Chromatography of metabolites in plasma and urine following oral administration of anthocyanin rich capsules

Master's thesis in Biological chemistryAnthocyanins (ACs) are powerful antioxidants widely distributed in fruits and vegetables. Several international studies suggest that anthocyanins have positive effects on the health, including various chronic diseases. To be able to study the possible anthocyanin mechanisms in the human body, it is of importance to know their metabolism. MEDOX® is an AC rich product made from bilberries and blackcurrant and consists of 17 different anthocyanins with delphinidin-3-O-β-glucopyranoside and cyanidin-3-O-β-glucopyranoside as the main constituents. There are only 5 different aglycone structures of these 17 anthocyanins; delphinidin, cyanidin, peonidin, petunidin and malvidin. An extensive metabolism of ACs is indicated following oral administration of this supplement. Free and conjugated benzoic acids (BAs) with functional groups corresponding to the anthocyanin B-ring structure have been recognized as metabolites in urine and plasma. Correspondingly, gallic acid, protochatechuic acids, vanillic acid, syringic acid and 3, 4-dihydroxy-5-methoxybenzoic acid have been suggested as metabolites of delphinidin, cyanidin, peonidin, malvidin and petunidin respectively. Hydroxy benzoic acids are relatively polar compounds, and derivatization is therefore usually necessary prior to gas chromatography mass spectrometry (GCMS) analysis. The objective of this thesis was to develop a robust analytical method for determination of these BAs in addition to 4-hydroxybenzoic acid which is a suggested metabolite of the AC pelargonidine. Liquid-liquid extraction (LLE) was chosen as the best sample extraction method as compared to solid phase extraction (SPE) and solid phase analytical derivatization (SPAD) with respect to recovery, reproducibility and sample purity. LLE was also less time consuming than SPE. The recoveries of BAs in urine ranged from 62 – 121 %, and recoveries in plasma from 37 – 158 %. All BAs were identified and quantified in urine and plasma after oral administration of MEDOX® in increased levels compared to before intake. All metabolites were detected as both free BAs and in conjugated form linked with glucuronic acid, in both urine and plasma. Results obtained from the work of this thesis suggest that the ACs in this product metabolize to their corresponding BAs