Rapid methods to determine procyanidins, anthocyanins, theobromine and caffeine in rat tissues by liquid chromatography-tandem mass spectrometry

Abstract

Rapid, selective and sensitive methods were developed and validated to determine procyanidins, anthocyanins and alkaloids in different biological tissues, such as liver, brain, the aorta vein and adipose tissue. For this purpose, standards of procyanidins (catechin, epicatechin, and dimer B2), anthocyanins (cyanidin- 3-glucoside and malvidin-3-glucoside) and alkaloids (theobromine, caffeine and theophylline) were used. The methods included the extraction of homogenized tissues by off-line liquid–solid extraction, and then solid-phase extraction to analyze alkaloids, or microelution solid-phase extraction plate for the analysis of procyanidins and anthocyanins. The eluted extracts were then analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, using a triple quadrupole as the analyzer. The optimum extraction solution was water/methanol/phosphoric acid 4% (94/4.5/1.5, v/v/v). The extraction recoveries were higher than 81% for all the studied compounds in all the tissues, except the anthocyanins, which were between 50 and 65% in the liver and brain. In order to show the applicability of the developed methods, different rat tissues were analyzed to determine the procyanidins, anthocyanins and alkaloids and their generated metabolites. The rats had previously consumed 1 g of a grape pomace extract (to analyze procyanidins and anthocyanins) or a cocoa extract (to analyze alkaloids) per kilogram of body weight. Different tissues were extracted 4 h after administration of the respective extracts. The analysis of the metabolites revealed a hepatic metabolism of procyanidins. The liver was the tissue which produced a greater accumulation of these metabolites.

This work was supported by the Spanish Ministry of Education and Science financing the project AGL2009-13517-C13-02 and the present work was also supported by the Catalan Government (Interdepartmental Commission for Research and Technological Innovation) through the A. Serra grant.