Simultaneous determination of 61 hormones in water by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry

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 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