Spectrophotometric and coulometric detection in the highperformance liquidchromatography of flavonoids and optimization of sample treatment for the determination of quercetin in orange juice

The capabilities of spectrophotometric and electrochemical detection techni ques were investigated for the high-performance liquid chromatographic dete rmination of flavonoids. Liquid chromatographic analyses were performed on eleven compounds belonging to three different classes of flavonoids: flavan one glycosides, flavone and flavonol aglycones. Separation of all compounds examined was carried out under reversed-phase conditions on a C-18 narrow- bore column for UV detection, whereas for electrochemical detection, a C-18 standard-bore column was used. UV analyses were carried out at 280 nm for flavanones and at 265 nm for flavones and flavonols, whereas controlled-pot ential coulometric measurements were performed using a porous graphite elec trode. Analytical performances of the methods were compared in terms of lin earity, limits of detection (LODs) and precision. Linearity over two orders of magnitude and LODs at low-ppm levels (0.06-1 mg/l) were demonstrated fo r all techniques considered. Instrumental precision in terms of relative st andard deviation was found to be between 0 and 5% for the liquid chromatogr aphy (LC)-UV system and between 0.6 and 10% for the LC-electrochemical dete ction (ED) system. The methods developed were applied to the analysis of fl avanones and flavonols in a real sample, such as an extract of orange juice . Even though quercetin glycoside is mostly present in orange juice as ruti n, other different glycosides of this flavonol could be present; on this ba sis, the hydrolysis of all glycosides to aglycone allows one to obtain more accurate data on the flavonol concentration in orange juice. To avoid samp le degradation and to increase extraction efficiency, quercetin hydrolysis was optimized using a central composite design to investigate the effects o f acid concentration and hydrolysis time on extraction recovery. (C) 2000 E lsevier Science B.V. All rights reserved.