HPLC Analysis of B-vitamins on Ascentis^® Express RP-Amide using UV detection

column

Ascentis Express RP-Amide, 10 cm x 3.0 mm I.D., 2.7 μm particles (53918-U)

mobile phase

[A] 20 mM potassium phosphate, pH 3; [B] methanol;

gradient

0.5% B held for 1.5 minutes, to 25% B in 1.0 minutes, held at 25% B for 1.7 minute, held at 0.5% B for 1.2 minutes

flow rate

0.75 mL/min

pressure

5584 psi (385 bar)

column temp.

30 °C

detector

UV, 210 nm

injection

0.5 μL

sample

100 μg/mL of all vitamins except 250 μg/mL of calcium pantothenate, biotin and riboflavin in water