Development and Validation of Methods for the Determination of Anthocyanins in Physiological Fluids via UHPLC-MSn

Abstract

Recent in vitro and in vivo studies on anthocyanins confirmed numerous health-promoting effects in humans. Daily anthocyanin intake can be estimated via food databases, but the amount absorbed by the organism still remains uncertain because anthocyanin bioavailability is yet to be elucidated in its entirety. For this purpose, suitable and validated methods of sample preparation and analysis are required. Therefore, a sample preparation method for anthocyanin metabolite analysis in plasma was successfully established and validated. The validation yielded acceptable results for the anthocyanins in terms of recovery (54-108%) and precision (coefficient of variation (CV) < 15%). The UHPLC-MS method used in the consecutive reaction monitoring (CRM) mode was sufficiently sensitive, resulting in limits of detection <2.3 ng/mL and limits of quantification < 8.1 ng/mL with associated repeatability of the MS system with CVs of <5.1%. In addition, a method for the sum parameter determination of anthocyanidins in urine comprising solely the evaporation of acidified samples was developed, validated, and successfully applied to real samples. The results showed that this method is applicable for the methylated anthocyanidins, but not for the hydroxylated anthocyanidins, due to the chosen CRM modes required for optimum selectivity.Molecules 2020, 25, 518 2 of 13 on visual health were discussed [7,8]. However, the particular mode of action and the effective dose are yet to be clarified regarding these health-promoting effects [3,4]. At least some effects, like the antioxidative and anti-inflammatory properties, are structure-dependent, specifically the substitution pattern of the anthocyanin B-ring. The solely hydroxylated delphinidin (Dp) and cyanidin (Cy) derivatives are, amongst others, effective in suppressing inflammatory peritonitis by inhibiting the expression of cyclooxygenase-2, whereas methoxylated anthocyanins like peonidin (Pn), petunidin (Pt), and malvidin (Mn) derivatives showed no effect on cyclooxygenase expression [4]. Since the availability of human target tissues is very limited, the determination of the concentration in the organism and bioavailability in total is primarily based on the measurements of metabolites in plasma or urine [2]. Recently, the analysis of low-molecular-weight phenolic acids, produced by the action of the colonic microbiota, gained importance; thus, feces samples were collected during several human studies and analyzed [9,10], as these products appeared to be the main metabolites of the anthocyanins occurring in the human organism [11,12]. Although these phenolic acids cause some beneficial effects on human health, the majority of the proven and discussed health-promoting effects are directly linked to anthocyanins and their phase II metabolites [3,4]. As mentioned before, both the concentration and the structure of the anthocyanins and their metabolites present in the organism are crucial for the understanding of their physiological effects [13].The anthocyanidin structure is pH-depend...

State of the Art of Anthocyanins: Antioxidant Activity, Sources, Bioavailability, and Therapeutic Effect in Human Health

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Evaluation of Poorly Soluble Drugs’ Dissolution Rate by Laser Scattering in Different Water Isotopologues

“…The repeatability of the technique is represented by the values of standard deviation, coefficient of variation, and confidence interval ( Table 2 ) [ 50 , 51 ].…”

“…The repeatability of the technique is represented by the values of standard deviation, coefficient of variation, and confidence interval (Table 2) [50,51]. Based on the assessment of the repeatability parameter, the intermediate precision studies were carried out.…”

Chemical Hemisynthesis of Sulfated Cyanidin-3-O-Glucoside and Cyanidin Metabolites

“…Samples were prepared according to a previously published protocol [ 26 ]. Briefly, 5 mL of a urine sample was evaporated at 40 °C under nitrogen flow to a final volume of 500–700 µL.…”