High Throughput Solid Phase Extraction and LC-MS/MS Determination of 27 Multiclass Synthetic and Natural Steroidal Hormones and Sterols in Untreated Wastewater Samples Using APCI

Importance of the Topic

Endocrine‐active steroidal hormones released into wastewater represent a growing environmental and public health concern. Even at trace levels, natural and synthetic androgens, estrogens, progestins, and sterols can disrupt aquatic ecosystems and pose risks to human and wildlife health. Effective monitoring of these compounds in complex wastewater matrices requires methods that are both highly sensitive and capable of handling large sample numbers with minimal manual steps.

Goals and Study Overview

This work aimed to develop and validate a streamlined, high‐throughput analytical method for the simultaneous detection of 27 multiclass steroidal hormones and sterols in untreated wastewater. Key objectives included minimizing sample volume, eliminating derivatization, reducing manual extraction steps, and ensuring compliance with regulatory sensitivity requirements.

Methodology and Instrumentation

A 5 mL aliquot of untreated wastewater was acidified and extracted using an Oasis HLB 96‐well SPE plate. Elution employed a mixture of ethyl acetate and n‐hexane to recover nonpolar analytes. Chromatographic separation was achieved on an ACQUITY Premier BEH C18 column (2.1 × 100 mm, 1.7 µm) at 65 °C with a water/methanol gradient containing ammonium fluoride. Detection used a Waters Xevo TQ‐XS tandem quadrupole mass spectrometer with atmospheric pressure chemical ionization (APCI) in positive/negative polarity switching mode. MRM transitions incorporated water‐loss precursor fragments for enhanced specificity.

Used Instrumentation

• UPLC system: ACQUITY UPLC I-Class Plus
• Column: ACQUITY Premier BEH C18, 130 Å, 1.7 µm, 2.1 × 100 mm
• Mass spectrometer: Xevo TQ-XS with APCI source
• Software: MassLynx v4.2 for acquisition; TargetLynx for quantitation

Main Results and Discussion

The method achieved limits of quantification between 0.2 and 40 µg/L across five linearity ranges tailored to environmental concentrations. Chromatographic resolution enabled baseline separation of critical isomers (e.g., 17α- vs. 17β-estradiol). Validation in a single laboratory demonstrated trueness of 74–103% at the lowest QC level, with repeatability and intermediate precision below 13% RSD. The approach maintained stable retention times and minimal carryover, supporting up to 100 samples per day.

Benefits and Practical Applications

• Low sample volume (5 mL) reduces solvent use and simplifies logistics
• No derivatization or liquid–liquid extraction accelerates turnaround
• Automated SPE minimizes manual error and increases throughput
• High sensitivity and specificity support regulatory monitoring and research

Future Trends and Opportunities

Advances may include integration with AI‐driven data analytics for real‐time surveillance, expansion to other micropollutant classes, improved sorbent chemistries for enhanced cleanup, and miniaturized flow systems for field deployments. Combining high‐throughput methods with multi‐omics approaches could further illuminate environmental fate and effect pathways.

Conclusion

The described UPLC–APCI-MS/MS workflow provides a validated, sensitive, and high‐throughput solution for quantifying 27 steroidal hormones in untreated wastewater. By reducing sample volume and streamlining extraction, this method aligns with regulatory guidelines while offering practical utility for routine surveillance programs.

Reference

• González A., Cerdà V. Development of an automatic sequential injection analysis-Lab on valve system exploiting molecularly imprinted polymers coupled with HPLC for estrogens in wastewater. Talanta. 2020.
• González A., et al. Steroid hormones and estrogenic activity in wastewater outfall and receiving waters of the Chascomús lake system. Sci Total Environ. 2020.
• Merlo F., et al. HPLC-MS/MS multiclass determination of steroid hormones in environmental waters after preconcentration on HA-C@Silica. Arabian J Chem. 2020.
• Sapozhnikova Y., et al. Analysis of selected natural and synthetic hormones by LC-MS/MS using EPA Method 1694. Anal Methods. 2011.
• US EPA Method 1698: Steroids and hormones in water, soil, sediment, and biosolids by HRGC/HRMS. 2007.