Rapid Determination of Antioxidant Polyphenols in Beverages and Herbal Supplements

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1 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

2 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

3 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

4 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

5 Processing of Camellia sinensis (Tea)

6 Pan Frying Green Tea

7 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)

8 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

The (B) ring is similar to catechol Two chiral centers at C2 and C3 Four

9 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

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

11 Analytical Methods To Determine Catechins INA Method USP Method HPLC-ED Our Method Column MetaChem Polaris Amide C18, 5 μm, mm C18 RP, 5 μm, mm C18, 5 μm, mm Acclaim 120 C18, 2.2 μm, ( 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

12 Separation of a Mixed Catechin Standards 100 AU Minutes Column: Thermo Scientific Acclaim 120, C18, 2.2 μm, analytical ( mm) Eluent: A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile Gradient: min, 100% A 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 Epigallocatechin Caffeine Catechin Epicatechin EGC Gallate Gallocatechin Gallate Epicatechin Gallate 100

13 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 Epigallocatechin Catechin Epicatechin Epigallocatechin Gallate Gallocatechin Gallate Epicatechin gallate EGC, Catechin, EGCG, EC, GCG, and ECG at concentrations of 15, 1, 30, 1, 2, and 3 μg/ml were used for precision studies.

14 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

15 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 ± Epigallocatechin ± Catechin ± Epicatechin ± Epigallocatechin Gallate ± Gallocatechin Gallate ± Epicatechin Gallate ± Total Catechins ±

16 Separation of Catechins in White Tea 10 mau Column: Thermo Scientific Acclaim 120, C18, 2.2 μm ( mm) Eluent: A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile Gradient: min, 100% A 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: Minutes Gallic Acid mg/g* 2. Gallocatechin Epigallocatechin Caffeine 5. Catechin Epicatechin EGC Gallate Gallocatechin Gallate Epicatechin Gallate 8.96 * Calculated concentration

17 Separation of Catechins in Green Tea 8.7 mau 3 6 Column: Thermo Scientific Acclaim 120, C18, 2.2 μm ( mm) Eluent: A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile Gradient: min, 100% A 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 Brand B Brand A Peaks: Brand A mg/g* Brand B mg/g* 1. Gallic Acid 2. Epigallocatechin Caffeine 4. Catechin Epicatechin EGC Gallate Gallocatechin Gallate Epicatechin Gallate Minutes * Calculated concentrations

18 Separation of Catechins in Black Tea 15 mau 3 Column: Thermo Scientific Acclaim 120, C18, 2.2 μm ( mm) Eluent: A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile Gradient: min, 100% A 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 Peaks: 1. Gallic Acid mg/g* 2. Epigallocatechin Caffeine 4. Catechin Epicatechin EGC Gallate GC Gallate Epicatechin Gallate Minutes * Calculated concentrations

19 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 Peak Area RSDs ranged from Between day Peak Area RSDs ranged from

20 Recovery of Catechins in Teas Sample Analytes Amount Spiked μg/ml % Recovery Gallocatechin EpiGallocatechin Catechin Green Tea Brand A Epicatechin Epigallocatechin Gallate Galocatechin Gallate Epicatechin Gallate Gallocatechin EpiGallocatechin Catechin White Tea Epicatechin Epigallocatechin Gallate Galocatechin Gallate Epicatechin Gallate Gallocatechin EpiGallocatechin Catechin White Tea and Green Tea Epicatechin Epigallocatechin Gallate Galocatechin Gallate Epicatechin Gallate

21 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

22 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

23 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

24 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

25 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

26 Analytical Methods to Determine Anthocyanins Thin-layer chromatography methods: to qualify the anthocyanidin content after anthocyanin hydrolysis AOAC Official Method : 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

Europe, and Asia Closely related to the native North American wild blueberry, but one

27 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.

28 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.

29 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

30 Analytical Conditions Method A Determination of Anthocyanins in PJ Column Thermo Scientific Acclaim RSLC 120, C18, 2.2 μm Analytical, mm Mobile phases A: 9% Acetonitrile, 10% formic acid B: 36% Acetonitrile, 10% formic acid Total run time 10 min 35 min Flow Rate ml/min ml/min Method B Determination of Anthocyanins in Bilberries Thermo Scientific Acclaim RSLC 120 C18, 2.2 μm Analytical, 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 ~ psi during the gradient ~ psi during the gradient

31 Separation of a Mixed Anthocyanin Standards Specific to PJ 10 mau Column: Thermo Scientific Acclaim 120, C18, 5.0 μm ( mm) Eluent: A: 10% MeCN, 10% Formic acid B: 40% MeCN, 10% Formic acid Gradient: min 100% A 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 Cyanidin 3,5-diglucoside Delphinidin 3-glucoside Pelargonidin 3,5-diglucoside Cyanidin 3-glucoside Pelargonidin 3-glucoside Minutes

32 Separation of a 15 Anthocyanin Standard Specific to Bilberries 8 mau Minutes Column: Thermo Scientific Acclaim 120, C18, 2.2 μm ( mm) Eluent: A: 10% Formic acid B: 10% Formic acid, 22.5% Methanol 22.5% MeCN Gradient: min 9% B min 35% B min 50% B min 9% B Temp.: 35 C Flow: ml/min Detection: Absorbance, Vis 520 nm Injection Volume: 2.0 µl Sample: 125 µg/ml15 Anthocyanin Standard Peaks: Conc. in μg/ml 1. Dp3Gal 2. Dp3Glu Cy3Gal Dp3Ara 5. Cy3Glu Pet3Gal 7. Cy3Ara 8. Delphinidin 9. Pet3Glu Peo3Gal Pet3Ara 12. Peo3Glu 13. Mal3Gal Peo3Ara Cyanidin 16. Mal3Glu 17. Mal3Ara 18. Petunidin 19. Peonidin 20. Malvidin

33 Separation of Anthocyanins in Pomegranate Juice mau Column: Thermo Scientific Acclaim 120, C18, 2.2 µm ( mm) Eluent: A: 10% CH 3 CN, 10% formic acid B: 40% CH 3 CN, 10% formic acid Gradient: min: 100% A min: 28.5% B Hold for 2 min at 28.5% B Temp: 30 C Flow: 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, Cy3, Dp Pg3, Cy Pg Minutes 6 * Calculated Concentrations 27831

34 3mAU Anthocyanins in Pomegranate/Wildberry Juice Column: Thermo Scientific Acclaim 120, C18, 2.2 µm ( mm) Eluent: A: 10% CH 3 CN, 10% formic acid B: 40% CH 3 CN, 10% formic acid Gradient: min: 100% A min: 28.5% B Hold for 2 min at 28.5% B Temp.: 30 C Flow: ml/min Detection: Absorbance, vis 540 nm Injection Volume: 0.5 µl Sample: 1:5 Dilute Pomegranate/wildberry juice Peaks: μg/ml* 1. Dp3, Cy3, Dp Unknown 5. Unknown 6. Cy Unknown * Calculated Concentrations Minutes 27833

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

36 Comparison of Anthocyanins in Simulated Adulterated PJ with Grape Juice mau 2 5 B 5 6 A Minutes Column: Thermo Scientific Acclaim 120, C18, 2.2 µm ( mm) Eluent: A: 10% CH 3 CN, 10% formic acid B: 40% CH 3 CN, 10% formic acid Gradient: min: 100% A min: 28.5% B Hold for 2 min at 28.5% B Temp: 30 C Flow: 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, Cy3, Dp Cy Pg3, Pg * Calculated Concentrations

37 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, Cy3, Dp Pg3, Cy Pg Method B Dp3Glu Cy3Gal Cy3Glu Pet3Glu Peo3Gal Peo3Ara Mal3Gal

38 1.1 mau Anthocyanins in Bilberry-Based Eye Antioxidants Column: Thermo Scientific Acclaim 120, C18, 2.2 µm ( mm) Eluent: A: 10% formic acid B: 10% formic acid, 22.5% methanol, 22.5% acetonitrile Gradient: min 9% B min 35% B min 50% B min 9% B Temp.: 35 C Flow: ml/min Detection: Absorbance, Vis 520 nm Injection Volume: 2.0 µl Sample: Eye antioxidant Peaks: Minutes Conc. in μg/ml 1. Dp3Gal 2. Dp3Glu Cy3Gal Dp3Ara 5. Cy3Glu Pet3Gal 7. Cy3Ara 8. Pet3Glu Peo3Gal Pet3Ara 11. Mal3Gal Peo3Ara Cyanidin 14. Mal3Glu

39 Anthocyanins in Bilberry Nutritional Supplement Brand A Column: T hermo Scientific Acclaim 120, C18, 2.2 µm ( mm) 12 Eluent: A: 10% formic acid B: 10% formic acid, 22.5% methanol, 22.5% acetonitrile Gradient: min 9% B min 35% B min 50% B min 9% B Temp.: 35 C Flow: ml/min Detection: Absorbance, Vis 520 nm Injection Volume: 2.0 µl Sample: Nutritional Supplement A Peaks: Conc. in μg/ml 1. Dp3Gal 4 2. Dp3Glu Cy3Gal Dp3Ara Cy3Glu Pet3Gal 7. Cy3Ara Delphinidin 9. Pet3Glu Peo3Gal Pet3Ara Peo3Glu Mal3Gal Peo3Ara Cyanidin 16. Mal3Glu Mal3Ara 28. Petunidin Peonidin Minutes 20. Malvidin mau

40 Anthocyanins in Bilberry-Based Nutritional Supplement Brand B 7 mau Column: Thermo Scientific Acclaim 120, C18, 2.2 µm ( mm) Eluent: A: 10% Formic Acid B: 10% Formic Acid, 22.5% methanol, 22.5% acetonitrile Gradient: min 9% B min 35% B min 50% B Hold for 5 min at 9% B Temp.: 35 C Flow: ml/min Detection: Absorbance, Vis 520 nm Injection Volume: 2.0 µl Sample: Nutritional Supplement B Peaks: Minutes Conc. in μg/ml 1. Dp3Gal 2. Dp3Glu Cy3Gal Dp3Ara 5. Cy3Glu Pet3Gal 7. Cy3Ara 8. Delphinidin 9. Pet3Glu Peo3Gal Pet3Ara 12. Peo3Glu 13. Mal3Gal Peo3Ara Cyanidin 16. Mal3Glu 17. Mal3Ara 28. Petunidin 19. Peonidin 20. Malvidin

41 Recovery of Anthocyanins in Various Juice Matrices Sample Analytes Amount μg/ml Amount Spiked μg/ml % Recovery Dp3, Cy3, Dp Pg3, Cy :5 dilute 100% pomegranate juice Pg Dp3,5 < LOD Cy3, Dp Pg3,5 < LOD Cy :5 dilute 100% grape juice Pg Dp3, Cy3, Dp Pg3, :5 dilute simulated adulterated pomegranate juice Cy Pg

42 Recovery of Anthocyanins in Herbal Supplement Matrices Sample Analytes Amount μg/ml Amount Spiked μg/ml % Recovery Dp3Glu Eye Antioxidant Brand A Bilberry-Based Nutritional Supplement Brand B Bilberry-Based Nutritional Supplement Cy3Gal Cy3Glu Peo3Gal Pet3Glu Mal3Gal Peo3Ara Dp3Glu Cy3Gal Cy3Glu Peo3Gal Pet3Glu Mal3Gal Peo3Ara Dp3Glu Cy3Gal Cy3Glu Peo3Gal Pet3Glu Mal3Gal Peo3Ara

43 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 Peak area RSDs ranged from Between-day peak area RSDs ranged from Method B: Three different samples were studied (n=3) Eye Antioxidant, Brand A and B Bilberry-Based Nutritional supplements Retention time RSDs ranged from Peak area RSDs ranged from Between-day peak area RSDs ranged from

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

44 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

45 Thank You

Rapid and Sensitive Determination of Anthocyanins in Bilberries Using UHPLC

Application Note 281 Rapid and Sensitive Determination of Anthocyanins in Bilberries Using UHPLC INTRODUCTION Bilberry (Vaccinium myrtillus L.) is a low-growing shrub that is native to temperate regions