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Microplastics (MPs) quantification in benthic marine sediments is typically performed by time-consuming and moderately accurate mechanical separation and microscopy detection. In this paper, we describe the results of our innovative Polymer Identification and Specific Analysis (PISA) of microplastic total mass, previously tested on either less complex sandy beach sediment or less demanding (because of the high MPs content) wastewater treatment plant sludges, applied to the analysis of benthic sediments from a sublittoral area north-west of Leghorn (Tuscany, Italy). Samples were collected from two shallow sites characterized by coarse debris in a mixed seabed of Posidonia oceanica, and by a very fine silty-organogenic sediment, respectively. After sieving at <2 mm the sediment was sequentially extracted with selective organic solvents and the two polymer classes polystyrene (PS) and polyolefins (PE and PP) were quantified by pyrolysis-gas chromatography-mass spectrometry (Pyr-GC/MS). A contamination in the 8–65 ppm range by PS could be accurately detected. Acid hydrolysis on the extracted residue to achieve total depolymerization of all natural and synthetic polyamides, tagging of all aminated species in the hydrolysate with a fluorophore, and reversed-phase high performance liquid chromatography (HPLC) (RP-HPLC) analysis, allowed the quantification within the 137–1523 ppm range of the individual mass of contaminating nylon 6 and nylon 6,6, based on the detected amounts of the respective monomeric amines 6-aminohexanoic acid (AHA) and hexamethylenediamine (HMDA). Finally, alkaline hydrolysis of the residue from acid hydrolysis followed by RP-HPLC analysis of the purified hydrolysate showed contamination by polyethylene terephthalate (PET) in the 12.1–2.7 ppm range, based on the content of its comonomer, terephthalic acid. [ABSTRACT FROM AUTHOR]