Concerns over the emergence of steroid hormones as pollutants in water have grown. Steroid hormone compounds present challenges in the simultaneous detection of total residual hormones owing to their analogous structures and diverse types. In this study, we established a rapid and high-throughput continuous online method based on solid phase extraction ( SPE ) coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) ) for the simultaneous determination of 61 hormone components, including 48 glucocorticoids, 1 mineralocorticoid, 4 androgens, and 8 progesterones, in water. Various SPE columns were investigated to assess their extraction efficiency for enriching and purifying target compounds in a large sample volume (1 L). ). An HC-C18 18 SPE column was selected because of its superior performance. Acetonitrile was used as a washing solution during SPE to ensure that the majority of the tested substances achieved recoveries exceeding 70% and effectively avoid interferences from water-soluble components. Various C8 and C18 columns were tested, and the optimal HPLC conditions for hormone retention were established. We systematically evaluated different UPLC columns and mobile phases, including methanol-water and acetonitrile-water systems with 0. 1% formic acid added to the aqueous phase. The optimized UPLC conditions were as follows: : BEH C 18 column (100 mm x2. 1 mm, 1.7 mu m); column temperature, 40 degree celsius; flow rate, 0.3 mL/ min; injection volume, 5 mu L; mobile phase A: : 0. 1% formic acid aqueous phase; mobile phase B: : acetonitrile. Gradient elution was performed as follows: : 0-0.5 min, 30% B; 0.5-15.0 min, 30% B-75% B; 15.0-18.0 min, 75% B-98% B; 18.0-19.0 min, 98% B; 19.0-19. 1 min, 98% B-30% B; 19.1-20.0 min, 30% B. Both positiveand negative-ion modes were explored in the UPLC-MS/ MS experiment to obtain the full scan of the parent ions, and positive mode was finally selected for electrospray ionization ( ESI ). Two product ions exhibiting strong signals and minimal interference were selected for quantitative and qualitative ion analyses, using an external standard method for quantification. MS/MS was performed in positive-ion ( ESI+ + ) mode with multiple reaction monitoring ( MRM) scanning. The MS/MS parameters were as follows: : atomizing gas pressure, 379 kPa; curtain air pressure, 241 kPa; spray voltage, 5 500 V; desolvation temperature, 550 degree celsius; collision exit voltage ( CXP ), 13 V; intake voltage (EP), 10 V; and residence time of each ion pair, 0. 5 ms. Other instrument settings, such as the collision energy and declustering voltage, were also optimized. The 61 hormones exhibited excellent linear relationships within their corresponding concentration ranges, with correlation coefficients greater than 0. 99. The method detection limits ( MDLs ) were in the range of 0. 05- 1. 50 ng/ L. The average recoveries of the 61 hormones across three spiked levels ranged from 62. 3% to 125. 2%, with relative standard deviations (RSDs, n=6) of 1.1%-10. 5%. Using this method, we successfully detected 10 hormone components (cortisone, fluticasone propionate, ciclesonide, betamethasone dipropionate, clobetasone butyrate, diflucortolone valerate, halobetasol propionate, isoflupredone, difluprednate, and hydroxyprogesterone caproate ) in various surface water and groundwater samples collected from the Taihu Basin region. The SPE-UPLC-MS/ MS method presented in this paper is simple, highly sensitivity, and exceptionally accurate. Thus, it exhibits promising potential for tracing targeted hormone residues in water and will be of great value in monitoring and ensuring water safety. Finally, a regional analysis was conducted on the hormone levels in water, and suggestions were made for the targeted treatment of hormone residues in future sewage treatment processes.
