Determination of phenolic compounds in nectar by solid phase extraction-capillary zone electrophoresis

VERONICA CAROLINA SOTO VARGAS; MARÍA DE LOS ÁNGELES FERNANDEZ; ISMAEL GATICA-HERNÁNDEZ; MARÍA FERNANDA SILVA

Buenos Aires

Simposio; XVIII Simposio Latinoamericano en Aplicaciones de la Electroforesis Capilar y Tecnología del Microchip en Biotecnología, Biomedicina , Biofarmacia e Industria; 2012

Flower attributes such as size, colour, flower organs, nectar volume and composition, and amount of pollen are considered to be important factors in the honeybee attractiveness to flowers. Nevertheless, very little is known about the origin of the metabolites that are commonly found in nectar. Phenolic compounds or polyphenols, are one of the most important groups of compounds occurring in fruits, vegetables, nuts, seeds, and flowers, where they are widely distributed, comprising at least 8000 different known structures. Phenolic substances are quite widespread in nectars. Their accumulation may make the nectar toxic, so that it then becomes repellent to some visitors. Phenolic substances are also relatively common scent compounds of flowers. As well as attracting pollinators or repelling nectar thieves, these scent compounds may have a defensive function. CE is becoming increasingly recognized as an important analytical separation technique due to its speed, efficiency, reproducibility, ultrasmall sample volume requirements, and ease of clearing the contaminants. However, so far, this technique has not been explored for the analysis of nectar. The aims of our work were to develop a method for routine analysis of phenolic compounds in nectar; and to isolate these components in the nectar of different plant species. Phenolic compounds for CZE analysis were extracted from nectar samples (250 µg), which were homogenized in Milli-Q water (1:5, w/v) at pH 2 adjusted with HCl until complete dissolution. The solutions were then extracted using a C18 cartridge, which was previously activated with methanol (5 mL) followed by acidic water (5 mL). The phenolic compounds remain in the column while sugars and other polar compounds elute with the aqueous solvent, resulting in a flavonoid recovery of >95%. The column was rinsed with 1 mL of acidic water (pH 2 with HCl). The whole phenolic fraction was eluted with 500 µL of pure methanol. The separations were undertaken in a 57cm total length, 50cm effective length, 75 mm ID, and 375 mm fused silica capillary. The running buffer was composed of 30mM boric acid; pH 9.5. The separation voltage was 25 kV and the capillary temperature was 25 ◦C. Samples were injected for 5 s. Electropherograms were recorded at 290 nm. Under the optimum conditions, the simultaneous determination of 13 phenolic components (catechin, naringenin, rutin, cinnamic acid, syringic acid, chlorogenic acid, apigenin, vanillic acid, luteolin, quercetin, caffeic acid, and gallic acid) could be well separated. The procedure was successfully used for the analysis and comparison of the phenolic content of nectar samples originating from different flowers.