Rapid determination of vitamin B2 and B12 in human urine by isocratic liquid chromatography

A simple and rapid method for the identification and quantification of vitamin B2 and B12 in human urine has been developed using reverse phase high performance liquid chromatography (HPLC) and the peaks identity were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). HPLC separation was performed in single wavelength detector (λ365) mode and separated isocratically using mobile phase methanol: 1 mM aqueous TFA (1:4) in C18 column. The calibration graphs plotted with different concentrations of vitamin B2 and B12 was linear with a correlation coefficients (r2) = 0.9975 and 0.9985, respectively. The recoveries of vitamin B2 and B12 were above 87% and 90%, respectively. The results of this present study suggest that the proposed method may be simple and convenient way of identifying and quantifying vitamin B2 and B12 from human urine.