Isotope dilution LC-orbitrap-HRMS with automated sample preparation for the simultaneous quantification of 11 antimycotics in human serum.
INTRODUCTION
The aim of this project was to develop and validate an isotope-dilution liquid chromatography high resolution mass spectrometry (LC-HRMS) method for the quantification of the 11 most widely used systemic antimycotics and to study whether HRMS is a feasible alternative for therapeutic drug monitoring (TDM) when compared to tandem MS (MS/MS) technology.


METHODS
After protein precipitation, followed by automated online sample clean-up the analytes were separated within 4 min on a C18 column using an acetonitrile-water gradient. Eleven antimycotics, namely 5-flucytosine, amphotericin B, anidulafungin, fluconazole, isavuconazole, itraconazole, ketoconazole, micafungin, OH-itraconazole, posaconazole and voriconazole were finally quantified in full MS scan mode using positive electrospray ionization (ESI +) with a mass range fromm/z 110-1300 using HRMS. The method was comprehensively validated on the basis of the European Medicines Agengy (EMA) method validation protocol using commercially available IVD kit components.


RESULTS
Good linear relationship between peak area responses and drug concentrations (R2 > 0.995) and excellent selectivity were observed for all antimycotics in this study. Inaccuracy and imprecision of all quality controls were consistently below ± 12.6% and ± 8.1%, respectively. Quantification results were in agreement with an IVD LC-MS/MS method.


CONCLUSION
HRMS was shown to be suitable for TDM of small molecules when compared to tandem mass spectrometry. The novel HRMS method is quickly installed and may be a robust and reliable tool for routine TDM of antimycotics in clinical laboratories.
['Schuster|Carina|C|', 'Paal|Michael|M|', 'Lindner|Johanna|J|', 'Zoller|Michael|M|', 'Liebchen|Uwe|U|', 'Scharf|Christina|C|', 'Vogeser|Michael|M|']
D000935:Antifungal Agents D002138:Calibration D002851:Chromatography, High Pressure Liquid D016903:Drug Monitoring D004355:Drug Stability D005240:Feasibility Studies D006801:Humans D057230:Limit of Detection D013058:Mass Spectrometry D012015:Reference Standards D015203:Reproducibility of Results D013048:Specimen Handling
Antimycotics Therapeutic drug monitoring (TDM) High resolution mass spectrometry (HRMS) Orbitrap