In-Sewer Stability Assessment of Anabolic Steroids and Selective Androgen Receptor Modulators

Abstract

Wastewater-based epidemiology is a potential complementary technique for monitoring the use of performanceand image-enhancing drugs (PIEDs), such as anabolic steroids and selective androgen receptor modulators (SARMs), within the general population. Assessing in-sewer transformation and degradation is critical for understanding uncertainties associated with wastewater analysis. An electrospray ionization liquid chromatography mass spectrometry method for the quantification of 59 anabolic agents in wastewater influent was developed. Limits of detection and limits of quantification ranged from 0.004 to 1.56 μg/L and 0.01 to 4.75 μg/L, respectively. Method performance was acceptable for linearity (R 2 > 0.995, few exceptions), accuracy (68−119%), and precision (1−21%RSD), and applicability was successfully demonstrated. To assess the stability of the selected biomarkers in wastewater, we used laboratory-scale sewer reactors to subject the anabolic agents to simulated realistic sewer environments for 12 h. Anabolic agents, including parent compounds and metabolites, were spiked into freshly collected wastewater that was then fed into three sewer reactor types: control sewer (no biofilm), gravity sewer (aerobic conditions), and rising main sewer (anaerobic conditions). Our results revealed that while most glucuronide conjugates were completely transformed following 12 h in the sewer reactors, 50% of the investigated biomarkers had half-lives longer than 4 h (mean residence time) under gravity sewer conditions. Most anabolic agents were likely subject to biofilm sorption and desorption. These novel results lay the groundwork for any future wastewater-based epidemiology research involving anabolic steroids and SARMs.

In-Sample Stability of 52 Performance- and Image-Enhancing Drug Biomarkers in Wastewater

“…This is consistent with initial cardarine and stenabolic concentrations found in spiked raw wastewater in our previous study. 10 In that study, cardarine losses through filtration and methanol content were observed, with no significant losses of the other biomarkers observed. 3′-Hydroxy stanozolol had the highest concentrations at t 0 (13− 13.8 μg/L) in unpreserved and Na 2 S 2 O 5 preserved wastewater (likely due to rapid deconjugation of its respective glucuronide) but had values close to the spiking level in HClpreserved wastewater (8.5−9.5 μg/L).…”

“…After vortexing, the vials were centrifuged at 4,750 rpm for 30 min at For statistical purposes, where at least one of the triplicate measurements was above the limit of detection (LOD), concentrations of the other measurements below the LOD and the limit of quantification (LOQ) were replaced with LOD/sqrt(2) and (LOD + LOQ)/2, respectively. 10 When all triplicate values were below the LOD, the concentration was set to 0.…”

“…After vortexing, the vials were centrifuged at 4,750 rpm for 30 min at 4 °C before they were analyzed via direct injection using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were analyzed by LC-MS/MS (Sciex QTRAP 6500+) as described elsewhere . Briefly, the aqueous mobile phase was 0.4 mM ammonium fluoride (NH 4 F) in 95:5 (v/v) ultrapure water/methanol and the organic mobile phase was 0.4 mM NH 4 F in 95:5 methanol/ultrapure water (v/v).…”

“…The aim of this study was to address that research gap and investigate the in-sample stability of 52 PIED biomarkers previously determined to be relevant for PIED use monitoring, many of which are novel within the field of wastewater analysis . This was achieved by determining (a) the stability of target PIEDs in influent wastewater at different temperatures and using different preservative methods, (b) the duration for which the PIEDs are stable under the investigated conditions, and (c) the best storage or archiving conditions for the investigated biomarkers.…”

“…The aim of this study was to address that research gap and investigate the in-sample stability of 52 PIED biomarkers previously determined to be relevant for PIED use monitoring, many of which are novel within the field of wastewater analysis. 10 This was achieved by determining (a) the stability of target PIEDs in influent wastewater at different temperatures and using different preservative methods, (b) the duration for which the PIEDs are stable under the investigated conditions, and (c) the best storage or archiving conditions for the investigated biomarkers. This will inform WBE researchers if the analyte concentrations found during analysis are representative of the concentrations at the time of collection, which archived samples can be targeted for analysis, and how to best preserve and store wastewater influent samples for future measurement of these biomarkers.…”