Polar Phenol Detection in Plasma and Serum: Insights on Sample Pre-Treatment for LC/MS Analysis and Application on the Serum of Corinthian Currant-Fed Rats

Abstract

Analysis of plasma and serum provides valuable information on the amounts of polar phenols' circulating after ingestion. In the present study, protein precipitation (PPT), liquid-liquid extraction (LLE), solid phase extraction (SPE), enzymatic hydrolysis and their combinations were meticulously evaluated for the extraction of a variety of polar phenolic moieties from plasma and serum. The recovery values of the above methods were compared; satisfactory recoveries (>60%) were attained for most analytes. Polar phenol aglycones undergo degradation with enzymatic hydrolysis; however, their extended phase II metabolism makes enzymatic hydrolysis a mandated process for their analysis in such biofluids. Hence, enzymatic hydrolysis followed by LLE was used for the identification of polar phenols in rats' serum, after the long-term oral consumption of Corinthian Currant. Corinthian Currant is a Greek dried vine product rich in bioactive polar phenolics. Flavonoids and phenolic acids, detected as aglycones, ranged from 0.57 ± 0.08 to 181.66 ± 48.95 and 3.45 ± 1.20 to 897.81 ± 173.96 ng/mL, respectively. The majority of polar phenolics were present as phase II metabolites, representing their fasting state in the blood stream. This is the first study evaluating the presence of polar phenolics in the serum of rats following a long-term diet supplemented with Corinthian Currant as a whole food.

Keywords: Corinthian Currant; LC-MS; Vitis vinifera; plasma; polar phenolics; rat; serum.

Figure 1

Extracted ion chromatograms (XICs) of polar phenolic compounds detected in rat serum from (a) the control group, (b) the control group that underwent enzymatic hydrolysis, (c) the Corinthian Currant supplemented group and (d) the Corinthian Currant supplemented group that underwent enzymatic hydrolysis. Peaks: (1) daidzein, (2) chrysin, (3) formononetin, (4) apigenin, (5) genistein, (6) hesperetin, (7) naringenin, (8) luteolin, (9) kaempferol, (10) isorhamnetin, (11) quercetin, (12) trans-cinnamic acid, (13) vanillic acid and (14) syringic acid.