Targeted quantitation of 6ppd-quinone in fish tissue samples with liquid chromatography-tandem mass spectrometry

The tire additive transformation product N-(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine-quinone (6PPD-quinone) has recently garnered global attention due to its acute toxicity to some salmonids, such as coho salmon (Oncorhynchus kisutch), and its ubiquitous presence in urban stormwater systems. In the present study, we developed and compared the extraction efficiency of two sample preparation methodologies for quantification of 6PPD-quinone among two fish tissue sample types that included fillet of smallmouth bass (Micropterus dolomieu) and whole-body samples of O. kisutch fry subjected to in vivo exposure tests with 6PPD-quinone. The two sample preparation methods tested included an accelerated solvent extraction (ASE) approach and a sonication extraction approach. Both sample preparation methods included identical purification steps for the crude sample extracts with enhanced matrix removal cartridges. The purified sample extracts were subjected to targeted analysis of 6PPD-quinone using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that extractions made with the reported ASE method demonstrated significantly higher absolute recovery (80–96%) of the extracted internal standard, [¹³C₆]-6PPD-quinone, than sonication-based extractions (74–80%) in both fish tissue sample types. The proposed ASE method shows acceptable limits of quantification (0.37–0.67 ng g⁻¹), linearity (R² > 0.996), and repeatability (RSD ≤ 9%). This work advances research capabilities for investigations on the toxicokinetic processes of 6PPD-quinone in biological samples.