WP1: Monitoring of Pharmaceutical Products and Organic Emerging Contaminants (D1.2 Updated Target Methods)

This deliverable (D1.2) reports the outcomes of Task 1.2 (M6–M9) within Work Package 1 (WP1) of the NBS4AQUAMISSION project. The work was conducted by Aarhus University (AU) and focuses on updating the existing targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) method based on the suspect-screening results reported in D1.1. Following the prioritisation of compounds identified across the project's demonstration sites, five wastewater-relevant organic micropollutants were selected for method development: tilmicosin, dipyridamole, protionamide, etofenprox, and chlorcyclizine. The updated method was developed using a SCIEX 7500+ triple-quadrupole LC-MS/MS system coupled with UHPLC, with direct injection of wastewater samples. The deliverable documents the analytical method development and implementation, including compound-specific multiple reaction monitoring (MRM) transitions, retention times, internal-standard assignments, calibration, limits of detection (LOD) and quantification (LOQ), blank and carry-over assessment, and recovery evaluation in spiked wastewater. Key findings: Four compounds—tilmicosin, dipyridamole, protionamide, and chlorcyclizine—were incorporated into the existing scheduled MRM method without changes to the established chromatographic run. The reported LODs for these four compounds were 0.10, 0.01, 0.02, and 0.02 µg/L, respectively, with corresponding LOQs of 0.25, 0.02, 0.05, and 0.05 µg/L. Recovery rates in spiked wastewater were 118% for tilmicosin, 102% for dipyridamole, 37% for protionamide, and 87% for chlorcyclizine. The relatively low recovery and lower linearity for protionamide indicate that further optimisation is needed. Etofenprox was incorporated with a limitation: a suitable second MRM transition for the parent compound could not be established. Further method development is required to strengthen analytical confirmation. Tetroxoprim, another compound prioritised in D1.1, was not included because an analytical standard was not commercially available at the time of assessment. Contents: Introduction to the NBS4AQUAMISSION project, its demonstration sites, and WP1 objectives Rationale for updating the targeted monitoring method based on D1.1 Instrumentation, chromatographic conditions, and mass spectrometry parameters Optimised MRM transitions and retention times for the selected compounds Calibration, detection, and quantification limits, quality control, and recovery assessment Updated target-method performance data and recommendations for further optimisation Annex containing analytical performance data for the existing and updated target compounds This deliverable provides the analytical basis for Task 1.3 routine quantification of pharmaceutical products and other organic micropollutants in wastewater from the NBS4AQUAMISSION demonstration sites. It supports SO1 (pollution characterisation) and SO3 (nature-based solution performance monitoring), contributing to the assessment of pollutant removal and the project's broader objectives for aquatic biodiversity protection.