Riboflavin

Riboflavin is vitamin B2. Vitamins are naturally occurring

substances necessary for many processes in the body.

Riboflavin is important in the maintenance of many tissues

of the body.

Riboflavin is used to treat or prevent deficiencies of

riboflavin.a
https://www.drugs.com/mtm/riboflavin.html
 Analysis of Vitamin B2

Liquid Chromatographic Analysis of Riboflavin Vitamers in

Foods Using Fluorescence Detection

Analysis of Ribo avin and Ribo avin Cofactor Levels in Plasma by

High-Performance Liquid Chromatography
 Liquid Chromatographic
using fluorescence detection
sample extraction
Weigh 12 g sample into extraction tube.

Add 10 mL acetonitrile and homogenize with the vibratory stirrer, 10 min.

Add 10 mL of 10 mM phosphate buffer, pH 5.0.

Centrifuge at 6000 rpm, 5 min.

Dilute supernatant to 25 mL with the phosphate buffer.

Filter through 0.45 m filter, inject.

 Chromatography
Column 15 cm 4.6 mm

Stationary phase Discovery RP-Amide C16, 5 m (end-capped with TMS)

Mobile phase MeCN:10 mM KH2PO4 (10:90), pH 5.0

Flow rate 1 mL min-1

Column temperature Ambient

Detection Fluorescence, Ex = 270, Em = 516

Calculation Standard addition, peak area

 Analysis of Ribo avin and Ribo avin Cofactor
Levels in Plasma by High-Performance Liquid
Plasma Extraction
Chromatography

To 1 mL plasma, add galactoflavin (IS) to final concentration of

206 nM.

Add 1 mL 15 mM magnesium acetate, incubate at 65C, 15 min.

Add 0.5 mL TCA.

Centrifuge, 3200 g, 15 min, 4C.

Rinse pellet with 1 mL magnesium acetate, combine supernatants.

Add supernatants onto a C18 Sep-Pack catridge (conditioned)

with 2 mL methanol and 2mL of a 10 mM KH PO and 15 mM magnesium acetate solution, pH 3.4.

2 4

Elute flavin with 2 mL of the KH PO magnesium acetate solution.

2 4
 Chromatography
column 25cm x 4mm

stationary phase C18

mobile phase 15% MeCN in the 10mM KH2PO4-15 mMmagnesium acetate solution

flow rate 1 mL min1

column temperature ambient

detection fluorescence, Ex = 445, Em = 530

calculation Internal standard, galactoflavin

Vitamin Analysis for the Health and Food Sciences 2nd Edition, page 353-354