Chromatography-Enabled Detection of Respiratory Medications in Wastewater

Despite the high prevalence of respiratory diseases, there is a lack of sensitive analytical methods for detecting associated medications in complex wastewater matrices. Therefore, researchers at the University of Louisville (Kentucky) developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring for 10 common respiratory pharmaceuticals. A paper based on their research was published in the journal Environmental Science; Water Research & Technology (1).

Wastewater-based epidemiology (WBE) has come to be considered as a reliable approach for community surveillance for both molecular (pathogens) and chemical (pharmacologic compounds and their metabolites) targets (2-4). In addition to its scientific utility, WBE has earned hearty public support, especially for the investigation of prescription medication usage (5). However, quantifying these compounds remains a challenge because of analyte stability, sample collection, storage conditions, and analytical performance (6).

Respiratory diseases such as asthma, chronic obstructive pulmonary disease, allergic rhinitis, sinus infections, and pneumonia are among the conditions most commonly requiring pharmacological treatment and result in a great burden on healthcare resources (7). Incidence of these diseases is strongly influenced by environmental exposures (8) and are frequently related to inconsistent prescribing practices, especially the use of antibiotics for viral respiratory infections. The inappropriate use of antibiotics can increase the danger of antimicrobial resistance, a major public health concern (9).

Detection and quantification limits achieved in the study ranged from 0.7 to 19 ng L^-1 and 3 to 125 ng L^-1, respectively, with recoveries of 82-194% and precision within 0.14-7.2% relative standard deviation. Matrix effects (64-228%) were effectively corrected using stable isotope-labeled internal standards (SILs). Application to 12 neighborhood-level wastewater samples detected 9 of the 10 target compounds, with 6 (albuterol, amoxicillin, azithromycin, cetirizine, diphenhydramine, and fexofenadine), detected above their quantification limits. Fexofenadine was the most abundant, reaching 3309 ng L^-1 Most analytes achieved baseline separation in LC; however, two sets of analytes (albuterol with amoxicillin and prednisolone with prednisone) co-eluted from the LC and entered MS together. Each pair of these molecules has different parent ion m/z values and daughter ions. They could still be differentiated and quantified in the first multiple reaction monitoring (MRM) quadrupole due to different parent ion selection (1).

The researchers believe that their work has resulted in “a robust and time-efficient LC-MS/MS with the MRM-validated method for quantifying 10 pharmaceuticals, associated with respiratory medication use, in wastewater.” They state that their method is the first to incorporate freeze-drying for preconcentration, online SPE for sample cleanup and reduced sample losses, and MRM for robust quantification. Furthermore, their use of stable isotope-labeled standards ensured accuracy and reproducibility in a complex matrix. In addition, the method demonstrated strong analytical performance, requiring only 1 mL of sample and achieving low detection limits (1).

Although their study focused on 10 respiratory-related pharmaceuticals, the developed method is readily extendable to other pharmaceuticals and metabolites. They also noted that the method’s scalability and suitability for high-throughput analysis highlight its potential for broader application in WBE, making population-level surveillance of pharmaceutical usage and supporting public health efforts possible (1).

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References

1. Gasparetto, R. L.; Bickel, S.; Yin, X. et al. Targeted LC-MS/MS Method for Quantifying Respiratory Pharmaceuticals in Wastewater. Environ. Sci. (Camb) 2025.DOI: 10.1039/d5ew00894h
2. Bowes D. A. Towards a Precision Model for Environmental Public Health: Wastewater-Based Epidemiology to Assess Population-Level Exposures and Related Diseases. Curr. Epidemiol. Rep. 2024, 11 , 131 —139. DOI: 10.1007/s40471-024-00350-6
3. Kilaru, P.; Hill, D.; Anderson, K. et al. Wastewater Surveillance for Infectious Disease: A Systematic Review. Am. J. Epidemiol. 2023, 192 , 305-322. DOI: DOI: 10.1093/aje/kwac175
4. Lorenzo, M.; Picó, Y. Wastewater-Based Epidemiology: Current Status and Future Prospects. Curr. Opin. Environ. Sci. Health 2019, 9, 77 —84. DOI: 10.1016/j.coesh.2019.05.007
5. Holm, R. H.; Anderson, L. B.; Ness, H. D. et al. Towards the Outbreak Tail, What is the Public Opinion About Wastewater Surveillance in the United States? J. Water Health 2024, 22 (8), 1409-1418. DOI: 10.2166/wh.2024.074
6. Oliveira, T. S.; Murphy, M.; Mendola, N. et al. Characterization of Pharmaceuticals and Personal Care Products in Hospital Effluent and Waste Water Influent/Effluent by Direct-Injection LC-MS-MS. Sci. Total Environ. 2015, 518-519, 459-478. DOI: 10.1016/j.scitotenv.2015.02.104
7. Duan KI, Birger M, Au DH, Spece LJ, Feemster LC, Dieleman JL. Health Care Spending on Respiratory Diseases in the United States, 1996-2016. Am J Respir Crit Care Med. 2023 Jan 15;207(2):183-192.
8. Khadke, S.; Khadke, V.; Kumar, A. et al. Environmental Justice Index and Prevalence of Asthma and COPD in US Neighborhoods- A Population-Based Study. Lancet Reg. Health Am. 2025, 49, 101195. DOI: 10.1016/j.lana.2025.101195
9. Llor, C.; Bjerrum, L. Antimicrobial Resistance: Risk Associated with Antibiotic Overuse and Initiatives to Reduce the Problem. Ther. Adv. Drug Saf. 2014, 5 (6), 229-241. DOI: 10.1177/2042098614554919