Rapid Determination of Antioxidant Polyphenols in Beverages and Herbal Supplements

Rapid Determination of Antioxidant Polyphenols in Beverages and Herbal Supplements Pranathi R. Perati, Brian De Borba, Deepali Mohindra, and Jeffrey Rohrer Oct 6 th 2011

Antioxidants Antioxidants protect cells from damage caused by free radicals Excess production of free radicals or decrease in antioxidants lead to oxidative stress, which is implicated in cancer, Alzheimer's disease, and aging The body's ability to produce antioxidants is controlled by genetic makeup and is influenced by exposure to environmental factors such as diet and smoking Increasing dietary intake of antioxidants helps mitigate damage due to oxidative stress

Flavonoids Flavonoids are polyphenolic compounds that are ubiquitous in plants The three classes of flavonoids that have attracted most attention in nutraceuticals and functional foods are: Flavanols or catechins Good sources are tea, chocolate, red wine, apples and berries Anthocyanins Responsible for the red, orange, and blue colors of most fruits and flowers Pomegranates, bilberries, grapes, blueberries are all good sources Pomegranates and bilberries have higher total polyphenol concentrations than most common fruit juices and berries Isoflavonoids Soy is a good source

Tea An agricultural product derived from the Camellia sinensis plant that is prepared and cured by various methods Four major varieties include: White tea Green tea Oolong tea Black tea Processing of teas White/green tea: not oxidized Oolong tea: partially oxidized Black tea: fully oxidized

Processing of Camellia sinensis (Tea) http://www.teazonline.com/tea-production.htm

Pan Frying Green Tea

Health Benefits of Catechins Strong antioxidant activity: superior to ascorbic acid (vitamin C) and tocopherol (vitamin E) Evaluated for its effectiveness in a number diseases: Cardiovascular Cancer (skin, breast, lungs, colon, and bladder) Neurodegenerative Some studies have characterized green tea as: Antiangiogenic (prevents tumor blood vessel growth) Antimutagenic Helping to prevent the development of atherosclerotic plaques Helping to protect against Parkinson s and Alzheimer s disease Providing antidiabetic effects (in animals)

Global Tea Market Import/export of tea Largest exporters: Consumption of green tea Largest consumers: China (largest producer/consumer), Hong Kong, Taiwan, Middle East, and Japan 8

Chemistry of Catechins Characteristics of catechins Two benzene rings (A & B) A dihydropyran heterocycle (C) with OH on C3 The (A) ring is similar to resorcinol The (B) ring is similar to catechol Two chiral centers at C2 and C3 Four diastereoisomers: two in trans config (catechin), two in cis config (epicatechin) 28471

Catechins In Green Tea Catechin Epicatechin Epicatechin Gallate Gallocatechin Gallate Epigallocatechin Gallocatechin Epigallocatechin Gallate EGCG: most abundant catechin (up to 65%, 100 200 mg) Catechin + GC: trace amounts 28472

Analytical Methods To Determine Catechins INA Method 111.002 USP Method HPLC-ED Our Method Column MetaChem Polaris Amide C18, 5 μm, 4.6 250 mm C18 RP, 5 μm, 4.6 250 mm C18, 5 μm, 4.6 150 mm Acclaim 120 C18, 2.2 μm, (2.1 150 mm) Mobile Phases A: 0.1% Phosphoric acid B: Acetonitrile A: 0.35% Phosphoric acid B: 95:5 Acetonitrile: CH 3 OH A: 50 mm Sodium phosphate with 1% CH 3 OH (ph 3.0) B: 50 mm Sodium phosphate with 10% CH 3 OH and 60% CH 3 OH (ph 3.45) A: 0.1%TFA, 5% ac et on itri le B: 0.1%TFA, in ac et on itri le Total Run Time 28 min 90 min 50 min 25 min Flow Rate 1.0 ml/min 0.8 ml/min 1.0 ml/min 0.45 ml/min Column Temperature 35 ºC 25 ºC 35 ºC 25 ºC Detection UV at 280 nm UV at 278 nm CoulArray UV at 280 nm Injection 10 μl 15 μl 50 μl 1 μl

Separation of a Mixed Catechin Standards 100 AU 1 2 3 4-10 0 2 4 6 8 10 12 15 Minutes 5 6 8 7 9 Column: Thermo Scientific Acclaim 120, C18, 2.2 μm, analytical (2.1 150 mm) Eluent: A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile Gradient: 0.0 1.2 min, 100% A 1.2 15.5 min, 28.5% B Hold for 1.5 min at 28.5% B Temp.: 25 C Flow: 0.45 ml/min Detection: Absorbance, UV, 280 nm Inj. Vol.: 1.0 µl Peaks: 1. Gallic Acid 50.0 µg/ml 2. Gallocatechin 100 3. Epigallocatechin 100 4. Caffeine 35.0 5. Catechin 100 6. Epicatechin 100 7. EGC Gallate 100 8. Gallocatechin Gallate 100 9. Epicatechin Gallate 100

Linearity, LOD, and LOQ of Catechins RSD Analyte Range (μg/ml) Correlation Coefficient (r 2 ) LOD (μg/ml) LOQ (μg/ml) Ret. Time* (n=30) Peak Area* (n=30) Gallocatechin 3.56 75 0.9993 1.17 3.56 0.11 1.17 Epigallocatechin 1.8 50 0.9993 0.59 1.8 0.18 1.45 Catechin 0.78 50 0.9992 0.20 0.78 0.13 1.19 Epicatechin 0.59 50 0.9999 0.20 0.59 0.06 1.51 Epigallocatechin Gallate 1.17 150 0.9994 0.39 1.17 0.04 1.00 Gallocatechin Gallate 1.2 10 0.9998 0.39 1.20 0.02 1.37 Epicatechin gallate 1.56 50 0.9995 0.39 1.56 0.02 1.30 EGC, Catechin, EGCG, EC, GCG, and ECG at concentrations of 15, 1, 30, 1, 2, and 3 μg/ml were used for precision studies.

NIST Collaborative Study: Catechins in Green Tea Samples Received from NIST: Controls: Spray-dried green tea extract Samples: Ground and homogenized green tea tablets Analytes of interest (7 catechins): C, EC, ECG, EGC, EGCG, GC, GCG NIST Sample and control Prep Procedure: Extraction solution: 70/30 H 2 O/CH 3 OH containing 0.05% formic acid Control: 20 mg/14 ml solvent + 5 min sonication Sample: 60 mg extracted with two portions of 5 ml of solvent + 90 min sonication All samples filtered with 0.45 mm cellulose acetate filter prior to analysis

Summary of Catechin Evaluation of NIST Control Material (SRM 3255) and Reference Samples (SRM 3256) Compared to Certified Values Analyte NIST Controls (mg/g) Certified values NIST Control (mg/g) NIST Sample (mg/g) Certified values NIST Sample (mg/g) Gallocatechin 22.8 24 ± 1 7.84 7.6 Epigallocatechin 84.7 88 ± 3 29.6 30.7 Catechin 9.7 9.8 ± 0.4 2.46 2.6 Epicatechin 47.3 46 ± 2 11.9 12 Epigallocatechin Gallate 427 417 ± 16 80.8 71.1 Gallocatechin Gallate 40.9 38 ± 3 4.46 4.6 Epicatechin Gallate 76.8 94 ± 5 17.4 17.1 Total Catechins 710 716.8 ± 27.4 154 145.7

Separation of Catechins in White Tea 10 mau -1 1 2 3 4 5 Column: Thermo Scientific Acclaim 120, C18, 2.2 μm (2.1 150 mm) Eluent: A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile Gradient: 0.0 1.2 min, 100% A 1.2 15.5 min, 28.5% B Hold for 1.5 min at 28.5% B Temp.: 25 C Flow: 0.45 ml/min Detection: Absorbance, UV 280 nm Inj. Vol.: 1.0 µl Sample: 1:20 Dilute white tea Peaks: 0 2 4 6 8 10 12 14 15 Minutes 6 7 8 9 1. Gallic Acid mg/g* 2. Gallocatechin 15.8 3. Epigallocatechin 16.5 4. Caffeine 5. Catechin 3.12 6. Epicatechin 2.73 7. EGC Gallate 42.6 8. Gallocatechin Gallate 8.83 9. Epicatechin Gallate 8.96 * Calculated concentration

Separation of Catechins in Green Tea 8.7 mau 3 6 Column: Thermo Scientific Acclaim 120, C18, 2.2 μm (2.1 150 mm) Eluent: A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile Gradient: 0.0 1.2 min, 100% A 1.2 15.5 min, 28.5% B Hold for 1.5 min at 28.5% B Temp.: 25 C Flow: 0.45 ml/min Detection: Absorbance, UV 280 nm Inj. Vol.: 1.0 µl Sample: 1:20 Dilute green tea 1 2 4 5 7 8 Brand B Brand A Peaks: Brand A mg/g* Brand B mg/g* 1. Gallic Acid 2. Epigallocatechin 45.1 43.5 3. Caffeine 4. Catechin 3.45 3.57 5. Epicatechin 6.14 6.08 6. EGC Gallate 64.0 60.6 7. Gallocatechin Gallate 6.74 6.65 8. Epicatechin Gallate 9.81 12.8-2.3 0 5 10 15 Minutes * Calculated concentrations

Separation of Catechins in Black Tea 15 mau 3 Column: Thermo Scientific Acclaim 120, C18, 2.2 μm (2.1 150 mm) Eluent: A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile Gradient: 0.0 1.2 min, 100% A 1.2 15.5 min, 28.5% B Hold for 1.5 min at 28.5% B Temp.: 25 C Flow: 0.45 ml/min Detection: Absorbance, UV 280 nm Inj. Vol.: 1.0 µl Sample: 1:20 Dilute black tea 1 2 4 5 6 7 8 Peaks: 1. Gallic Acid mg/g* 2. Epigallocatechin 27.8 3. Caffeine 4. Catechin 4.35 5. Epicatechin 2.25 6. EGC Gallate 12.3 7. GC Gallate 9.20 8. Epicatechin Gallate 7.47-1 0 5 10 15 Minutes * Calculated concentrations

Sample Analysis Precision Five different samples were studied (n=3) White tea, white and green tea blend, two commercial brands of green tea, and black tea Retention time RSDs ranged from 0.01 0.36 Peak Area RSDs ranged from 0.33 1.63 Between day Peak Area RSDs ranged from 1.04 2.15 28265

Recovery of Catechins in Teas Sample Analytes Amount Spiked μg/ml % Recovery Gallocatechin 1.0 85.4 EpiGallocatechin 15.0 100 Catechin 1.0 101 Green Tea Brand A Epicatechin 1.5 90.9 Epigallocatechin Gallate 15.0 100 Galocatechin Gallate 2.0 95.5 Epicatechin Gallate 3.0 96.5 Gallocatechin 4.0 104 EpiGallocatechin 4.0 92.1 Catechin 1.0 99.8 White Tea Epicatechin 1.0 112 Epigallocatechin Gallate 10.0 93.3 Galocatechin Gallate 3.0 105 Epicatechin Gallate 3.0 84.2 Gallocatechin 1.0 93.4 EpiGallocatechin 6.5 91.3 Catechin 1.0 94.3 White Tea and Green Tea Epicatechin 1.0 96.4 Epigallocatechin Gallate 10.0 100 Galocatechin Gallate 1.0 97.7 Epicatechin Gallate 2.0 93.6

Basic Structure of Anthocyanins R3 OH HO O + R5 OH OH R3 R5 Anthocyanidin H H Pelargonidin OH H Cyanidin OH OH Delphinidin OCH 3 OH Petunidin OCH 3 H Peonidin OCH 3 OCH 3 Malvidin 28447

Anthocyanins in Pomegranates Punica granatum, (pomegranates) belong to the family Punicaceae Pomegranates first originated in central Asia, and spread to the rest of the world Today pomegranates are cultivated in Asia, Europe, North Africa, and North America in subtropical and tropical areas and in varied climatic conditions Pomegranates are still a minor horticultural crop

Health Benefits of Consuming PJ Fights breast cancer Helps prevent lung cancer Slows prostate cancer Keeps prostate-specific antigen (PSA) levels stable Helps protect the neonatal brain Helps protect the arteries Helps prevent Alzheimer's disease Lowers cholesterol Lowers blood pressure Helps provide dental protection Helps prevent osteoarthritis http://www.healthdiaries.com

Incentives to Adulterate Pomegranate Juice Global supply shortage encourages manufacturers to stretch the finite quantity further Sellers have higher profit margin with lower production cost Inexpensive fillers like apple juice and grape juice are more readily available Bottlers are able to enter the market at competitive prices

Chemical Profiling Methods to Determine the Authenticity of Pomegranate Juice (PJ) Polyphenols: anthocyanins Sugar profiles Glucose:mannitol ratios of 4:15 Glucose:fructose ratios of 0.8:1.0 Sucrose, sorbitol, and maltose not found in pomegranate juice Organic acids Predominantly citric and malic acid present Oxalic and tartaric acid present in low amount Potassium content

Analytical Methods to Determine Anthocyanins Thin-layer chromatography methods: to qualify the anthocyanidin content after anthocyanin hydrolysis AOAC Official Method 2005.02: ph Differential Method Liquid chromatography (LC): the best technique for standardization of anthocyanin extracts Other methods do not allow complete separation of all the constituents Samples used in other methodologies require acid hydrolysis

Bilberry Vaccinium myrtillus Bilberry: low-growing deciduous shrub belonging to the family Ericaceae Found in woods and on heaths, chiefly in hilly districts of Great Britain, northern Europe, and Asia Closely related to the native North American wild blueberry, but one characteristic difference is that they produce single or paired berries on the bush, unlike blueberries that grow in clusters.

Bilberry Vaccinium myrtillus Bilberries are also smaller, darker, hard, less juicy, easier to transport, and have a different flesh color than blueberries. The two berries also have different phytochemical profiles with the anthocyanin content of fresh bilberry fruits being almost four times higher than blueberries. The high price of the extract makes it more susceptible to adulteration.

Health Benefits of Bilberries Chronic venous insufficiency - veins in the legs that carry blood to the heart are damaged Promotes urinary tract health Diabetes - Bilberry leaves have traditionally been used to control blood sugar levels in people with diabetes Atherosclerosis - strengthen blood vessels, improve circulation, and prevent the oxidation of LDL ("bad") cholesterol, a major risk factor for atherosclerosis (plaque that blocks blood vessels, leading to heart attack and stroke) Diarrhea and wounds - Used in European medicine for nearly a thousand years to treat diarrhea Anti-inflamatory tannins act as astringent (constricting and tightening) Vision - British fighter pilots in World War II reported improved night-time vision after eating bilberry jam Treatment for retinopathy Prevent cataracts

Analytical Conditions Method A Determination of Anthocyanins in PJ Column Thermo Scientific Acclaim RSLC 120, C18, 2.2 μm Analytical, 2.1 150 mm Mobile phases A: 9% Acetonitrile, 10% formic acid B: 36% Acetonitrile, 10% formic acid Total run time 10 min 35 min Flow Rate 0.475 ml/min 0.475 ml/min Method B Determination of Anthocyanins in Bilberries Thermo Scientific Acclaim RSLC 120 C18, 2.2 μm Analytical, 2.1 150 mm A: 10% Formic Acid B: 10% Formic Acid, 22.5% Methanol, 22.5% Acetonitrile Column temperature 30 C 35 C Detection Vis at 540 nm Vis at 520 nm Injection Volume 0.5 μl 2 μl System Backpressure ~6025 6200 psi during the gradient ~6700 7400 psi during the gradient

Separation of a Mixed Anthocyanin Standards Specific to PJ 10 mau 1 2 3 4 5 6 Column: Thermo Scientific Acclaim 120, C18, 5.0 μm (4.6 250 mm) Eluent: A: 10% MeCN, 10% Formic acid B: 40% MeCN, 10% Formic acid Gradient: 0.0 2.5 min 100% A 2.5 30.00 min 28.5% B Hold for 15 min at 28.5% B Temp.: 30 C Flow: 1.0 ml/min Detection: Absorbance, Vis 540 nm Injection Volume: 2 µl Peaks: Conc. (μg/ml) 1. Delphinidin 3,5-diglucoside 20.0 2. Cyanidin 3,5-diglucoside 20.0 3. Delphinidin 3-glucoside 20.0 4. Pelargonidin 3,5-diglucoside 20.0 5. Cyanidin 3-glucoside 20.0 6. Pelargonidin 3-glucoside 20.0-0.5 0 2 4 6 8 10 12 14 16 18 20 Minutes

Separation of a 15 Anthocyanin Standard Specific to Bilberries 8 mau 0 1 2 3 4 5 6 7 8-0.5 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes 9 11 10 12 13 14 15 16 17 Column: Thermo Scientific Acclaim 120, C18, 2.2 μm (2.1 150 mm) Eluent: A: 10% Formic acid B: 10% Formic acid, 22.5% Methanol 22.5% MeCN Gradient: 0.0 12.0 min 9% B 12.0 25.0 min 35% B 25.0 30.0 min 50% B 30.0 35.0 min 9% B Temp.: 35 C Flow: 0.475 ml/min Detection: Absorbance, Vis 520 nm Injection Volume: 2.0 µl Sample: 125 µg/ml15 Anthocyanin Standard 18 19 20 Peaks: Conc. in μg/ml 1. Dp3Gal 2. Dp3Glu 20.1 3. Cy3Gal 11.0 4. Dp3Ara 5. Cy3Glu 12.3 6. Pet3Gal 7. Cy3Ara 8. Delphinidin 9. Pet3Glu 9.91 10. Peo3Gal 3.91 11. Pet3Ara 12. Peo3Glu 13. Mal3Gal 8.76 14. Peo3Ara 12.7 15. Cyanidin 16. Mal3Glu 17. Mal3Ara 18. Petunidin 19. Peonidin 20. Malvidin

Separation of Anthocyanins in Pomegranate Juice mau 6 1 2 3 5 Column: Thermo Scientific Acclaim 120, C18, 2.2 µm (2.1 150 mm) Eluent: A: 10% CH 3 CN, 10% formic acid B: 40% CH 3 CN, 10% formic acid Gradient: 0.0 0.9 min: 100% A 0.9 8.0 min: 28.5% B Hold for 2 min at 28.5% B Temp: 30 C Flow: 0.475 ml/min Detection: Absorbance, vis 540 nm Injection Volume: 0.5 μl Sample: 1:5 Dilute 100% Pomegranate juice Peaks: Conc. (μg/ml)* 1. Dp3,5 65.5 2. Cy3,5 123 3. Dp3 27.5 4. Pg3,5 8.00 5. Cy3 121 6. Pg3 4.45 0-0.2 4 0 1.3 2.5 3.8 5 Minutes 6 * Calculated Concentrations 27831

3mAU Anthocyanins in Pomegranate/Wildberry Juice 1 2 4 6 Column: Thermo Scientific Acclaim 120, C18, 2.2 µm (2.1 150 mm) Eluent: A: 10% CH 3 CN, 10% formic acid B: 40% CH 3 CN, 10% formic acid Gradient: 0.0 0.9 min: 100% A 0.9 8.0 min: 28.5% B Hold for 2 min at 28.5% B Temp.: 30 C Flow: 0.475 ml/min Detection: Absorbance, vis 540 nm Injection Volume: 0.5 µl Sample: 1:5 Dilute Pomegranate/wildberry juice 3 5 7 Peaks: μg/ml* 1. Dp3,5 39.0 2. Cy3,5 54.0 3. Dp3 10.0 4. Unknown 5. Unknown 6. Cy3 50.5 7. Unknown * Calculated Concentrations 0-0.1 0 1.3 2.5 3.8 5.0 Minutes 27833

Anthocyanins in Pomegranate/Cherry Juice mau 7 2 Column: Thermo Scientific Acclaim 120, C18, 2.2 µm (2.1 150 mm) Eluent: A: 10% CH 3 CN, 10% formic acid B: 40% CH 3 CN, 10% formic acid Gradient: 0.0 0.9 min: 100% A 0.9 8.0 min: 28.5% B Hold for 2 min at 28.5% B Temp: 30 C Flow: 0.475 ml/min Detection: Absorbance, vis 540 nm Injection Volume: 0.5 µl Sample: Pomegranate/Cherry Peaks: μg/ml 1. Dp3,5 1.50 2. Unknown 3. Unknown 4. Unknown 1 3 4 * Calculated Concentrations 0-0.25 0 1.3 2.5 3.8 5 Minutes 27832

Comparison of Anthocyanins in Simulated Adulterated PJ with Grape Juice 3.5 3 4 2 3 4 1 mau 2 5 B 5 6 A 0-0.1 0 2.5 5 7.5 10 Minutes 27834 Column: Thermo Scientific Acclaim 120, C18, 2.2 µm (2.1 150 mm) Eluent: A: 10% CH 3 CN, 10% formic acid B: 40% CH 3 CN, 10% formic acid Gradient: 0.0 0.9 min: 100% A 0.9 8.0 min: 28.5% B Hold for 2 min at 28.5% B Temp: 30 C Flow: 0.475 ml/min Detection: Absorbance, vis 540 nm Injection Volume: 0.5 µl Sample: A: 1:5 dilute Simulated adulterated pomegranate juice (PJ) B: 1:5 dilute Grape juice Peaks: A B (μg/ml)* (μg/ml)* 1. Dp3,5 33.5 2. Cy3,5 61.0 4.95 3. Dp3 26.0 34.0 4. Cy3 73.0 37.1 5. Pg3,5 3.10 7.03 6. Pg3 2.10 * Calculated Concentrations

Data for Linearity, LOD and LOQ of Anthocyanins for Methods A and B Analyte Range μg/ml Coefficient of Determination r 2 LOD (μg/ml) LOQ (μg/ml) Ret. Time (n=30) RSD Peak Area (n=30) Method A Dp3,5 0.31 160 0.9992 0.21 0.66 0.06 1.82 Cy3,5 0.31 160 0.9995 0.19 1.25 0.12 1.60 Dp3 0.31 160 0.9996 0.12 0.63 0.06 1.45 Pg3,5 0.31 160 0.9984 0.37 1.25 0.07 1.80 Cy3 0.31 160 0.9994 0.15 1.25 0.06 1.46 Pg3 0.31 160 0.9996 0.20 0.63 0.09 1.70 Method B Dp3Glu 0.39 50 0.9997 1.56 6.25 0.15 0.60 Cy3Gal 0.39 25 0.9996 0.78 3.13 0.12 0.97 Cy3Glu 0.39 25 0.9999 0.78 3.13 0.14 1.29 Pet3Glu 0.39 25 0.9999 0.20 0.78 0.08 1.51 Peo3Gal 0.39 10 0.9988 0.56 2.34 0.03 1.05 Peo3Ara 0.39 25 0.9997 0.78 3.13 0.03 0.50 Mal3Gal 0.39 25 0.9993 0.20 0.78 0.01 0.72

1.1 mau 0-0.1 Anthocyanins in Bilberry-Based Eye Antioxidants 1 2 3 4 5 Column: Thermo Scientific Acclaim 120, C18, 2.2 µm (2.1 150 mm) Eluent: A: 10% formic acid B: 10% formic acid, 22.5% methanol, 22.5% acetonitrile Gradient: 0.0 12.0 min 9% B 12.0 25.0 min 35% B 25.0 30.0 min 50% B 30.0 35.0 min 9% B Temp.: 35 C Flow: 0.475 ml/min Detection: Absorbance, Vis 520 nm Injection Volume: 2.0 µl Sample: Eye antioxidant Peaks: 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes 6 7 8 9 10 11 12 13 14 Conc. in μg/ml 1. Dp3Gal 2. Dp3Glu 5.15 3. Cy3Gal 2.23 4. Dp3Ara 5. Cy3Glu 2.02 6. Pet3Gal 7. Cy3Ara 8. Pet3Glu 1.89 9. Peo3Gal 0.28 10. Pet3Ara 11. Mal3Gal 4.27 12. Peo3Ara 1.49 13. Cyanidin 14. Mal3Glu

Anthocyanins in Bilberry Nutritional Supplement Brand A Column: T hermo Scientific Acclaim 120, C18, 2.2 µm (2.1 150 mm) 12 Eluent: A: 10% formic acid B: 10% formic acid, 22.5% methanol, 22.5% acetonitrile Gradient: 0.0 12.0 min 9% B 12.0 25.0 min 35% B 1 25.0-30.0 min 50% B 30.0 35.0 min 9% B Temp.: 35 C Flow: 0.475 ml/min Detection: Absorbance, Vis 520 nm Injection Volume: 2.0 µl Sample: Nutritional Supplement A 2 3 9 15 Peaks: Conc. in μg/ml 1. Dp3Gal 4 2. Dp3Glu 21.7 3. Cy3Gal 11.9 4. Dp3Ara 5 12 6 5. Cy3Glu 10.6 13 6. Pet3Gal 7. Cy3Ara 7 11 8. Delphinidin 9. Pet3Glu 8.76 16 10. Peo3Gal 3.31 8 10 11. Pet3Ara 14 12. Peo3Glu 17 19 20 13. Mal3Gal 6.26 18 14. Peo3Ara 8.88 0 15. Cyanidin 16. Mal3Glu -1 17. Mal3Ara 28. Petunidin 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 19. Peonidin Minutes 20. Malvidin mau

Anthocyanins in Bilberry-Based Nutritional Supplement Brand B 7 mau 0 1 2 3 4 5 Column: Thermo Scientific Acclaim 120, C18, 2.2 µm (2.1 150 mm) Eluent: A: 10% Formic Acid B: 10% Formic Acid, 22.5% methanol, 22.5% acetonitrile Gradient: 0.0 12.0 min 9% B 12.0 25.0 min 35% B 25.0 30.0 min 50% B Hold for 5 min at 9% B Temp.: 35 C Flow: 0.475 ml/min Detection: Absorbance, Vis 520 nm Injection Volume: 2.0 µl Sample: Nutritional Supplement B Peaks: -0.5 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes 6 7 8 9 10 11 12 13 14 15 16 17 20 19 18 Conc. in μg/ml 1. Dp3Gal 2. Dp3Glu 20.8 3. Cy3Gal 10.2 4. Dp3Ara 5. Cy3Glu 9.99 6. Pet3Gal 7. Cy3Ara 8. Delphinidin 9. Pet3Glu 8.40 10. Peo3Gal 1.12 11. Pet3Ara 12. Peo3Glu 13. Mal3Gal 3.27 14. Peo3Ara 6.96 15. Cyanidin 16. Mal3Glu 17. Mal3Ara 28. Petunidin 19. Peonidin 20. Malvidin

Recovery of Anthocyanins in Various Juice Matrices Sample Analytes Amount μg/ml Amount Spiked μg/ml % Recovery Dp3,5 13.2 15.0 101.8 Cy3,5 23.8 25.0 98.0 Dp3 6.50 5.0 106.9 Pg3,5 1.04 1.0 104.1 Cy3 23.6 25.0 102.2 1:5 dilute 100% pomegranate juice Pg3 0.88 1.0 108.3 Dp3,5 < LOD 2.5 104.6 Cy3,5 1.05 1 105.1 Dp3 5.12 5 89.5 Pg3,5 < LOD 1 87.3 Cy3 4.24 5 85.5 1:5 dilute 100% grape juice Pg3 0.86 1 102.2 Dp3,5 6.5 10.0 102.6 Cy3,5 12.8 10.0 80.1 Dp3 5.48 5.0 103.4 Pg3,5 0.56 0.75 102.0 1:5 dilute simulated adulterated pomegranate juice Cy3 14.9 10.0 97.2 Pg3 0.43 0.75 87.6

Recovery of Anthocyanins in Herbal Supplement Matrices Sample Analytes Amount μg/ml Amount Spiked μg/ml % Recovery Dp3Glu 6.24 10 89.3 Eye Antioxidant Brand A Bilberry-Based Nutritional Supplement Brand B Bilberry-Based Nutritional Supplement Cy3Gal 2.53 3.0 94.8 Cy3Glu 2.45 3.0 81.1 Peo3Gal 0.28 0.5 96.2 Pet3Glu 2.25 3.0 92.6 Mal3Gal 0.53 0.5 94.2 Peo3Ara 1.78 3.0 91.2 Dp3Glu 24.3 25 90.2 Cy3Gal 13.5 10 82.8 Cy3Glu 12.01 10 78.7 Peo3Gal 3.26 4.0 98.1 Pet3Glu 9.94 10 78.4 Mal3Gal 6.47 10 107 Peo3Ara 10.3 10 104 Dp3Glu 20.1 20 97.6 Cy3Gal 9.86 10 84.0 Cy3Glu 9.47 10 93.6 Peo3Gal 1.64 2.0 105 Pet3Glu 8.01 10 83.7 Mal3Gal 2.76 3.0 101 Peo3Ara 6.07 5.0 108

Sample Analysis Precision Method A: Five different samples were studied (n=3) Pomegranate juice, grape juice, pomegranate/wildberry, pomegranate/cherry, and simulated adulterated pomegranate juice Retention time RSDs ranged from 0.004 0.178 Peak area RSDs ranged from 0.85 1.71 Between-day peak area RSDs ranged from 1.18 2.68 Method B: Three different samples were studied (n=3) Eye Antioxidant, Brand A and B Bilberry-Based Nutritional supplements Retention time RSDs ranged from 0.01 0.17 Peak area RSDs ranged from 0.65 6.00 Between-day peak area RSDs ranged from 1.5 6.93

Conclusions The three methods discussed are simple, sensitive, and accurate methods to separate and quantify catechins and anthocyanins with minimal sample preparation Commercially available teas with varying concentrations of catechins ranging from 2.25 mg/g to 427 mg/g were determined The method described in the study was used to determine and quantify catechins in NIST control material (SRM 3255) and sample (SRM 3256) The values determined using the method were in agreement with the NIST certified values Several herbal supplements and juices with varying anthocyanin concentrations ranging from 0.2 μg/ml to 123 μg/ml were determined

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