Fast and Simple Analysis of Polyphenols In
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TN-1166 APPLICATIONS Fast and Simple Analysis of Polyphenols in Red Wine Using Luna® 3 µm C8(2) by LC/MS/MS Matt Trass, Sueki Leung, and Allen Misa Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA A simple and fast method for the analysis of polyphenols in red Figure 1. wines has been developed. The method can be used for wine Structures of Abundant Red Wine Polyphenols authentication, as the polyphenol character of a wine can indicate both its varietal and region of origin. Gallic acid (MW: 170.12) Coumaric acid (MW: 164.16) HO O HO O Introduction Polyphenols in red wines are widely recognized for the health benefits from their antioxidant properties. Studies have shown that moderate red wine consumption has been attributed to a decreased risk of heart attacks and certain cancers. Red wine is also thought to lower cholesterol and blood pressure. HO OH In addition to the important health benefits, the polyphenol profile OH of a red wine contributes to the flavor, mouth-feel and color. Aside from these aesthetic qualities, the polyphenol character of a wine indicates the region and variety of grapes used to produce the wine. OH The locality of the grapes used to produce a wine is significant with regards to labeling. In fact, most major wine making regions Resveratrol (MW: 228.24) Caffeic acid (MW: 180.16) have stringent labeling regulations regarding the percentage and OH HO O origin of grapes used to produce a wine. In extreme cases, cheaply produced wines are labeled fraudulently as being from a more highly-regarded wine region. Subsequently, there is a lot of interest in monitoring polyphenols in wine for research, quality control, and fraudulent investigation. The most sensitive way to monitor polyphenols is by using high performance liquid chromatography coupled with a mass- spectrometer. This present method utilizes a simple dilute-and- shoot sample preparation procedure, followed by LC/MS/MS HO analysis in the negative mode using a Phenomenex Luna 3 µm C8(2) column. Several different wine varietals from different wine making HO OH OH regions were analyzed. In each wine the observed polyphenolic compounds (Figure 1) were quantified against a calibration curve. Ferulic acid (MW: 194.06) Catechin (MW: 290.27) The quantitative results were then used to compare the polyphenolic H O profile of each wine with respect to its variety and location. 3 O OH H3O HO HO O HO O OH HO OH OH O O Myricetin (MW: 318.24) Quercetin (MW: 302.24) OH OH OH HO O HO O OH OH OH OH OH O OH O For additional technical notes, visit www.phenomenex.com Page 1 of 8 TN-1166 Materials and Methods Results and Discussion Reagents and Chemicals Figure 2. LC/MS Ion Chromatogram of Red Wine Polyphenols All reagents and solvents were HPLC or analytical grade. HPLC 1.00e6 1 Grade methanol was purchased from Honeywell, Burdick & Jackson (Muskegon, MI), Milli-Q® water was used for to prepare 9.00e5 the LC mobile phase and for diluting samples. Analytical grade 8.00e5 standards were purchased from Sigma-Aldrich. 7.00e5 4 Equipment and Materials 6.00e5 2 5,6 Agilent® 1200 Series HPLC (Agilent Technologies Inc., Santa Clara, 5.00e5 CA, USA) was interfaced with AB SCIEX API 4000™ MS/MS with Intensity, cps Intensity, 4.00e5 ESI TurboIonSpray® (AB SCIEX, Framingham, MA, USA). 3.00e5 3 Sample Preparation 2.00e5 10 The wine samples were prepared as follows: 1.00e5 7 8 9 APP ID 22019 1. In an autosampler vial, dilute 10 µL of red wine sample to 0.00 1 2 3 4 min 1000 µL with D.I. water (100:1 dilution). Note: Jacob’s Creek Pinot Noir (2007) diluted 100:1 2. Add internal standard. This study involved LC/MS/MS analysis of 27 red wines from 4 different wine making regions. Of the 27 wines, 3 different varietals: LC/MS/MS Conditions Merlot, Pinot Noir and Cabernet Sauvignon were evaluated. For Column: Luna® 3 μm C8(2) 100 Å each wine, 10 polyphenols were quantified by comparison to a neat Dimensions: 50 x 2.0 mm calibration curve (Figures 5 & 6). The quantitative data was then Part No.: 00B-4248-B0 used to create a polyphenol profile for each wine(Figure 9.). The Mobile Phase: A: 5 mM Ammonium acetate in Water polyphenol profile of each wine was then evaluated with respect to with 0.5 % Acetic acid its varietal and location. B: 5 mM Ammonium acetate in Methanol with 0.5 % Acetic acid Figure 2 shows a chromatogram for a Pinot Noir sample (Jacob’s Flow Rate: 0.5 mL/min Creek®, Australia) monitoring 10 different polyphenols. These 10 Gradient: Time (min) %B polyphenols were chosen due to their abundance in red wines and 0.00 2 3.00 80 they represent different classes of polyphenols. All analyte peaks 5.00 80 elute in less than 4 minutes with a total analysis time of 8 minutes 5.01 2 - including column equilibration. Aside from the fast analysis time, 8.00 2 Luna 3 µm C8(2) is chosen for this analysis because it provides Injection Volume: 5 µL high efficiency and good peak shape. Peak shape and efficiency Temperature: Ambient are important for separation of the isomeric polyphenols such MS/MS Detection: API 4000 MS/MS, ESI negative (ESI-) as catechin/epicatechin and trans-resveratrol/cis-resveratrol (as shown in Figures 3 & 4.). The ability to separate and monitor polyphenol isomer ratios gives key information about the location Peak No. Analyte Q1 Q3 RT (min) that a wine was produced. 1 Gallic acid 169.1 125/78.6 0.89 2 Catechin 289.0 245/203 2.08 3 Epicatechin 289.0 245/203 2.38 4 Caffeic acid 179.0 135/117 2.46 5 p-Coumaric acid 163.1 119/93 2.85 13 5 p-Coumaric- C3 (I.S.) 165.8 120.7/119 2.85 6 Ferulic acid 193.0 134/178 2.86 7 trans-Resveratrol 227.0 143/159 3.09 13 7 trans-Resveratrol- C6 (I.S.) 233.0 190.7/164.8 3.09 8 Myricetin 317.0 151/179 3.15 9 cis-Resveratrol 227.0 143/159 3.38 10 Quercetin 301.0 151/179 3.43 Page 2 of 8 TN-1166 Figure 3. Separation of trans-Resveratrol and cis-Resveratrol Figure 5. trans-Resveratrol Calibration curve, 0.9998 R2 isomers 1 8.0 4.0e4 2 7.0 3.0e4 6.0 5.0 4.0 2.0e4 Intensity, cps Intensity, 3.0 Analyte Area / IS Area Analyte Area 2.0 1.0e4 1.0 APP ID 22020 0.0 0.0 100 200 300 400 500 600 700 800 900 1000 1 2 3 4 min Analyte Conc. / IS Conc. Figure 4. Separation of Catechin and Epicatechin Isomers Figure 6. p-Coumaric acid Calibration curve, 0.9994 R2 6.0e4 1 9.0 5.0e4 8.0 7.0 4.0e4 6.0 5.0 3.0e4 2 4.0 Intensity, cps Intensity, 2.0e4 3.0 Analyte Area / IS Area Analyte Area 2.0 1.0e4 1.0 APP ID 22021 0.0 0.0 1 2 3 4 min 100 200 300 400 500 600 700 800 900 1000 Analyte Conc. / IS Conc. For additional technical notes, visit www.phenomenex.com Page 3 of 8 TN-1166 Figure 7 shows the polyphenol profiles of three representative Merlots from three different wine making regions. Derived from the quantitative results for the 27 wines, certain polyphenol profile features were linked to both the varietal and the location of the winery. Figure 7a shows a typical polyphenol profile for a Merlot produced in Chile. The profile displays high levels of myricetin and quercetin. In comparison, the Californian and French Merlots (Figures 7b & 7c) display lower levels of myricetin and quercetin. However, the Californian merlots contain approximately equal levels of myricetin and quercetin, where as the French and Chilean merlots typically contain at least a 2:1 myricetin:quercetin ratio. In most instances gallic acid levels were high. Gallic tannins are largely present in red wines due to external addition for stabilization or are extracted into the wine from aging in wooden casks1. Therefore, while gallic acid levels are important, they cannot be relied upon to identify the location or varietal of a wine. Figure 7. Polyphenol profiles of three representative Merlots from three different wine making regions a. Representative Chilean Merlot c. Representative French Merlot Vistamar 220 Saint Antoine 200 170 150 120 µg/mL 100 µg/mL 70 50 20 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Polyphenol Polyphenol b. Representative Californian Merlot Canyon Oaks® 200 150 µg/mL 100 50 0 1 2 3 4 5 6 7 8 9 10 Polyphenol Page 4 of 8 TN-1166 Figure 8 compares the polyphenol profiles of the 3 different varietals studied. Glass Mountain is shown in Figure 8a as a representative Merlot. It has moderate levels of most polyphenols with low levels of resveratrol and ferulic acid. In contrast, Figure 8b the Pinot Noirs analyzed, display characteristically high levels of the catechin isomers and caffeic acid with relatively low levels of myricetin and quercetin. Finally, Figure 8c shows the polyphenol profile for a typical Cabernet Sauvignon. This wine varietal displays relatively low levels of catechin and epicatechin, but relatively elevated myricetin and quercetin levels. Figure 8. Polyphenol profiles of 3 different varietals studied a. Representative Merlot polyphenol profile c.