## 4 Conclusions  
A comprehensive and sensitive analytical method, based on SPE followed by LC-MS/MS detection, has been developed for the quantitative confirmatory analysis of 38 anthelmintic compounds in raw, unfiltered, environmental water samples and screening analysis for a further two anthelmintic residues. The method has been extensively validated over a broad range of concentration levels, in-line with expected concentration in the environment, based on review of currently available literature. This method is advantageous compared to existing analytical methods for environmental samples because it allows for analysis of a wider range of anthelmintic residues (40), from different structural classes. Of these 40 compounds, 13 of them are metabolites/transformation products, for which currently available methods are lacking. This provides a more comprehensive application to improve the understanding of the environmental occurrence of anthelmintics. The method development work carried out showed the impact of sample modification prior to extraction, which in this case aided desorption of some analytes from the sample container. The matrix effect study demonstrated the importance of assessing ion enhancement/suppression due to matrix, as part of method development and validation stages. The results of this study highlighted the significance of incorporating deuterated internal standards into the analytical methodology, which was shown to improve the overall accuracy and precision for the majority of analytes. This work incorporates deuterated or surrogate internal standards for all 40 compounds. The overall method presented was validated according to appropriate guidelines and deemed to be fit for the purpose intended.  
**Supplementary Materials:** The following are available online. Figure S1: Structures of anthelmintic compounds, Figure S2: Mean recovery and precision (%RSD, presented as error bars) for assessment of sorbent mass (200 mg vs. 500 mg) and elution volume (10, 15 and 20 mL), Figure S3a: RSM optimiser graph for the 17 analytes selected for assessing the effect of percentage modifier (0-40%) and sample pH (4-10) on extraction, Figure S3b: RSM optimiser graph demonstrating predicted recoveries for the remaining 23 analytes, Figure S4: Increase (green bar) or decrease (red bar) in recoveries for all analytes, when the 20% MeOH sample modifier is incorporated, in comparison to the use of no modifier. The acceptable recovery range is as shown by the upper (120%) and lower (70%) recovery lines, Table 1: Physicochemical data for the studied anthelmintics [40,41] (where available), Table S2: Optimised UHPLC-MS/MS conditions optimised and refined from [PERSON] et al., 2010 [30], Table S3: Summary of 13 experimental combinations, including 5 center points, generated using MiniTab, for response surface methodology assessing sample modifier and pH.  
**Author Contributions:** Conceptualization, D.M., C.C., K.G.R., M.D., L.G. and P.-E.M.; methodology, D.M. and M.D.; validation, D.M.; investigation, D.M.; resources, M.D. and P.-E.M.; writing--original draft preparation, D.M.; writing--review and editing, M.D., C.C., K.G.R., L.G. and P.-E.M.; visualization, D.M., C.C. and M.D.; supervision, M.D., C.C. and K.G.R.; project administration, D.M. and C.C.; funding acquisition, C.C., K.G.R., L.G. and M.D.  
**Funding:** This work has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under Grant Number 13/RC/2092 and is co-funded under the European Regional Development Fund and by iCRAG industry partners.  
**Acknowledgments:** This work was carried out as part of a joint project between Trinity College Dublin and Teagasc through the Walsh Fellowship scheme, and it forms part of the Groundwater Challenge of the Irish Centre for Research in Applied Geosciences (iCRAG).  
**Conflicts of Interest:** The authors declare no conflict of interest.