Using UHPLC-MS/MS to Detect Illicit Drugs in Wastewater

A study done by researchers at China Pharmaceutical University in Nanjing, China developed a new ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based method for analyzing compounds in wastewater. Their findings were published in the Journal of Chromatography A (1).

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The presence of new psychoactive substances (NPS), such as cannabis, cocaine, and LSD, has increased significantly within illicit drug markets (2). To address the evolving issues around drug abuse, governments worldwide have been dynamically monitoring patterns, scales, and trends of drug abuse.

Wastewater-based epidemiology (WBE), which involves testing raw sewage for pathogens, employs wastewater composition analysis, showing an effective approach for monitoring illicit drug usage (3). WBE, first introduced in 2001, involves detecting concentrations of drugs or metabolites in wastewater to estimate drug abuse in communities. It can account for many substances, including illicit addictive substances, such as NPS.

The most prevalent wastewater analysis method involves ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) preceded by sample preparation that involves solid-phase extraction (SPE). SPE cartridges concentrate target compounds and reduce background interference to meet quantitative requirements. However, using a single cartridge to enrich all compounds is impractical, and using multiple SPE cartridges could result in increased expenses, longer processing times, and more complex pretreatment procedures. Scientists have explored using direct injection (DI) techniques for detecting NPS and other drugs in wastewater. This method involves adding internal standards to a small volume of wastewater samples, which are subsequently analyzed using UHPLC-MS/MS following membrane filtration.

For this study, the scientists developed a DI technique for UHPLC-MS/MS, which enabled the detection of 77 compounds, which encompassed metabolites of human biomarkers, illicit drugs, and new psychoactive substances. The DI method went through rigorous optimization and validation, showing a lower limit of quantitation (LLOQ) ranging from 1 ng L^–1 to 100 ng L^–1 and a limit of detection (LOD) ranging from 0.5 ng L^–1 to 80 ng L^–1. A SPE method, which comprised two common SPE cartridges, and the DI method were compared in terms of matrix effects, recoveries, and accuracies through analyzing spiked wastewater samples. The DI method showed superior capability in detecting a wider range of compounds while more time-efficient, while also having better recovery, lower matrix effects, and lower relative error in spiked samples.

After the method comparisons, the DI method was used to analyze wastewater samples from 25 wastewater treatment plants (WWTPs) across China. Among the 77 compounds, 39 were detected in influent samples. 14 compounds exhibited detection rates of 100%, serving as biomarkers for alcohol, tobacco, endogenous metabolites, and more. The concentrations of opiates, including morphine, codeine, tramadol, and oxycodone, ranged from 0 ng L^–1 to 82.95 ng L^–1. While this is effective, legal medicinal morphine use poses challenges in monitoring heroin abuse, as it can potentially influence concentration levels and introduce uncertainties.

Overall, the developed method was concluded to be optimized and reliable, showing more effectiveness in detecting spiked samples than SPE methods. Combined with successful application in analyzing wastewater samples, this new method was displayed as a high-throughput method that improves upon the accuracy, reliability, and detection efficiency of wastewater drug monitoring.

References

(1) Tao, W.; Che, X.; Liu, P.; et al. Direct Injection UPLC-MS/MS Method for Analysing 77 Compounds Including Human Biomarkers, Illicit Drugs, New Psychoactive Substances and Metabolites in Wastewater. J. Chromatogr. A 2025, 1741, 465596. DOI: 10.1016/j.chroma.2024.465596

(2) New Psychoactive Substances. Alcohol and Drug Foundation 2025. https://adf.org.au/drug-facts/new-psychoactive-substances/ (accessed 2025-1-21)

(3) Wastewater-Based Epidemiology. Discovery Partners Institute 2025. https://dpi.uillinois.edu/applied-research/wastewater-epidemiology/ (accessed 2025-1-21)