Abstract: The ubiquitous occurrence of emerging contaminants across environmental compartments, coupled with their biomagnification potential in trophic chains, presents considerable ecological and public health risks. These factors necessitate continuous environmental biomonitoring strategies and the development of reliable bioanalytical techniques for health risk assessment. Consequently, optimizing sample preparation methodologies is an important research focus, particularly for the simultaneous determination of organophosphate esters (OPEs) and their diester metabolites (di-OPEs). Quantifying these trace residues in complex biological matrices remains analytically challenging due to the high lipid and protein content in tissues. Conventional analytical protocols often suffer from methodological limitations, primarily encountering matrix effects that can compromise the accuracy and reliability of subsequent toxicological evaluations. To establish a streamlined and objective analytical framework, this study developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. The method is designed to address existing analytical bottlenecks, enabling the simultaneous quantification of 20 OPEs and 7 di-OPEs in biological tissues. Methodological development involved a systematic comparison of different cleanup procedures, with parameters such as extraction solvents and reconstitution volumes objectively optimized. The finalized protocol utilized a triple ultrasound-assisted extraction (UAE) procedure with high-purity acetonitrile to ensure stable extraction efficiency. The extracted supernatant fractions were consolidated and subjected to matrix cleanup utilizing ENVI-18 solid-phase extraction (SPE) cartridges. Following direct loading of the crude tissue extract, a two-step elution procedure—collecting the primary target fraction and applying a secondary 5 mL methanol elution—was conducted to improve overall analyte recovery. The pooled eluates were concentrated to near-dryness by vacuum centrifugation and reconstituted with 1.0 mL methanol. Instrumental analysis was performed using a reversed-phase liquid chromatography system coupled with a triple quadrupole mass spectrometer, aiming to simplify the operational workflow while maintaining analytical sensitivity. Chromatographic separation was achieved using optimized gradient elution conditions of the mobile phase. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode, with positive electrospray ionization for parent OPEs and negative ionization for the more polar di-OPEs. Quantitative calibration was conducted using deuterium-labeled internal standards to compensate for extraction losses and matrix-induced ionization suppression. Method validation confirmed good linearity over the calibration range of 0.05-20 ng/mL (R>0.991). The calculated limits of detection (LODs), based on sample wet weight, ranged from 0.006β11 to 0.075β4 ng/g. Comprehensive spiked recovery tests yielded recoveries of 54.1-91.1%, with relative standard deviations (RSDs) consistently below 18.4%, validating the precision and reproducibility of the developed method for trace analysis. To investigate the accumulation and distribution characteristics of these compounds under environmentally relevant conditions, the validated method was applied to tissue samples from a mouse model after 63 days low-dose oral exposure (280 ng/kg·day). Internal standard recoveries in these biological samples ranged from 54.3% to 133.8%, indicating the practical applicability of the method for complex in vivo matrix analysis. The analysis revealed varied total target concentrations across different organs. Measured contents were 1.25 ng/g in muscle, 1.99 ng/g in lungs, 5.47 ng/g in spleen, and 4.86 ng/g in heart tissues, with 6, 5, 7, and 9 distinct compounds detected, respectively. Tri(1-chloro-2-propyl) phosphate (TCIPP) constituted the predominant contaminant in the muscle, spleen, and heart. Conversely, triethyl phosphate (TEP) was the primary accumulated compound in the lungs. These tissue-specific distribution profiles indicate distinct toxicokinetic behaviors, reflecting compound-specific biodistribution mechanisms driven by physicochemical properties and differential metabolic clearance among target organs. This methodological approach provides reliable quantitative data to support the investigation of organ-specific metabolic pathways and future environmental health risk assessments.

Key words: organophosphate esters (OPEs), metabolites, solid phase extraction (SPE), high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), biological tissues