Quantification Of Trans-resveratrol Research Articles

Development and Validation of a Stability-Indicating HPLC Method for the Simultaneous Determination of trans-Resveratrol and cis-Resveratrol in an Injectable Solution.

trans-Resveratrol, a phytochemical compound with antioxidant power and various therapeutic effects, such as cardioprotective, chemopreventive, and neuroprotective, among others, has disadvantages of poor solubility and limited stability, creating difficulties for the development of new strategies for its quantification. This study developed and validated an analytical stability method for trans-resveratrol by high-pressure liquid chromatography with photodiode-array detection (HPLC-PDA), which allowed its quantification in the presence of its degradation products. The quantification of trans-resveratrol occurred at a retention time of 2.6 min, with ammonium formate (10 mM, pH = 4)/acetonitrile, 70/30 v/v, as mobile phase. The validation met the ICH Q2 criteria of specificity, method linearity (2.8–4.2 μg/ml), precision and accuracy, robustness, quantification limit (0.176 μg/ml), and detection (0.058 μg/ml). As degradation products, cis-resveratrol was observed at 3.9 min, which could be resveratrone in 3.2 min and five unidentified products in 0.7, 1.0, 1.4, 1.8, and 5 min. Some solutions subjected to temperature stress of 40 and 60°C, UV light, and acidic and basic hydrolysis exhibited colour changes. An analytical method was obtained by HPLC-PDA, which allowed quantifying the stability of trans-resveratrol in a fast and specific manner in the presence of its degradation products.

Quantification of trans-resveratrol and its metabolites in human plasma using ultra-high performance liquid chromatography tandem quadrupole-orbitrap mass spectrometry

Trans-resveratrol is a stilbene polyphenol with a large spectrum of biological activities. This is why it is widely studied in terms of activities, bioavailability and quantitation in different foods, beverages and biological matrices. Different analytical methods are employed for its quantitation. In this study a quadrupole-orbitrap tandem mass spectrometer coupled to a reverse phase ultra-high performance liquid chromatography is applied to a quantitation of trans-resveratrol and its metabolites trans-resveratrol-3-O-β-d-glucuronide, trans-resveratrol-4′-O-β-d-glucuronide, trans-resveratrol-3-O-sulfate, a,b-dihydroresveratrol, a,b-dihydroresveratrol-glucuronide, a,b-dihydroresveratrol-glucuronide-sulfate, a,b-dihydroresveratrol-sulfate, trans-resveratrol-3,5-O-β-d-diglucuronide, trans-resveratrol-3,4′-O-d-β-diglucuronide, trans-resveratrol-3-O-β-d-glucuronide-sulfate and trans-resveratrol-4′-O-β-d-glucuronide-sulfate in human plasma. MS/MS experiments coupled to a high resolving power and accurate mass measurements as well as the use of labeled internal standards enabled the achievement of linear calibration curves across the four orders of magnitude concentration ranges. The method was validated in terms of specificity and selectivity, accuracy and precision, sensitivity and matrix effect and can be now applied to pharmacokinetic studies or routine analysis. In addition, the application of quadrupole-orbitrap mass spectrometer to the quantitation of trans-resveratrol and its metabolites provides acquisition of full collision induced dissociation spectra of analyzed compounds giving place to the structural characterization and sensitivity and linear concentration ranges respecting the accuracy and precision, specificity and selectivity requirements.

Quantification of Trans-resveratrol in Red Wines Using QuEChERS Extraction Combined with Liquid Chromatography-Tandem Mass Spectrometry.

Resveratrol is one of representative ingredient in red wine, but its quantification is a challenge because of a complex and abundant matrix. In this study, two sample pretreatments, direct dilution and QuEChERS extraction, coupling LCMS analysis were examined for resveratrol quantification. Similar recoveries of 106.4 to 93.7% were obtained for direct dilution and QuEChERS, respectively. With the aid of condition optimization, QuEChERS extraction could concentrate the resveratrol from red wines to improve the detection sensitivity with a LOD value of 2.5 ng/mL, which is four-times greater than the direct dilution approach. As a result, the QuEChERS method can provide a high linearity within the concentration range of 5 - 500 ng/mL, in which direct dilution produced the linear calibration curve within the concentrations of 25 - 500 ng/mL. A high consistency was obtained for both approaches in which intra-day precisions were within 0.5 to 7.2% (n = 3), and the inter-day precisions were within 7.8 to 16.0% (n = 9). Overall, the sample pretreatment of QuEChERS can effectively reduce the matrix effect, which leads LCMS to quantify the low resveratrol abundance of 8.0 ppb in each red wine sample, which is not achieved with the direct dilution approach.

Development of new ultrasonic–solvent assisted method for determination of trans-resveratrol from red grapes: Optimization, characterization, and antioxidant activity (ORAC assay)

Abstract This work was aimed at developing an ultrasonic-assisted extraction of trans-resveratrol from red grape wastes using a new combination of solvents and response surface method- Box–Behnken design. The experiment was carried out on the industrial waste, skin, and pulp parts of 18 different types of Iranian red grapes. Ultrasound-assisted solvent extraction was carried out using different ratios of ethanol:polyethylene glycol (PEG):water (v/v), ultrasonication time, and temperature. The optimized conditions were achieved as a combination of 19.4 min, 53.6 °C, and 48:32:20 (v/v), which remarkably improved the extraction of trans-resveratrol content. The presence of PEG in the extraction medium enhanced the extraction efficiency by 39.48%. Reverse phase high performance liquid chromatography ultra-violent (RP-HPLC-UV) was applied for the quantification. The amounts of trans-resveratrol varied between 68.36 ± 3.06 and 862 ± 4.67 µg/g-dry material of the initial samples based on optimized conditions. Retention time of trans-resveratrol was obtained in less than 5 min, which indicates the high accuracy of developed HPLC method. There was a significant relationship between the amount of resveratrol and antioxidant activity. Antioxidant activity of samples extracted by optimized method was about four times higher than conventional method (80:20 ethanol:water (v/v), 30 min sonication, and 60 °C temperature) measured by oxygen radical absorbance capacity (ORAC) assay. This research provided food grade extraction method due to reduction in the use of ethanol in extraction process. Furthermore, the results can provide a new HPLC elution method for easy and fast detection and quantification of trans-resveratrol in industrial scale.

Monitoring trans-Resveratrol in Grape Berry Skins During Ripening and in Corresponding Wines by HPLC.

Trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) is naturally present in the skin of grapes and therefore is expected to occur in grape products. Recently, there has been a renewal of interest in wine as medical treatment for various disorders. In this study, berry skins of five red grape varieties were analysed at weekly intervals for trans-resveratrol production, during 2012 and 2013 harvest. The obtained wines were also evaluated. Quantification of trans-resveratrol in grape skin extracts and wines was carried out by high-performance liquid chromatography. Favourable viticultural climate during harvest in 2012 resulted in a higher content of trans-resveratrol. The highest trans-resveratrol content was in Pinot Noir and Feteasca Neagra grape varieties, both in berry skins and wines, while the lowest amounts were identified in Cabernet Sauvignon variety. We can conclude that there was a significant correlation between the content of trans-resveratrol in grape skins and that in the respective wine.

Development and validation of an RP-HPLC method for quantification of trans-resveratrol in the plant extracts

New, simple, cost effective, accurate and reproducible RP-HPLC method was developed and validated for the quantification of trans-resveratrol in the extracts of grape exocarp and seeds. The method has proved to be simpler and faster than available methods. Methanol was used as a mobile phase with a flow rate of 1.0 cm3 min-1, while the quantification was effected at 306 nm. The separation was performed at 35?C using a C18 column. The results showed that the peak area response was linear in the concentration range of 1-40 ?g cm-3. The values of LOD and LOQ were found to be 0.125 and 0.413 ?g cm-3, respectively. The antioxidant activity of the extracts was determined using DPPH assay. The ability of DPPH radicals inhibition decreases in the following order: the extract of grape exocarp > trans-resveratrol standard > the extract of grape seeds.

In vivo pharmacokinetic applicability of a simple and validated HPLC method for orally administered trans-resveratrol loaded polymeric nanoparticles to rats

A new formulation of trans-resveratrol nanoparticles (t-RVT NPs) with potential stealth properties was prepared by nano-precipitation method in our laboratory. To investigate the pharmacokinetic of t-RVT NPs, a simple and rapid high performance liquid chromatographic method was developed for the quantification of trans-resveratrol in plasma of rats treated with t-RVT NPs. Catechin (0.2 μg/ml) was used as an internal standard (IS). The chromatographic separation was achieved on a reversed-phase C18 column using isocratic elution with methanol: 10 mM potassium dihydrogen phosphate buffer (pH 6.8): 3 % acetic acid solution (70:28:2, v/v/v) at a flow rate of 1 ml/min with a total run time of 6 min. The column effluent was monitored by UV detector at 306 nm. Calibration curve in plasma spiked with varying concentration of trans-resveratrol were linear over the concentration range of 0.010–3.2 μg/ml with correlation coefficient of (r2 > 0.9987). The system was found to construct sharp peaks for trans-resveratrol and IS with retention times of 3.82 and 5.49 min, respectively. The method was sensitive with a limit of quantification of 0.007 μg/ml. The method showing accuracy, precision (inter-day relative standard deviation (RSD) and intra-day RSD values <15.0 %. The method was applied for determining trans-resveratrol concentration in plasma after oral administration of 20 mg/kg of free trans-resveratrol and t-RVT NPs to rats. The plasma concentrations of trans-resveratrol and t-RVT NPs at 30 min and 10 h after oral administration were quantified as 0.1771 ± 0.38 and 5.64 ± 0.436 μg/ml. Results established selectivity and suitability of the method for pharmacokinetic studies of trans-resveratrol from t-RVT NPs.

QUANTIFICATION OF TRANS-RESVERATROL IN RAT PLASMA BY A SIMPLE AND SENSITIVE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD AND ITS APPLICATION IN PRE-CLINICAL STUDY

The intention of this study was to develop and validate a simple, sensitive, and accurate high performance liquid chromatography (HPLC) method for quantification of trans-resveratrol in rat plasma. Trans-resveratrol was recovered from plasma samples by liquid-liquid extraction. A reverse phase HPLC column was used and the assay was performed at 35°C through isocratic delivery (1 mL · min−1) of acetonitrile and phosphate buffer solution, pH 7.0 (30:70 v/v). UV absorbance was monitored at 320 nm. Plasma samples were stable (>99%) at −80°C for 3 months. The recovery of trans-resveratrol from plasma was >95%. The HPLC method was validated. The retention times of trans-resveratrol and carbamazepine were 5.5 and 10.4 min, respectively. The calibration curve was linear (R2 = 0.9999) within the range of 5–1,000 ng · mL−1. The lower limits of detection and quantification of trans-resveratrol were 2.5 and 5 ng · mL−1, respectively. The intra-day and inter-day variations were <5% and accuracy was >95%. This simple HPLC method was subsequently applied to measure trans-resveratrol concentration in plasma following oral administration of trans-resveratrol suspension in rats. The plasma concentration of trans-resveratrol at 30 min after oral administration was quantified as 151.66 ± 120.53 ng · mL−1.

Resveratrol Analysis and Degradation Study in Blueberry Samples by HPLC with Fluorescence Detection

Separation and quantification of trans-resveratrol in blueberry extracts were achieved using high performance liquid chromatography (HPLC). Trans-resveratrol in blueberry juice was found to be at a high level of 16.60 ± 0.19 µg/mL. This antioxidant polyphenolic compound was also quantified, in two brands of blueberry powder, to be 236.4 ± 7.5 µg/g or 269.9 ± 5.7 µg/g. Degradation of trans-resveratrol was observed in standard solutions and in blueberry samples (juice and powder extracts). It was found that degradation of trans-resveratrol was more severe when it was exposed to room light at room temperature. Therefore, it is advisable to store blueberry juice at 4°C in the dark, and consume it within 1–3 days in order to benefit from adequate intake of the antioxidant in the juice.

Quantification of trans-resveratrol and its metabolites in rat plasma and tissues by HPLC

trans-Resveratrol, a polyphenol from grapes, is being recognized as a bioactive agent with potential beneficial effects on health. However, little is known about its distribution in the organism mainly because of the lack of accurate and precise detection methods. Consequently the aim of the present study was to develop a methodology of extraction and quantification of trans-resveratrol and its metabolites in plasma, brain, testis, liver, lungs and kidney by HPLC. To this end, the time of homogenization and liquid extraction were adapted to the different tissues. The methods were validated using homogenized tissues spiked with pure trans-resveratrol. The precision (% R.S.D.) ranged from 3.7% in testis to 13.2% in lungs. Recoveries were 98.5 ± 3.2% (liver), 100.1 ± 1.8% (kidney), 96.5 ± 7.6% (lungs), 99.0 ± 0.7% (brain) and 103 ± 2.7% (testicle). The limits of detection ranged from 5.5 nM in testis to 11.2 nM in kidney. After validation, the methods were applied to the assessment of the bioavailability and distribution of trans-resveratrol in rats after the intravenous administration of 15 mg/kg. At 90 min, trans-resveratrol and its glucuronide and sulfate conjugates were widely distributed in all the tissues studied. The highest concentrations (nmol/g tissue) were found in kidney (resveratrol: 1.45 ± 0.35; glucuronide: 2.91 ± 0.19; sulfate: not detected), and the lowest in brain (resveratrol: 0.17 ± 0.04; glucuronide: not detected; sulfate: 0.04 ± 0.01). In conclusion, accurate and reproducible methods have been described to identify target tissues of resveratrol as a first step to understand its mechanisms of action in vivo.

Identification and Quantification of Trans-Resveratrol in Dietary Supplements by a Rapid and Straightforward RP-HPLC Method

This paper reports the results of a study performed to develop a rapid and straightforward chromatographic method for the identification and quantification of trans-resveratrol in commercial trans-resveratrol containing dietary supplements. The method employs an Alltech Platinum EPS 100 A C-18 column operated under isocratic elution mode with a 70:30 (v/v) water-acetonitrile mixture as the mobile phase, at flow rate of 1.0 mL min−1. Peak detection is performed at 306 nm with limit of detection (LOD) of 0.27 µ g mL−1 and limit of quantification (LOQ) of 0.95 µ g mL−1 and linear calibration graph within the concentration range of 2.63 to 10.53 µ g mL−1. Other data related to the successful method validation include representative linear regression equation and correlation coefficient of the calibration graph for quantification of trans-resveratrol, as well as the results of replicate analysis of real samples and of a recovery study, carried out to evaluate precision and accuracy, respectively. The method is successfully applied to study the stability of trans-resveratrol in methanol solution under various storage conditions and to analyse commercial preparations of trans-resveratrol containing dietary supplements.

Development of a reverse-phase high-performance liquid chromatography method for analyzing trans-resveratrol in peanut kernels

A reverse-phase high-performance liquid chromatography (HPLC) method for identification and quantification of trans-resveratrol from peanut extracts, using phenolphthalein as internal standard, was developed. The HPLC system consisted of a C 18 column (250 l × 4.6 i.d. mm, 5 μm particle size), with PDA detection at 307 nm and mobile phase consisting of 0.1% acetic acid in water and 100% acetonitrile. Gradient elution increased acetonitrile linearly from 5% to 41.8% over 23 min (GS = 1.6) followed by an increase of acetonitrile to 77% over 5 min (GS = 7.04), returning to initial conditions over 1 min and held for an additional 5 min with a flow rate of 1.5 ml/min. A lower column temperature resulted in higher peak heights and better baseline separation, therefore 25 °C was selected over 40 or 60 °C to simplify the method. Accuracy, precision, linearity, limit of detection and limit of quantitation were consistent or better than reported previously in the literature for related studies.

Direct analysis of trans-resveratrol in red wine by high performance liquid chromatography with chemiluminescent detection

A novel high-performance liquid chromatography with chemiluminescence detection has been developed for the determination of trans-resveratrol in red wines based on the enhancement by trans-resveratrol of the chemiluminescence from luminol–potassium ferricyanide system in alkaline medium. Samples were separated on a C 8 column using isocratic elution of methanol/water (35:65). Quantification of trans-resveratrol in Chinese red wines was performed without any sample pretreatment. It allows for the determination of trans-resveratrol in the range 0.5–750 μg/L with a detection limit of 0.166 μg/L (S/N=3). The relative standard deviation (RSD) is 1.16% for 7.5 μg/L trans-resveratrol ( n=11). trans-Resveratrol was detected in Chinese red wines with the recoveries of 92.2–114.7%. Concentrations ranged from 0.072 to 1.607 mg/L in Chinese red wines.

Direct HPLC analysis of quercetin and trans-resveratrol in red wine, grape, and winemaking byproducts.

A simple and fast reversed-phase HPLC method using diode array detection was developed and validated for the simultaneous determination of trans-resveratrol and quercetin in Sicilian red wine from the Nero d'Avola red grape variety. Investigation was also extended to the quantitative determination of resveratrol and quercetin in grape skins and winemaking byproducts obtained from the same cultivar. Samples were eluted using a C18 narrow-bore column under isocratic conditions in less than 20 min. Quantification of trans-resveratrol and quercetin in red wine was performed without any sample pretreatment, whereas the determination of these phenolic compounds in grape skins and wine pomage required a solvent extraction procedure. Linearity was demonstrated over the 0.39-12.5 and 0.45-57.6 microg/mL range for trans-resveratrol and quercetin, respectively. Detection limits in real samples were in the low ppm level (0.07 mg/L for trans-resveratrol and 0.12 mg/L for quercetin). The HPLC-UV/DAD method was applied for the routine analyses of red wine and grape skin and winemaking byproduct extracts to evaluate their trans-resveratrol and quercetin content. In particular, a very high content of quercetin was found in wine pomace, suggesting the use of this wine byproduct as a potential source of this health-promoting phenolic compound.