HPLC Analysis of Water-Soluble B-Vitamins on Ascentis^® Express F5

column

Ascentis Express F5, 5 cm x 3.0 mm I.D., 2.7 μm particles (53576-U)

mobile phase

[A] 20 mM potassium phosphate, dibasic, pH 6.2; [B] acetonitrile

gradient

0% B for 1.5 min, 0-10% over 0.5 min; to 15% B in 1.5 min, 2 min re-equilibration at 0% B

flow rate

1.5 mL/min

pressure

4931 psi (340 bar)

column temp.

35 °C

detector

UV, 210 nm

injection

5 μL

sample

all vitamins in water, 10 μg/mL for nicotinic acid and folic acid, 12 μg/mL for thiamine hydrochloride and cyanocobalamin, (20 μg/mL for pyridoxine, 30 μg/mL for riboflavin, 40 μg/mL for niacineamide, 100 μg/mL for calcium pantothenate, 120 μg/mL for biotin)