Rapid determination of vitamin B₂ (riboflavin) in plasma by HPLC.
BACKGROUND
Riboflavin (vitamin B₂), as the exclusive source for the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in humans, is a water-soluble vitamin critical for metabolism and energy production. In its coenzyme forms, riboflavin is involved in essential oxidation-reduction reactions. Deficiency leads to skin and mucosal disorders. Measurement of plasma riboflavin can be used to assess vitamin B₂ status in at-risk individuals.


METHODS
Proteins are removed from plasma by acid precipitation. An aliquot of the resulting supernatant is analyzed by reversed-phase HPLC. Impurities are separated from riboflavin isocratically and the target material is detected fluorometrically (excitation 450 nm; emission 520 nm).


RESULTS
The method was validated for linearity, limit of quantification, accuracy, precision, and interference. The method was accurate and correlated well (R² = 0.993) to expected concentrations of spiked pooled plasma samples. Imprecision was < 10%. Riboflavin concentrations were determined in samples obtained from self-reported healthy adults who were not taking vitamin supplements. The reference interval established by nonparametric analysis was 6.7-50.1 nmol/l.


CONCLUSIONS
This HPLC method allows separation and measurement of riboflavin in plasma in 7 min. Results from the assay may be used for clinical diagnosis of deficiency and to monitor therapeutic vitamin supplementation regimes.
Petteys
Brian J
BJ
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA.
Frank
Elizabeth L
EL
D000328:Adult; D000368:Aged; D001774:Blood Chemical Analysis / Q000379:methods* / Q000592:standards; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D012016:Reference Values; D012256:Riboflavin / Q000097:blood*; D012997:Solvents / Q000737:chemistry; D013048:Specimen Handling; D013997:Time Factors; D055815:Young Adult
D012997:Solvents; D012256:Riboflavin