Combating New Psychoactive Substances (NPS) Abuse: Adapting a Forensic Toxicology Screening Solution for Environmental Wastewater Surveillance with ACQUITY UPLC H-Class PLUS Coupled with Xevo G2- XS QTof Instrument

Importance of the Topic

New psychoactive substances (NPS) are an emerging global concern, characterized by continuously evolving chemical structures and unpredictable health and social consequences. Traditional monitoring techniques often fail to detect these low-level, diverse compounds in complex environmental matrices. Wastewater surveillance provides an indirect, population-level approach to assess community exposure, early warning for new trends, and to inform public health responses.

Objectives and Study Overview

This application note aims to adapt a forensic toxicology screening solution for environmental wastewater surveillance. Key objectives include:

• Developing a single solid-phase extraction protocol suitable for complex wastewater samples.
• Integrating a high-throughput UPLC-HRMS workflow to screen for a broad library of NPS and related compounds.
• Evaluating method performance, limits of detection (LOD), and false positive reduction using certified reference materials and a system suitability mix.

Methodology

Wastewater samples (n=100) were collected from diverse locations and subjected to a tailored SPE protocol using Oasis MCX cartridges. After conditioning and washing, analytes were eluted with an alkaline organic solvent mixture. Chromatographic separation was achieved on an ACQUITY UPLC HSS C18 column (2.1×150 mm, 1.8 µm) at 50 °C with a 15 min gradient from aqueous ammonium formate/formic acid to acetonitrile with formic acid. High resolution accurate mass data were acquired on a Xevo G2-XS QTof in positive electrospray MSE mode, capturing both precursor and fragment ions in a single injection.

Instrumentation Used

• ACQUITY UPLC H-Class PLUS System
• ACQUITY UPLC HSS C18 Column, 100 Å, 1.8 µm, 2.1 × 150 mm
• Xevo G2-XS QTof Mass Spectrometer
• waters_connect Software with UNIFI Application

Main Findings and Discussion

The method successfully identified all 10 compounds in the system suitability mix within ±0.35 min retention time and <5 ppm mass error. Internal validation with over 100 certified reference materials established LODs down to 1 µg/L and reduced false positives by enforcing intensity and fragment ion criteria. In real samples, 42 controlled substances—including cannabinoids, benzodiazepines, fentanyls, stimulants, and antidepressants—were detected with high confidence. The time-aligned MSE acquisition provided rich fragment ion data, enhancing specificity and streamlining library matching.

Benefits and Practical Applications

• Comprehensive screening of ~1,975 analytes in a single injection.
• Automated workflows minimize manual data review and increase throughput.
• Enhanced sensitivity and specificity reduce false positive rates.
• Applicable to forensic, public health, and environmental monitoring laboratories.

Future Trends and Potential Applications

Advancements in high-resolution mass spectrometry, expanding spectral libraries, and integration with data analytics will further improve early detection of emerging psychoactive compounds. Coupling wastewater surveillance with geographical and temporal mapping can refine epidemiological insights. Continued development of automated suspect and non-targeted screening workflows will support rapid response to novel substances.

Conclusion

This study demonstrates a robust UPLC-HRMS workflow, combining an optimized SPE protocol and MSE acquisition, for broad-scope wastewater surveillance of NPS. The integrated solution delivers high sensitivity, specificity, and efficiency, enabling laboratories to track community drug use patterns and support public health initiatives.

References

• Conlan XA, Theakstone AG. CHAPTER 1 Detection Strategies for Traditional Illicit Substances. RSC Publishing; 2021.
• Lee R, Wood M. Using the Waters Forensic Toxicology Screening Application Solution with UNIFI to Determine Diuretics in Urine. Waters Application Note 720005391; 2015.
• Lee R, Wood M. Screening for Cannabinoids using the Waters Forensic Toxicology Application Solution with UNIFI. Waters Application Note 720005413; 2015.
• Mistry NS, Carlton LJ, Cooper J. The Utility of MSE for Toxicological Screening with waters_connect and the Xevo G3 QTof Mass Spectrometer. Waters Application Brief 720008045; 2023.