Py-GC-MS/MS quantification of microplastics in vertebrate tissues: Addressing false positives of polyethylene

As plastic production continues to rise and waste accumulates, animal species across the planet are being increasingly exposed. However, the fate of plastics within the vertebrate body, and particularly of small microplastics (SMPs, 10-1000 µm), remains largely unknown due to challenges in reliably quantifying their loads. To address this gap, we developed a method based on pyrolysis gas chromatography coupled with tandem mass spectrometry (Py-GC/MS-MS) to quantify SMPs in the Yellow-legged Gull (Larus michahellis), an opportunistic seabird highly exposed to plastic pollution. Five gull carcasses were necropsied under controlled conditions, isolating four organs of the gastrointestinal tract. Sample preparation was undertaken in seven steps, including a potassium hydroxide digestion. Interference from lipids in quantifying polyethylene (PE), a recognized problem for this type of quantification, was addressed by selecting markers with carbon atom chains longer than the aliphatic chains of common lipids (i.e., >22 atoms), enabling us to effectively distinguish PE from biological matrix components. The use of long markers for PE raised the limit of quantification (LOQ) to 50 µg/g, while the LOQs for PMMA (poly(methyl methacrylate)), PP (polypropylene), and PS (polystyrene) were 1 µg/g. For PET (polyethylene terephthalate), the LOQ was 10 µg/g due to the relatively high level of airborne contamination in negative controls. Using this method, microplastic concentrations in gull digestive tissues was found to range over several 100 µg/g dry weight, with one individual showing particularly high exposure to PS and PE. Our method enables a reliable quantification of microplastics and provides a basis for analyzing their impacts on vertebrates.