Simultaneous determination of seven azole antifungal drugs in serum by ultra-high pressure liquid chromatography and diode array detection

Azole antifungals are a group of fungistatic agents that can be administered orally or parenterally. The
determination of the concentrations of these antifungals (miconazole, fluconazole, ketoconazole,
posaconazole, voriconazole, itraconazole, and its major active metabolite, hydroxy-itraconazole) in serum
can be useful to adapt the doses to pharmacological ranges because of large variability in the absorption
and metabolism of the drugs, multiple drug interactions, but also potential resistance or toxicity. A method
was developed and validated for the simultaneous determination of these drugs in serum utilizing ultra-high
pressure liquid chromatography and diode array detection (UHPLC-DAD). After a simple and rapid liquid–
liquid extraction, the pre-treated sample was analysed on an UHPLC-DAD system (Waters CorporationH).
The chromatographic separation was carried out on an Acquity BEH C18 column (Waters Corporation) with
a gradient mode of mobile phase composed of acetonitrile and aqueous ammonium bicarbonate 10.0 M
pH10. The flow rate was 0.4 ml/min and the injection volume was 5 ml. The identification wavelength varied
according to the drug from 210 to 260 nm. The method was validated by the total error method approach
by using an analytical validation software (eNnoval V3.0 ArlendaH). The seven azole antifungals were
identified by retention time and specific UV spectra, over a 13-minute run time. All calibration curves
showed good linearity (r2.0.99) in ranges considered clinically adequate. The assay was linear from 0.05
to 10 mg/l for voriconazole, posaconazole, itraconazole, hydroxy-itraconazole, and ketoconazole, from 0.3
to 10 mg/l for fluconazole, and from 0.1 to 10 mg/l for miconazole. The bias and imprecision values for intraand
inter-assays were lower than 10% and than 15%, respectively. In conclusion, a simple, sensitive, and
selective UHPLC-DAD method was developed and validated to determine seven azole antifungal drugs in
human serum. This method is applicable to patient samples, and can be applied successfully to clinical
applications and therapeutic drug monitoring.