Journal of Chromatography B: Biomedical Sciences and Applications
Validated method for the quantitation of quercetin from human plasma using high-performance liquid chromatography with electrochemical detection
Introduction
The flavonoids are a group of polyphenols widely occurring in the plant kingdom. One of the flavonoid subclasses, the flavonols, and particularly quercetin, has received much attention during the past few years. Quercetin has been reported to have many biological activities, such as antioxidant, antithrombotic, anti-inflammatory and anticarcinogenic activities [1]. Epidemiological studies suggest that dietary flavonols, including quercetin, could prevent coronary heart disease [2] and cancer [3]. Quercetin is one of the most abundant flavonoids in the diet with an intake estimated as 2.6–38.2 mg per day [4]. The compound is mainly present as glycosides in foods, with the aglycone form being quite rare. The structures of quercetin aglycone and quercetin glycosides present in onions (spireaoside) and tea (rutin), are shown in Fig. 1.
Although in vitro studies and animal experiments show that quercetin and other flavonoids are biologically active in many respects, it has been difficult to draw any definite conclusions about their relevance to human health. Analytical methods suitable for the measurement of quercetin from human tissues have only been available for a few years. They have mainly been based on HPLC with UV-detection [5], [6], [7], [8], [9], which lacks both the selectivity and sensitivity required, when analyzing low levels of quercetin in plasma. Recently, analytical methods with lower detection limits, based on HPLC with fluorometric [10] or electrochemical [11], [12] detection, have been introduced. Little validation data, however, or data about the stability of quercetin during the different steps of analysis, have been presented. Previous reports [13], [14], [15] and preliminary experiments in our laboratory have shown that quercetin is unstable under various conditions.
Information about the different forms of quercetin present in plasma is scarce. This information is relevant, because conjugation can considerably affect the biological activity of a compound. It has been assumed that quercetin, as many other compounds, is conjugated with glucuronide and/or sulfate groups in the liver [16]. Quercetin could also be present in plasma as glycosides, as proposed by Hollman et al. [17]. In one study [18], large amounts of rutin and other quercetin glycosides were found in plasma of unsupplemented subjects, by using HPLC with diode-array detection, and UV-spectra for identification.
This paper describes a reproducible, sensitive and selective method for the quantitation of trace levels of quercetin aglycone in plasma. The method can be used in supplementation studies and when analyzing quercetin in plasma of unsupplemented subjects. In this report, the method was applied to a small scale study of quercetin pharmacokinetics. We also attempted to assess whether unconjugated quercetin or quercetin glycosides are present in plasma.
Section snippets
Reagents and chemicals
Quercetin, rutin, epicatechin, (+)-catechin, tert.-butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), dithiothreitol (DTT), ubiquinone (Q10), β-glucosidase, β-glucuronidases (type HP-2, B and IX-A) and sulfatase (type V) were obtained from Sigma (St. Louis, MA, USA). Spireaoside was purchased from Extrasynthese (Genay, France). Trolox, (R)-(+)-trolox and (S)-(−)-trolox were obtained from Aldrich Chem. Co. (Milwaukee, WI, USA). Ascorbic acid, oxalic acid,
Chromatography
Representative chromatograms of low-quercetin and high-quercetin plasma extracts are shown in Fig. 2. The retention time of quercetin was 7.8 min. The HPLC column used, Inertsil ODS-3, was superior to the other HPLC columns tested, giving a quercetin peak with least tailing.
The detector was set to +100 mV, although the maximum response for a quercetin standard was achieved at much higher potentials. When the oxidation was done at a potential this low, the method was extremely selective and
Conclusions
This paper describes a validated method for the quantitation of trace levels of quercetin in human plasma. The method has excellent precision, reproducibility and sensitivity. Special attention has been paid to the stability of quercetin during the different steps of analysis. The method can be used when measuring quercetin in plasma of non-supplemented and supplemented subjects, and is therefore suitable for both supplementation studies and biomarker studies.
The method was applied to a small
Acknowledgements
The excellent technical assistance of Eva Kammiovirta is acknowledged. The authors also want to thank the volunteers participating in the study. This work was supported by the Finnish Heart Research Foundation, Juho Vainio Foundation, Yrjö Jahnsson Foundation and the project ‘Nutritional quality and safety of Finnish foods’.
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