Validated LC–MS/MS method for 26 prescription BZDs was applied to influent wastewater in Belgium, Latvia, Lithuania and Romania, revealing spatial consumption differences. Findings highlight the challenge of interpreting BZD trends due to overlapping metabolic pathways, emphasising the need to distinguish biomarker classes to ensure accurate and comparable results across studies.

ABSTRACT

Benzodiazepines and related z-drugs (BZDs) are widely used pharmaceuticals but require careful monitoring due to limited efficacy, tolerance development and the risk of dependence. Wastewater-based epidemiology (WBE) is a valuable approach to gather population-wide information on consumption patterns through analysis of influent wastewater. This study examines how overlapping metabolic pathways within the BZD drug class can lead to misinterpretation if not carefully considered. An analytical method is developed and validated for 26 biomarkers of prescription BZDs in influent wastewater using solid-phase extraction and liquid chromatography–tandem mass spectrometry and applied to influent wastewater samples for the first time in Belgium (13 locations), Latvia (1), Lithuania (3) and Romania (1). The countries were chosen for their differing prescription BZD market registration, allowing testing of different hypotheses. To aid in complex data interpretation, this study presents a structured categorisation of BZDs into direct biomarkers, which directly reflect consumption, and downstream biomarkers, which are influenced by multiple BZDs, and further discusses the implication this has on the interpretation. Spatial differences were noted among direct biomarkers such as higher consumption of zolpidem in Belgium and zopiclone in Riga (LVA). Downstream biomarkers had higher population-normalised mass loads but cannot be interpreted in isolation, for example, lorazepam (ranging 4–25 mg/day/1000 inhabitants over all locations), lormetazepam (8–30), oxazepam (10–50) and temazepam (1–12). These findings highlight challenges in interpreting BZD consumption trends and emphasise the need of distinguishing different biomarker classes to ensure accurate and comparable results across studies needed to guide informed decision-making.