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

column

Ascentis Express RP-Amide, 5 cm x 3.0 mm I.D., 2.7 μm particles (53916-U)

mobile phase

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

gradient

0% B for 0.5 min, to 15% B over 0.3 min; up to 35% B in 1.2 min, 1 min equilibration at 0% B

flow rate

2.0 mL/min

pressure

5076 psi (350 bar)

column temp.

35 °C

detector

UV, 210 nm

injection

5 μL

sample

9 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)