Separation of both glucuronide and sulfate steroids was successfully achieved on BEH and BEH 2-EP columns, but with relatively long run time (approximately 40 min; data not shown). Interestingly, the polar BEH phase mainly promoted interactions with the conjugated moiety and therefore provided class separation of conjugates (sulfated first, followed by glucuronated), whereas BEH 2-EP SP mainly interacted with the steroid base regardless of the conjugated form. However, the objective of this study being to provide high-throughput analysis of both types of compound, it seemed more relevant to consider them separately.¹

Sulfate-steroid separation was achieved on an Acquity UPC² BEH 2-EP. A mixture of MeOH–H₂O–ammonium formate (95:5:30, v/v/mmol L⁻¹) (B) was used as co-solvent with CO₂ (A), in gradient mode (A:B): 95:5 during 0–1 min, 82:18 at 4 min, 80:20 at 6 min, 78:22 at 7 min, 77:23 at 8.5 min, 70:30 during 10.5–13 min, and 95:5 during 13.1–15 min. The back pressure was set at 130 bar. The flow was 2 mL min−1, the column temperature was 50 °C, and the injection volume was 2 μL. Glucuronide steroids were separated on an Acquity UPC2 BEH. A mixture of MeOH–H₂O–ammonium formate (95:5:30, v/v/mmol L⁻¹) (B) was used as co-solvent with CO₂ (A), in gradient mode (A:B): 95:5 during 0–1 min, 72:28 during 4–7 min, 70:30 during 7.5–9.5 min, 95:5 during 9.6–12 min. The back pressure was 130 bar. The flow was 2 mL min⁻¹, the column temperature was 40⁰C, and the injection volume was 2 μL.