TN-1166 APPLICATIONS Fast and Simple Analysis of Polyphenols in Red 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. 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 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 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 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

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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 (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 , Pinot Noir and 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

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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.

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Figure 7 shows the polyphenol profiles of three representative 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

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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. Representative Cabernet Sauvignon polyphenol profile

220 200 Glass Mountain® Razor’s Edge

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 Pinot Noir polyphenol profile 220 Cloud Break®

170

120 µg/mL

70

20

1 2 3 4 5 6 7 8 9 10 Polyphenol

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Figure 9 shows the polyphenol profile summary of all 27 wines evaluated with respect to its varietal and location. Based on the mean values, all 3 varietals contain similar amounts of gallic acid. Pinot Noir is the most abundant in catechin, epicatechin, caffeic acid, and cis/trans resveratrol.

Figure 9. Polyphenol content of 27 red wines from 4 different wine making regions in µg/mL

Gallic Caffeic p-Coumaric Ferulic trans- cis- Wine Varietal Region Brand acid Catechin Epicatechin acid acid acid Resveratrol Resveratrol Myrcetin Quercetin Merlot California Glass Mountain® 146.00 46.80 43.10 15.60 8.44 0.90 0.41 0.45 69.80 58.30 California Canyon Oaks® 124.00 49.00 40.70 0.15 2.95 0.28 0.46 0.71 24.00 15.90 California Double Dog Dare® 114.00 45.70 37.30 6.03 3.54 0.38 1.35 1.73 32.70 15.70 Chile Vermonte 161.00 38.50 24.70 12.40 5.48 0.58 2.03 1.21 102.00 63.50 Chile Vistamar 156.00 33.70 20.80 18.00 3.25 0.24 0.66 0.76 113.00 45.40 Chile Vistamar 184.00 55.50 29.90 22.70 10.70 0.47 0.85 0.98 193.00 77.00 France Mordoree 220.00 59.00 32.70 18.00 6.74 0.52 0.57 0.80 157.00 36.70 France Saint Antoine 150.00 58.30 44.60 8.94 7.12 0.18 4.83 4.17 81.70 15.00 France Luc Pirlet 118.00 49.70 31.30 1.06 2.41 0.34 3.55 2.45 90.50 11.60 Mean 152.56 48.47 33.90 11.43 5.63 0.43 1.63 1.47 95.97 37.68 St. Dev. 338.49 85.64 81.96 79.19 28.43 2.20 15.69 11.83 542.76 246.23 Pinot Noir California Castle Rock® 126.00 95.90 51.40 32.30 6.09 1.01 4.04 5.12 45.10 51.90 California Cloud Break® 155.00 97.90 68.60 34.80 4.60 0.99 2.29 3.46 9.20 15.60 California Crane Lake® 123.00 102.00 67.70 16.40 2.44 0.55 1.24 1.17 13.90 30.20 France Ropiteau® 123.00 76.40 41.00 10.60 3.67 0.56 2.00 1.18 65.20 35.60 France D'autrefois 144.00 105.00 57.50 22.10 5.12 0.51 5.05 3.36 69.10 37.40 France Ropiteau® 126.00 88.30 51.30 18.10 4.15 0.44 4.39 2.69 36.00 23.50 Australia Jacob's Creek® 154.00 93.10 64.90 5.01 2.67 0.62 3.74 0.90 36.40 35.10 Australia Little Penguin® 134.00 37.30 24.80 3.09 4.70 0.67 1.85 1.90 50.80 24.40 New Zealand Brancott® 140.00 101.00 61.10 10.40 2.68 1.00 7.19 4.99 24.20 10.80 Mean 136.11 88.54 54.26 16.98 4.01 0.71 3.53 2.75 38.88 29.39 St. Dev. 127.81 210.44 142.19 111.82 12.52 2.31 18.94 16.06 209.81 124.40 Cabernet Sauvignon Australia Razor's Edge 122.00 21.00 13.00 0.92 5.04 0.55 1.62 0.57 151.00 55.70 Australia Pillar 89.70 24.60 14.60 5.81 8.99 0.68 1.20 0.75 264.00 47.50 Australia Yellow Tail® 126.00 26.20 15.70 0.35 3.51 0.38 0.59 0.68 214.00 46.80 California BV® 139.00 39.40 24.90 11.80 3.07 0.65 0.19 0.43 112.00 24.30 California Dancing Bull® 85.90 28.60 15.70 3.93 4.28 0.63 0.19 0.57 121.00 32.30 California Beringer® 123.00 30.40 19.70 6.62 5.11 0.82 0.24 0.52 82.50 16.80 Chile Aresti® 197.00 43.00 27.90 12.70 6.57 0.51 1.37 0.63 136.00 51.40 Chile Los Vascos® 123.00 31.20 18.10 8.55 3.66 0.37 0.38 0.50 135.00 36.60 Chile Root 1® 190.00 48.10 28.40 14.80 6.27 0.69 1.24 0.74 144.00 55.50 Mean 132.84 32.50 19.78 7.28 5.17 0.59 0.78 0.60 151.06 40.77 St. Dev. 384.77 90.61 58.67 51.27 18.72 1.48 5.73 1.09 552.06 140.07

Conclusion References Polyphenol analysis of red wines gives important information about 1. Jaitz, L., Siegl, K., Eder, R., Rak, G., Abranko, L. Gunda, a wines varietal and region. These compounds are best analyzed K., Hann, S., (2010). LC-MS/MS analysis of phenols for using a high efficiency HPLC media such as Luna® 3 µm C8(2). The classification of red wine according to geographic origin, Luna 3 µm C8(2) results in a fast analysis time (8 minutes including variety and vintage. Food Chemistry., 122, p366-372. column equilibration) and resolution of the isomeric polyphenols.

The analysis of 27 red wines gave quantitative data, resulting in unique polyphenol profiles for each wine. Through comparison of the polyphenol profiles, common features can be linked to both the varietal and location of each wine. Along with other methods of authentication such as isotope ratio and multi-element based classification, the polyphenol profiles can be used to support a polyphenol investigation.

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Ordering Information Luna® HPLC Columns 3 μm Microbore and Minibore Columns (mm) SecurityGuard Cartridges (mm) Phases 50 x 1.0 150 x 1.0 30 x 2.0 50 x 2.0 100 x 2.0 150 x 2.0 4 x 2.0* /10pk C8(2) 00B-4248-A0 00F-4248-A0 00A-4248-B0 00B-4248-B0 00D-4248-B0 00F-4248-B0 AJ0-4289 for ID: 2.0-3.0 mm

SecurityGuard™ 3 μm Narrow Bore and Analytical Columns (mm) Cartridges (mm) Phases 30 x 3.0 50 x 3.0 150 x 3.0 30 x 4.6 50 x 4.6 75 x 4.6 100 x 4.6 150 x 4.6 4 x 2.0* 4 x 3.0* /10pk /10pk C8(2) 00A-4248-Y0 00B-4248-Y0 00F-4248-Y0 00A-4248-E0 00B-4248-E0 00C-4248-E0 00D-4248-E0 00F-4248-E0 AJ0-4289 AJ0-4290 for ID: 2.0-3.0 mm 3.2-8.0 mm

SecurityGuard™ 5 μm Microbore and Minibore Columns (mm) Cartridges (mm) Phases 50 x 1.0 150 x 1.0 30 x 2.0 50 x 2.0 150 x 2.0 250 x 2.0 4 x 2.0* /10pk C8(2) 00B-4249-A0 00F-4249-A0 00A-4249-B0 00B-4249-B0 00F-4249-B0 00G-4249-B0 AJ0-4289 for ID: 2.0-3.0 mm

5 μm Narrow Bore and Analytical Columns (mm) SecurityGuard™ Cartridges (mm) Phases 50 x 3.0 150 x 3.0 250 x 3.0 30 x 4.6 50 x 4.6 75 x 4.6 4 x 2.0* 4 x 3.0* /10pk /10pk C8(2) 00B-4249-Y0 00F-4249-Y0 00G-4249-Y0 00A-4249-E0 00B-4249-E0 00C-4249-E0 AJ0-4289 AJ0-4290 for ID: 2.0-3.0 mm 3.2-8.0 mm

* SecurityGuard Analytical Cartridges require holder, Part No.: KJ0-4282.

If Phenomenex products in this technical note do not provide at least an equivalent separation as compared to other products of the same phase and dimensions, return the product with comparative data within 45 days for a FULL REFUND.

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Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions. Trademarks Luna is a registered trademark and SecurityGuard is a trademark of Phenomenex. Agilent is a registered trademark of Agilent Technologies Inc. TurbolonSpray is a registered trademark and API 4000 is a trademark of AB SCIEX Pte. Ltd. AB SCIEX™ is being used under license. Milli-Q is a registered trademark of Merck KGaA, Darmstadt, Germany. Glass Mountain is a registered trademark of Glass Mountain LLC. Canyon Oaks is a registered trademark of ASV Wines, Inc. Corporation. Double Dog Dare is a registered trademark of The Wine Group LLC. Castle Rock is a registered trademark of Albert L. DBA ALG Enterprises. Cloud Break is a registered trademark of O’Neill Beverage Co., Ltd. Crane Lake is a registered trademark of Bronco Wine Company Corporation. Ropiteau is a registered trademark of Maison Ropiteau Freres “S.A.R.F.” Corporation. Jacob’s Creek and Brancott is a registered trademarks of Pernod Ricard Winemakers PTY LTD. Little Penguin is a registered trademark of Southcorp Brands Pty Limited. Yellow Tail is a registered trademark of Casella Wines Pty Limited Corporation. BV is a registered trademark of Diageo North America, Inc. Dancing Bull is a registered trademark of E. & J. Gallo Winery Corporation. Beringer is a registered trademark of Treasury Wine Estates Americas Company Corporation. Aresti is a registered trademark of Sociedad de Inversiones de la Produccion Limitada Corporation. Los Vascos is a registered trademark of Agricola Vina Los Vascos LTD LLC. Root 1 is a registered trademark of Winebow, INC. Corporation. Disclaimer Phenomenex is in no way affiliated with Agilent Technologies. SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362 © 2014 Phenomenex, Inc. All rights reserved.

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