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Quantitation of Flavanols, Proanthocyanidins, Isoflavones, Flavanones, Dihydrochalcones, Stilbenes, Benzoic Acid Derivatives Using UV Absorbance after Identification by LC-MS Longze Lin, and James M. Harnly J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf3006905 • Publication Date (Web): 11 May 2012 Downloaded from http://pubs.acs.org on May 23, 2012

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Quantitation of Flavanols, Proanthocyanidins, Isoflavones, Flavanones,

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Dihydrochalcones, Stilbenes, Benzoic Acid Derivatives Using UV Absorbance

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after Identification by LC-MS

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LONG-ZE LIN* AND JAMES M. HARNLY

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Food Composition and Methods Development Laboratory, Beltsville Human Nutrition Research

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Center, Agricultural Research Service, USDA, 10300 Baltimore Ave., Beltsville, MD 20705-

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3000

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Running Head: Quantitation Method for Plant Phenolic Components

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*Corresponding author: Telephone: (301)-504-9136; Fax: (301)-504-8314; E-mail:

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[email protected].

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ABSTRACT

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A general method was developed for the systematic quantitation of flavanols, proanthocyanidins,

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isoflavones, flavanones, dihydrochalcones, stilbenes, and hydroxybenzoic acid derivatives

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(mainly hydrolyzable tannins) based on UV band II absorbance arising from the benzoyl

28

structure. The compound structures and the wavelength maximum were well correlated and

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were divided into four groups: the flavanols and proanthocyanidins at 278 m, hydrolyzable

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tannins at 274 nm, flavanones at 288, and isoflavones at 260 nm. Within each group, molar

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relative response factors (MRRF) were computed for each compound based on the absorbance

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ratio of the compound and the group reference standard. Response factors were computed for

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the compounds as purchased (MRRF), after drying (MRRFD), and as the best predicted value

34

(MRRFP). Concentrations for each compound were computed based on calibration with the

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group reference standard and the MRRFP. The quantitation of catechins, proanthocyanidins and

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gallic acid derivatives in white tea was used as an example.

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Keywords: Quantitation; hydroxybenzoic acids, flavanols, proanthocyanidins, flavanones;

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isoflavones; hydrolyzable tannins, UV, response factors; green tea.

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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Polyphenols are widely distributed in foods and have potential health effects in the

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prevention of human chronic diseases.1-4 Biological, epidemiological, and clinical studies aimed

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at establishing the relationship between dietary polyphenols and health outcomes require

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accurate analytical methods to determine the concentration of individual polyphenols in foods.

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While new liquid chromatography (LC) and mass spectrometry (MS) technology has made

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identification of polyphenols relatively routine (with the exception of isomer identification),

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quantitation of the many phenolic compounds is still problematic. This difficulty arises from the

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large variety of compounds and the lack of standards.

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Previous research presented a general approach for quantifying hydroxycinnamic acid

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derivatives and glycosides of flavonols and flavones based on the strong UV band I absorbance

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(310-380 nm) of their cinnamoyl structure. Using an LC with diode array and MS detection

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(LC-DAD-MS), the phenolic compounds were first identified and then quantified. Five general

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groups were established based on the wavelength maximum (λmax) of the band I absorbance

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peak. Inexpensive, commercially available compounds were selected as reference standards for

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each group. Molar relative response factors (MRRFs) were computed for each compound based

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on the ratio of the peak areas of authentic standards and the group reference standards. Accuracy

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based on calibration with the group reference standards was found, in the worst case, to be ±13%

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for more than 80 tested compounds. 5

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This study presents a similar approach for quantitating flavanols, proanthocyanidins,

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isoflavones, flavanones, dihydrochalcones, stilbenes, and hydroxybenzoic acids and their

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derivatives (mainly hydrolyzable tannins) (Figure 1) based on the UV band II absorbance of their

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benzoyl structure after identification by LC-MS. The band II maxima (λmax) ranged from 250-

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300 nm. Since these compounds do not contain a cinnamoyl structure, they lack a band I

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absorbance peak. 6

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The compounds discussed in this study are widely found in foods. Flavanols, mainly

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catechins, exist in their free forms at high concentrations in a variety of green teas, green tea

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products, chocolates, and others foods. In addition, oligomers of the flavanols

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(proanthocyanidins) are distributed widely in many plant-derived foods. 2, 4, 6-8 Hydroxybenzoic

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acids, especially gallic acid, are found in their free form, as simple esters, and as more complex

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forms called hydrolyzable tannins in many plants. 2, 9-12 Isoflavones are less well distributed but

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no less important. 6, 14 They are found mainly in plants from the Leguminosae family, especially

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soybean and soybean products. 4, 13 Flavanones exist primarily in citrus fruits, juices, and peels.

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4, 15

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absorbance profiles to the flavanones.

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important grape polyphenols, have also been studied. 2, 9

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Dihydrochalcones, such as phloretin and phloridzin from apples, have similar UV 2, 4

Trans- and cis-resveratrol and their glycosides, the

In this study we examined the structure, λmax, and MRRF values of authentic standards

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for more than 55 compounds. Based on the band II λmax, which strongly correlated with

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structure, the compounds were divided into 4 major groups and a group reference standard was

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selected for each group. Calibration using the group reference standards was extended to the

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other compounds using the MRRFs. This general approach permits quantitation of flavanols,

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proanthocyanidins, isoflavones, flavanones, dihydrochalcones, and hydrolyzable tannins in plant

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materials using a few inexpensive and commercially available standards. The quantitation of the

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catechins, proanthocyanidins, and gallic acid derivatives of white green tea was used as the

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example.

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MATERIALS AND METHODS

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Standards. (+)-Catechin, (-)-epicatechin, (-)-catechin-3-O-gallate, (-)-epicatechin-3-O-gallate,

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(-)-gallocatechin, (-)-epigallocatechin, (-)-gallocatechin-3-O-gallate, (-)-epigallocatechin-3-O-

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gallate, phloretin, phloridzin dihydrate, trans-resveratrol, daidzein, daidzin, puerarin,

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formononetin, genistein, genistin, glycetein, glycetin, biochanin A, prunetin, trans-resveratrol,

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phloretin, phloridzin dehydrate, gallic acid, salicylic acid, gentisic acid, protocatechuic acid,

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vanillic acid, 2,4,6-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid and syringic acid were

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obtained from Sigma-Aldrich Chemical Co. (Saint Louis, MO). 2′,6-Dihydroxyflavanone and

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procyanidin A2 were obtained from Indofine Chemical Co. (Somerville, NJ). Theaflavin,

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theaflavin 3- and 3'-O-gallate mixture, procyanidin B1, procyanidin B2, procyanidin C1, (+)-

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catechin, (-)-epicatechin, (-)-epicatechin-3-O-gallate, (-)-gallocatechin, (-)-epigallocatechin, (-)-

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epigallocatechin-3-O-gallate, (+)-taxifolin , daidzin, genistein, genistin, and glycetin were

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obtained from Chromadex, Inc. (Irvine, CA). Pinocembrin, liguiritigenin, 5-hydroxy-7-

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methoxyflavanone, naringenin, naringin, sukuranetin, isosukuranetin, didymin, poncitrin,

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eriodictyol, eriodictyol-7-O-glucoside, neoeriocitrin eriocitrin, hesperetin, hesperidin,

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neohesperidin, (-)-homoeriodictyol and flavanomarein, and the remaining compounds in the

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tables were purchased from Extrasynthese (Genay, Cedex, France).

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The purities of commercial standards are generally categorized as reagent or HPLC

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grade. In some cases, the moisture content was listed or the molecular weight was presented

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with water of hydration. Few had both purity and moisture content. As in our previous study,

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moisture (or solvent) content was determined when sufficient quantities of the compound were

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available. 5. To remove the water of crystallization or absorbed compounds were dried under

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vacuum at 110 ˚C until a constant weight was reached (around 24 hours).

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Other chemicals. HPLC grade solvents (methanol, acetonitrile), formic acid and dimethyl

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sulfoxide (DMSO) were purchased from VWR International, Inc. (Clarksburg, MD). HPLC

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water was prepared from distilled water using a Milli-Q system (Millipore Lab., Bedford, MA).

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Standard solutions. Standards were prepared by weighing 3.00-6.00 mg standards into 10 mL

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volumetric flasks. The individual standards were first dissolved in 2 mL of DMSO and then

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brought to volume with aqueous methanol (60/40, v/v). These standards were analyzed by

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HPLC-MS to check for impurities.

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Mixtures of 3-5 standards and the group reference standard were then prepared with the

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same molar concentration and injected in HPLC three times. Each mixed solution was prepared

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at 3 concentration levels (as prepared and diluted1:4 and1:16) to provide a range of signals

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suitable for determining the MRRF values. The standard deviation (RSDs) for the peak areas

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were all ≤5%. Peak areas for peaks whose height fell in the linear range were used to compute

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the MRRF and MRRFD values. 5 This was done for all compound standards as purchased and,

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when sufficient material was available, after drying.

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Plant material and Quantitative extract. One dried white tea (WT-1) was powdered and

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passed through 60 mesh sieves prior to extraction. 5 The ground powders (100.0 mg for tea)

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were extracted with methanol-water (10.000 mL, 60:40, v/v) using an FS30 Ultrasonic sonicator

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(Fisher Scientific, Pittsburg, PA, USA) at 40 KHz and 100 W for 60 min. at room temperature,

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respectively. The slurry mixture was centrifuged at 2500 rpm for 15 min, the supernatant was

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filtered through a 17mm (0.45 µm) PVDF syringe filter (VWR Scientific, Seattle, WA, USA),

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and 50 µL of the extract was injected into the HPLC, and contents for the polyphenols expressed

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as mg/100 g dried food, are then calculated. 5

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HPLC and MS. The high-performance liquid chromatography-diode array detector-

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electrospray ionization/mass spectrometer (HPLC-DAD-ESI/MS) was previously described as

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were the conditions for identifying the phenolic compounds in food samples. 16 The same

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conditions were used to check for the UV and mass detectable impurities of the standards. Entire

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UV spectra were archived from the DAD for the entire chromatographic run. The wavelengths

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at 260, 274, 278, 288 and 354 nm were monitored in real time to obtain chromatograms for each

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group of compounds. Absorbances around each λmax were acquired at 2 nm intervals in both

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directions ( ±6 nm). 5

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Concentration Calculation. Simplistically, the molar concentration for each phenolic compound can be computed as: C = Creference standard/MRRF More specifically, the concentration in units of mg/100 g dry plant materials was calculated as: C (mg/100g) = [1000 x Ax x MWx x Ws x Vs]/[As x MWs x Wx x Vx x MRRF] or the weight percent as:

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C (%, w/w) = [100 x Ax X MWx x Ws x Vs]/[As x MWs x Wx x Vx x MRRF]

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Where Ax, MWx, Wx, Vx and As, MWs, Ws, Vs are the peak area (µAu or mAu), molecular

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weight, and weight (mg) of the testing sample (subscripted x) and the standard (subscripted s) in

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same volume of the extract or solution, respectively. Depending on the calculation, MRRFD and

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MRRFP were substituted for MRRF.

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RESULTS AND DISCUSSION

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Molar Relative Response Factor (MRRF). The molar relative response factor (MRRF) of a

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compound is ratio of the molar absorptivity of the compound to that of a reference compound. In

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this study, the response factors were computed based on the peak areas from chromatograms of

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authentic standards of comparable molar concentration. Any standard that had an impurity

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observable in the chromatogram was discarded. Tables 1 and 3-5 show response factors for

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compounds as purchased (MRRF) and, when sufficient compound was available, after

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vacuuming drying (MRRFD). The predicted response factors (MRRFP) represent our best

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estimate of the “true” value of all the factors.

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Differences between the MRRF and MRRFD values arose from differences in the

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moisture (or solvent) content of the standards before drying. In general, increased water of

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hydration was observed with increased polarity of the compound. In Tables 1, 3-5, compounds

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for which sufficient standard were available for drying are shaded in gray. A strong indicator

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that the difference in the MRRF values arises from differences in the solvent content, and not a

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difference in molar absorptivity, is the agreement of the wavelength maximum (λmax) of the band

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II absorbance. Changes in molar absorptivity are usually accompanied by a shift of λmax. A

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previous study showed that, for the flavonoid band I absorbance, shifts of less than 10 nm were

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not accompanied by significant changes in molar absorptivity. 5

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Flavanols, catechins and proanthocyanidins (Group 1). As shown in Figure 1, the flavanols

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have two chiral centers (C-2 and C-3), which can form four stereoisomers. Thus, eight common

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catechins, (+)-catechin (C) and (-)-epicatechin (EC), (-)-gallocatechin (GC or 5'-hydroxycatechin)

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and (-)-epigallocatechin (EGC), (-)-catechin 3-O-gallate (CG) and (-)-epicatechin 3-O-gallate

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(ECG), (-)-gallocatechin 3-O-gallate (GCG) and(-)- epigallocatechin 3-O-gallate (EGCG), exist

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as four stereoisomer pairs. All have the same 2R configuration, but different configurations at

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their 3 positions. In each pair, the first compound (no prefix) has an S configuration at C3 (i.e.,

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3S), while the second compound, with the “epi” prefix, has a 3R configuration. Each compound

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also has a 2S configuration isomer denoted by adding an “ent-” prefix to the name of their 2R

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isomers. Besides the 8 common catechins, afzelechin (i.e., 3'-deoxycatechin), epiafzelechin,

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fisetinidol (i.e., 5-deoxycatechin), epifisetinidol, robinetinidol (i.e., 5-deoxygallocatechin),

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epirobinetinidol, and their gallates and glycosides have also been reported in some plants.

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Afzelechin has been detected in green tea and strawberries in trace amounts. 2, 4, 6-8, 17

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Standards were purchased for each of the eight common catechins; some were quite

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expensive resulting in limited quantities. Five of the standards were also purchased from a

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second supplier. Table 1 presents MRRF and MRRFD values using catechin as a reference

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standard. These values were measured at 278 nm although their peak maxima are at the

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wavelengths noted in Table 1. In general, we observed no difference in response for the isomers.

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An extra hydroxyl in the B ring induced reduced response for GC and EGC, and substitution of

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gallate at the 3 position appears to add equally to the absorbance of CG, ECG, GCG, and EGCG.

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The MRRFD value for procyanidin C1 also suggests that the response factors are additive.

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Table 2 compares the MRRF values obtained in the current study with values reported previously. 18, 19 None of the standards from the previous studies were dried. Responses for the

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vacuum dried standards (MRRFD values) for the current study are denoted with an asterisk. If

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we assume that C and EC have a similar response based on values from the current study and

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previous work, 18 and that the earlier C value19 is biased low (so response factors in Table 2 were

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computed relative to EC rather than C), then the MRRF values shown in Table 2 are consistent.

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Wang et al.18 reported differences in responses between CG and ECG and between GCG and

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EGCG, whereas this study (Tables 1, 3-5) and Pelillo et al. 19 did not. Based on this data, we felt

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comfortable in assuming that there was no systematic difference between the responses of the

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isomers.

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The assigned MRRFP values for the stereoisomer pairs were 3.8, 0.3, and 3.1,

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respectively (Table 1). These values were consistent with the response factors from previous

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studies and with the MRRFD values of 2.8 and 3.0 for gallic acid and procyanidin C1,

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respectively. 18, 19 Compared to the MRRFP values, the average MRRFD values were 0.98 ±0.04,

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indicating that the assigned values were in close agreement with the experimental data. The 4%

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standard deviation was consistent with the uncertainty observed for repeat determinations of the

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moisture content in this study (endnotes for Tables 1, 3-5) and our previous study on cinnamoyl-

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based band I absorbance. 5 The average MRRF value was 0.96 ±0.10, indicating that failure to

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account for the solvent content biased the results slightly lower and introduced greater

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uncertainty.

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The assumed additive property of the response factors suggests that the experimental

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MRRFD value for theaflavin 3-O-gallate might be low. There is no absolute assurance that the

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chemical bond between two compounds, such as theaflavin and gallic acid, does not have a slight

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influence on the molar absorptivity of the new compound. However, since there was no

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significant shift in λmax, the assumption that the molar absorptivities are additive seems

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reasonable. Thus, theaflavin gallate was assigned an MRRFP value of 8.2 and not 7.2. Similarly,

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we assigned theaflavin 3,3'-digallate an MRRFP value of 11.0 due to the addition of a second

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gallate moiety. These MMRFP values represent a reasonable approximation until a suitable

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standard becomes available.

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Proanthocyanidins (condensed tannins) are the various length polymers (oligomers) of

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flavanols (catechins and their ent- isomers) and have been well studied. 4, 6-8, 10, 20 They are

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linked mainly through single (B-type) or double (A-type) bonds. The number of flavanol units in

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a compound is described by its degree of polymerization (DP). Only a few proanthocyanidin

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standards, such as procyanidin B1 (epicatechin-(4β→8)-catechin), B2 (epicatechin-(4β→8)-

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epicatechin), A2 (epicatechin-(4β→8, 2β→O→7)-epicatechin), and C1 (epicatechin-(4β→8)-

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epicatechin-(4β→8)-epicatechin), are available.

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Table 1 shows that there was no shift in λmax for the dimer standards, compared to the

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monomers, and their MRRF values ranged from 1.7 to 2.0. The MRRFD value for procyanidin

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C1 was 3.0, three times that of catechin. Previous studies reported relative molar absorptivity

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response ratios for the monomers, dimers, and trimers of 1.0, 1.7, and 2.9 at 280 nm. 8, 21 These

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data are also supported the assignment of the MRRFP values of 2.0 and 3.0 to the dimers and

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trimers, respectively. Thus, it can be assumed that the MRRFP values of the oligomers

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consisting only of catechin or epicatechin are equal to their DP values.

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Isoflavonoids (Group 2). To date, more than 1400 isoflavonoids (only 10 % glycosylated), with

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a wide variety of structures, have been found in plants. However, quantitative methods and

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analytical values are primarily limited to those found in soybeans and soybean products.

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Consequently, there is a need for the development of more comprehensive quantitative methods.

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2, 4, 13

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and glycosides, at 260 nm using genistein as the group reference standard (Table 3). When

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possible, standards were obtained from two suppliers and, when sufficient standard was available,

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dried to provide MRRFD values. Compared to genistein, the MRRF values of genistin (genistein

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7-O-glycoside), prunetin (genistein 7-methoxy), and biochanin A (genistein 4'-methoxy) were

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very close to 1.00 and all had a λmax of 260 nm. This indicated that methylation and

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glycosylation at the 7- or 4'-position did not affect the molar absorptivities and the MRRF values

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significantly. The MRRFD values were also close to 1.0. The agreement of the MRRF and

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MRRFD values is not surprising since the moisture content for genistein and many of the other

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isoflavonoids was only about 1%.

In this study, MRRF values were computed for 11 isoflavonoid standards, both aglycones

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Daidzein, another common isoflavonoid, differs structurally from those described above,

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lacking a hydroxyl group at the 5-position. The MRRF and MRRFD values were 0.66 and there

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was a shift of λmax to 248 nm. Glycosylation at the 7 and 8 positions (daidzin and puerarin) and

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methylation at the 4’ position (formononetin) had no effect on the λmax and little effect on the

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MRRF values. Consequently, all were assigned MRRFP values of 0.66.

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Addition of a methoxy group at the 6 position shifted the λmax to 256 nm and the MRRF

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values to 0.80 for glycetein and glycetin, back towards the values for genistein. Although the

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shift of λmax from the group reference standard was only 4 nm, the MRRF and MRRFD values

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were consistent at 0.80. Visual inspection showed that the absorbance profile of band II was

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relatively narrow compared band I. Consequently, the assigned MRRFP values were 0.80. Since

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MS provides little information regarding isomeric structure, differentiation between glycetein

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and bochanin A, both with a mass of 284 Da, will have to be based on retention time in order to

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assign the correct value for MRRFP.

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Flavanones, dihydrochalcones, chalcones and stilbenes (Group 3). Table 4 lists structures

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and response factors for flavanone, dihydrochalcone, chalcone and stilbene standards recorded at

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288 nm using hesperetin as the group reference standard. The flavanone standards included

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common aglycones and glycosides found in citrus fruits and juices. 9, 16, 22 In general, all the

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flavanones had a λmax at 288 nm ±4 nm and MRRF and MRRFD values close to 1.0. These data

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indicated that methylation and glycosylation at the 4’ and 7 positions did not affect the molar

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absorptivity.

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The flavanones with MRRF values significantly different from 1.0 were glycosylated

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(flavanomarein) or prenylated (isoxanthohumol) at the 8 position or had significantly different

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hydroxylation sites (liquiritigenin and 2′,6-dihydroxyflavanone). However, only flavanomarein

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had a λmax within 4 nm of hesperitin. Isoxanthohumol is the extracted flavanone form of

280

xanthohumol, a 3-prenylated chalcone with a dramatically different λmax and an MRRFD value of

281

0.22. Both compounds were from Humulus lupulus L. and exist in beer produced using this

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plant. 23

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Two dihydrochalcones (phloretin and phloretin 6-O-glucoside) had a λmax close to

284

hesperitin. The glycosylated form had an MRRF value of 0.99 and the aglycone had an MRRFD

285

value of 1.17. These compounds were assigned MRRFP values of 1.00 and 1.19, respectively.

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The two stilbene standards (resveratrol and resveratrol 3-glucoside) had λmax at 306 nm

287

and 318 but MRRFD values close to 1.00. These compounds are found in grapes, wines and

288

some herbs. 2, 9 The λmax would not immediately suggest calibration using hesperitin. However,

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upon identification such a calibration strategy would provide accurate results.

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Benzoic acids and hydrolysable tannins (Group 4). Various forms of benzoic acids are

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commonly found in fruits. Hydrolysable tannins are polyesters of a sugar moiety (or other non-

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aromatic polyhydroxy compounds) and a benzoic acid, usually gallic acid or

294

hexahydroxydiphenic (HHDP) acid (in S- or R- form). In most cases, the sugar component is

295

glucose, but fructose, xylose, saccharose, hamamelose are also found. If the acid component is

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gallic acid, these tannins are called gallotannins, while the esters with hexahydroxydiphenic acid

297

are called ellagitannins because ellagic acid is formed by lactonization of hexahydroxydiphenic

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acid when the tannins are hydrolyzed. However, most ellagitannins are mixed esters with both

299

hexahydroxydiphenic and gallic acids. Gallo- and ellagitannins can be further linked by C-C and

300

C-O-C bonds to form extensive polymers. In general, the hydrolysable tannins have many

301

isomeric forms and are found in many plant derived foods.10-12

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Table 5 shows a list of common hydroxybenzoic acids with gallic acid as the group

303

reference standard. In almost every case, the addition or removal of a hydroxyl or methoxy

304

group has a pronounced effect on the λmax and the molar absorptivity. Thus, gallic acid will have

305

limited use as a group reference standard. The λmax and the mass of the hydroxybenzoic acids

306

are distinctive and standards are available for those listed in Table 5. Thus, quantitation of these

307

acids and their derivatives will be more reasonably accomplished by using authentic standards

308

that are very common and very inexpensive.

309

The fact that hexahydroxydiphenic acid had the same λmax as gallic acid and an MRRF

310

value of 2.00, double that of gallic acid, suggests that the MRRF values of the gallotannins and

311

simple ellagitannins are additive and can be predicted for smaller molecules. For example,

312

monogalloylglucose has one galloyl and its MRRF should be 1.00 and trigalloylglucose has 3

313

galloyls and its MRRF should be 3.00. Similarly, 1-galloyl-4,6-HHDP-glucose has one galloyl

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314

and one HHDP and should have an MRRF value of 3.00 (Table 6). This approach, however,

315

cannot predict MRRF values for tannins containing different conjugated systems (other than

316

those for galloyl or HHDP) that would have different λmax and molar absorptivities.

317 318

Accuracy of MRRFP Values. The ideal approach using response factors is to identify the

319

compound using MS and/or MSn data, determine which group the compound belongs to, and

320

then use the correct MRRFP value with calibration for the appropriate group reference standard

321

for quantitation. Difficulties arise if a compound has not been previously analyzed and no

322

MRRFP value exists. There are two possible solutions to this problem. The first is to identify

323

the group the compound belongs to based on λmax and assume an MRRFP value of 1.00. The

324

second approach is to match the compound as closely as possible with a compound in the

325

database based on structure and to use its group reference standard and value for MRRFP.

326

In our previous study of cinnamoyl containing compounds, we considered the first

327

possibility. We divided the hydroxycinnamic derivatives and the glycosides of flavonols and

328

flavones into five groups. When the λmax of a compound fell within ±2 nm of that of a group

329

reference standard, the MRRFD values for 17 compounds were 1.01 ±0.03. These data suggested

330

there were no shifts in the molar absorptivities since the uncertainty associated with repeat

331

moisture determinations was ±3%. Alternatively, if the λmax of a compound fell within 10 nm of

332

that of a group reference standard, the undried MRRF values for 51 compounds were 0.96 ±0.13.

333

This result indicated that the assumption of an MRRFP value of 1.00 for any compound not

334

previously analyzed, but with a λmax within 10 nm of that of a group reference standard, could be

335

analyzed with an accuracy of ±13%.

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336

The approach just described, which assumes an MRRFP value of 1.0, is not applicable to

337

all the benzoyl containing compounds examined in the current study. Band II was found to be

338

narrower that band I and the λmax for the 4 group reference standards were much closer together;

339

a range of 260-288 nm for band II compared to 326-368 nm for band I. Shifts of as little as 2 nm

340

were found to indicate significant changes in the molar absorptivities of the flavanols and

341

isoflavones (Tables 1 and 3). For example, MRRFP values for EGC, EGCG, and ECG were 0.3,

342

3.1, and 3.8 with shifts of λmax of 10, 4, and 2 nm, respectively. For the isoflavones, MRRFP

343

values of 0.66 and 0.8 were observed for daidzein and glycetin with shifts of 12 and 4 nm,

344

respectively, for λmax. In both cases, accurate quantitation can only be achieved through the

345

second approach, i.e. match the new compound as closely as possible with a compound in the

346

database based on MS data and use its group reference standard and value for MRRFP.

347

The need for specific MRRFP values is also obvious for the hydroxybenzoic acids (Table

348

5). Significant changes in the MRRF values are accompanied by large shifts in the λmax.

349

Conversely, the flavanones, dihydrochalcones, and stilbenes (Table 4) were similar to the

350

previously reported cinnamoyl containing compounds. The average MRRF value for 20

351

standards with a λmax within 4 nm of that of hesperitin was 1.01 ±0.09. This suggests that the

352

assumption of an MRRFP value of 1.0 for these compounds would not be unreasonable.

353

However, accuracy for the quantitation of the dihydrochalcones improves with the use of specific

354

MRRFP values.

355

The need for specific MRRFP values for the benzoic acids, catechins, flavanones, and

356

isoflavones is not particularly problematic since the numbers of these compounds are much less

357

extensive. As more standards become available, the database (Tables 1 and 3) can be expanded.

358

Thus, quantitation of all but the most exotic compounds will be possible.

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359 360

Quantitation of Catechins, Proanthocynidins, and Gallic Acid Derivatives in White Tea.

361

The LC-MS study of white tea identified 11 catechins, 3 proanthocyanidins, gallic acid, 3

362

galloylquinic acids, and 13 gallotannins (Figure 2, Table 6). These phenolic compounds also

363

were also found in various green teas 20, 24-27 The catechins have been quantified in two ways:

364

using authentic standards, when available, and using a group reference standard. The catechins

365

were quantified at 278 nm using (+)-catechin as the group reference standard. The MRRFP

366

values for the stereoisomer pairs were 1.0, 3.8, 0.3 and 3.1, as previously discussed.

367

Concentrations based on authentic and group reference standards were in good agreement (Table

368

6). Both sets of values fell within the range of concentrations previously published for white teas.

369

24, 25

370

Numerous galloylglucoses were identified in the white tea (Figure 2). All have been

371

previously reported. 20, 25, 26 They were quantified at 274 nm using gallic acid as the group

372

reference standard. 1- and 6-monogalloylglucoses (peaks 1 and 2, Figure 2A and 2E) were

373

quantified at 274 nm using MRRFP values of 1.00. 1,3-, 1,2-,1,6-, and 2,6- digalloylglucosides

374

have been previously reported in tea, 20,24-27. Three of them can be were found in white tea,

375

peaks 7, 8, and 11 ([M-H]- at m/z 483, Figures 2A and B) with peak area ratios of 7: 8:11 of

376

1.6:1.0:43.7 in the extracted ion chromatograms (EIC). They were quantified at 274 nm using an

377

MRRFP value of 2.00. Peak 7 co-eluted with caffeine (Figure 2A) but was resolved using an

378

EIC (Figure 2B). Similarly, trigalloylglucoses (1,2,6- and 1,4,6-tri-galloylglucosides) ([M-H]- at

379

m/z 635) (peaks 14, 17, 18, and 20, Figure 2C) had very small and partially overlapping peaks at

380

274 nm (Figure 2A). They were cleanly resolved using EIC (Figure 2C) and quantified at 274

381

nm using MRRFP values of 3.00 based on the ratio of EIC peaks.

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382

Stricitinin (1β-galloyl-4,6-(S)-HHDP-glucose, [M-H]-at m/z 633) (peak 13) and two

383

isomers (peaks 6 and 10, Figure 2D) and 1,2-digalloyl-4,6-HHDP-glucose (peak 22, [M-H]- at

384

m/z 787) were detected in white tea by both UV and MS (EIC for peak 22 not shown).

385

Stricitinin has been reported as a main tea polyphenol and 1,2-digalloyl-4,6-HHDP-glucose has

386

been reported in some Chinese green teas. 25-27 Two strictinin isomers, isostrictinin (1β-galloyl-

387

2,3-(S)-HHDP-glucose) and gemin D (3-galloyl-4,6-(S)-HHDP-glucose), 26 ,27 have been

388

reported, but positive identification cannot be made at this time using only LC-MS data. These

389

compounds were quantified using MRRFP values of 5.00 and 6.00 respectively. This may be the

390

first report of some of the trigalloylglucosides and three isomeric stricitinins co-existing in green

391

tea samples.

392

Theasinensin A contained two EGCG units and was quantified using an MRRFP value of

393

6.2. Similarly, the MRRFP for catechin-(4α→8)-epicatechin 3-O-gallate was the sum of values

394

for catechin (1.00) and ECG (3.80), or 4.80. The concentration of 3,5-di-O-gallates of

395

epigallocatechin and gallocatechin were calculated using an MRRFP value of 5.9. Some 3,5-

396

gallates of flavanols have been previously reported in Chinese green teas. 20, 26

397

As indicated above, after checking the purity of available commercial standards, selecting

398

suitable group reference standards, and determining the MRRFP values of individual compounds,

399

this approach can provide an easy and convenient method for the quantitation of phenolic

400

compounds in foods.

401 402

ACKNOWLEDGEMENTS

403

This research is supported by the Agricultural Research Service of the U.S. Department of

404

Agriculture and an Interagency Agreement with the Office of Dietary Supplements of the

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Journal of Agricultural and Food Chemistry

National Institutes of Health.

406

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LITERATURE CITED

408

(1)

409 410

the prevention of diseases. Crit. Rev. Food Sci. Nutr. 2005, 45, 287-306. (2)

411 412

Alan Crozier, A.; Jaganath, I. B.; Clifford, M. N. Dietary phenolics: chemistry, bioavailability and effects on health. Nat. Prod. Rep. 2009, 26, 1001-1043.

(3)

413 414

Scalbert, A.; Manach, C.; Morand, C.; Rémé sy, C.; Jiménez, L. Dietary polyphenols and

Manach, C.; Scalbert, A; Morand, C.; Remesy, C.; Jimenez, C. Bioavailability and bioefficacy of polyphenols in humans. Am. J. Clin. Nutr. 2004, 79, 727-747.

(4)

Harborne, J. B.; Baxter, H. (Editors), Introduction, flavanones and dihydroflavonols,

415

flavans and proanthocyanidins, isoflavones and newflavonoids and the indexes, in Vol. 2

416

of The handbook of natural flavonoids. eds by Harborne, J. B. and Baxter. H. John Wiley

417

& Sons, Inc.: New York, 1999.

418

(5)

Lin, L.-Z.; Harnly, J.; Zhang, R.-W. ; Fain, X.-E.; Chen, H.-J. Quantitation of the

419

hydroxycinnamic acid derivatives and the glycosides of flavonols and flavones by UV

420

absorbance after identification by LC-MS. J. Agric. Food Chem. 2012, 60, 544-553.

421

(6)

422 423

food anthocyanins, isoflavones and flavanols. J. Chromatogr. A 2009, 1216, 7143-7172. (7)

424 425 426

Valls, J.; Millán, S.; Martí, M. P.; Borràs, E.; Arola, L. Advanced separation methods of

Aron, P. M.; Kennedy, J. A. Flavan-3-ols: nature, occurrence and biological activity. Mol Nutr Food Res. 2008, 52, 79-104.

(8)

Hümmer, W.; Schreier, P. Analysis of proanthocyanidins. Mol Nutr Food Res. 2008, 52, 1381-1398.

20

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Page 20 of 34

Page 21 of 34

427

Journal of Agricultural and Food Chemistry

(9)

Regos, I.; Urbanella, A.; Treutter, D. Identification and quantitation of phenolic

428

compounds from the forage legume sainfoin (Onobrychis viciifolia ). J Agric Food Chem.

429

2009, 57, 5843-5852.

430

(10)

Serrano, J.; Puupponen-Pimiä, R.; Dauer, A.; Aura, A.-M.; Saura-Calixto, F. Tannins:

431

Current knowledge of food sources, intake, bioavailability and biological effects. Mol.

432

Nutr. Food Res. 2009, 53, S310 - S329.

433

(11)

Buzzini, P.; Arapitsas, P.; Goretti, M.; Branda, E.; Turchetti, B.; Pinelli, P.; Ieri, F.;

434

Romani, A. Antimicrobial and antiviral activity of hydrolysable tannins. Mini Rev. Med.

435

Chem. 2008, 8, 1179-1187.

436

(12)

437

Feldman, K. S. Recent progress in ellagitannin chemistry. Phytochemistry 2005, 66, 1984-2000.

438

(13)

Veitch, N. C. Isoflavonoids of the leguminosae. Nat Prod Rep. 2009, 26, 776-802.

439

(14)

Mortensen, A.; Kulling, S. E.; Schwartz, H.; Rowland, I.; Ruefer, C. E.; Rimbach, G.;

440

Cassidy, A.; Magee, P.; Millar, J.; Hall, W. L. Birkved, F. K.; Sorensen, I. K.; Sontag, G.

441

Analytical and compositional aspects of isoflavones in food and their biological effects.

442

Mol Nutr Food Res. 2009, 53, S266-309.

443

(15)

Benavente-García, O.; Castillo, J. Update on uses and properties of citrus flavonoids: new

444

findings in anticancer, cardiovascular, and anti-inflammatory activity. J Agric Food

445

Chem. 2008, 56, 6185-6205.

446

(16)

Lin, L.-Z.; Harnly, J. M. A screening method for the identification of glycosylated

447

flavonoids and other phenolic compounds using a standard analytical approach for all

448

plant materials. J. Agric.Food Chem. 2007, 55, 1084-1096.

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449

(17)

Papagiannopoulos, P.; Wollseifen, H. R.; Mellenthin, A.; Haber, B.; Galensa, R.

450

Identification and quantitation of polyphenolis in carob fruit (Ceratonia siliqua L.) and

451

derived products by HPLC-UV-ESI/MSn. J. Agric. Food Chem. 2004, 52, 3784-3791.

452

(18)

453 454

Wang, H.; Provan, G. J.; Helliwell, K. HPLC determination of catechins in tea leaves and tea extracts using relative response factors. Food Chem. 2003, 81, 307-312.

(19)

Pelillo, M.; Cuvelier, M. E.; Biguzzi, B, T.; Toschi, G.; Berset, C.; Lercker, G.

455

Calculation of the molar absorptivity of polyphenols by using liquid chromatography

456

with diode array detection: the case of carnosic acid. J. Chromatogr. A 2004, 1023, 225–

457

229.

458

(20)

459 460

Lin, L.-Z.; Chen, P.; Harnly, J. M. New phenolic components and chromatographic profiles of green and fermented teas. J. Agric. Food Chem. 2008, 56, 8130-8140.

(21)

Papagiannopoulos, P.; Wollseifen, H. R.; Mellenthin, A.; Haber, B.; Galensa, R.

461

Identification and quantitation of polyphenolis in caros fruit (Ceratonia siliqua L.) and

462

derived products by HPLC-UV-ESI/MSn. J. Agric. Food Chem. 2004, 52, 3784-3791.

463

(22)

Redeuil, K.; Bertholet, R.; Kussmann, M.; Steiling, H.; Rezzi, S.; Nagy, K. Quantitation

464

of flavan-3-ols and phenolic acids in milk-based food products by reversed-phase liquid

465

chromatography–tandem mass spectrometry. J. Chromatogr. A 2009, 1216, 8362-8370.

466

(23)

Intelmann, D.; Haseleu, G.; Hofmann, T. LC-MS/MS quantitation of hop-derived bitter

467

compounds in beer using the ECHO technique. J. Agric. Food Chem. 2009, 57, 1172-

468

1182.

469

(24)

470 471

Hilal, Y.; Engelhardt, U. Characterization of white tea – comparison to green and black tea. J. Verbr. Lebensm. 2007, 2, 414 – 421.

(25)

Wang, D.; Lu, J.; Miao, A.; Xie, Z.; Yang, D. HPLC-DAD-ESI-MS/MS analysis of

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Journal of Agricultural and Food Chemistry

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polyphenols and purine alkaloids in leaves of twenty-two tea cultivars in China. J. Food

473

Comp. Anal. 2008, 21, 361-369.

474

(26)

Gao, D.F.; Zhang, Y.-J.; Yang, C.-R,; Chen, K.-K.; Jiang, H.-J. Phenolic antioxidants

475

from green tea produced from Camellia taliensis. J. Agric. Food Chem. 2008, 56, 7517–

476

7521.

477

(27)

Scoparo, C. T.; de Souza, L. M.; Dartora, N.; Sassaki, G. L.; Gorin, P.A.J.; Iacomini, M.

478

Analysis of Camellia sinensis green and black teas via ultra high performance liquid

479

chromatography assisted by liquid-liquid partition and two-dimensional liquid

480

chromatography (size exclusion x reversed phase). J. Chromatogr. A, 2012, 1222, 29-37.

481

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FIGURE LEGENDS Figure 1. The Strctures of some food polyphenol standards. Figure 2. The LC (274 nm, A and E) and extracted ion chormatograms (EIC) (B, C, D)

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Table 1. Catechins and Proanthocyanidins at 278 nm*

Compound

3'

catechin (1) OH catechin (2) OH epicatechin (1) OH epicatechin (2) OH catechin-3-O-gallate OH epicatechin-3-O-gallate (1) OH epicatechin-3-O-gallate (2) OH gallocatechin OH epigallocatechin (1) OH epigallocatechin (2) OH gallocatechin-3-O-gallate OH epigallocatechin-3-O-gallate (1) epigallocatechin-3-O-gallate (2) procyanidin B1 OH procyanidin B2 OH procyanidin C1 OH procyanidin A2 OH

4' OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH

Position 5' 2

3

4

5

7

- OH - OH OH - OH - OH OH - OH - OH OH - OH - OH OH - Ga - OH OH - Ga - OH OH - Ga - OH OH OH - OH - OH OH OH - OH - OH OH OH - OH - OH OH OH - Ga - OH OH OH OH - Ga - OH OH OH - Ga OH OH - OH C8′ OH OH - OH C8′ OH OH - OH C8′ OH OH - OC7′ OH C8′ OH OH

gallic acid see Table 5 theaflavin omitted theaflavin 3- and 3'-O-gallate omitted

λmax MRRRF MRRFD RRFP MW (nm) 278 nm 278 nm 278 nm 290 290 290 290 442 442 442 306 306 306 458 458 458 578 578 866 576

280 280 280 280 278 278 278 270 270 270 274 274 274 280 280 280 278

1.00 1.02 0.97 1.08 3.91 3.79 3.91 0.31 0.29 0.30 3.42 3.00 3.10 1.67 1.85 2.24 1.95

1.00 1.02 0.98 0.96 ----3.74 ----0.29 --3.14 ------3.01 ---

1.00 1.00 1.00 1.00 3.80 3.80 3.80 0.30 0.30 0.30 3.10 3.10 3.10 2.00 2.00 3.00 2.00

170 274 2.60 564 272 4.65 716 274 6.07

2.80 5.34 7.15

2.80 5.34 8.14

*The average moisture content for (+)-catechin (1) was 4.22% (3.91% to 4.63% for 3 determinations). Functional Groups: OH=hydroxyl, C=catechin, Ga=gallate, C8′=the C8 of the second monomer, OC7′= the O-C7 of the second monomer

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Table 2. Comparison of Catechin Molar Relative Response Factors. Compound

Wang et al. 280 nm

Pelillo et al. 270 nm

Current Study 278 nm

C EC GC EGC CG ECG GCG EGCG Gallic acid

1.00 1.03 0.29 0.24 4.70 3.94 3.56 2.66 ---

0.58 1.00 --0.47 ----2.55 2.77 2.60

1.00* 0.97* 0.31 0.29* 3.91 3.74* 3.41 3.14* 2.80*

*Values based on vacuum dried standards.

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Table 3. Isoflavones and Their 7-O-glucosides at 260 nm*

Compound

4'

genistein (1) OH genistein (2) OH genistin (1) OH genistin (2) OH prunetin OH biochanin A OCH3 daidzein (1) OH daidzein (2) OH daidzin OH puerarin OH formononetin OCH3 glycetein OH glycetin (1) OH glycetin (2) OH

5

Position 6 7

8

OH - OH OH - OH OH - OGl OH - OGl OH - OCH3 OH - OH - OH - OH - OGl - OH CGl - OH - OCH3 OH - OCH3 OGl - OCH3 OGl -

λmax MRRF MRRFD MW (nm) 260nm 260nm 270 270 432 432 284 284 254 254 416 416 268 284 446 446

260 260 260 260 260 260 248 248 248 248 248 256 256 256

1.00 1.03 0.93 1.06 0.98 0.99 0.66 0.66 0.66 0.76 0.70 0.81 0.80 0.79

1.00 1.04 ------1.00 --0.66 ----------0.80

MRRFP 260nm 1.00 1.00 1.00 1.00 1.00 1.00 0.66 0.66 0.66 0.66 0.66 0.80 0.80 0.80

*The average moisture content for genistein (1) was 1.25% (1.15 to 1.35 for 4 determinations). Functional Groups: OH=hydroxyl, OCH3=methoxy, OGl=O-glucoside, CGl=C-glucoside.

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Table 4. Flavanones, Chalcones and Resveratrols at 288 nm*

Compound

2'

hesperitin hesperidin neohesperidin pinocembrin 5-hydroxy-7-methoxyflavanone naringenin naringin sukuranetin isosukaranetin didymin poncitrin eriodictyol eriodictyol-7-O-glucoside eriocitrin neoeriucutrin (+)-taxifolin (-)-homoeriodictyol flavanomarein isoxanthohumol liguiritigenin 2',6-dihydroxyflavanone OH phloretin phloridzin xanthohumol

-

Position 3 4

3'

4'

5'

2

OH OH OH OH OH OH OH OH OH OH OH -

OCH3 OCH3 OCH3 OH OH OH OCH3 OCH3 OH OH OH OH OH OH OCH3 OH OH -

OH -

-

-

OH OH OH

-

OH OH OH

OH - OH P

OH OH OH

5 OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OCH3 -

6

7

- OH - OGl - OGl - OH OH OCH3 OH - OGl - OCH3 - OH - OGl - OGl - OH - OGl - OGl - OGl - OH - OH - OH OGl - OH P - OH OH -

- OH - CGl - OCH3 -

28

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-

MW

λmax MRRF MRRFDMRRFP (nm) 288nm 288nm 288nm

302 610 610 256 272 580 286 286 584 584 288 450 596 596 304 302 450 354 256 256

288 284 284 290 270 290 284 288 290 284 284 288 284 284 284 288 288 284 290 276 258

1.00 0.98 1.04 0.98 286 0.94 0.97 1.06 1.05 1.04 0.96 1.03 1.08 1.07 1.09 1.04 1.10 0.82 0.80 0.63 0.13

1.00 0.97 1.00 0.98 0.98 0.99 0.95 1.06 1.05 --0.97 1.05 1.03 --1.02 ------0.87 0.60 ---

1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 --1.00 0.60 ---

274 286 1.17 436 288 0.99 354 224,382 0.20

1.19 --0.22

1.19 1.19 ---

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trans-resveratrol piceid

-

-

OH OH

-

-

OH OGl

-

OH OH

-

-

-

228 306,318 --390 306,318 ---

1.12 1.00

1.12 1.00

*The average moisture content for hesperetin was 1.58 % (1.40% to1.81% for 3 determinations). Functional Groups: OH=hydroxyl, OCH3=methoxy, OGl=O-glycoside, CGL=C-glycoside, P=prenylated.

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Table 5. Hydroxybenzoic Acids and Hydrolysable Tannins at 274 nm*

Compound

1

2

Position 3 4

gallic acid (1) COOH - OH gallic acid (2) COOH - OH salicylic acid COOH OH gentisic acid COOH OH protocatechuic acid COOH - OH vanillic acid COOH - OCH3 2,4,6-trihydroxybenzoic acidCOOHOH 2,3,4-trihydroxybenzoic acidCOOHOH OH syringic acid COOH - OCH3

OH OH OH OH OH OH OH

5

6

OH OH OH OCH3

OH -

λmax (nm)

MW

MRRF MRRFDMRRFP 274 nm274 nm274 nm

170 274 1.00 170 274 0.98 138 302 0.08 154 328 0.04 154 260,294 0.60 168 260,294 0.79 170 256,294sh 0.32 170 266 0.95 198 276 1.30

1.00 -----------------

1.00 1.00 0.08 0.04 0.60 0.79 0.32 0.95 1.30

The average moisture content for gallic acid was 2.65% (2.58% to 2.75% for 3 determinations). Functional Groups: OH=hydroxyl, OCH3-methoxy, and COOH=carboxyl and. No standard for HHDP.

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Table 6. Concentration of catechins, proanthocyanidins, and gallic acid derivatives in white tea. Concentration (mg/100g) [M-H]- λmax Group Ref. Authentic (m/z) (nm) GroupMRRFP Standard Standard

Peak No. Compound 1 1-galloylglucose* 2 6-galloylglucose* 3A 3-galloylquinic acid 3B 4-galloylquinic acid 3C 5-galloylquinic acid (theogallin ) 4 gallic acid 6 stricitinin isomer 7 digalloylglucose 8 digalloylglucose 10 stricitinin isomer 11 digalloylglucose 13 stricitinin 14 trigalloylglucoses 17 trigalloylglucoses 18 trigalloylglucoses 20 trigalloylglucose 22 1,2-di-galloyl-4,6-HHDP-glucose Total tannins and qalloylquinic acids

331 331 343 343 343 169 633 483 483 633 483 633 635 635 635 635 785

274 274 274 274 274 274 274 274 274 274 274 274 274 274 274 274 274

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

1.0 1.0 1.0 1.0 1.0 1.0 3.0 2.0 2.0 3.0 2.0 3.0 3.0 3.0 3.0 3.0 4.0

30.9±1.2 247±10 107±4 150±4 1890±76 219±9 30.0±1.2 46.7±1.9 35.4±1.4 25.1±1.0 133±5 999±37 97.8±3.9 48.8±2.0 172±7 740±30 73.2±2.9 5045±202

----------219±9 -------------------------

5 gallocatechin (GC) 9 epigallocatechin (EGC) 12 catechin [C] 15 epicatechin (EC) 19 epigallocatechin 3-O-gallate (EGCG) 23 gallocatechin 3-O-gallate (GCG) 25 epicatechin 3-O-gallate (ECG)* 26 gallocatechin 3,5-di-O-galltate* 27 epigallocatechin 3,5-di-O-galltate 28 catechin 3-O-gallate (CG) 29 epiafzelechin 3-O-gallate (EAG) Total Catechins

305 305 289 289 457 457 441 609 609 441 425

270 270 278 278 274 274 274 274 274 274 274

1 1 1 1 1 1 1 1 1 1 1

0.3 0.3 1.0 1.0 3.1 3.1 3.8 5.9 5.9 3.8 3.8

136±5 820±33 20.5±0.8 67.6±2.7 9971±399 683±27 4346±174 217±9 363±16 173±7 638±26 17436±697

132±5 806±32 20.5±0.8 67.6±2.7 9950±398 675±27 4315±173 ----170±7 -----

16 procyanidin B2 (19.95/12.96) 577 21 theasinensin A or D [EGCG-(2'→2')-EGCG] 913 24 catechin-(4α→8)-epicatechin 3-O-gallate 729 Total proanthocyanidins

278 278 278

1 1 1

2.0 6.2 4.8

11.6±0.5 212±8 130±5 354±14

11.6±0.5 -------

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Figure 1

R2

COOH

OH 1

2' 3'

HO 7 6

A

O C

5

4

8

B5'4'

6' 2

6 5

R3

2 4 3

HO

OR1

3

OR2

R4

R5 R1 Catechins (R=H, OH, or OGalloyl) Procyanidins: Polymers (B or A types) connected through single (4-6 or 4-8) or double (additional 2-7) bonds.

Benzoic acids Gallic acid (GA): R1=R2=H Hyxahydroxydiphenic acid (HHDP or GA-(2→6)-GA)

O 7 8 6 A 5

C 4

O

2' 3' 4' B 5' 6'

2

1'

O

3 2' 6'

B

7 8 6 A 5

3' 4'

5'

C 4

Isoflavones

2 3

R

O Flavanones (R=H or OH)

5 3

Trans-Stilbenes: Trans-Resveratrol (3,4′,5-trihydroxystilben) Piceid (Trans-resveratrol 3-O-glucoside)

4' 4' 5'

3'

A

B

OH 2'

β α

6'

O Chacones Dihydrochalcones (α,β single bond)

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Journal of Agricultural and Food Chemistry

Figure 2

mAU

caffeine+7

3 1000

1,2

4

0

9 8 10 11

5 6

5

10

13 14 12

15 16 17

15

18

20

22 24 21 23

20

25

30

26 35

29

27 28 40

min

B

8

7

20000

25

WT-2, EIC of NI m/z 483

11

40000

A

WT-2, SymmetryShield column

19

0 5

10

15

20

25

20 18

60000 40000

14

20000

30

17

35

40

min

C

WT-2, EIC of NI m/z 635

0 5

10

15

20

25

30

35

40

min

"

13

100000 50000

D

WT-2, EIC of NI m/z 633

10

6

0 5 mAU

1000 0

3B+3C 3A 5* 4 5

10

15

caffeine+7 13

9 10

20

25

35

40

min

E

25

20 15

30

WT-2, Symmetry column

19

29 20

25

33

30

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40

min

Journal of Agricultural and Food Chemistry

TOC Picture

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