Development of artificial wastewater matrix for the investigation of legal and illegal drug consumption

Wastewater-Based Epidemiology (WBE) studies are based on the principle that psychoactive substances consumed by individuals are excreted as metabolites into wastewater, which are then detected and analyzed. However, real wastewater is complex, variable, and microbiologically active, containing organic and inorganic contaminants, pharmaceuticals, detergents, pathogens, and numerous other substances. This complicates optimization in laboratory studies, impacts analytical sensitivity and reproducibility, and can limit method validation. For this reason, artificial wastewater formulations that can represent real domestic wastewater are essential for conducting controlled laboratory experiments. This thesis aimed to develop artificial (synthetic) domestic wastewater matrix that provides controlled and reproducible experimental conditions to mimic real wastewater in order to evaluate the behavior of psychoactive substances and develop/validate analytical methods in wastewater matrix. To achieve this, a total of 22 artificial domestic wastewater formulations, containing various ingredients and concentrations, were studied. These recipes were evaluated in terms of physicochemical properties, and throughout the experimental studies, key wastewater parameters such as chemical oxygen demand (COD), biological oxygen demand (BOD), total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), total suspended solids (TSS), pH, temperature, odour and color were monitored. The formulation showing the closest similarity to real domestic wastewater was found to have COD of 384.0 mg/L, BOD of 151.5 mg/L, TN of 44.0 mg/L, TP of 35.5 mg/L, TOC of 46.2 mg/L, TSS of 38.2 mg/L, and pH of 7.3. Its content included: 1 L tap water, 25 mg/L yeast, 20 mg/L glucose, 50 mg/L ammonium chloride, 200 mg/L potassium phosphate, 20 mg/L sodium acetate, 40 mg/L urea, 45 mg/L starch, 50 mg/L milk powder, 50 mg/L meat extract, 0.05 mL sunflower oil, and 1 mL detergent mixture. The developed artificial domestic wastewater matrix was evaluated for its applicability for wastewater epidemiology analyses, its potential to correct for matrix effects in analytical studies, and its similarity to real wastewater samples in terms of its association with psychoactive substances. For this purpose, reference standards for illicit psychoactive substances commonly used in our country and worldwide, such as morphine, amphetamine, methamphetamine, and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), were spiked into tap water, real wastewater, and developed artificial wastewater matrix. These samples were analyzed by the solid phase extraction – liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS), which was previously validated at the Forensic Toxicology Laboratory of the Istanbul University-Cerrahpaşa, Institute of Forensic Sciences and Legal Medicine and routinely applied in WBE studies. Thus, it was aimed to obtain a more controlled matrix for psychoactive substance analyses in WBE studies and to increase reproducibility in method development and validation studies. The results of the analyses were evaluated according to verification parameters such as selectivity, accuracy (trueness and precision), matrix effect and stability. To observe matrix effect, matrix-matched calibration were compared with the solvent calibration, and linearity, working range, limits of detection and quantification were evaluated accordingly. In the selectivity study, the instrument response was consistent with the retention time for all substances. The linearity studies with matrix-matched calibration yielded working ranges of 0.3–20 ng/mL for morphine and amphetamine, 0.1–20 ng/mL for methamphetamine, and 1–20 ng/mL for THC-COOH, with correlation coefficients>0.99 for all compounds. Accuracy and precision results (% recovery 70–120% and %RSD <20%) met acceptance criteria for all substances monitored. Stability results observed that the degradation rates of all substances were ≤30 %, which is consistent with the acceptance criteria. In conclusion, within the scope of this doctoral thesis, the artificial wastewater matrix developed for the first time in Turkey for use in psychoactive substance analyses stands out as an alternative sample that can exhibit matrix effects similar to real wastewater and provide a controlled experimental environment. Thus, in the long term, the aim has been to establish a standardized matrix that enhances the reliability, comparability, and sustainability of studies conducted in the fields of forensic toxicology and public health, while improving accuracy and reliability during method development stages.