Bio-analytical liquid chromatographic-based method with a mixed mode online solid phase extraction for drug monitoring of fluconazole in human serum

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2021-12, Vol.1187, p.123045-123045, Article 123045
• Main Authors: Zarad, Walaa, El-Gendy, Heba, Bazan, Lamyaa, Ali, Ahmed, Aboulella, Yasmine, Kamal, Maha, Emara, Samy, Shawky, Ahmed
• Format: Article
• Language: English
• Subjects: Chromatography, High Pressure Liquid - methods; Chromatography, Reverse-Phase - methods; Drug Monitoring - methods; Fluconazole; Fluconazole - blood; Fluconazole - chemistry; Fluconazole - isolation & purification; Fluconazole - pharmacokinetics; Human serum; Humans; Limit of Detection; Linear Models; Male; Pharmacokinetic study; protein-coated µBondapak CN silica column; Reproducibility of Results; Solid Phase Extraction - methods
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2021.123045

•An automated bio-analytical HPLC-based method for measuring fluconazole in serum.•Utilization of small mixed mode protein-coated column for on-line SPE.•Size-exclusion mode for protein extraction from untreated serum.•Reversed-phase mode for fluconazole trapping and enrichment.•The developed method was applied for pharmacokinetic study of fluconazole in human. A simple, cost-effective and sensitive liquid chromatography-based bio-analytical method has been developed and validated for therapeutic drug monitoring of fluconazole (FLUC) in human serum. Integration of online mixed-mode solid-phase extraction (SPE) into the analytical system was the key for direct injection of untreated serum samples. A short protein-coated (PC) µBondapak CN silica column (PC-µB-CN-column) as a SPE tool and phosphate buffer saline (PBS) (pH 7.4) as an eluent were applied in the extraction step. PC-µB-CN-column operates in two different chromatographic modes. Using PBS, proteins were extracted from serum samples by size-exclusion liquid chromatography, while FLUC trapping was reversed-phase liquid chromatography dependent. FLUC was then eluted from the PC-µB-CN-column onto the quantification position using a mixture of acetonitrile-distilled deionized water (20:80, v/v) as an eluent and ODS analytical column. FLUC was separated at ambient temperature (22 ± 1 °C) and detected at 260 nm. The method was linear over the range of 200–10000 ng/mL. FLUC recovery in untreated serum samples ranged from 97.8 to 98.8% and showed good accuracy and precision. The reliability of the developed method was evaluated by studying the pharmacokinetic profile of FLUC in humans after an oral administration of a single 150 mg tablet.