Título: Medición de Resveratrol, Kaempferol, Quercetina e Isorhamnetina en Plasma Humano.
Título original: Measurement of Resveratrol, Kaempferol, Quercetin and Isorhamnetin in Human Plasma
Autor: Paul Gamache and Ian Acworth Thermo Fisher Scientific, Chelmsford, MA, USA
Tipo de recurso: Nota de Aplicación.
There has been a great deal of interest in the potential health benefits of flavonoids and phenolic compounds present in fruits and vegetables. The most widely studied compounds include catechins, flavonols and phytoestrogens. The biological effects of these compounds, as shown by numerous in vitro and animal studies, suggest that they may be protective against cancer, cardiovascular, inflammatory and other diseases. Several in vitro studies have also suggested pro-oxidant and mutagenic effects. Although there have been great strides in understanding the occurrence, bio-availability and activities of these compounds, relatively little is known of their in vivo effects in humans. Analytical methods that are capable of measuring low levels of these compounds and their metabolites in biological tissues are therefore needed. Several methods based on HPLC with electrochemical detection (ECD) have recently been shown to be useful for these purposes primarily due to its high sensitivity and selectivity for detection of compounds with phenolic substituents. The present study used HPLC with coulometric array electrochemical detection. This technique utilizes multiple sensors that can be optimized for more than one chemical class. Easily oxidized compounds can be selectively detected at upstream, low potential sensors, while higher oxidizing compounds respond at downstream higher potential sensors.
1.0 mL of serum was incubated at 37 °C overnight with 10 μL of 10 μg/mL estriol-3-(β-glucuronide) [internal standard], 0.1 mL of 0.1 M ascorbic acid, 0.25 mL of 1.0 M ammonium acetate buffer, pH 5.0 and 1,000 units of β-glucuronidase from Helix pomatia. After addition of 0.1 mL of glacial acetic acid, hydrolysates were washed with 5.0 mL of hexane and extracted with two 3.0 mL volumes of diethyl ether. Combined extracts were evaporated to near dryness under N2 and the resulting residue dissolved by sonication for 1 min in 0.2 mL of methanol followed by addition of 0.2 mL of water. Samples were then kept at 4 °C for 30 min and centrifuged at 14,000 rpm for 10 min prior to HPLC-ECD
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