised.
Methods:
After protein precipitation by adding methanol/ZnSO(4) containing ketoconazole as internal standard (IS) the analytes were isolated from matrix and enriched by solid-phase extraction (SPE) and were separated on an Allure PFP HPLC column, maintained at 60°C. The retention times were 1.12, 1.09, 1.09, 1.15 and 1.94 min for fluconazole, voriconazole, posaconazole, itraconazole and ketoconazole, respectively. Cycle time was 3 min, injection to injection. The analytes were monitored by means of a tandem mass spectrometer. Selected reaction monitoring (SRM) in positive ion mode was used for quantitation.
Results:
The presented method has limits of detection of 2-10 ng/mL, a lower limit of quantitation of 14 ng/mL and linearities ≥0.999 for the different analytes. The intra- and interday correlation coefficients of variation were <8.1 and <9.8%, respectively. The accuracy ranged from 85 to 106%.
Conclusions:
A rapid, sensitive, and robust HPLC-MS/MS method for quantifying fluconazole, itraconazole, posaconazole and voriconazole levels in human plasma was validated and successfully applied to samples from antifungal treated patients.
Insights
A new HPLC-MS/MS method accurately measures levels of four antifungal triazoles in plasma. This validated assay is crucial for therapeutic drug monitoring in patients with invasive fungal infections.
Area of Science:
- Analytical Chemistry
- Pharmacology
Background:
- Triazole antifungals like fluconazole, itraconazole, posaconazole, and voriconazole are vital for treating invasive fungal infections.
- Therapeutic drug monitoring (TDM) of these antifungals in human plasma is recommended to ensure efficacy and safety.
Purpose of the Study:
- To develop and validate a rapid, sensitive, and robust high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method.
- To quantify fluconazole, itraconazole, posaconazole, and voriconazole concentrations in human plasma.
Main Methods:
- Analytes were extracted from plasma using solid-phase extraction (SPE) after protein precipitation.
- Separation was performed on an Allure PFP HPLC column at 60°C with a 3-minute cycle time.
- Quantification was achieved using tandem mass spectrometry with selected reaction monitoring (SRM) in positive ion mode.
Main Results:
- The method demonstrated excellent linearity (R² ≥ 0.999) and sensitivity with limits of detection between 2-10 ng/mL.
- Low limits of quantitation (14 ng/mL) and high accuracy (85-106%) were achieved.
- Intra- and interday coefficients of variation were below 8.1% and 9.8%, respectively.
Conclusions:
- A validated HPLC-MS/MS method provides a reliable tool for quantifying key triazole antifungal drugs in human plasma.
- The method's speed, sensitivity, and robustness make it suitable for routine clinical use.
- Successful application to patient samples underscores its utility in managing invasive fungal infections.
