113645 PN HPLC Antioxident GrTea CrnbrryExtrt 21Jun2012 PN70138

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Rapid Determination of PolyphenolAntioxidants in Green Tea and Cranberry

Extract Using Core Shell ColumnsPranathi P. Perati, Brian M. De Borba, and Jeffrey S. Rohrer,

Thermo Fisher Scientific, Sunnyvale, CA, USA


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IntroductionPolyphenols are a large family of compounds that are widely distributed in plants.

The main sources of dietary polyphenols are fruits, fruit juices, tea, coffee, and redwine. Studies suggest that diets abundant in fruits and vegetables provide protection

against cardiovascular disease and cancer. Flavonoids are a subclass of polyphenolsthat include anthocyanins and catechins. Major anthocyanin sources include

blueberries, pomegranates, and cranberries. The popularity of cranberries is

primarily associated with their reported ability to combat urinary tract infection andtheir high antioxidant content, making these products amongst the fastest-growing

herbal dietary supplements in the U.S. Green tea is another good source of

antioxidants from its high concentrations of catechins. In North America, theconsumption of green tea products has increased due to its reported health benefits.

This paper describes two sensitive, fast, and accurate HPLC methods to determineanthocyanins in cranberry extract and catechins in green tea extract using the

Thermo Scientific Accucore C18 HPLC column. Here we demonstrate how this

column, a core-shell column, can increase separation efficiency without increasingpressure, and therefore does not require specialized equipment.

Methods A Thermo Scientific Dionex UltiMate 3000 Rapid Separation LC (RSLC) System was

used for this study. For individual components of the system and preparation ofsolutions, standards, and samples refer to Dionex (now part of Thermo Scientific)

Application Notes 275 and 281.1,2

Method ADetermination of

Catechins in Tea

Method BDetermination of

Anthocyanins in Cranberry

Columns Accucore™, C18, 2.6 µm Analytical, 2.1 x 150 mm

Accucore, C18, 2.6 µm Analytical, 2.1 x 150 mm

Mobile Phases A: 2.5% Acetonitrile, 0.1% TFAB: 0.1% TFA in Acetonitrile

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

Methanol, 22.5% Acetonitrile

Gradient

0–1 min 0% B

1–5 min 10% B5–6 min 0% B

0–15 min 10% B (Isocratic)

Total Run Time 6 min 15 min

Flow Rate 0.8 mL/min 0.65 mL/min

ColumnTemperature

42 ºC 42 ºC

Detection UV at 280 nm Vis at 520 nm

InjectionVolume

2 µL 2 µL

SystemBackpressure

~8700 psi ~7800 psi

Table 1. Method Conditions


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ResultsThis study demonstrates two HPLC methods to determine polyphenols in tea and

cranberry extract.

Method A Standard and Sample Analysis

Figure 1. Separation of a mixed catechins standard specific to tea using Method A

Column: Accucore C18, 2 .6 µmAnalytical (2.1 × 150 mm)

Mobile Phase: A: 0.1% TFA, 2.5% Acetonitrile

B: 0.1% TFA in Acetonitrile

Gradient: 0.0–1.0 min, 100% A

1.0–5.0 min, 10% B

5.0–6.0 min, 100% A

Flow Rate: 0.8 mL/min

Inj. Volume: 2.0µL

Temp.: 42 °C

Detection: Absorbance, UV 280 nm

Peaks: Conc

1. Gallic Acid 50.0 µg/mL

2. Gallocatechin 100.0

3. Epigallocatechin 100.0

4. Catechin 100.0

5. Caffeine 35.0

6. Epicatechin 100.0

7. Epigallocatechin

Gallate 100.0

8. GallocatechinGallate 100.0

9. Epicatechin

Gallate 100.0

0 1 2 3 4 5 6

-5

0

6

12

18

24

28

34

40

1

23

4

mAU

Min

5

6

7

8

9

Figure 1 shows a chromatogram of a mixed standard containing the predominantcatechins in tea. In addition, free gallic acid and caffeine are naturally present in tea,

and therefore were included in the mixed standard. All catechins are well separatedand the total analysis time is less than 6 min.

Several different brands of teas were evaluated for their catechin content. The samples

investigated in this study included green, white, and black teas. White tea is minimallyprocessed; it is expected to be very high in catechin content and therefore to have the

greatest health benefits. However, in our experiments green tea showed the highest

catechin content. This was probably due to the low quality of the white tea purchased,and its adulteration with more processed varieties of tea.



Column: Accucore C18, 2 .6 µmAnalytical (2.1 × 150 mm)

Mobile Phase: A: 2.5% Acetonitrile in water

B: 0.1% TFA in acetonitrile

Gradient: 0.0–1.0 min, 0% B

1.0–5.0 min, 10% B

5.0–6.0 min, 0% B


Inj. Volume: 2.0µL

Temp.: 42 °C

Detection: Absorbance, UV 280 nm

Sample: 1 :5 Di luted Te a

Peaks:

1. Gallic Acid

2. Gallocatechin

3. Epigallocatechin

4. Catechin

5. Caffeine

6. Epicatechin

7. Epigallocatechin Gallate

8. Gallocatechin Gallate

9. Epicatechin Gallate 0 1 2 3 4 5 6

-18

-10

-5

0

5

1

min

mAU

Black Tea

White Tea

Green Tea

134

5

6

7 8

9

Figure 2. Separation of catechins in 1:20 diluted samples of commerciallyavailable white, green, and black teas using Method A

Method B Method Performance

The linearity, limits of detection (LODs), and limits of quantification (LOQs) wereevaluated to characterize the method for this analysis. The anthocyanins delphinidin

3-galactose (Dp3Gal), delphinidin 3-glucoside (Dp3Glu), cyanidin 3-galactoside

(Cy3Gal), cyanidin 3-glusocide (Cy3Glu), peonidin 3-galactoside (Peo3Gal),peonidin 3-glucoside (Peo3Glu), peonidin 3-arabinoside (Peo3Ara), and Malvidin 3-

Galactoside (Mal3Gal) showed a linear peak response in the ranges chosen andproduced correlation coefficients between 0.9763–0.9974 (Table 2). The LODs

ranged from 0.1 µg/mL for Dp3Gal to 1.56 µg/mL for Peo3Ara, and the LOQs

ranged from 0.28 µg/mL for Dp3Gal to 3.12 µg/mL for Peo3Ara.

Table 2: Data for LODs and LOQs of Anthocyanins in Cranberry

Analyte Range µg/mL

Coefficient ofDetermination

r 2

LOD (µg/mL)

LOQ (µg/mL)

Dp3Gal 0.2–12.5 0.9933 0.10 0.28

Dp3Glu 0.2–50.0 0.9965 0.20 0.78

Cy3Gal 0.2–50.0 0.9821 0.80 1.56

Cy3Glu 0.2–50.0 0.9953 0.26 0.78

Peo3Gal 0.2–50.0 0.9951 0.78 1.56

Peo3Glu 0.2–50.0 0.9763 0.20 0.56

Peo3Ara 0.2–50.0 0.9981 1.56 3.12

Mal3Gal 0.2–50.0 0.9974 0.20 0.78

In 2007, the National Institute of Standards and Technology (NIST) initiated the

Dietary Supplements Quality Assurance Program (DSQAP) to help improve theaccuracy of measurements in dietary supplements. The program includes themeasurements of nutritional elements, marker compounds, contaminants, and fat- and

water-soluble vitamins in foods, botanical supplement ingredients, and finishedproducts. As part of a DSQAP study with the NIST, we determined the anthocyanin

content in cranberry powder.


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Figure 3: Separation of Anthocyanins in Cranberry Powder using Method A

Method B Sample Analysis

Figure 3 shows the separation of anthocyanins in cranberry extract investigated in

this study. Ten different anthocyanins were identified in this sample, but only ninewere quantified due to the availability of standards for the rest of the anthocyanins.

The nine anthocyanins quantified ranged from 0.021 mg/g for Dp3Gal to 0.176 mg/g

for Peo3Gal.

This study included a total of 14 participating laboratories that submitted results to theNIST. An overall consensus of the average reported concentrations was observed,

indicating validity of the data and the method.

Table 3. Comparison of the experimentally determined anthocyanin values tothe average values determined by the NIST collaborative study

Column: Accucore C18, 2 .2 µm Analytical(2.1 × 150 mm)

Mobile Phase: A: 10% Formic Acid

B: 10% Formic Acid, 22.5% Methanol,22.5% MeCN

Gradient: 0.0–15.0 min, 10% B


Inj. Volume: 2.0µL

Temp.: 42 °C

Detection: Absorbance, Vis 520 nm

Sample: 25 mg/mL Cranberry Extract

Peaks: *Conc

1. Dp3Gal 0 .021 mg/g

2. Dp3Glu 0.037

3. Dp3Ara —

4. Cy3Gal 0.090

5. Cy3Glu 0.041

6. Cy3Ara 0.157

7. Peo3Gal 0.176

8. Peo3Glu 0.032

9. Peo3Ara 0.170

10. Mal3Gal 0.088

* Calculated Concentration

0 1 2 3 4 5 6 7 8

-1

0

1

2

3

4

5

6

7

1 23

4

5

6

7

8 9

10

min

mAU

AnalyteExperimental Values

(mg/g)

Average Values Reportedby the collaborative study

(mg/g)

Dp3Gal 0.021 0.019 ± 0.029

Dp3Glu 0.037 0.057 ± 0.062

Cy3Gal 0.090 0.130 ± 0.039

Cy3Glu 0.041 0.059 ± 0.078

Cy3Ara 0.157 0.200 ± 0.060

Peo3Gal 0.176 0.200 ± 0.043

Peo3Glu 0.032 0.047 ± 0.045

Peo3Ara 0.170 0.150 ± 0.048

Mal3Gal 0.088 0.060 ± 0.087



ConclusionTwo sensitive and rapid methods to separate and quantify catechins and

anthocyanins in different commercially available teas and cranberry powder weredemonstrated .

References1. Dionex (now part of Thermo Scientific) Application Note 275: Sensitive

Determination of Catechins in Tea by HPLC. [Online] www.dionex.com/en-us/

webdocs/110783-AN275-HPLC-Catechins-Tea-19May2011-LPN2799.pdf

(accessed May 21, 2012).

2. Dionex (now part of Thermo Scientific) Application Note Note 241: Rapid andSensitive Determination of Anthocyanins in Bilberries Using UHPLC. [Online]

www.dionex.com/en-us/webdocs/83393-AN241-LC-SteviolGlycosides-25Nov2009-LPN2371.pdf (accessed May 21, 2012).


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113645 PN HPLC Antioxident GrTea CrnbrryExtrt 21Jun2012 PN70138