Accurate determination of fluoxetine and its metabolites in Mediterranean mussel organs based on solid phase extraction clean-up and HPLC-HRMS

Fluoxetine, a widely prescribed antidepressant, is frequently detected in marine environments, exposing marine invertebrates to this emerging contaminant. Due to its potential to bioaccumulate and undergo biotransformation, it is essential to quantify both the parent compound and its metabolites for accurate risk assessment. Furthermore, tissue-specific measurements are crucial for identifying organ-specific accumulation that may cause localized toxic effects. An analytical method was then developed to quantify fluoxetine and three of its metabolites in individual mussel tissues (soft tissues, gills, digestive glands) using a minimal sample size (40 or 400 mg). Four solid-phase extraction sorbents were evaluated: MCX, HLB, C18, and Phree phospholipid removal. The Phree sorbent was selected as the optimal cleanup method. Further optimization involved testing two extraction solvents (acetonitrile and methanol) and two extraction techniques: ultrasonic-assisted extraction and bead-based extraction. Identification and quantification were performed using HPLC-Orbitrap-MS, relying on accurate mass measurements of selected MS/MS fragments. The method demonstrated satisfactory Method Quantification Limits (MQL) for fluoxetine (1.2, 10.5, and 10.4 μg/kg dw) and its main metabolite, norfluoxetine (5.1, 25.6, and 24.3 μg/kg dw) in soft tissues, digestive glands, and gills, respectively. These MQL values align with literature data, especially given the low sample size and the expression of concentrations on a dry weight basis. The final method allows the sensitive quantification of fluoxetine and its metabolites from small volumes, facilitating the assessment of their distribution across three target organs. These advancements support the development of toxicokinetic/toxicodynamic (TK/TD) models, contributing to environmental risk assessment of emerging contaminants.