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Human biomarkers for measuring riboflavin intake and status

Riboflavin status can be assessed by different methods, as seen in the table below.


Table 2: Assessment methods of riboflavin status (4, 5, 7)

 Biomarker Analysis type Sample Benefits Intricacies
 EGRAC Indirect analysis  RBC  Only a marker for B2 deficiency  Does not reflect status, fresh RBS are needed
 FAD Direct analysis  RBC (plasma)  Reflects status (reflects intake)  Does not reflect intake (status)
 Urinary flavin Direct analysis  Urine  Reflects recent intake  Not a marker for low B2 levels
 EPPOA Indirect analysis  Plasma  Suitable for G6PD deficient individuals  Not readily available

EGRAC: erythrocyte glutathione reductase activity coefficient; EPPOA: erythrocyte pyridoxine phosphate oxidase activity FAD: Flavin adenine dinucleotide; RBC: red blood cells


Erythrocyte glutathione reductase activity coefficient (EGRAC)

This assay has been commonly used to determine riboflavin adequacy. The enzyme activity of the erythrocyte glutathione reductase is measured before and after exposure to FAD. The results are expressed as EGRAC; an EGRAC of 1.0 indicates no stimulation by FAD due to more than adequate riboflavin status (4, 7). The IOM suggest the following interpretation of EGRAC: <1.2 is acceptable, 1.2-1.4 is low, >1.4 is deficient. This assay cannot be used for individuals with glucose 6-phosphate dehydrogenase  (G6PD) deficiency due to the resulting increased avidity of EGR towards FAD (4).


Erythrocyte Glutathione Reductase Activity Coefficient (EGRAC), see method in reference (9): Becker et al.  International Journal for Vitamin and Nutrition Research 1991;61:180-7


FAD analysis

Vitamin B2 vitamers (riboflavin, FMN, FAD) can be simultaneously analysed using chromatographic techniques, such as HPLC with fluorescence detection, or LC-MS. Riboflavin in plasma is accepted as general clinical analysis of status and supplementation monitoring (7).


Riboflavin metabolites in plasma by:

HPLC-FLD, see method in reference (4): IOM DRIs for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic acid, Biotin, and Choline. Washington, DC: National Academy Press

LC-MS, see method in reference (10): Meisser Redeuil et al. Journal of Chromatography A 2015.Nov 27;1422:89-98


Urinary vitamin B2

Status can be assessed by analysis of urinary excretion in a random 24h specimen, expressed as either total riboflavin excreted or in relation to creatinine excretion (5). Since little riboflavin is stored in the body, urine measurements are a good proxy for dietary intake (7).


Riboflavin in urine, see method in reference (11): Chen et al. Journal of Chromatography B 2005;820:147-50.


Erythrocyte pyridoxine phosphate oxidase activity (EPPOA)

This assay has been described for plasma samples and appears to be suitable for population with high prevalence of G6PD deficiency (5, 8). However, isolation and purification of the riboflavin-apoprotein prior to analysis is required (8).


Erythrocyte pyridoxine phosphate oxidase activity (EPPOA), see method in reference (8): Kodentsova et al. Annals of Nutrition and Metabolism 1995;39:355-60.

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Technical assistance

For questions on riboflavin methods or for technical assistance, please or write to:

Dr Daniela Hampel, PhD
Project Scientist
USDA/ARS Western Human Nutrition Research Center
Department of Nutrition, University of California Davis

Email: or