Multi-residue extraction to determine organic pollutants in mussel hemolymph

This study assesses the extraction of eleven pharmaceuticals, five pesticides, five perfluoroalkyl substances, and two illicit drugs in hemolymph from (Mytilus Galloprovincialis). Four extraction procedures using Phree™ Phospholipid Removal cartridges were tested using different volumes of methanol (400 and 600 μL) and acetonitrile (300 and 450 μL). The pollutants were determined by high-performance liquid chromatography-tandem mass spectrometry. The use of methanol gave several problems during the extraction procedure, such as longer times and sample loss. Three methods (acetonitrile 300 and 450 μL; and methanol 600 μL) were validated. Recoveries at three concentration levels (5, 50, and 100 ng/mL) ranged 35.1-129.0 and 29.3-133.0% for acetonitrile 300 and 450 μL, respectively, while recoveries for methanol 600 μL ranged 52.2-166.0%. Limits of detection were < 10 ng/mL for most analytes using any of the methods. Methanol 600 μL was the only method capable to extract the illicit drug 4-methoxyphencyclidine and provided a better peak shape and higher signal-noise ratio. When applied to non-spiked samples from local markets salicylic acid and diclofenac were detected at 33.50-97.79 and 28.30-30.31 ng/mL respectively. To our knowledge, there are no methods to determine organic contaminants in hemolymph and this is the first application of Phree™ cartridges for mussel hemolymph extraction.