Quantitation of Resveratrol in Red Wines by Means of Stable Isotope

ARTICLE pubs.acs.org/ac

Quantitation of Resveratrol in Red Wines by Means of Stable Isotope Dilution Analysis
Ultra-Performance Liquid Chromatography
Quan-Time-of-Flight Mass Spectrometry and Cross Validation Timo Stark,* Nadine Wollmann, Sofie L€osch, and Thomas Hofmann Chair of Food Chemistry and Molecular Sensory Science, Technische Universit€at M€unchen, Lise-Meitner Str. 34, D-85354 Freising, Germany ABSTRACT: A stable isotope dilution analysis (SIDA) was developed for the quantitative analysis of the health-promoting phytoalexin (E)-resveratrol in red wines by means of UPLCQuanTOF-MS. After hemisynthetic preparation of (E)-3,5,40 trihydroxy-2,4,6-trideuterostilbene ((E)-[2H3]-resveratrol) as the stable isotope labeled internal standard, validation experiments revealed recovery rate of 96.2 ( 0.8% RSD, thus demonstrating the robustness and accuracy of the SIDAUPLC-QuanTOF-MS method. Repeatability and reproducibility expressed as RSD showed excellent values of 3.0% and 4.0% for (E)-[2H3]-resveratrol. Cross validation against a SIDAHPLC-MS/MS analysis using a triple quadrupole mass spectrometer revealed comparable data, but the SIDA-UPLC-QuanTOF-MS was four times faster, thus making the latter method preferential for an accurate high-throughput analysis of wine samples. Comparison of the SIDA data to those obtained by quantitation using a standard addition method and external calibration, respectively, revealed 97.7% and 32.4% of the resveratrol concentration determined by means of SIDA-UPLC-QuanTOF-MS and 101.0% and 12.7% of the resveratrol levels found by using SIDA-HPLC-MS/MS.

T

he desirable taste and the alluring aroma of red wines have been attracting consumers for more than thousands of years. Since the discovery of the phytoalexins (E)-resveratrol (1, Figure 1) and (Z)-resveratrol (2) in 1940,1
3 a series of studies were performed on putative health benefits of these 3,5,40 trihydroxystilbenes including antioxidative, anti-inflammatory, platelet aggregation inhibitory, antiestrogenic, anticancer, as well as chemopreventive activities.4
15 Quantitative analysis of (E)-resveratrol (1), commonly done by means of HPLC connected to a diode array detector or a triple quadrupole mass spectrometer, revealed concentrations of up to 21 mg/L in red wines depending on the variety, origin, and growing conditions of the grapes.16
27 As the analytical procedures used so far for resveratrol quantitation involve different sample workup procedures and the mass spectrometric analysis of red wine samples is heavily influenced by severe matrix effects,28 a stable isotope dilution analysis (SIDA), using a stable isotopologue of resveratrol as the internal standard, is expected to enable the correction of compound discrimination during extraction, clean up, chromatographic separation, and MS detection. Therefore, Di Donna et al. prepared (E)-d4-resveratrol from d6-phenol via a laborious six-step reaction sequence, but the overall synthesis is rather laborious and gives the target compound in comparatively low yield of less than 25%.29 r 2011 American Chemical Society

The purpose of the present investigation was to produce a deuterated internal standard by means of protium/deuteriumexchange of (E)-resveratrol (1), to confirm its structure and stability by means of TOF-MS and 1H NMR spectroscopy, and to develop a versatile and reliable SIDA for the accurate high-throughput quantitation of (E)-resveratrol in red wines by means of a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer enabling acquisition rates compatible with UPLC separations.30

’ MATERIALS AND METHODS Chemicals. The following compounds were obtained commercially: (E)-resveratrol (1), tartaric acid, Amberlyst 15 (Sigma-Aldrich, Steinheim, Germany); (Z)-resveratrol (2, in 5 mg/mL ethanol, Santa Cruz Biotechnology, Santa Cruz, CA). Water for chromatographic separations was purified with a MilliQ Gradient A10 system (Millipore, Schwalbach, Germany), and solvents used were of HPLC grade (Merck, Darmstadt, Germany). Deuterated solvents were obtained from Euriso-Top (Gif-surYvette, France). Acetonitrile and ethanol were dried over a molecular sieve (0.3 nm, Merck, Darmstadt, Germany) prior to use. Received: December 21, 2010 Accepted: March 25, 2011 Published: March 25, 2011 3398

dx.doi.org/10.1021/ac103305s | Anal. Chem. 2011, 83, 3398–3405

Analytical Chemistry

ARTICLE

Figure 1. Chemical structures of (E)-resveratrol (1), (Z)-resveratrol (2), and deuterated (E)-resveratrol ([2H3]-1).

The following red wines were obtained from a local wine shop (Munich, Germany): Cabernet Sauvignon (2006, Paarl, South Africa; 2007, Valle de Colchagua, Chile; 2008, California), Bardolino (2009, Gambellara, Italy), Merlot (2009, Southwest Australia), Barbera d'Asti (2005, Piemont, Italy), Spaetburgunder (Pinot Noir) (2007, Baden; 2008, Breisgau, Germany), Lemberger (Blauer Limberger) (2008, W€urttemberg, Germany), Bordeaux (2003, C^otes de Castillon; 2007, Graves, France), Shiraz (2005, Southeast Australia; 2009, Southwest Australia), Zinfandel (2007, California; 2008, California). According to the producer, Cabernet Sauvignon (2006 South Africa) was aged in small French oak barrels, Bordeaux (2003, C^otes de Castillon) was aged in oak barrels, Bordeaux (2007, Graves) is a mixture of Merlot (76%), Cabernet Sauvignon (20%), and Cabernet Franc (4%) and was aged in oak barrels, and Shiraz (2005) was matured in a mixture of oak barrels and stainless steel for 9 months. In addition, Dornfelder red wine samples were obtained from a German vineyard (Rheinhessen): Dornfelder sample (2005) was aged in barrique barrels for 15 months. These barrels (225 L) were made of French oak wood and were used twice prior to this vintage. Another Dornfelder sample (year 2005) was matured for 15 months in oak-wood barrels, which were already in use for more than 10 years. Finally, a Dornfelder sample from the year 2007 (Germany) was not matured in wooden barrels. Purity Control of (E)- and (Z)-Resveratrol, 1 and 2. Analysis of 1 and 2 by means of HPLC-DAD, UPLC-TOF-MS, and 1 H/13C NMR spectroscopy confirmed the identity of the compounds and revealed a purity of 98% (HPLC-DAD). Characterization data for (E)- and (Z)-resveratrol, (1 and 2, Figure 1) follow. UPLC-TOF-MS (ESI
): m/z 227.0704 (calculated for [C14H11O3
H]
: m/z 227.0708, Δ
0.4 mDa). 1 H and 13C NMR data were identical to those reported in literature.31 Preparation of Deuterated (E)-Resveratrol, [2H3]-1. Following a procedure for H/D exchange32 with some modifications, a portion of Amberlyst-15 catalyst resin (10 mg), which was dried for 24 h at 120 °C prior to use, was added to a solution of 1 (0.04 mmol) in acetonitrile/deuterium oxide (2/1, v/v; 4.5 mL) in a centrifugation tube (Schott AG, Germany). After closing, the mixture was stirred with a magnetic stirrer for 24 h at 90 °C in the dark, the suspension was cooled to room temperature, and, after membrane filtration, acetonitrile was removed under a stream of nitrogen. The remaining aqueous solution was applied onto the top of a C18-SPE cartridge (6 mL; 1000 mg; Strata C18-E, Phenomenex, Aschaffenburg, Germany), and after rinsing with water (4 mL) and drying under vacuum, the title compound [2H3]-1 was eluted with methanol (1 mL). The organic effluent was diluted with water (9 mL), and after freeze-drying, the H/D exchange was checked by MS-electrospray ionization (ESI
) as well as 1H NMR spectroscopy. Characterization data for (E)-[2H3]-resveratrol ([2H3]-1, Figure 1) follow. UV-DAD purity >98%. UPLC-TOF-MS (ESI
): m/z

230.0892 (calculated for [C14H8D3O3
H]
: m/z 230.0896, Δ
0.4 mDa). 1H NMR (500 MHz, d3-MeOD): δ 6.15 [s, 0.01 H, H
C(4)], 6.44 [s, 0.09 H, H
C(2, 6)], 6.76 [d, 2H, J = 8.6 Hz, H
C(30 , 50 )], 6.79 [d, 1H, J = 16.3 Hz, H
C(7)], 6.95 [d, 1H, J = 16.3 Hz, H
C(8)], 7.34 [d, 2H, J = 8.6 Hz, H
C(20 , 60 )]. Development of a Stable Isotope Dilution Assay (SIDA) for the Analysis of (E)-Resveratrol (1) by Means of UPLC-TOFMS. A sample of red wine (1 mL) was spiked with a solution of [2H3 ]-1 in ethanol (100 μL, 56 μg/mL) and vortexed for 3 min, and an aliquot (2 μL) was injected into the UPLCTOF-MS system. Each wine sample was prepared three times and analyzed using three replicates, respectively. Wine samples were kept at 10 °C in the autosampler in the dark until analysis. Calibration. For quantitation, the analytes (E)-(1) and (Z)resveratrol (2) and the internal standard (E)-[2H3]-resveratrol ([2H3]-1) were mixed in five ratios from 0.1 to 10 (0.5
50 μg/ mL in EtOH), vortexed, and analyzed by means of UPLC-TOFMS in triplicates. Calibration solutions were stored in the autosampler in the dark at 10 °C until analysis. Calibration curves were prepared by plotting peak area ratios of analytes to internal standard against concentration ratios of each analyte to the internal standard using linear regression. The equation obtained was y = 1.3993x
0.0087 (1/[2H3]-1, R2 = 0.9992), y = 0.5884x
0.0413 (1/2, R2 = 0.9991), and y = 2.7055x þ 0.0577 (2/[2H3]-1, R2 = 0.9997). Method Validation. Linearity was investigated using mixtures of 1 and [2H3]-1 in five ratios from 0.1 to 10. To study the repeatability (intraday precision) of the method, Dornfelder (2005, aged in oak barrels) was analyzed in three independent repetitions using four replicates in one day. The average repeatability over 14 wines was calculated from three independent workups using three replicates for each concentration in one day (n = 128). Reproducibility (interday precision) was studied with Bordeaux (2003), in which two bottles of this wine were independently analyzed with a difference of two months in three independent workups using three replicates in one day (n = 18). The determination of the recovery was performed using a sample (9.9 mL) of Cabernet Sauvignon (2007), which was spiked with increasing amounts of 1 dissolved in ethanol (100 μL), to give wine solutions (10 mL) containing additional 1, 2, 3, 4, and 5 mg/L of the analyte. All samples were analyzed by means of the SIDA as described above in three replicates, respectively. Limit of detection (LOD) and limit of quantitation (LOQ) were calculated as the concentrations for which signal-to-noise ratios were 3 and 10, respectively. Studies on the D/H Re-Exchange of (E)-[ 2H3]-Resveratrol 2 ([ H3]-1). (E)-[2H3]-Resveratrol was dissolved in dried ethanol (1 mg/10 mL) and added to a wine model solution (15% ethanol in water adjusted to pH 3.5 with tartaric acid) in a brown glass 3399

dx.doi.org/10.1021/ac103305s |Anal. Chem. 2011, 83, 3398–3405

Analytical Chemistry vial. After vortexing for 3 min, the brown glass vial was kept for 2, 20, and 40 h either at room temperature or at 10 °C in the autosampler in the dark until analysis. Over a time period of 2 h in the autosampler, the sample was injected 12 times, and the ratio of area ([2H3]-1, m/z 230.0896)/area (1, m/z 227.0708) was determined in [2H3]-1 with QuanLynx (Waters, Manchester, U.K.) as described below. Investigation of Matrix Effects. To study possible matrix effects during the UPLC-TOF-MS analysis of red wine, an aliquot (2 μL) of Bordeaux (2003) was analyzed using the same parameters as described above. A constant flow of 10 μL/min of a solution of (E)-resveratrol (1, 1 mg/L) was introduced to the solvent flow via the fluidics system (flow state: combined). UPLC-TOF-MS Quantitation of (E)-Resveratrol (1) by Means of External Calibration and Standard Addition. Quantitation by means of external calibration was performed by comparing the peak area obtained for the exact mass of 1 in red wine (Cabernet Sauvignon, 2007) with those of defined standard solutions (0.1
25.0 mg/L) of 1 dissolved in ethanol. The experiment was repeated in triplicate, with triple injection of each sample. Calibration curve, obtained by linear regression analysis of the peak area versus concentration, showed a linear response with a correlation coefficient of R2 = 0.9962. Standard addition was carried out by spiking aliquots (9.9 mL) of Cabernet Sauvignon (2007) with increasing amounts of 1 dissolved in ethanol (100 μL), to give wine solutions (10 mL) containing no additional trans-resveratrol (zero sample, ∼1 mg/L) or containing additional 0.1, 0.2, 0.3, 0.4, and 0.5 mg/L of 1, respectively. Each experiment was repeated three times with triple injection. Calibration curve, obtained by linear regression analysis of the peak area versus concentration, showed a linear response with a correlation coefficient of R2 = 0.9969. Stable Isotope Dilution Assay (SIDA) for the Analysis of (E)-Resveratrol (1) by Means of HPLC-MS/MS. An ethanolic solution of (E)-[2H3]-resveratrol (100 μL, 50 mg/L) was added to red wine samples (1 mL), equilibrated for 3 min on a vortexer at room temperature, then 1:10 diluted with aqueous ethanol (13% ethanol in water), and, finally, aliquots (5 μL) were analyzed by means of HPLC-MS/MS. The peak areas obtained from the mass transition m/z 226.9f143.0 for (E)-resveratrol (1) and m/z 229.9f143.9 for (E)-[2H3]-resveratrol ([2H3]-1) were used for quantitation. Calibration. Ethanolic solutions of (E)-resveratrol (5 mg/L) and (E)-[2H3]-resveratrol (5 mg/L) were mixed in five molar ratios from 0.1 to 10 (0.5
50 μg/mL in EtOH), vortexed, and 1:10 diluted with aqueous ethanol (13% ethanol in water). Then, aliquots (5 μL) were analyzed by means of HPLC-MS/MS. Calibration curves were prepared by plotting peak area ratios of analyte to internal standard against concentration ratios of each analyte to the internal standard using linear regression. The equation obtained was y = 1.5288x
0.0610 (1/[2H3]-1, R2 = 0.9999). Method Validation. For recovery experiments, a sample of Cabernet Sauvignon (2007) was diluted 1:25 with aqueous ethanol (13% ethanol in water), and aliquots (9.9 mL) were spiked with ethanolic solutions (100 μL) containing (E)-resveratrol in different concentrations to give wine samples with spiked (E)-resveratrol levels of 0.05, 0.1, and 0.2 mg/L. Aliquots of these spiked wine samples (950 μL) were mixed with ethanolic internal standard solution of (E)-[2H3]-resveratrol (50 μL, 1 mg/L), vortexed for 3 min, and analyzed by means of HPLC-MS/MS. For repeatability of the method, a sample of Cabernet Sauvignon

ARTICLE

(2007) was analyzed in three independent workups as described above using three replicates in one day. Relative standard deviation of these nine replicates was (7.1%. Limit of detection (LOD) and limit of quantitation (LOQ) were calculated as described above. HPLC-MS/MS Quantitation of (E)-Resveratrol (1) by Means of External Calibration and Standard Addition. For external quantitation, a sample of Cabernet Sauvignon (2007) was diluted (1:10 or 1:100) with a mixture of ethanol and water (13/87, v/v), and aliquots (10 μL) of the diluted or the nondiluted red wine samples were analyzed via HPLC-MS/MS. Each dilution was carried out and injected in triplicate. The amount of (E)resveratrol was calculated using a seven-point response curve which had been determined by analysis of solutions containing defined amounts of (E)-resveratrol (1) in different concentrations ranging from 0.0003 to 0.3 mg/L. Calibration curve, obtained by linear regression analysis of the peak area versus concentration, showed a linear response with a correlation coefficient of R2 > 0.9996. Standard addition was carried out exactly as described above. Diluted wine (5 μL, 1:10) was analyzed via HPLC-MS/MS. Calibration curve, obtained by linear regression analysis of the peak area versus concentration, showed a linear response with a correlation coefficient of R2 > 0.9896. Ultra-Performance Liquid Chromatography/Time-ofFlight Mass Spectrometry (UPLC/TOF-MS). Mass spectra of the compounds were measured on a Waters Synapt G2 HDMS mass spectrometer (Waters, Manchester, U.K.) coupled to an Acquity UPLC core system (Waters, Bedford, MA) consisting of a binary solvent manager, sample manager, and column oven. Aliquots (1 μL) of ethanolic solutions of the analytes 1 and 2 and the internal standard [2H3]-1 or red wine samples (2 μL) were injected into the UPLC-TOF-MS system equipped with a BEH C18, 2 mm  150 mm, 1.7 μm, column (Waters, Manchester, U.K.). Operated with a flow rate of 0.3 mL/min at a temperature of 40 °C, the following gradient was used for chromatography: starting with a mixture (35/95, v/v) of acetonitrile (0.1% HCOOH) and aqueous formic acid (0.1% HCOOH), the acetonitrile content was increased to 95% within 3 min and, then, kept constant for 1 min. Scan time for the MSE method (centroid) was set to 0.1 s. Measurements were performed using negative ESI and the resolution mode consisting of the following ion source parameters: capillary voltage
2.0 kV, sampling cone 20, extraction cone 4.0, source temperature 150 °C, desolvation temperature 450 °C, cone gas 30 L/h, and desolvation gas 850 L/h. Data processing was performed by using MassLynx 4.1 SCN 779 (Waters) and the elemental composition tool for determining the exact mass. QuanLynx (Waters) was used for peak detection and integration of the exact mass of (E)-resveratrol (1, m/z 227.0708) and (E)-[2H3]-resveratrol ([2H3]-1, m/z 230.0896) with a chromatogram mass window of 20mDa. All data were lock mass corrected on the pentapeptide leucine enkephaline (Tyr-Gly-Gly-Phe-Leu, m/z 554.2615, [M
H]
) in a solution (2 ng/μL) of acetonitrile/0.1% formic acid (1/1, v/v). Scan time for the lock mass was set to 0.3 s, an interval of 15, and 3 scans to average with a mass window of (0.3 Da. Calibration of the Synapt G2 in the range m/z 50
600 was performed using a solution of sodium formate (5 mmol/L) in 2-propanol/water (9/1, v/v). High Performance Liquid Chromatography
Triple Quadrupole Mass Spectrometry (HPLC-MS/MS TripleQuad). HPLC-MS/MS analysis was performed using a Dionex UltiMate 3400

dx.doi.org/10.1021/ac103305s |Anal. Chem. 2011, 83, 3398–3405

Analytical Chemistry 3000 HPLC-System (Dionex, Idstein, Germany), consisting of a binary pump system, an autosampler, and a column compartment connected to the API 4000QTrap triple quadrupole mass spectrometer (AB SCIEX, Darmstadt, Germany). Running in the negative electrospray ionization (ESI
) mode, the ion source parameters were set as follows: ion spray voltage
4500; source temperature 400 °C; zero grade air served as nebulizer gas (45 psi) and as turbo gas for solvent drying (55 psi); nitrogen served as curtain (20 psi) and collision gas (8.7  10
7 psi). Both quadrupoles were set at unit resolution. The MS/MS parameters were tuned for each individual compound, detecting the fragmentation of the [M
H]
molecular ions into specific product ions after collision with N2 (8.7  10
7 psi). By means of the multiple reaction monitoring (MRM) mode, resveratrol (m/z 226.9f185.0; DP,
65 V; EP,
10 V; CE,
26 V; CXP,
11 V; m/z 226.9f143.0; DP,
65 V; EP,
10 V; CE,
38 V; CXP,
7 V) and trans-[2H3]-resveratrol (m/z 229.9f143.9; DP,
70 V; EP,
10 V; CE,
36 V; CXP,
7 V; m/z 229.9f186.9; DP,
70 V; EP,
10 V; CE,
26 V; CXP,
11 V) were analyzed for a duration of 150 ms using the mass transitions, declustering potential (DP), entrance potential (EP), collision energy (CE), and cell exit potential (CXP), each given in parentheses. The column utilized for separation was a Synergi-Fusion RP-18 column, 2 mm  150 mm, 5 μm (Phenomenex, Aschaffenburg, Germany) with a flow rate of 0.25 mL/min. For gradient elution, a binary system of 0.1% aqueous formic acid (solvent A) and methanol containing 0.1% formic acid (solvent B) was applied. Chromatography was performed starting with 40% solvent B for 1 min, followed by a linear gradient to 75% within 6 min. Then, solvent B was increased to 100% within 1 min, and after keeping for 2 min, solvent B was decreased to 40% within 4 min. Temperature of the column compartment was set at 25 °C and autosampler temperature was 8 °C. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR spectroscopy was performed on an Avance III 500 MHz spectrometer with a TCI probe (Bruker, Rheinstetten, Germany). For quantitative measurement of remaining protium abundance in (E)-[2H3]-resveratrol, relaxation delay [D1] was set to 10 s with a P1 of 90° and 7.74 μs for full relaxation of all protons. Chemical shifts were referenced to TMS. Data processing was performed by using Topspin version 1.3 (Bruker, Rheinstetten) and MestReNova version 5.2.3 software (Mestrelab Research, Santiago de Compostela, Spain).

’ RESULTS AND DISCUSSION To enable a robust and accurate method for the high-throughput quantitation of (E)-resveratrol (1) in foods and wine, a stable isotope dilution assay (SIDA) was developed using ultra-performance liquid chromatography (UPLC) coupled to an hybrid quadrupole orthogonal acceleration time-of-flight (oa-TOF) mass spectrometer employing QuanTOF technology.30

Hemisynthetic Preparation of (E)-[2H3]-resveratrol, [2H3]-1. For the hemisynthesis of (E)-[2H3]-resveratrol by means of protium/deuterium exchange, a solution of 1 in acetonitrile/deuterium oxide was thermally treated in the presence of Amberlyst 15 as a catalyst. After cleanup by means of solid-phase extraction, the incorporation of deuterium into the target molecule was confirmed by means of mass spectrometry and 1H NMR spectroscopy. Comparison of the high-resolution MS spectrum (ESI
) of (E)-resveratrol (Figure 2A) with the MS data recorded for [2H3]1 (Figure 2B) showed a mass increase of the pseudomolecular

ARTICLE

Figure 2. UPLC-TOF-MS spectrum (ESI
) of (A) (E)-resveratrol (1) and (B) (E)-[2H3]-resveratrol ([2H3]-1).

ion m/z 227.0708 ([M
H]
) by three deuterium atoms to m/z 230.0913, thus confirming (E)-[2H3]-resveratrol as the predominant isotopologue. The isotopic distribution of [2H3]-1 detected by high-resolution MS was found to be 0.3% natural [2H]abundant, 0.6% [2H1]-1, 6.3% [2H2]-1, and 92.8% [2H3]-1. In order to confirm these data and to locate the deuterium atoms in the structure of the target molecule, the 1H NMR spectrum of 1 (Figure 3A) was compared with the spectrum recorded after H/D exchange. As displayed in Figure 3B, the presence of three deuterium atoms were clearly identified in the A-ring of (E)-3,5,40 -trihydroxy-2,4,6-trideuterostilbene, [2H3]-1, showing only minor proton abundance of 1% at position 4 and 9% at positions 2 and 6, respectively. Because only 0.3% natural [2H]-abundant 1 was found to be present in the synthesized d3-1, less than 0.2% of the fourth isotopic signal of 1 (m/z 230.0780) was detectable, and the difference of the fourth isotopic signal of 1 and the monoisotopic signal of the 3-fold labeled d3-1 was calculated to be >18 mDa and measured to be at least >11 mDa. The calibration of 1 for quantitative UPLC-QuanToF-MS via high-resolution mass spectrometry was achieved using the monoisotopic exact mass m/z 227.0708 ([M
H]
) of nonlabeled (E)-resveratrol (1) and the exact mass m/z 230.0913 ([M
H]
) of the 3-fold deuterated (E)-[2H3]-resveratrol ([2H3]-1). To check the stability of (E)-[2H3]-resveratrol in a winelike acidic hydroalcoholic medium, a solution of [2H3]-1 in water/ ethanol acidified with tartaric acid to pH 3.5 was kept at room temperature or in the autosampler at 10 °C in the dark and was, then, analyzed by means of UPLC-TOF-MS. Storage at room temperature for 2, 20, and 40 h induced a slight decrease of the signals recorded for (E)-[2H3]-resveratrol by 0.4%, 6.0%, and 3401

dx.doi.org/10.1021/ac103305s |Anal. Chem. 2011, 83, 3398–3405

Analytical Chemistry

ARTICLE

Figure 3. 1H NMR spectra (500 MHz; d3-MeOD) of (A) (E)-resveratrol (1) and (B) (E)-[2H3]-resveratrol.

9.0%, whereas the signals of the 2-fold deuterated isotopologue for (E)-[2H2]-resveratrol increased in intensity by 0.4%, 5.0%, and 7.5%, respectively. The same trend could be observed for the singly deuterated (E)-resveratrol isotopologue increasing by 0.0%, 1.0%, and 1.5%, respectively. The intensity for the nonlabeled (E)-resveratrol in [2H3]-1 stayed constant. These data indicated a partial H/D re-exchange of [2H3]-1 when stored at room temperature for more than 2 h under winelike conditions. In comparison, incubation of [2H3]-1 for 2 h in the autosampler cooled to 10 °C gives a constant signal intensity of all isotopic signals with 12 repetitive injections over 120 min with a constant ratio of area (2.9% RSD ([2H3]-1, m/z 230.0896)/area (1, m/z 227.0708). These data demonstrate the stability of [2H3]-1 under winelike conditions for at least 2 h at 10 °C and confirm the suitability of [2H3]-1 as an internal standard to be used for SIDA. Development of a Stable Isotope Dilution Analysis (SIDA). Optimization of the chromatographic conditions enabled sufficient separation of (Z)- and (E)-resveratrol during UPLC analysis in less than 3 min (Figure 4) and their sensitive detection by means of the QuanTOF technology.30 To convert the measured ion intensities into the mass ratios of the internal standard [2H3]-1 and the analyte (E)-resveratrol (1), a graph was calculated from calibration mixtures of known mass ratios and the corresponding peak area ratios in UPLC-QuanTOF-MS. In order to check the performance of the developed UPLC-QuanTOF-MS method linearity, trueness, intraday and interday precision, sensitivity, and selectivity were investigated. Calibration curve, obtained by linear regression analysis of the peak area versus concentration, showed a linear response with a correlation coefficient of >0.999. Cabernet Sauvignon (2007) was spiked with a defined amount of [2H3]-1 as the internal standard,

followed by equilibration at room temperature. UPLC-QuanTOFMS analysis revealed a concentration of 1.08 ( 0.02 RSD mg/L (Table 1). To check the trueness of the analytical method, recovery experiments were performed in the following. Defined amounts of (E)-resveratrol (1) were added to the red wine sample Cabernet Sauvignon (2007) in five different concentrations prior to analysis by means of UPLC-QuanTOF-MS, and the amounts determined were compared with those found in the blank wine sample as the control (Table 1). The average recovery rate, calculated on the basis of the content of 1 added to the wine prior to analysis, was found to be 96.2% ( 0.8% RSD. These data demonstrate the developed SIDA in combination with UPLCQuanTOF-MS as a reliable tool enabling a rapid and accurate quantitative determination of (E)-resveratrol (1) in wine. Cross-Validation of the UPLC-QuanTOF-MS SIDA Method. To further validate the developed UPLC-QuanTOF-MS method, a cross-validation experiment was performed by comparing the quantitative data obtained for (E)-resveratrol (1) in four different wines using the UPLC-QuanTOF-MS-based SIDA developed above with the data obtained by SIDA using an HPLC-triple quadrupole mass spectrometer and with those found by means of external calibration and standard addition, respectively, on both mass spectrometers (Table 2). Comparing the results obtained by SIDA on both mass spectrometers revealed similar concentrations of 1 in the red wines, e.g., from 5.6% (Cabernet Sauvignon 2007) to 25.4% (Shiraz 2005) difference calculated in relation to the higher concentration of 1. Quantitation based on the standard addition method performed either with the triple quadrupole or the TOF/MS system revealed 101.6% or 97.7% of 1 when compared to the data obtained by means of the SIDA. In conclusion, standard addition 3402

dx.doi.org/10.1021/ac103305s |Anal. Chem. 2011, 83, 3398–3405

Analytical Chemistry

ARTICLE

in combination with HPLC-MS/MS or TOF/MS revealed a similar concentration as found with the SIDA, thus strengthening the accuracy of the method. Quantitative analysis of 1 by means of external calibration revealed only 12.7% or 32.4% of the concentrations found by means of the SIDA, thus demonstrating ion suppression by severe matrix interferences. In order to visualize such matrix effects, a constant flow of a solution of (E)-resveratrol (1) was introduced into the UPLC-TOF-MS system via a syringe pump during the analysis of red wine. As shown in Figure 4A,B severe influence of eluting matrix compounds on the ionization of (E)resveratrol (1) could be observed depending on the retention time, thus indicating that the use of an isotopic labeled internal

standard is highly recommended to compensate for matrix effects during LC-MS analysis. Furthermore, such matrix influences could be observed by comparing the amount of 1 obtained by means of SIDA and external calibration using HPLC-MS/MS. The matrix influences were found to be strongly depending on the dilution of the injected red wine. When compared to the data obtained by means of SIDA, only 12.7%, 58.8%, and 83.3% of the concentrations of 1 were found in the nondiluted, the 1:10, and the 1:100 diluted red wine samples, respectively (Table 2). As these matrix constituents are expected to be different for each wine sample depending on variety of the grapes, soil and climate conditions, and oak maturation, the use of the stable isotope labeled internal standard helps to overcome these challenges, thereby enabling a high-throughput analysis of (E)-resveratrol in red wines. In summary, this cross validation confirmed again the suitability of the internal standard and the capability of accurate quantitation by means of UPLC-QuanTOF-MS. The repeatability of the UPLC-QuanTOF-MS method for 1 was studied by analyzing Dornfelder (2005) aged in oak barrels in three independent workups using four replicates in one day with a relative standard deviation of (3.0% (Table 2). Reproducibility for 1 was studied with Bordeaux (2003), in which two bottles of this wine were independently analyzed with a difference of two months in three independent workups using three replicates for each concentration in one day with a relative standard deviation of (4.0% (Tables 2 and 3). The base peak ion (BPI) chromatogram of Bordeaux (2003) is shown in Figure 5A, whereas the mass chromatograms showing the exact mass for the analyte 1 and [2H3]-1 are given in Figure 5B,C. These data clearly demonstrate the developed UPLC-QuanToF-MS SIDA method as a reliable tool enabling a rapid and accurate quantitative determination of (E)resveratrol (1) in wine. In order to check as to whether the calibration curve obtained for 1/[2H3]-1 is enabling also the quantitation of (Z)-resveratrol (2), the mass spectrometric response of the two geometric isomers was investigated. Therefore, fixed amounts of 1 and 2 were mixed in ratios of 0.1
10 on a molar basis, and the ratios of areas obtained by UPLC-QuanTOF-MS were plotted against the quotient of concentrations to give the equation y = 0.5884x
0.0413 (1/2), clearly demonstrating a 1.7-fold increased MS-response of 1 when compared to 2. Consequently, quantitation of diastereomer 2 was performed via a new calibration curve (2/[2H3]-1). Quantitation of (E)-(1) and (Z)-Resveratrol (2) in Red Wines by Means of SIDA-UPLC-QuanTOF-MS. The developed UPLC-QuanTOF-MS SIDA method was applied to the analysis

Figure 4. (A) UPLC-TOF-MS base peak ion chromatogram (ESI
) and (B) exact mass trace (mass window = 10mDa) of (E)-resveratrol recorded for Bordeaux (2003), while a continuous flow of (E)-resveratrol (1) was introduced into the UPLC system by means of a syringe pump.

Table 1. Determination of the Recovery Rates for the Quantitative Analysis of (E)-Resveratrol (1) in Fortified Red Wine (Cabernet Sauvignon, 2007) by Means of a Stable Isotope Dilution Analysis (SIDA) amount added [mg/L] tQ

TOF

tQ

0.05

1.38

0.10 0.22

a

b

conc calcd [mg/L]

a

a

conc determined [mg/L] b

a

b

TOF

recovery [%] a

tQ

TOF

recovery [%] av b

TOF

tQ

0.039 ( 0.002

1.08 ( 0.02

0.089 ( 0.002

2.46 ( 0.02

0.085 ( 0.002

2.38 ( 0.00

95.9 ( 4.1

96.7 ( 0.1

2.22

0.140 ( 0.002

3.30 ( 0.02

0.131 ( 0.006

3.25 ( 0.05

93.6 ( 3.9

98.4 ( 1.4

3.39

0.259 ( 0.002

4.47 ( 0.02

0.246 ( 0.004

4.30 ( 0.02

95.2 ( 1.8

tQa

TOFb

94.9 ( 3.1

96.2 ( 0.8

96.2 ( 0.5

4.38

5.46 ( 0.02

5.16 ( 0.02

94.5 ( 0.4

5.25

6.33 ( 0.02

6.02 ( 0.09

95.1 ( 1.5

tQ = triple quadrupole mass spectrometry. b TOF = time-of-flight mass spectrometry. 3403

dx.doi.org/10.1021/ac103305s |Anal. Chem. 2011, 83, 3398–3405

Analytical Chemistry

ARTICLE

Table 2. Quantitation of (E)-Resveratrol (1) via Different MS-Instruments and Techniques concentration (mg/L) SIDAa

Cabernet Sauvignon (2007)

standard addition

external calibration

tQb

TOFc

tQb

TOFc

tQb

TOFc

1.02 ( 0.07

1.08 ( 0.02

1.03 ( 0.10

1.05 ( 0.02

not diluted 0.13 ( 0.01

0.35 ( 0.01

Δ5.6%d 1:10 dil:e 0.60 ( 0.01 1:100 dil:e 0.85 ( 0.05 Dornfelder (2007)

1.72 ( 0.04

1.43 ( 0.06

Δ17.3%d Bordeaux (2003)

5.51 ( 0.21

5.04 ( 0.14

Shiraz (2005)

Δ8.5%d 1.06 ( 0.03

0.79 ( 0.04

Δ25.4%d LODf

0.0009

0.04

LOQf

0.0030

0.14

repeatability

7.1%

3.0%

reproducibility

n.d.g

4.0%

a SIDA = stable isotope dilution analysis. b tQ = triple quadrupole mass spectrometry. c TOF = time-of-flight mass spectrometry. d Differences were calculated in relation to the higher concentration. e Diluted with aqueous ethanol (13%, v/v). f Limit of detection (LOD) and limit of quantitation (LOQ) were calculated as the concentrations for which signal-to-noise ratios were 3 and 10, respectively. g n.d. indicates not determined.

Table 3. Quantitation of (E)- (1) and (Z)-Resveratrol (2) via SIDA-UPLC-Quan-TOF-MS conc (mg/L) 1

2

Dornfelder barique, 2005

1.26 ( 0.04

0.41 ( 0.02

Dornfelder, 2005

0.61 ( 0.02