HPLC Analysis of Caffeine and Metabolites on Ascentis^® RP-Amide

column

Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 μm particles (565324-U)

mobile phase

[A] 0.1% ammonium acetate in water, pH 4.5 with acetic acid; [B] acetonitrile

gradient

2 to 8% B in 3 min; to 20% B in 9 min; held for 2 min; to 2% B in 0.1 min; held for 0.9 min

flow rate

1.0 mL/min

column temp.

30 °C

detector

UV, 275 nm

injection

10 μL

sample

as indicated in 0.1% ammonium acetate in water (pH 4.5 with acetic acid)