Development and Validation of a HPLC Method Using a Monolithic
Column for Quantification of trans-Resveratrol in Lipid Nanoparticles for Intestinal Permeability Studies
Ana Rute Neves, Salette Reis, and Marcela A. Segundo*
UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal *S Supporting Information
ABSTRACT: The development of nanodelivery systems that protect trans-resveratrol is extremely important to preserve its bioactive properties in the development of further applications as nutraceuticals to supplement foods and beverages. In this work, a validated HPLC method was developed for the quantification of trans-resveratrol in lipid nanoparticles for application in studies of in vitro intestinal permeability. The chromatographic separation was achieved in a C18 monolithic column connected to a fluorometric detector (330/374 nm), by isocratic elution consisting of 2% acetic acid/acetonitrile (80:20). Two calibration ranges were established (0.020−0.200 and 0.200−2.00 μmol L−1), and low quantification limits (2−6 nmol L−1, 23−69 pg) were achieved. Stability studies showed that trans-resveratrol is stable for 24 h at 4 °C, and storage at room temperature and freeze− thaw cycles are not recommended. The proposed method was applied to in vitro intestinal permeability studies, in which values between 0.05 ± 0.01 and 1.8 ± 0.3 μmol L−1 were found.
KEYWORDS: trans-resveratrol, lipid nanoparticles, nutraceutical, intestinal permeability, bioavailability ■ INTRODUCTION
In recent decades, there was a rising interest from health professionals and scientists in nutraceuticals such as resveratrol, which is a natural polyphenol present in a wide variety of plants including fruits, vegetables, seeds, and roots.1 Grapes are probably the most important source of resveratrol for humans, because the compound is also found in one of the end products of grapes, wine.2 In fact, resveratrol is pointed out as a possible contributor to the cardiovascular protection conferred by red wine consumption, the so-called “French paradox”.3 Moreover, interest in resveratrol has increased due to several other beneficial effects, such as chemopreventive capacity, antiinflammatory properties, antioxidant activity, neuroprotection, antiaging, and diabetes and obesity prevention.4
Resveratrol is found in nature as both cis and trans isomers (Supporting Information Figure S1) and is highly photosensitive; >80% of the trans-resveratrol in solution is converted to cis-resveratrol if exposed to light for 1 h.5 The biological activity of the compound has been attributed mainly to the trans isomer,6 and despite the therapeutic effects of transresveratrol, its pharmacokinetic properties are not so favorable.
In fact, trans-resveratrol is poorly soluble in water, is chemically unstable, and is rapidly and extensively metabolized inside the body, which leads to low bioavailability.7−9
Given the favorable prophylactic and therapeutic effects of trans-resveratrol, protection from premature metabolism and from degradation, with increase of its lifetime within the body, are of utmost importance, particularly during the process of intestinal absorption. For this reason, nanodelivery systems have been developed for the encapsulation of trans-resveratrol, which protect the compound during its transport inside the organism while enhancing its bioavailability after oral administration.10 The development of resveratrol-loaded nanoparticles is essential to further applications as nutraceuticals to supplement juices, yogurts, milk, or cheese with health benefits similar to those attributed exclusively to red wine consumption.
In this context, the objective of this work was the development and validation of a simple, sensitive, and selective high-performance liquid chromatography (HPLC) method, according to the International Conference on Harmonization (ICH) guidelines on bioanalytical method validation,11 for the quantification of trans-resveratrol incorporated in lipid nanoparticles, comprehending both solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs). Considering the methods found in the literature (Table S1) for the quantification of trans-resveratrol in wines, grapes, and plasma using spectrophotometric,5,12−31 fluorometric,12,28,32−34 mass spectrometry,14,20,31,35 or electrochemical detection,16 high detection limits, within the micromolar per liter range, were generally attained. As our research aim includes the evaluation of in vitro intestinal permeability of trans-resveratrol inside lipid nanoparticles, by using Caco-2 cell monolayers, a detection limit close to nanomolar per liter of resveratrol is required.
Furthermore, three different media to mimic the intestinal conditions will be applied: Hanks’ balanced salt solution (HBSS) as the control medium, fasted-state simulated intestinal fluid (FaSSIF), and fed-state simulated intestinal fluid (FeSSIF). These media mimic the fasted-state and fed-state intestinal juices because they contain natural surfactants (bile
Received: November 5, 2014
Revised: March 5, 2015
Accepted: March 10, 2015
Article pubs.acs.org/JAFC © XXXX American Chemical Society A DOI: 10.1021/acs.jafc.5b00390
J. Agric. Food Chem. XXXX, XXX, XXX−XXX salts and lecithin) which generate micelles that may enhance the dissolution rate and solubility of lipophilic resveratrolloaded lipid nanoparticles.36,37 Hence, a monolithic C18 column, consisting of micro- and mesopores,38,39 was selected with regard to its suitability for bioanalysis,40 avoiding further sample treatment. ■ MATERIALS AND METHODS
Chemicals. trans-Resveratrol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and acetic acid (LiChrosolv HPLC grade) were obtained from Merck (Darmstadt, Germany). Water from an arium water purification system (resistivity > 18 MΩ cm, Sartorius,
Goettingen, Germany) was used for the preparation of all solutions.