RP-HPLC Separation and (1)H NMR Identification of a Yellow Fluorescent Compound—Riboflavin (Vitamin B(2))—Produced by the Yeast Hyphopichia wangnamkhiaoensis

The yeast Hyphopichia wangnamkhiaoensis excretes a brilliant yellow fluorescent compound into its growth culture. In this study, we isolated and identified this compound using reverse-phase high-performance liquid chromatography-diode array detector (RP-HPLC-DAD) as well as (1)H NMR and UV–Vis spectroscopy. Two of the three RP-HPLC-DAD methods used successfully separated the fluorescent compound and involved (1) a double separation step with isocratic flow elution, first on a C18 column and later on a cyano column, and (2) a separation with a linear gradient elution on a phenyl column. The wavelengths of maximum absorption of the fluorescent compound-containing HPLC fractions (~224, 268, 372, and 446 nm) are in good agreement with those exhibited by flavins. The (1)H NMR spectra revealed methyl (δ 2.30 and 2.40) and aromatic proton (δ 7.79 and 7.77) signals of riboflavin. The (1)H NMR spectra of the samples spiked with riboflavin confirmed that the brilliant yellow fluorescent compound is riboflavin. The maximum excitation and emission wavelengths of the fluorescent compound were 448 and 528 nm, respectively, which are identical to those of riboflavin.