HPLC Analysis of Tetracycline Antibiotics on Ascentis^® Express C18

column

Ascentis Express C18, 15 cm x 3.0 mm I.D., 2.7 μm particles (53816-U)

mobile phase

[A] 10 mM oxalic acid in water; [B] 5 mM oxalic acid in methanol:acetonitrile (1:1)

gradient

15% B for 1 min; to 20% B in 0.1 min, held at 20% for 4 min; to 80% B in 5 min; to 15% B in 0.1 min, held at 15% B for 3 min

flow rate

0.5 mL/min

pressure

3597 psi (248 bar)

column temp.

30 °C

detector

UV, 355 nm

injection

50 μL

sample

100 ng/mL of each compound in acetontrile:water:formic acid (5:94.9:0.1)