Bioanalytical determination of azole antifungals for mycetoma treatment in biological fluids: Advances, challenges, and implementation strategies

Mycetoma is a chronic and debilitating neglected tropical disease caused by fungi or filamentous bacteria, with Madurella mycetomatis representing the most common fungal agent worldwide. Current treatment relies primarily on azole antifungals such as itraconazole and posaconazole, yet their clinical effectiveness is frequently compromised by variable pharmacokinetics, limited tissue penetration, and the requirement for prolonged therapy. These challenges underscore the importance of therapeutic drug monitoring (TDM) to ensure adequate systemic exposure and to guide individualized therapy. This review provides a critical synthesis of bioanalytical methods used for azole quantification in biological matrices, highlighting liquid chromatography–tandem mass spectrometry (LC–MS/MS) as the current gold standard. We summarize sample preparation strategies ranging from traditional solid-phase and liquid–liquid extraction to more pragmatic protein precipitation approaches, and we discuss their relative advantages and limitations in the context of mycetoma. Key analytical issues such as matrix effects, analyte stability, and regulatory validation requirements are addressed, with particular emphasis on the constraints faced in resource-limited endemic regions. Recent innovations in microsampling, including dried blood spots (DBS) and volumetric absorptive microsampling (VAMS), are also reviewed. These techniques offer significant benefits for decentralized TDM by minimizing sample volume, reducing reliance on cold-chain logistics, and simplifying transport and storage. However, issues such as hematocrit effects, assay standardization, and stability must be carefully validated before clinical implementation. Taken together, the integration of robust LC–MS/MS platforms with innovative field-adapted microsampling approaches represents a promising strategy to optimize antifungal therapy and improve long-term outcomes for patients with mycetoma in endemic settings. • LC–MS/MS remains the gold standard for azole antifungal quantification in bioanalysis. • Protein precipitation offers a rapid, low-cost extraction method with acceptable sensitivity. • Microsampling tools (DBS, VAMS) enable decentralized therapeutic drug monitoring. • Field-adapted workflows address challenges in mycetoma-endemic, resource-limited settings. • Integration of LC–MS/MS with microsampling improves individualized antifungal therapy.