Article pubs.acs.org/JAFC
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
■
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
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 © XXXX American Chemical Society
Received: November 5, 2014 Revised: March 5, 2015 Accepted: March 10, 2015
A
DOI: 10.1021/acs.jafc.5b00390 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
resveratrol in all three media against the nominal concentration of six standard solutions, for each low-level and high-level calibration curve. Linear regression analysis was performed using the least-squares method. The detection limit (DL) and the quantification limit (QL) of the developed HPLC method were determined by the signal to-noise ratio. The DL and QL were the concentrations of trans-resveratrol generated by a peak 3 times and 10 times, respectively, higher than the baseline noise. Precision and accuracy were estimated by analyzing five replicates of the three different QC levels (0.020, 0.200, and 1.50 μmol L −1 of trans-resveratrol) for each matrix. Repeatability and reproducibility were assessed by analyzing five replicates of the three QC levels on three different runs within the same day (intraday) and on three different days (interday), respectively. Stability studies were performed by maintaining the samples inside the autosampler of the HPLC instrument at 4 °C for 24 h, at room temperature for 24 h, and after three freeze−thaw cycles, by thawing at room temperature for 2 h and freezing again for 24 h. Five replicates of each QC sample were analyzed for each storage condition in all matrices. Application to in Vitro Intestinal Permeability Studies. The developed method was targeted to application in studies of intestinal permeability of trans-resveratrol carried inside lipid nanoparticles (SLNs and NLCs), after oral administration. Resveratrol-loaded lipid nanoparticles were prepared by a modified hot homogenization technique and characterized as described elsewhere.10 The results showed spherical and uniform nanoparticles with a smooth surface, a Z-average of 150−250 nm, a highly negative zeta potential of −30 mV, and a resveratrol entrapment efficiency of 70%.10 The recovery of resveratrol from lipid nanoparticles corresponded to 93%, and it was determined using 4.0 mL of a 1.25 mg mL−1 suspension of transresveratrol-loaded nanoparticles, where 1.0 mL of acetonitrile was added to promote the disruption of the lipid matrix and the release of trans-resveratrol. Next, trans-resveratrol was quantified by direct spectrophotometric detection at 306 nm, using standards prepared in 20% (v/v) acetonitrile/water, after subtraction of signal scattering by blank nanoparticles. In vitro intestinal permeability assays were performed using Caco-2 monolayers in transwell devices that mimic the intestinal barrier.41 Three different transport media were used to mimic intestinal fluids: HBSS as the control medium, FaSSIF as the fasted state medium, and FeSSIF as the fed state medium. Resveratrolloaded lipid nanoparticles were incubated during 4 h in the apical side of the intestinal barrier, and aliquots of 100 μL were withdrawn at regular intervals of 30 min from the basolateral side for quantification to determine the intestinal permeability in all three media. Sample treatment was minimal, involving the addition of 25 μL of acetonitrile/ acetic acid (92:8, v/v) solution to 100 μL of sample to promote the disruption of the lipid matrix and the release of trans-resveratrol to be quantified by HPLC and also to avoid band broadening effects due to differences from mobile phase composition. Statistical Analysis. Statistical analyses were performed using SPSS software (v 18.0; IBM, Armonk, NY, USA). Permeability measurements were repeated at least six times, and data were expressed as the mean ± SD. Data were analyzed using one-way analysis of variance (one-way ANOVA), followed by Bonferroni, Tukey, and Dunnett post hoc tests. A p value 18 MΩ cm, Sartorius, Goettingen, Germany) was used for the preparation of all solutions. Acetic acid aqueous solution (2% (v/v)) was filtered through a 0.22 μm Millipore-type GVWP filter. Prior to use, the mobile phase was degassed in an ultrasonic bath for 15 min. The transport medium HBSS was purchased from Gibco (Paisley, UK), whereas FaSSIF and FeSSIF were prepared by using SIF instant powder (Phares Drug Delivery AG, Muttenz, Switzerland) according to the instructions of the manufacturer. Table S2 summarizes the compositions of HBSS, FaSSIF, and FeSSIF media. For the nanoparticle synthesis, cetyl palmitate was kindly supplied by Gattefossé (Nanterre, France), polysorbate 60 (Tween 60) was provided by Merck, and miglyol-812 was from Acofarma (Madrid, Spain). For cell culturing, the Caco-2 cell line purchased from the American Type Culture Collection (ATCC, Wesel, Germany) between passages 35 and 55 was maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% fungizone (amphotericin B, 250 μg mL−1), and 1% Pen Strep (penicillin, streptomycin), all obtained from Gibco. Equipment and Chromatographic Conditions. The samples were injected (50 μL) into a reversed-phase monolithic column (Chromolith RP-18e, 100 mm × 4.6 mm i.d., Merck), connected to a Jasco (Easton, MD, USA) HPLC system (pump PU-2089, autosampler AS-2057, and LC-Net II/ADC controller) coupled to a fluorometric detector (Jasco FP-2020, λexc = 330 nm, λem = 374 nm). Chromatographic separation was achieved by isocratic mode consisting of 80:20 (v/v) aqueous solution (acetic acid 2%, v/v) and acetonitrile at a flow rate of 1.0 mL min−1. Standard Solutions and Quality Control Samples. Stock solutions of trans-resveratrol were daily prepared in mobile phase, HBSS, FaSSIF, and FeSSIF at 10 μmol L−1. Standard solutions were accurately prepared by dilution in the same transport media to final concentrations of 0.020, 0.050, 0.100, 0.150, 0.200, 0.400, 0.700, 1.00, 1.50, and 2.00 μmol L−1. The standard solutions were divided in two calibration curves for each transport media, the low-level calibration curve between 0.020 and 0.200 μmol L−1 and the high-level calibration curve between 0.200 and 2.00 μmol L−1. The quality control (QC) samples at three different levels (low, middle, and high) were prepared at concentrations of 0.020, 0.200, and 1.50 μmol L−1 for each transport medium, within three batches of five samples each. The standards containing 0.020 and 0.200 μmol L−1 were interpolated in the low-level calibration curve, whereas the 1.50 μmol L−1 standard was interpolated in the high-level calibration curve. Acetonitrile was added to all standard solutions and samples immediately after preparation or collection to attain a 20% (v/v) concentration (100 μL of standard or sample +25 μL of acetonitrile:acetic acid (92:8, v/v)) to match the mobile phase composition, avoiding peak broadening effects. Validation of HPLC Method. The chromatographic method developed was validated for selectivity, concentration range, detection and quantification limits, accuracy, precision, and stability according to the ICH guidelines on bioanalytical method validation.11 Selectivity concerns the ability to differentiate the analyte of interest (transresveratrol) from endogenous components in the matrix or in the sample and was assessed by analyzing six different samples of blank HBSS, FaSSIF, and FeSSIF, which were evaluated for interference. To evaluate linearity, calibration curves were acquired in triplicate, on three consecutive days, by plotting the integrated peak area of trans-
■
RESULTS AND DISCUSSION Study of Chromatographic Conditions. Considering the method application to studies of intestinal permeability of transresveratrol inside lipid nanoparticles, the main goal to be achieved in the development of this chromatographic method was low detection and quantification limits. Furthermore, because there was a constraint in the available sample volume, it was also important to develop a method that needed minimal sample treatment. Therefore, a monolithic column coupled to fluorescence detection was chosen to meet these needs. In this work, the development of a simplified HPLC method with isocratic elution was aimed for. The choice of the organic
B
DOI: 10.1021/acs.jafc.5b00390 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Chromatograms of trans-resveratrol (10 μmol L−1) using different mobile phase compositions containing acetic acid (2%, v/v)/ acetonitrile.
to 20% (v/v), whereas only a 2% enhancement in peak area was observed when acetonitrile was increased from 20 to 23% (v/ v). Therefore, a mobile phase composed of 20% of acetonitrile and 80% of acetic acid (2%, v/v) was selected. This composition permitted the elution of trans-resveratrol with a good compromise between the trans-resveratrol retention time and the peak symmetry factor, allowing a sharp peak separation. Using this isocratic condition, trans-resveratrol was eluted with a retention time of 6.5 min and with the lowest peak symmetry factor of 1.22, which reflects a reduced peak tailing in these conditions (Table 1). The injection volume was also tested to increase the sensitivity of the developed method. Injection volumes of 50 and 100 μL were tested, providing a signal area enhancement of ca. 2 as expected. The injection volume chosen was 50 μL as the volume collected during the permeability assay should be
