journal article·2025-09-12Open access

Advancing Wastewater Surveillance: Development of High-Throughput Green Robotic SPE-UPLC-MS/MS Workflow for Monitoring of 27 Steroids and Hormones

Bhaskar Karubothula, Chaitanya Devireddy, Dnyaneshwar Shinde, Rizwan Shukoor, Ghenwa Hafez, Raghu Tadala, Samara Bin Salem, Wael Elamin, Grzegorz Brudecki

Applied Sciences· Volume 15· Issue 18

Abstract

Conventional methods for testing steroids and hormones (SHs) in environmental samples are exhaustive, complex, and score poorly in sustainability matrices. Therefore, this study evaluates the automated sample preparation approach using the modular Biomek i7 Workstation for the analysis of 27 SHs in wastewater. Method development involved optimizing Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS) parameters, preparing wastewater matrix blank, and assessing extraction efficiency using three solid phase extraction (SPE) cartridges. Extraction efficiency trials showed suitability in the order of Hydrophilic–Lipophilic Balance (HLB) > Mixed-Mode Cation Exchange (MCX) > Mixed-Mode Anion Exchange (MAX). The method demonstrated specificity for all targeted SHs, with Cholesterol showing a maximum interfering peak of 17.71% of the quantification limit (LOQ). The method met matrix effect tolerance of ±20% for 26 SHs, while Epi Coprostanol (34.92%) showed signal enhancement >20%. The 8-point calibration curve plotted using automated extraction demonstrated acceptable linearity across the tested range. Spiked studies at low (LQC), middle (MQC), and higher (HQC) quality control (QC) levels (n = 6, repeated on three separate occasions) demonstrated % RSD values within 20% and recoveries ranging from 71.54% to 115.00%. The method met validation criteria, showing reliability in Intra-Laboratory Comparison (ILC) and Blind Testing (BT). The method outperformed the conventional approach in greenness assessment (Complex Modified Green Analytical Procedure Index) and practicality evaluation (Blue Applicability Grade Index), offering an effective and sustainable protocol for environmental testing laboratories.
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Research context

OpenAlex topics: Pharmaceutical and Antibiotic Environmental Impacts · Effects and risks of endocrine disrupting chemicals · Analytical chemistry methods development

SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.

Affiliations

RASID Laboratory of Abu Dhabi Quality & Conformity Council (ADQCC) & M42 Environmental Sciences, Abu Dhabi P.O. Box 853, United Arab Emirates

Abu Dhabi Quality and Conformity Council, Abu Dhabi P.O. Box 853, United Arab Emirates

References (40)

Show source-provided references
1. The Environmental and Health Impacts of Steroids and Hormones in Wastewater Effluent, as Well as Existing Removal Technologies: A Review10.3390/ecologies3020016 ↗
2. Determination of Selected Steroid Compounds in Sediment Samples from Golden Horn Estuary (the Sea of Marmara, Turkey) Using LC-ESI/MS-MS
3. Steroid Hormones in Wastewater: Sources, Treatments, Environmental Risks, and Regulations10.1016/j.emcon.2023.100210 ↗
4. Determination of Estrogens and Progestogens by Mass Spectrometric Techniques (GC/MS, LC/MS and LC/MS/MS)10.1002/jms.529 ↗
5. Analysis of Steroid Hormones and Their Conjugated Forms in Water and Urine by On-Line Solid-Phase Extraction Coupled to Liquid Chromatography Tandem Mass Spectrometry10.1186/s13065-016-0174-z ↗
6. Assessment of Automated Off-Line Solid-Phase Extraction LC-MS/MS to Monitor EPA Priority Endocrine Disruptors in Tap Water, Surface Water, and Wastewater10.1016/j.talanta.2022.123216 ↗
7. Current Knowledge on the Environmental Fate, Potential Impact, and Management of Growth-Promoting Steroids Used in the US Beef Cattle Industry10.2489/jswc.68.4.325 ↗
8. Occurrence and Fate of Androgens, Estrogens, Glucocorticoids and Progestagens in Two Different Types of Municipal Wastewater Treatment Plants10.1039/C1EM10783F ↗
9. Validation of a Quantitative Assay Using GC/MS for Trace Determination of Free and Conjugated Estrogens in Environmental Water Samples10.1002/jssc.200390000 ↗
10. Treatment Strategies for Enhancing the Removal of Endocrine-Disrupting Chemicals in Water and Wastewater Systems10.1016/j.jwpe.2021.102017 ↗
11. An Overview of the Glucocorticoids’ Pathways in the Environment and Their Removal Using Conventional Wastewater Treatment Systems10.3390/pollutants1030012 ↗
12. Simultaneous Determination of Corticosteroids, Androgens, and Progesterone in River Water by Liquid Chromatography–Tandem Mass Spectrometry10.1016/j.chemosphere.2009.12.025 ↗
13. Persistence and Impact of Steroidal Estrogens on the Environment and Their Laccase-Assisted Removal10.1016/j.scitotenv.2019.07.025 ↗
14. Steroid Hormones in the Aquatic Environment10.1016/j.scitotenv.2021.148306 ↗
15. Clinical Use of Androgens10.1146/annurev.me.35.020184.001231 ↗
16. Rocha, M., and Rocha, E. (2022). Synthetic Progestins in Waste and Surface Waters: Concentrations, Impacts and Ecological Risk. Toxics, 10.10.3390/toxics10040163 ↗
17. Analysis and Distribution of Estrogens and Progestogens in Sewage Sludge, Soils and Sediments10.1016/j.trac.2004.08.007 ↗
18. U.S. Environmental Protection Agency (2024, December 19). Contaminant Candidate List 4-CCL, Available online: https://www.epa.gov/ccl/contaminant-candidate-list-4-ccl-4-0.
19. Trace Analysis of 28 Steroids in Surface Water, Wastewater and Sludge Samples by Rapid Resolution Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry10.1016/j.chroma.2011.01.014 ↗
20. Analytical Methods for Detection of Selected Estrogenic Compounds in Aqueous Mixtures10.1021/es981294f ↗
21. Analysis of Estrogenic Hormones in Municipal Wastewater Effluent and Surface Water Using En-zyme-Linked Immunosorbent Assay and Gas Chromatography/Tandem Mass Spectrometry10.1002/etc.5620200114 ↗
22. Determination of Steroid Estrogens in Wastewater by Immunoaffinity Extraction Coupled with HPLC−Electrospray-MS10.1021/ac010327y ↗
23. Detection and Distribution of Endogenous Steroids in Human Stratum Corneum10.1016/j.dsi.2013.07.003 ↗
24. (2024, December 19). U.S. EPA Method 1698: Steroids and Hormones in Water, Soil, Sediment, and Biosolids by HRGC/HRMS, Available online: https://www.epa.gov/sites/default/files/2015-10/documents/method_1698_2007.pdf.
25. Enhancement of Chemical Derivatization of Steroids by Gas Chromatography/Mass Spectrometry (GC/MS)10.1016/j.jchromb.2009.08.005 ↗
26. LC-MS/MS Determination of Potential Endocrine Disruptors of Cortico Signalling in Rivers and Wastewaters10.1007/s00216-014-8206-9 ↗
27. Analysis of Selected Natural and Synthetic Hormones by LC-MS-MS Using the US EPA Method 169410.1039/c0ay00748j ↗
28. Determination of 17β-Estradiol and 17α-Ethinylestradiol in Water at Sub-Ppt Levels by Liquid Chromatography Coupled to Tandem Mass Spectrometry10.1039/c4ay00479e ↗
29. High-Sensitivity Analysis of Female-Steroid Hormones in Environmental Samples10.1016/j.trac.2011.11.008 ↗
30. (2024, December 19). SANTE 11312/2021v2. Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed, EURL for Residues of Pesticides. Available online: http://food.ec.europa.eu/system/files/2023-11/pesticides_mrl_guidelines_wrkdoc_2021-11312.pdf.
31. Joint Committee for Guides in Metrology (2008). Evaluation of Measurement Data Guide to the Expression of Uncertainty in Measurement. JCGM, 100, 1–116.
32. Ellison, S.L.R., and Williams, A. (2012). EURACHEM/CITAC Guid: Quantifying Uncertainty in Analytical Measurement, EURACHEM. [3rd ed.]. Available online: https://www.eurachem.org/index.php/publications/guides/quam.
33. Introduction of a Small Volume Ethyl Acetate Based Liquid-Liquid Extraction Procedure for Analysis of Polycyclic Aromatic Hydrocarbons in Wastewater by Atmospheric Pressure Gas Chromatography-Mass Spectrometry and Evaluation of Method Greenness10.1016/j.chroma.2024.465563 ↗
34. (2017). International Organization for Standardization, 2017 General Requirements for the Competence of Testing and Calibration Laboratories (Standard No. ISO/IEC 17025:2017). Available online: https://www.iso.org/standard/66912.html.
35. A Total Scoring System and Software for Complex Modified GAPI (Com-plexMoGAPI) Application in the Assessment of Method Greenness10.1016/j.greeac.2024.100126 ↗
36. Blue Applicability Grade Index (BAGI) and Software: A New Tool for the Evaluation of Method Practicality10.1039/D3GC02347H ↗
37. Analytical Key Issues and Challenges in the LC-MS/MS Determination of Antibiotics in Wastewater10.1016/j.aca.2022.340739 ↗
38. Determination of Sterols and Steroid Hormones in Surface Water and Wastewater Using Liquid Chromatography-Atmospheric Pressure Chemical Ionization-Mass Spectrometry10.1016/j.microc.2017.07.011 ↗
39. (2022). Statistical Methods for Use in Proficiency Testing by Interlaboratory Comparison (Standard No. ISO 13528:2022). Available online: https://www.iso.org/standard/78879.html.
40. United Nations (2015). Transforming Our World: The 2030 Agenda for Sustainable Development, United Nations. Available online: https://sdgs.un.org/publications/transforming-our-world-2030-agenda-sustainable-development-17981.