DETERMINATION OF RIBOFLAVIN,FMN,AND FAD IN HUMAN PLASMA AND ERYTHROCYTES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY / 营养学报

ABSTRACT

Objective: To develop a high performance liquid chromatographic (HPLC) method for determination of riboflavin, FMN, and FAD in human plasma and erythrocytes. Method: Waters 600 model HPLC pump and an Atlantis TM C18 column (150 mm?4.6 mm i.d. 5 ?m) were used. The mobile phase consisted of 35% methanol and 65% 5 mmol/L ammonium acetate solution. The flow rate was 1.0ml/min. The spectro-photofluorimeter was set at wavelength of 450 nm for excitation and 520 nm for emission. Plasma and erythrocyte hemolyzed samples were treated with acetonitrile and chloroform. and the supernatant was analyzed. Results: A good linear correlation existed between riboflavin, FMN and FAD concentration (from 1 to 400 nmol/L) and fluorescence intensity. The detection limits were 2.0 nmol/L, 2.5nmol/L and 2.5nmol/L at a signal to noise ratio of 3, respectively. The intra-assay and inter-assay relative standard deviations were in the range of 1.3% to 3.7%. The recoveries for riboflavin, FMN and FAD in both plasma and erythrocytes were satisfactory. Conclusion:This method for determination of riboflavin,FMN, and FAD in human plasma and erythrocytes is sensitive, rapid and accurate.