Quantitation of trans-resveratrol and detection of its metabolites in human plasma and urine by high performance liquid chromatography
Introduction
In recent years there has been a growing interest in trans-resveratrol (3,5,4′-trihydroxystilbene, referred to in this document as resveratrol), a phytochemical occurring naturally in high to moderate quantities in various foods including grapes [1], peanuts [2] and wine [3]. Resveratrol has antioxidant properties, and as a constituent of red wine it has been implicated in the “French Paradox” that the incidence of coronary heart disease is relatively low in southern France despite high dietary intake of saturated fats, and suggested to mediate the cancer chemoprevention properties of red wine [4]. It has also been reported to possess a variety of anti-inflammatory, anti-platelet, and both pro- and anti-estrogenic effects [5], [6], [7], [8].
Resveratrol inhibits proliferation of a variety of cancer cell lines (reviewed in [9]), formation of preneoplastic lesions in the DMBA-induced mouse mammary organ culture model [10] and benzo(a)pyrene-induced transformation of rat tracheal epithelial cells [11]. In rodent carcinogenesis models, resveratrol interfered with the formation of azoxymethane-induced aberrant crypt foci in rat colon [12], decreased the number of adenomas in the small intestine and suppressed tumor formation in the colon of APC Min+/− mice [13] although the latter finding has not been confirmed [14]. It also reduced mammary tumor formation in N-methyl-N-nitrosourea-treated rats when given at a relatively high dose (100 mg/kg) [10].
Preclinical studies in mice, rats and dogs suggest that resveratrol is readily absorbed and rapidly glucuronidated and sulfated both in the liver and in intestinal epithelial cells [15], [16], [17].
Administration of red wine to rats by intragastric intubation, resulted in measurable concentrations of resveratrol in plasma, heart, liver and kidneys [15]. In rats treated with an oral dose of 2 mg/kg resveratrol, plasma peak concentrations of 2.6 μM were achieved 10 min after dosing [18]. Experiments using radiolabelled trans-resveratrol suggest that at least 50–75% of the orally administered dose is absorbed in Wistar rats [16]. Earlier in vitro studies indicate that resveratrol readily undergoes glucuronidation and sulfation in the liver and gut of both humans and rats [19], [20]. In human liver and duodenal tissue, dietary flavonoids, particularly quercetin, inhibited sulfation and glucuronidation of resveratrol; thus improving its bioavailability [20].
Resveratrol has been previously shown to be well absorbed in humans when given at levels commensurate with that available in red wine [24], [29] and at low doses (25 mg orally) [27], with high phase II conjugation appearing to be the rate limiting step in resveratrol bioavailability, although no studies have been carried out at higher (gram level) doses of this compound.
As part of ongoing investigations in this laboratory into the pharmacokinetics, bioavailability and anti-oxidant effects of high dose resveratrol, our goal was to develop a protein precipitation extraction and analytical methodology that combine separation of the major metabolites of resveratrol and allows quantitation of the parent compound in a reasonably short run time. Part of the development and refinement of the analytical assay included the validation of a gradient elution HPLC assay for plasma and urine. Several methods have been published for the analysis of resveratrol and some of its metabolites [21], [24], [25], [26] This method is an improvement on the UV-HPLC method developed by Zhu et al. [21] as it allows resolution of the several human phase II metabolites of resveratrol, by direct means rather than by deconjugation (many of which are very polar and difficult to resolve in gradient modifications of the previous method) in addition to trans-resveratrol and uses a mobile phase that can be directly transferred to LC–MSMS for identification of these metabolites. Wang et al. [26] published an HPLC–MSMS method for identification of major metabolites in rat urine, this method identified several conjugated metabolites as well as dihydroresveratrol and its monosulfate conjugate that were found in humans by Walle et al. [27]. This method was however not quantitative but provided structural characterisation of the metabolites of resveratrol. A recent publication by He et al. [28] presents a method for analysing resveratrol in rat plasma for pharmacokinetic analysis, although chromatographically rapid, this method does not resolve any metabolites of resveratrol and has a relatively higher limit of detection for resveratrol. The fast and non-intensive sample preparation method of protein precipitation simplifies large sample processing for clinical trials, whilst having better or comparable limits of quantitation for resveratrol (5 ng/mL), and resolving the major conjugate metabolites.
Section snippets
Chemicals
Resveratrol (99.9%, Fig. 1) was obtained from Royalmount Pharma (Montreal, Canada) and the capsule preparation carried out in house. HPLC gradient grade methanol, water, ammonium acetate and hydrochloric acid were obtained from Fisher Scientific Ltd. (Loughborough, UK).
Mobile phase
The mobile phase consisted of A (aqueous) 5 mM ammonium acetate in distilled water (pH unaltered ∼6.7) containing 2% propan-2-ol. B (organic) consisted of methanol with 2% propan-2-ol. These solutions were degassed by sonication
Resveratrol HPLC analysis and detection
In order to resolve the more polar metabolites of resveratrol successfully whilst still retaining a relatively short run-time, the Atlantis C18 column was chosen, due to its characteristic ability to run in 100% aqueous conditions. No endogenous peaks in plasma or urine co-eluted with resveratrol under our chromatographic conditions.
Several major metabolites of resveratrol were detected in both plasma and urine and separated at various time points post-dose (Fig. 2, Fig. 3). Under these UV-HPLC
Conclusion
In the present paper, a reliable, reproducible extraction procedure and sensitive HPLC separation coupled to UV detection was described allowing quantification of resveratrol and separation and identification of at least six major conjugate metabolites in human plasma and urine. Reproducible recovery of resveratrol down to 5 ng/mL was achieved, with the potential for accurate assessment of concentrations down to 1 ng/mL in both urine and plasma. This HPLC system coupled to mass spectrometry could
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