MS³ acquisition and μ-SPE cleanup enable sensitive quantification of PPG1000 in zebrafish tissues: systematic...

Polypropylene glycol 1000 (PPG1000) is a hydrophilic polymer widely used in pharmaceuticals and industry, but quantifying it in complex biological samples remains challenging. This study develops and validates a sensitive UPLC-MS/MS method for PPG1000 measurement in zebrafish tissues. The method combines micro-solid-phase extraction (μ-SPE) cleanup with a 2.5 min chromatographic run and MS³ detection for PPG1000-equivalent quantification in zebrafish tissues. We systematically compared three mass spectrometric acquisition modes: multiple reaction monitoring (MRM), MS³, and in-source collision-induced dissociation (CID). The ammonium adduct [M + NH₄]⁺ of the 15-mer oligomer (m/z 906.6) served as the precursor ion. MS³ using a linear ion trap (m/z 906.6 → 175.1 → 117.1) delivered the highest signal intensity (3.6 × 10⁶ cps), approximately 3.3-fold and 40-fold higher than MRM and CID, respectively. Integrating μ-SPE further improved method performance over conventional protein precipitation. The method showed excellent linearity (100 to 10 000 ng mL^-1, r > 0.997) and was applied to zebrafish tissue distribution after aqueous exposure to 200 μg mL^-1 PPG1000. All three tissues (gill, liver, and brain) exhibited increasing concentrations from 2 to 4 h, with the gill showing the highest accumulation. Detectable brain levels suggest partial blood-brain barrier penetration. This work provides the first systematic comparison of MRM, MS³, and in-source CID for PPG quantification, establishing a sensitive analytical platform with complementary greenness and practical applicability assessments using AGREE, AGSA, CACI, and GEMAM, for polymer bioanalysis in complex matrices.