HPLC Analysis of Conjugated Estrogens on Discovery^® HS C18

column

Discovery HS C18, 15 cm × 4.6 mm I.D., 5 μm particles (568522-U)

mobile phase

[A] 10 mM potassium phosphate (pH 7.01 unadjusted); [B] acetonitrile; [C] methanol

gradient

80% A, 15% B, 5% C, held for 5 min; to 40% A, 45% B, 15% C in 20 min; held for 5 min

flow rate

1 mL/min

column temp.

30 °C

detector

UV, 220 nm

injection

10 μL

sample

10 μg/mL each in 60/40, water:acetonitrile