Matrix-independent quantification of paracetamol in complex aquatic matrices using quadruple isotope dilution Orbitrap mass spectrometry

Accurate quantification of trace organic compounds in complex aquatic matrices is frequently limited by matrix effects, analyte losses, and uncertainties associated with conventional calibration strategies. In this study, a quadruple isotope dilution (ID⁴) approach coupled with Orbitrap high-resolution mass spectrometry was developed as a reference-grade analytical method. The method was applied to wastewater (WW) and river water (RW) samples, using paracetamol as the model analyte. The LC-ESI-ID⁴-HRMS method demonstrated excellent accuracy, yielding recoveries between 99.7% and 101.7% across three different aqueous matrices. The robustness of the ID⁴ strategy was evaluated through two targeted pairwise comparisons. At low concentration levels, the values determined using the ID⁴ strategy (4.04 ± 0.03 μg kg⁻¹ for the effluent WW sample and 3.06 ± 0.05 μg kg⁻¹ for the RW sample) showed close agreement with those obtained by the standard addition calibration method (4.02 ± 0.04 μg kg⁻¹ for the effluent WW sample and 3.26 ± 0.04 μg kg⁻¹ for the RW sample). At high analyte levels in the influent water, results obtained by the LC-ESI-ID⁴-HRMS and direct infusion NanoESI-ID⁴-HRMS were in close agreement (1490.6 ± 9.3 μg kg⁻¹ and 1457.6 ± 48.3 μg kg⁻¹, respectively), demonstrating reliable quantification even without chromatographic separation. These results demonstrate that the combination of high-resolution isotope ratio measurements with the ID⁴ experimental design enables matrix-independent quantification across different concentration levels and analytical configurations.