Optimizing separation conditions for riboflavin, flavin mononucleotide and flavin adenine dinucleotide in capillary zone electrophoresis with laser-induced fluorescence detection.
A method was developed for the quantitative determination of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), using free solution capillary zone electrophoresis in uncoated fused-silica capillaries with laser-induced fluorescence (LIF) detection. Various factors influencing the separation and detection of flavin vitamers were investigated, including pH (5.5-10.5), concentration and nature of the run buffer (phosphate, borate and carbonate), applied voltage (15-30 kV), temperature (15-30 degrees C) and injection time. Optimal resolution and detection were obtained with a pH 9.8, 30 mM aqueous phosphate buffer at 15 degrees C and 30 kV of applied voltage. LIF detection was obtained with a He-Cd laser source using an excitation wavelength at 442 nm and lambda(em) > or = 515 nm. Riboflavin could be determined in the concentration ranges 0.5-350 microg/l with a rather low detection limit (LOD) down to 50 amol. The LODs of FAD and FMN were slightly higher, 300 and 350 amol, respectively. Combined with a simple clean-up procedure, the practical utility of this method is illustrated by the measurements of flavin derivates in foods and beverages, such as wines, milk, yoghurt and raw eggs.
Catald
Tommaso R I
TR
Dipartimento di Chimica, Università degli Studi della Basilicata, Potenza, Italy. cataldi@unibas.it
Nardiello
Donatella
D
De Benedetto
Giuseppe E
GE
Bufo
Sabino A
SA
D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods*; D005486:Flavin Mononucleotide / Q000302:isolation & purification*; D005182:Flavin-Adenine Dinucleotide / Q000302:isolation & purification*; D005504:Food Analysis; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D009994:Osmolar Concentration; D012015:Reference Standards; D012256:Riboflavin / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D002021:Buffers; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D012256:Riboflavin