Rapid Determination of Polyphenol Antioxidants in Green Tea and Cranberry Extract Using Core Shell Columns

Transcription

1 Rapid Determination of Polyphenol Antioxidants in Green Tea and Cranberry Extract Using Core Shell Columns Pranathi P. Perati, Brian M. De Borba, and Jeffrey S. Rohrer, Thermo Fisher Scientific, Sunnyvale, CA, USA

2 Introduction Polyphenols 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 red wine. Studies suggest that diets abundant in fruits and vegetables provide protection against cardiovascular disease and cancer. Flavonoids are a subclass of polyphenols that 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 and their 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, the consumption of green tea products has increased due to its reported health benefits. This paper describes two sensitive, fast, and accurate HPLC methods to determine anthocyanins in cranberry extract and catechins in green tea extract using the Thermo Scientific Accucore C8 HPLC column. Here we demonstrate how this column, a core-shell column, can increase separation efficiency without increasing pressure, 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 of solutions, standards, and samples refer to Dionex (now part of Thermo Scientific) Application Notes 27 and 28.,2 Table. Method Conditions Columns Mobile Phases Method A Determination of Catechins in Tea Accucore, C8, 2. µm Analytical, 2. x 0 mm A: 2.% Acetonitrile, 0.% TFA B: 0.% TFA in Acetonitrile Method B Determination of Anthocyanins in Cranberry Accucore, C8, 2. µm Analytical, 2. x 0 mm A: 0% Formic Acid B: 0% Formic Acid, 22.% Methanol, 22.% Acetonitrile Gradient 0 min 0% B min 0% B min 0% B 0 min 0% B (Isocratic) Total Run Time min min Flow Rate 0.8 ml/min 0. ml/min Column Temperature 42 ºC 42 ºC Detection UV at 280 nm Vis at 20 nm Injection Volume System Backpressure 2 µl 2 µl ~8700 psi ~7800 psi 2 Rapid Determination of Polyphenol Antioxidants in Green Tea and Cranberry Extract Using Core Shell Columns

3 Results This study demonstrates two HPLC methods to determine polyphenols in tea and cranberry extract. Method A Standard and Sample Analysis Figure. Separation of a mixed catechins standard specific to tea using Method A 40 Column: Accucore C8, 2. µm Analytical (2. 0 mm) 34 Mobile Phase: A: 0.% TFA, 2.% Acetonitrile B: 0.% TFA in Acetonitrile Gradient: min, 00% A.0.0 min, 0% B.0.0 min, 00% A 28 Flow Rate: 0.8 ml/min 24 Inj. Volume: 2.0 µl Temp.: 42 C Detection: Absorbance, UV 280 nm 8 mau Min 8 9 Peaks: Conc. Gallic Acid 0.0 µg/ml 2. Gallocatechin Epigallocatechin Catechin Caffeine 3.0. Epicatechin Epigallocatechin Gallate Gallocatechin Gallate Epicatechin Gallate 00.0 Figure shows a chromatogram of a mixed standard containing the predominant catechins 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 separated and the total analysis time is less than 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 minimally processed; 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. Thermo Scientific Poster Note PN7038_e 0/2S 3

4 Figure 2. Separation of catechins in :20 diluted samples of commercially available white, green, and black teas using Method A Column: Accucore C8, 2. µm Analytical (2. 0 mm) Mobile Phase: A: 2.% Acetonitrile in water B: 0.% TFA in acetonitrile Gradient: min, 0% B.0.0 min, 0% B.0.0 min, 0% B Flow Rate: 0.8 ml/min 0 Inj. Volume: 2.0 µl Temp.: 42 C Detection: Absorbance, UV 280 nm - mau Black Tea 9 8 Sample: : Diluted Tea Peaks:. Gallic Acid 2. Gallocatechin -0 White Tea 3. Epigallocatechin 4. Catechin. Caffeine Green Tea min. Epicatechin 7. Epigallocatechin Gallate 8. Gallocatechin Gallate 9. Epicatechin Gallate In 2007, the National Institute of Standards and Technology (NIST) initiated the Dietary Supplements Quality Assurance Program (DSQAP) to help improve the accuracy of measurements in dietary supplements. The program includes the measurements of nutritional elements, marker compounds, contaminants, and fat- and water-soluble vitamins in foods, botanical supplement ingredients, and finished products. As part of a DSQAP study with the NIST, we determined the anthocyanin content in cranberry powder. Method B Method Performance The linearity, limits of detection (LODs), and limits of quantification (LOQs) were evaluated 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 and produced correlation coefficients between (Table 2). The LODs ranged from 0. µg/ml for Dp3Gal to. µg/ml for Peo3Ara, and the LOQs ranged from 0.28 µg/ml for Dp3Gal to 3.2 µg/ml for Peo3Ara. Table 2: Data for LODs and LOQs of Anthocyanins in Cranberry Analyte Coefficient of Range LOD LOQ Determination µg/ml r 2 (µg/ml) (µg/ml) Dp3Gal Dp3Glu Cy3Gal Cy3Glu Peo3Gal Peo3Glu Peo3Ara Mal3Gal Rapid Determination of Polyphenol Antioxidants in Green Tea and Cranberry Extract Using Core Shell Columns

5 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 nine were quantified due to the availability of standards for the rest of the anthocyanins. The nine anthocyanins quantified ranged from 0.02 mg/g for Dp3Gal to 0.7 mg/g for Peo3Gal. Figure 3: Separation of Anthocyanins in Cranberry Powder using Method A Column: Accucore C8, 2.2 µm Analytical (2. 0 mm) 7 Mobile Phase: A: 0% Formic Acid B: 0% Formic Acid, 22.% Methanol, 22.% MeCN Gradient: min, 0% B Flow Rate: 0. ml/min Inj. Volume: 2.0 µl Temp.: 42 C 4 mau 3 4 Detection: Absorbance, Vis 20 nm Sample: 2 mg/ml Cranberry Extract Peaks: *Conc 7. Dp3Gal 0.02 mg/g Dp3Glu Dp3Ara 4. Cy3Gal Cy3Glu Cy3Ara Peo3Gal min 8. Peo3Glu Peo3Ara Mal3Gal * Calculated Concentration This study included a total of 4 participating laboratories that submitted results to the NIST. 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 to the average values determined by the NIST collaborative study Analyte Experimental Values (mg/g) Average Values Reported by the collaborative study (mg/g) Dp3Gal ± Dp3Glu ± 0.02 Cy3Gal ± Cy3Glu ± Cy3Ara ± 0.00 Peo3Gal ± Peo3Glu ± 0.04 Peo3Ara ± Mal3Gal ± Thermo Scientific Poster Note PN7038_e 0/2S

6 Conclusion Two sensitive and rapid methods to separate and quantify catechins and anthocyanins in different commercially available teas and cranberry powder were demonstrated. References. Dionex (now part of Thermo Scientific) Application Note 27: Sensitive Determination of Catechins in Tea by HPLC. [Online] webdocs/0783-an27-hplc-catechins-tea-9may20-lpn2799.pdf (accessed May 2, 202). 2. Dionex (now part of Thermo Scientific) Application Note Note 24: Rapid and Sensitive Determination of Anthocyanins in Bilberries Using UHPLC. [Online] SteviolGlycosides-2Nov2009-LPN237.pdf (accessed May 2, 202). Rapid Determination of Polyphenol Antioxidants in Green Tea and Cranberry Extract Using Core Shell Columns

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