Optimising QuEChERS for the analysis of mussels: efficient extraction of Emerging Contaminants in environmental samples

1 Introduction Emerging contaminants (ECs) are chemical compounds continuously released in the environment, whose dispersion may cause significant environmental and human health damage. Despite their potential adverse effects, these compounds have yet to be internationally regulated [1]. The ECs definition encompasses a wide range of chemical species, including pesticides, pharmaceuticals and personal care products, and hormones, some of which are characterised by poor degradability and pseudo-persistence [2,3]. Filter-feeding organisms, such as bivalves, are frequently used in the literature as biomonitoring species due to their capacity to accumulate pollutants through their feeding habits. These organisms are robust sentinels, due to their characteristics: they are filter feeders with limited mobility, possess wide spatial distribution, demonstrate tolerance to diverse environmental conditions, and are easily sampled [4]. Additionally, mussels are widely consumed as food around the world. In the EU, the average annual per capita consumption of mussels is 1.28 kg. Therefore, the presence of contaminant residues in mussels and other edible marine species therefore represents a critical public health concern that necessitates rigorous monitoring through specific analyses [5]. Our study aimed to develop a method to detect extremely low concentrations of contaminants. We utilised Adamussium colbecki, an Antarctic bivalve filter-feeding organism known for minimal contamination, to develop and optimise this method. Subsequently, we applied the method to Mytilus galloprovincialis samples, which are not only used for monitoring but are also edible. Developing analytical methods to detect pollution residues in various edible aquatic species is crucial, as it combines environmental monitoring with the assessment of dietary intake risks. 2 Experimental Samples of A. colbecki, an Antarctic bivalve filter-feeding organism, underwent a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) pre-treatment procedure optimised using the experimental design approach. The steps of the QuEChERS method were studied, by varying solvent volume, shaking mode, and clean-up phases amount. All the resulting extracts were dried and reconstituted with methanol-water (1:1) before analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After optimising the experimental procedure with Antarctic Adamussium samples, the refined method was applied to local mussel samples (M. galloprovincialis) to further assess its robustness and applicability. 3 Results The procedure was designed to detect a wide range of ECs, characterised by different polarity, acidic/basic properties, and molecular mass. Obtaining analysis accuracy for all of them strictly requires a multivariate approach, also due to the several variables involved in the sample treatment strategy. A Plackett-Burman screening design was used to identify significant parameters, which resulted to be the amount of dispersive solid-phase extraction (dSPE) phase, the clean-up time, and the solvent-to-sample ratio. These parameters were subsequently optimised using a Dohelert design. The preliminary results obtained from applying the refined method to environmental samples successfully detected the presence of ECs such as ketoprofen, gemfibrozil, nicotine, ethylhexyl salicylate (EHS), ethyl hexyl methoxy cinnamate (EHMC), and octyl dimethyl p-aminobenzoate (OD-PABA) in M. galloprovincialis samples. Still, further analysis is required for EHMC and OD-PABA confirmation, due to the detection of traces of these contaminants in the procedural blank. Ketoprofen was detected at the LOD level; however, its presence could not be confirmed due to the absence of a qualifier ion in the mass spectrum. These results confirmed the method’s effectiveness and robustness, although some results necessitate additional scrutiny to fully validate the contaminants’ presence. 4 Conclusions Optimising the QuEChERS method marks a significant advancement in the detection and analysis of ECs in mussel samples. Our study aims to improve the sensitivity and accuracy of detection methods for many contaminants, of utmost importance for food safety and environmental monitoring. This method has the potential to provide valuable data for regulatory and scientific efforts to safeguard public health and preserve environmental quality. By validating the technique under different environmental conditions and sample matrices, we ensured its reliability and effectiveness for detecting emerging contaminants in diverse contexts. Future research will focus on the expansion of the set of mussel samples, including those from various geographical locations and different aquatic environments, to investigate contamination in multiple areas of our territory.