Optimization of solid phase microextraction for non-lethal in vivo determination of selected pharmaceuticals in fish muscle using liquid chromatography–mass spectrometry

A new thin film microextraction (TFME) configuration based on C18 thin film was optimized to improve solid phase microextraction (SPME) sensitivity and extraction kinetics for in vivo determinations of trace pharmaceuticals in fish tissue. Optimization of SPME involved increasing sampler surface area and volume of extraction phase to improve performance within in vitro applications such as agarose gel matrix and spiked fish tissue, as well as in vivo determinations of pharmaceuticals in fish exposed to municipal wastewater. Rainbow trout (Oncorhynchus mykiss) were exposed for 4 d to effluents from a municipal wastewater plant that was treated under three different configurations of a pilot plant. Fathead minnow (Pimephales promelas) were caged for 14 d upstream and downstream of municipal wastewater effluent discharges along Grand River at near Kitchener, ON. TFME and regular C18 fibers were inserted into the dorsal-epaxial muscle of rainbow trout and fathead minnow, respectively, for 30 min sampling intervals. Sample extracts obtained from fibers were desorbed in methanol:water (3:2) for 90 min under 1500 rpm agitation and analyzed using liquid chromatography coupled with tandem mass spectrometry (LC/MS–MS) to determine pharmaceutical bioconcentration. Most target pharmaceuticals were detected in the wastewater with the exception of norfluoxetine and paroxetine. C18 TFME phase successfully quantified fluoxetine, venlafaxine, sertraline, paroxetine, and carbamazapine in muscle of living fish at concentrations ranging from 1.7 to 259 ng/g. Reproducibility of the method in spiked fish muscle was 9–18% RSD with limits of detection and quantification ranging from 0.08 to 0.21 ng/g and 0.09 to 0.64 ng/g (respectively) for the analytes examined. Ibuprofen and gemfibrozil were not detected in fish tissues, likely due to rapid excretion leading to low rates of bioconcentration. This study demonstrates improved in vivo SPME sensitivity (recovery) and extraction rates during pre-equilibrium sampling of target pharmaceuticals in fish tissue due to the improved C18 extraction phase geometry.

► A new in vivo SPME technique based on thin film geometry is developed.
► Strategies to improve sensitivity and extraction kinetics are demonstrated.
► Application of the method is demonstrated in live fish.
► Bioconcentration of pharmaceuticals in fish is successfully determined.