Assessment of phenolic compounds and their metabolites in biological samples: comparison of different extraction procedures and identification by HPLC/nanoLC-ESI-TOF (MS)

Over last decade an increasing interest for antioxidants in foods has arisen. The healthy properties of antioxidants related to the prevention of degenerative diseases are the main cause of this boom. An antioxidant prevents the oxidation process, the initial step of development of degenerative diseases, cancer and many others. The study of the bioavailability of some antioxidant compounds, such as phenolic compounds, and the concentration of its metabolites in biological samples is necessary to understand its effects on health. It is important to know which metabolites are present, since they are the compounds that reach the target cell or tissues and their activity could be different from that observed by the polyphenols present in food. The aim of this work is the determination of phenolic compounds in serum and plasma of rats that have been overfed with a lippia citrodora extract. The previous extraction of the phenolic compounds from the samples is a critical step because of the complexity of these biological samples. For this purpose the comparison of different extraction systems were studied for the serum and plasma samples: different solid phase extractions and enzymatic hydrolysis were checked to clean up the samples from proteins ad other substances. To evaluate the recovery of the different extraction systems samples of fetal bovine serum spiked with some phenolic standards were used (naringenin, luteolin, apigenin, rutin, p-coumaric acid, syringic acid and catechin). The analyses of the samples were performed by HPLC-ESI-MS (TOF). The optimum extraction system was used to study the metabolism of the rats that had been overfed with a lippia citrodora extract. These extracts had been described to be very rich in phenylpropanoids, flavonoids and iridoids that present a very high antioxidant and antiinflammatory activity. Plasma from nine Wistar rats were analyzed; 3 of the rats (control samples) had not been fed with the phenolic extract, while the other 6 rats had eaten 1440 mg of extract/ Kg of rat weight right 20 min before taking the sample. The samples were analyzed by HPLC/nanoLC-ESI-MS (TOF). The advantages of nanoLC are its high sensitivity and the low quantity of sample required. This methodology has allowed detecting in the biological samples 11 phenolic compounds that are present in the lippia citrodora extract and also some of its possible metabolites. Notwithstanding, this is the first time that nanoLC has been used to determine phenolic compounds in biological samples

Evaluation of different extraction approaches for the determination of phenolic compounds and their metabolites in plasma by nanoLC-ESI-TOF-MS

Sample preparation is an important step for the determination of phenolic compounds in biological samples. Different extraction methods have been tested to determine phenolic compounds and their metabolites in plasma by nano-liquid chromatography coupled to electrospray ionization-time of flight mass spectrometry (nanoLC-ESI-TOF-MS). The sample treatment optimisation was performed using commercial foetal bovine serum spiked with representative phenolic standards, namely naringenin, luteolin, verbascoside, apigenin, rutin, syringic acid and catechin. Different protein-precipitation conditions were evaluated as well as enzymatic digestion with trypsin and solid-phase extraction using different phases such as C-18, ABN and ENV+, working at different pH values. The optimum extraction procedure consisted of a previous protein-precipitation step using HCl 200 mmol/L in methanol for 2.5 h at 50\ubaC followed by a solid-phase extraction using C-18 cartridges at pH 2.5. This procedure was finally applied to the plasma of rats overfed with a phenolic-rich Lippia citriodora extract. These samples were analysed by nanoLC-ESI-TOF-MS, enabling the identification of five compounds previously found in the administered Lippia citriodora extract and one metabolite