RP-HPLC Separation and <sup>1</sup>H NMR Identification of a Yellow Fluorescent Compound—Riboflavin (Vitamin B<sub>2</sub>)—Produced by the Yeast <i>Hyphopichia wangnamkhiaoensis</i>
Raziel Arturo Jiménez-Nava,
Luis Gerardo Zepeda-Vallejo,
Fortunata Santoyo-Tepole,
Griselda Ma. Chávez-Camarillo,
Eliseo Cristiani-Urbina
Affiliations
Raziel Arturo Jiménez-Nava
Departamento de Ingeniería Bioquímica, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Avenida Wilfrido Massieu s/n, Unidad Profesional Adolfo López Mateos, Ciudad de Mexico 07738, Mexico
Luis Gerardo Zepeda-Vallejo
Departamento de Química Orgánica, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Prolongación de Carpio y Plan de Ayala s/n, Colonia Santo Tomás, Ciudad de Mexico 11340, Mexico
Fortunata Santoyo-Tepole
Departamento de Microbiología, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Prolongación de Carpio y Plan de Ayala s/n, Colonia Santo Tomás, Ciudad de Mexico 11340, Mexico
Griselda Ma. Chávez-Camarillo
Departamento de Microbiología, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Prolongación de Carpio y Plan de Ayala s/n, Colonia Santo Tomás, Ciudad de Mexico 11340, Mexico
Eliseo Cristiani-Urbina
Departamento de Ingeniería Bioquímica, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Avenida Wilfrido Massieu s/n, Unidad Profesional Adolfo López Mateos, Ciudad de Mexico 07738, Mexico
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 1H 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 1H NMR spectra revealed methyl (δ 2.30 and 2.40) and aromatic proton (δ 7.79 and 7.77) signals of riboflavin. The 1H 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.
