Simultaneous determination of diclofenac, its human metabolites and microbial nitration/nitrosation transformation products in wastewaters by liquid chromatography/quadrupole-linear ion trap mass spectrometry

• Quantitative determination of five diclofenac metabolites in wastewater.
• Instrument-dependant acquisition on QLIT-MS applied for confirmatory purposes.
• Chemical synthesis of several metabolites and transformation products.
• Absence of nitrated/nitrosated DCF in untreated sewage while present in effluents.

An analytical method was developed and validated for the first determination of five major human metabolites of the non-steroidal anti-inflammatory drug diclofenac as well as two microbial transformation products in wastewater. The method was based on the extraction of diclofenac and the chemically synthetized compounds by solid-phase extraction (SPE), using a hydrophilic–lipophilic balanced polymer followed by liquid chromatography (LC) coupled to hybrid quadrupole-linear ion trap mass spectrometry (QqLIT-MS). Quantitation was performed by the internal standard approach, to correct for matrix effects. The accuracy of the method was generally higher than 40% for raw and treated wastewater with a precision below 12%. In wastewater influent and effluent samples the detection limits for the majority of target compounds were 0.3–2.5 ng L−1 and 0.1–3.1 ng L−1, respectively. The method was applied to the analysis of influent and effluent wastewater samples from urban wastewater treatment plants. Moreover, to obtain an extra tool for confirmation and identification of the studied diclofenac-derived compounds, Information-Dependent Acquisition (IDA) experiments were performed, with selected reaction monitoring (SRM) as the survey scan and an enhanced product ion (EPI) scan as the dependent scan. Diclofenac and its major human metabolite, 4′-hydroxydiclofenac were detected in all samples at concentrations of 331–1150 ng L−1 and 585–6000 ng L−1, respectively. Neither microbial transformation product of diclofenac was detected in any of the influent samples analyzed, but in effluents, their concentrations ranged from 4 to 105 ng L−1.