UHPLC Analysis of Conjugated Estrogens on Ascentis^® Express C18

column

Ascentis Express C18, 10 cm x 2.1 mm I.D., 2 μm particles (50813-U)

mobile phase

[A] 10 mM potassium phosphate, pH 7.0; [B] acetonitrile; [C] methanol

gradient

90:5:5 (A:B:C) to 75:20:5 (A:B:C) in 4.35 min; to 40:45:15 (A:B:C) in 6.75 min; to 90:5:5 (A:B:C) in 7 min

flow rate

0.6 mL/min

pressure

9500 psi (655 bar)

column temp.

35 °C

detector

UV, 220 nm

injection

2 μL

sample

50 μg/mL each in water:acetonitrile 60:40