UHPLC Analysis of B-vitamins on Titan C18 using UV detection

column

TITAN C18, 50 mm x 2.1 mm I.D., x 1.9 μm particles (577122-U)

mobile phase

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

gradient

0.5% B held for 0.5 minutes, to 30% B in 1.3 minutes, held at 30% B for 1 minute, held at 0.5% B for 0.8 minutes

flow rate

0.5 mL/min

pressure

7977 psi (550 bar)

column temp.

30 °C

detector

UV, 210 nm

injection

0.5 μL

sample

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