Rapid Determination of Antioxidant Polyphenols in Beverages and Herbal Supplements

Pranathi R. Perati, Brian De Borba, Deepali Mohindra, and Jeffrey RohrerOct 6th

2011

●

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

Antioxidants

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

http://www.teazonline.com/tea-production.htm

Processing of Camellia sinensis (Tea)

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)

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

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

Epicatechin Gallate

Epigallocatechin

Epigallocatechin Gallate

Gallocatechin

Gallocatechin GallateCatechin Epicatechin

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 acidB: Acetonitrile

A: 0.35% Phosphoric acidB: 95:5 Acetonitrile: CH3

OH

A: 50 mM Sodium phosphate with 1% CH3

OH (pH 3.0)B: 50 mM Sodium phosphate with 10% CH3

OH and 60% CH3

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 Volume

10 μL 15 μL 50 μL 1 μL

Column:

Thermo Scientific Acclaim™ 120, C18, 2.2 μm, analytical

(2.1 × 150 mm)Eluent:

A: 0.1% TFA, 5% AcetonitrileB: 0.1% TFA in Acetonitrile

Gradient:

0.0–1.2 min, 100% A1.2–15.5 min, 28.5% BHold for 1.5 min at 28.5% B

Temp.:

25 °CFlow: 0.45 mL/minDetection: Absorbance, UV, 280 nmInj. Vol.: 1.0 µL

Peaks: 1. Gallic Acid

50.0 µg/mL2. Gallocatechin

1003. Epigallocatechin

1004. Caffeine

35.05. Catechin

100 6. Epicatechin

1007. EGC Gallate

1008. Gallocatechin Gallate

1009. Epicatechin Gallate

1000 2 4 6 8 10 12 15

-10

100

1

2 3

4

5 6

7 8

9

Minutes

Separation of a Mixed Catechin Standards

AU

EGC, Catechin, EGCG, EC, GCG, and ECG at concentrations of 15, 1, 30, 1, 2, and 3 μg/mL were used for precision studies.

Linearity, LOD, and LOQ of Catechins

Analyte Range(μg/mL)

Correlation Coefficient (r2)

LOD (μg/mL)

LOQ (μg/mL)

RSD

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

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 H2

O/CH3

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

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

Summary of Catechin Evaluation of NIST Control Material (SRM 3255) and Reference Samples (SRM 3256) Compared to Certified Values

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% A1.2–15.5 min, 28.5% BHold for 1.5 min at 28.5% B

Temp.: 25 °CFlow: 0.45 mL/minDetection: Absorbance, UV 280 nmInj. Vol.: 1.0 µLSample: 1:20 Dilute white tea

Peaks: 1. Gallic Acid

—mg/g*2. Gallocatechin

15.83. Epigallocatechin

16.5

4. Caffeine

—5. Catechin 3.126. Epicatechin 2.737. EGC Gallate 42.68. Gallocatechin Gallate 8.839. Epicatechin Gallate 8.96

* Calculated concentrationMinutes0 2 4 6 8 10 12 14 15

-1

1

2 3

4

6

7

8

9

5

10

mAU

Separation of Catechins in White Tea

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% BHold for 1.5 min at 28.5% B

Temp.: 25 °CFlow: 0.45 mL/minDetection: Absorbance, UV 280 nmInj. Vol.: 1.0 µLSample: 1:20 Dilute green tea

Peaks: Brand A mg/g* Brand B mg/g*1. Gallic Acid —

—2. Epigallocatechin 45.1

43.53. Caffeine

——4. Catechin

3.45 3.575. Epicatechin 6.14 6.086. EGC Gallate

64.0

60.67. Gallocatechin Gallate 6.74 6.658. Epicatechin Gallate 9.81

12.8

* Calculated concentrations0 5 10 15Minutes

-2.3

8.7

12

3

5

6

7

8

4

Brand A

Brand B

mAU

Separation of Catechins in Green Tea

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% BHold for 1.5 min at 28.5% B

Temp.: 25 °CFlow: 0.45 mL/minDetection:

Absorbance, UV 280 nmInj. Vol.:

1.0 µLSample:

1:20 Dilute black tea

Peaks: 1. Gallic Acid

—

mg/g*2. Epigallocatechin

27.83. Caffeine

—4. Catechin 4.355. Epicatechin 2.256. EGC Gallate

12.37. GC Gallate 9.208. Epicatechin Gallate

7.47

* Calculated concentrations

0 5 10 15-1

15

12

3

5

6

7

8

4

mAU

Minutes

Separation of Catechins in Black Tea

28265

●

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

Sample Analysis Precision

Recovery of Catechins in TeasSample Analytes Amount Spiked

μg/mL % Recovery

Green Tea Brand A

Gallocatechin 1.0 85.4

EpiGallocatechin 15.0 100

Catechin 1.0 101

Epicatechin 1.5 90.9

Epigallocatechin Gallate 15.0 100

Galocatechin Gallate 2.0 95.5

Epicatechin Gallate 3.0 96.5

White Tea

Gallocatechin 4.0 104

EpiGallocatechin 4.0 92.1

Catechin 1.0 99.8

Epicatechin 1.0 112

Epigallocatechin Gallate 10.0 93.3

Galocatechin Gallate 3.0 105

Epicatechin Gallate 3.0 84.2

White Tea and Green Tea

Gallocatechin 1.0 93.4

EpiGallocatechin 6.5 91.3

Catechin 1.0 94.3

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′ R5′ Anthocyanidin

H H Pelargonidin

OH H Cyanidin

OH OH Delphinidin

OCH3 OH Petunidin

OCH3 H Peonidin

OCH3 OCH3 Malvidin

28447

HO

OH

O+

R3′

R5′

OH

OH

•

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

Anthocyanins in Pomegranates

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 prevent osteoarthritis

•

Helps protect the arteries

•

Helps prevent Alzheimer's disease

•

Lowers cholesterol

•

Lowers blood pressure

•

Helps provide dental protection

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

•

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

Chemical Profiling Methods to Determine the Authenticity of Pomegranate Juice (PJ)

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: 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

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.

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 jamTreatment for retinopathy Prevent cataracts

Health Benefits of Bilberries

Method A Determination of Anthocyanins in

PJ

Method B Determination of Anthocyanins in

BilberriesColumn Thermo Scientific Acclaim RSLC 120,

C18, 2.2 μm Analytical, 2.1 × 150 mm

Thermo Scientific Acclaim RSLC 120 C18, 2.2 μm Analytical, 2.1 × 150 mm

Mobile phases A: 9% Acetonitrile, 10% formic acidB: 36% Acetonitrile, 10% formic acid

A: 10% Formic AcidB: 10% Formic Acid, 22.5% Methanol, 22.5% Acetonitrile

Total run time 10 min 35 min

Flow Rate 0.475 mL/min 0.475 mL/min

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

Analytical Conditions

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% A2.5–30.00 min –

28.5% BHold for 15 min at 28.5% B

Temp.:

30 °CFlow: 1.0 mL/minDetection: Absorbance, Vis 540 nmInjection Volume:

2 µL

Peaks:

Conc. (μg/mL)1. Delphinidin 3,5-diglucoside

20.02. Cyanidin 3,5-diglucoside

20.03. Delphinidin 3-glucoside

20.0

4. Pelargonidin 3,5-diglucoside

20.05. Cyanidin 3-glucoside 20.0 6. Pelargonidin 3-glucoside 20.0

0 2 4 6 8 10 12 14 16 18 20-0.5

10

mA

U

Minutes

12

3

4

5

6

Separation of a Mixed Anthocyanin Standards Specific to PJ

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% B12.0–25.0 min –

35% B25.0–30.0 min –

50% B30.0–35.0 min –

9% BTemp.:

35 °CFlow:

0.475 mL/minDetection: Absorbance, Vis 520 nmInjection Volume: 2.0 µLSample: 125 µg/mL15 Anthocyanin Standard

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30-0.5

0

8

Minutes

mAU

Separation of a 15 Anthocyanin Standard Specific to Bilberries

Peaks: Conc. in μg/mL1. Dp3Gal

—2. Dp3Glu

20.13. Cy3Gal

11.0 4. Dp3Ara

—5. Cy3Glu

12.36. Pet3Gal —7. Cy3Ara

—8. Delphinidin

—9. Pet3Glu

9.9110. Peo3Gal

3.91 11. Pet3Ara

—12. Peo3Glu

—13. Mal3Gal

8.7614. Peo3Ara

12.715. Cyanidin

—16. Mal3Glu —17. Mal3Ara

—18. Petunidin

—19. Peonidin

—20. Malvidin

—

12 3

4

5

67

8

9

1011

12

1314

15

1617

18

19

20

Column:

Thermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1 × 150 mm)

Eluent:

A: 10% CH3

CN, 10% formic acidB: 40% CH3

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% BTemp: 30 °CFlow: 0.475 mL/minDetection:

Absorbance, vis 540 nmInjection Volume:

0.5 μLSample: 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

1216. Pg3 4.45

* Calculated Concentrations

mA

U

Minutes0 1.3 2.5 3.8 5

-0.2

6

1

2

3

4

5

6

0

27831

Separation of Anthocyanins in Pomegranate Juice

Column:

Thermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1×150 mm)

Eluent:

A: 10% CH3

CN, 10% formic acidB: 40% CH3

CN, 10% formic acidGradient: 0.0–0.9 min: 100% A

0.9–8.0 min: 28.5% B

Hold for 2 min at 28.5% BTemp.: 30 °CFlow: 0.475 mL/minDetection:

Absorbance, vis 540 nmInjection Volume:

0.5 µLSample: 1:5 Dilute Pomegranate/wildberry juice

Peaks:

μg/mL*1. Dp3,5

39.02. Cy3,5 54.03. Dp3

10.04. Unknown ––5. Unknown ––6. Cy3 50.57. Unknown

––

* Calculated Concentrations

Minutes0 1.3 2.5 3.8 5.0

-0.1

3

mA

U

1 2

3

6

4

5

7

0

27833

Anthocyanins in Pomegranate/Wildberry Juice

Column:

Thermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1 × 150 mm)

Eluent:

A: 10% CH3

CN, 10% formic acidB: 40% CH3

CN, 10% formic acid Gradient:

0.0–0.9 min: 100% A0.9–8.0 min: 28.5% B

Hold for 2 min at 28.5% BTemp: 30 °CFlow: 0.475 mL/minDetection:

Absorbance, vis 540 nmInjection Volume: 0.5 µLSample: Pomegranate/Cherry

Peaks: μg/mL1. Dp3,5

1.502. Unknown —3. Unknown

—4. Unknown

—

Minutes

0 1.3 2.5 3.8 5-0.25

7

mA

U

1

2

34

0

27832

* Calculated Concentrations

Anthocyanins in Pomegranate/Cherry Juice

Column:

Thermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1 × 150 mm)

Eluent:

A: 10% CH3

CN, 10% formic acid

B: 40% CH3

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% BTemp:

30 °CFlow:

0.475 mL/minDetection:

Absorbance, vis 540 nmInjection Volume:

0.5 µLSample: 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

4.953. Dp3 26.0

34.04. Cy3

73.0

37.15. Pg3,5 3.10

7.036. Pg3 2.10

––

* Calculated Concentrations

0 2.5 5 7.5 10-0.1

3.5

mA

U

Minutes

B

A0

1

2

3

4

5 6

2

3

4

5

27834

Comparison of Anthocyanins in Simulated Adulterated PJ with Grape Juice

Analyte Rangeμg/mL

Coefficient of Determination

r2

LOD(μg/mL)

LOQ(μg/mL)

RSD

Ret. Time(n=30)

Peak Area(n=30)

Method A

Dp3,5 0.31–160 0.9992 0.21 0.66 0.06 1.82Cy3,5 0.31–160 0.9995 0.19 1.25 0.12 1.60Dp3 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 1.80Cy3 0.31–160 0.9994 0.15 1.25 0.06 1.46Pg3 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 0.60Cy3Gal 0.39–25 0.9996 0.78 3.13 0.12 0.97Cy3Glu 0.39–25 0.9999 0.78 3.13 0.14 1.29Pet3Glu 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 0.50Mal3Gal 0.39–25 0.9993 0.20 0.78 0.01 0.72

Data for Linearity, LOD and LOQ of Anthocyanins for Methods A and B

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% B12.0–25.0 min –

35% B25.0–30.0 min –

50% B30.0–35.0 min –

9% BTemp.:

35 °CFlow:

0.475 mL/minDetection:

Absorbance, Vis 520 nmInjection Volume:

2.0 µLSample: Eye antioxidant

Peaks: Conc. in μg/mL1. Dp3Gal

—2. Dp3Glu

5.153. Cy3Gal

2.23 4. Dp3Ara

—5. Cy3Glu

2.026. Pet3Gal —7. Cy3Ara

—8. Pet3Glu

1.899. Peo3Gal

0.2810. Pet3Ara

—11. Mal3Gal

4.2712. Peo3Ara

1.4913. Cyanidin

—14. Mal3Glu

—0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

-0.1

0

1.1

Minutes

mAU

Anthocyanins in Bilberry-Based Eye Antioxidants

1

2

34

5 67

8

9

10

1112

13

14

Anthocyanins in Bilberry Nutritional Supplement Brand A Column:

T hermo 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% B12.0–25.0 min –

35% B25.0-30.0 min –

50% B30.0–35.0 min –

9% BTemp.:

35 °CFlow: 0.

475 mL/minDetection:

Absorbance, Vis 520 nmInjection Volume:

2.0 µLSample:

Nutritional Supplement A

Peaks:

Conc. in μg/mL1. Dp3Gal

—2. Dp3Glu

21.73. Cy3Gal

11.9 4. Dp3Ara

—5. Cy3Glu 10.66. Pet3Gal —7. Cy3Ara

—8. Delphinidin

—9. Pet3Glu 8.7610. Peo3Gal 3.31 11. Pet3Ara

—12. Peo3Glu

—13. Mal3Gal 6.2614. Peo3Ara 8.8815. Cyanidin

—16. Mal3Glu —17. Mal3Ara

—28. Petunidin

—19. Peonidin

—20. Malvidin

—0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

-1

0

12

Minutes

mAU

1

2 3

4

5 6

7

8

9

10

11

12

13

14

15

16

17 181920

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% B12.0–25.0 min –

35% B25.0–30.0 min –

50% BHold for 5 min at 9% B

Temp.:

35 °CFlow:

0.475 mL/minDetection:

Absorbance, Vis 520 nmInjection Volume:

2.0 µLSample:

Nutritional Supplement B

Peaks:

Conc. in μg/mL1. Dp3Gal

—2. Dp3Glu

20.83. Cy3Gal

10.24. Dp3Ara

—5. Cy3Glu

9.996. Pet3Gal —7. Cy3Ara —8. Delphinidin

—9. Pet3Glu

8.4010. Peo3Gal

1.12 11. Pet3Ara

—12. Peo3Glu

—13. Mal3Gal

3.2714. Peo3Ara

6.9615. Cyanidin

—16. Mal3Glu —17. Mal3Ara

—28. Petunidin

—19. Peonidin

—20. Malvidin

—

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30-0.5

0

7

Minutes

mAU

Anthocyanins in Bilberry-Based Nutritional Supplement Brand B

1

2

3

45

6

7

8

9

10

11

12

13

14

15

16

1718

1920

Sample Analytes Amount μg/mL Amount Spiked μg/mL % Recovery

1:5 dilute 100% pomegranate juice

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

Pg3 0.88 1.0 108.3

1:5 dilute 100% grape juice

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

Pg3 0.86 1 102.2

1:5 dilute simulated adulterated pomegranate juice

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

Cy3 14.9 10.0 97.2

Pg3 0.43 0.75 87.6

Recovery of Anthocyanins in Various Juice Matrices

Sample Analytes Amountμg/mL

Amount Spiked μg/mL % Recovery

Eye Antioxidant

Dp3Glu 6.24 10 89.3Cy3Gal 2.53 3.0 94.8Cy3Glu 2.45 3.0 81.1Peo3Gal 0.28 0.5 96.2Pet3Glu 2.25 3.0 92.6Mal3Gal 0.53 0.5 94.2Peo3Ara 1.78 3.0 91.2

Brand A Bilberry-Based Nutritional Supplement

Dp3Glu 24.3 25 90.2Cy3Gal 13.5 10 82.8Cy3Glu 12.01 10 78.7Peo3Gal 3.26 4.0 98.1Pet3Glu 9.94 10 78.4Mal3Gal 6.47 10 107Peo3Ara 10.3 10 104

Brand B Bilberry-Based Nutritional Supplement

Dp3Glu 20.1 20 97.6Cy3Gal 9.86 10 84.0Cy3Glu 9.47 10 93.6Peo3Gal 1.64 2.0 105Pet3Glu 8.01 10 83.7Mal3Gal 2.76 3.0 101Peo3Ara 6.07 5.0 108

Recovery of Anthocyanins in Herbal Supplement Matrices

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

Thank You