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Development of a Thiolysis HPLC Method for the Analysis of Procyanidins in Cranberry Products Chi Gao, David Cunningham, Haiyan Liu, Christina Khoo, and Liwei Gu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04625 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 16, 2018

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Journal of Agricultural and Food Chemistry

Development of a Thiolysis HPLC Method for the Analysis of Procyanidins in Cranberry Products

Chi Gao#, David G. Cunningham^, Haiyan Liu^, Christina Khoo^, and Liwei Gu#, * #

Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences,

University of Florida, Gainesville, Florida, 32611, United States ^

Ocean Spray Cranberries, Inc. Lakeville‐Middleboro, Massachusetts, 02349, United States

* Corresponding author: Phone: 352‐294‐3730; Fax: 352‐392‐9467; Email: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT

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The objective of this study was to develop a thiolysis HPLC method to quantify total

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procyanidins, ratio of A‐type linkages, and A‐type procyanidin equivalents in cranberry products.

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Cysteamine was utilized as a low‐odor substitute of toluene‐α‐thiol for thiolysis

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depolymerization. Reaction temperature of 70 °C and reaction time of 20 min, in 0.3 M of HCl

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were determined to be an optimum depolymerization condition. Thiolytic products of cranberry

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procyanidins were separated on a RP‐HPLC and identified using high resolution mass

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spectrometry. Standards curves of good linearity were obtained on thiolyzed procyanidin dimer

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A2 and B2 external standards. The detection and quantification limits, recovery, and precision of

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this method were validated. The new method was applied to quantitate total procyanidins,

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average degree of polymerization, ratio of A‐type linkages, and A‐type procyanidin equivalents

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in cranberry products. Results showed that the method was suitable for quantitative and

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qualitative analysis of procyanidins in cranberry products.

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KEYWORDS: cranberry, procyanidin, flavan‐3‐ol, HPLC, thiolysis, cysteamine

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INTRODUCTION

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American cranberries (Vaccinium macrocarpon) possess various health benefits due to a

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high content of bioactive procyanidins 1. Procyanidins, also known as condensed tannins, are the

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oligomers or polymers of (epi)catechin linked by A‐type or/and B‐type inter‐flavan bonds.

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Cranberries contain both A‐type and B‐type procyanidins. Previous research have been reported

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that an A‐type procyanidin in cranberries contain one or more A‐type interflavan linkages. The

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position of A‐type linkage can be present between any adjacent (epi)catechin units 2. A‐type

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procyanidins in cranberries were known to inhibit the adhesion of E. coli and may prevent urinary

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tract infection whereas B‐type had no such activity 3.

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Several colorimetric methods have been applied to quantify total procyanidins. These

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methods include the Folin‐Ciocalteu method, hydrochloric acid/butanol assay, vanillin assay, and

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4‐(dimethylamino)‐cinnamaldehyde (DMAC) assay 4. DMAC assay is the most commonly used for

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cranberry procyanidins because of its simplicity and better specificity towards flavan‐3‐ol

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compared with other methods. However, like all other colorimetric methods, DMAC assay does

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not distinguish A‐type procyanidins from B‐type or total procyanidins 5. A recent study showed

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that the absorbance spectra of DMAC‐procyanidin complex vary considerably per the degree of

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polymerization (DP) and presence of A‐type linkages 6. This study rejected the assumption that

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molar absorbance coefficients of DMAC‐procyanidin complex are similar across the various

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procyanidin species.

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Normal phase HPLC has the capacity to separate procyanidins from monomers to

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decamers per DP. Procyanidins larger than decamers elute as an unresolved peak following

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decamers 4. Procyanidin monomers and dimers are commercially available whereas standards

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for procyanidins with higher DP are not commonly attainable. Robbins et al (2012) developed a

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method to quantify procyanidins with different DPs using Response Factors relative to

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epicatechin as an external standard. However, this method is not applicable to cranberries

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because A‐type oligomers cannot be separated from B type due to peak overlapping 7.

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Depolymerization method was used to determine the average degree of polymerization

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of procyanidins. The commonly used nucleophile to depolymerize procyanidins is toluene‐α‐

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thiol. It was observed that A‐type interflavan linkage remain stable during thiolysis while B‐type

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interflavan linkages are cleaved by toluene‐α‐thiol 8. However, toluene‐α‐thiol is impractical for

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routine use in a lab due to its extremely repulsive odor. Cysteamine is a low‐odor substitute of

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toluene‐α‐thiol for thiolysis depolymerization of procyanidins. It was used to depolymerize B‐

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type procyanidins in grapes but not A‐type procyanidins as those found in cranberries 9.

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Currently there is no method that can quantify the total amount of procyanidins in

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cranberry products and obtain DP value and ratio of A‐type linkage. It is important to distinguish

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A‐type procyanidins and determine the DP because both impact their bioactivities 10. The

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objective of this study was to develop a new quantitative method to simultaneously determine

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total procyanidins, average degree of polymerization, and the ratio of A‐type procyanidins in

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cranberry products. This HPLC method uses cysteamine‐based thiolysis of procyanidins. A single‐

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lab validation of this new method was performed according to criteria established by the AOAC

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International for dietary supplements and botanicals 11.

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MATERIALS AND METHODS

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Reagents. HPLC‐grade methanol, methylene chloride, 95% ethanol, acetone, and acetic

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acid were purchased from Fischer Scientific Co. (Pittsburgh, PA). Milli‐Q water was used as 4


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extraction solvent and HPLC mobile phase. Cysteamine hydrochloride, 2‐furfurylthiol,

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hydrochloric acid, N, N‐dimethylformamide, and Sephadex LH‐20 were obtained from

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Sigma−Aldrich (St. Louis, MO). Standards of procyanidin A2 and B2 were purchased from

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PhytoLab GmbH & Co. KG (Dutendorfer, Germany). Partially purified cranberry procyanidins (90%

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w/w, estimated using DMAC method) were provided by Ocean Spray Cranberries, Inc. It was

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purified on Sephadex LH‐20. Cransins® dried cranberry and cranberry juice are products of Ocean

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Spray Cranberries, Inc. (Middleborough, MA). A dietary supplement containing cranberry

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concentrate is a product of Nature’s Bounty, Inc. (Bohemia, NY).

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Thiolysis of partially purified cranberry procyanidins. Ten mg of partially purified

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cranberry procyanidins was dissolved in 1.0 mL 95% ethanol to prepare 10 mg/mL cranberry

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procyanidins solution. Eighty microliters of this solution was depolymerized using 80 µL of 500

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mg/mL cysteamine dissolved in 95% ethanol after acidity was adjusted to 0.4 M using 12 M HCl.

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The mixture was placed in 60 °C water bath for 20 min and immediately transferred into ‐20 °C

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freezer after reaction. The solution was filtered through polypropylene filter unit (0.45 µm)

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before injected for HPLC analysis.

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Instrumentation and HPLC methods. Procyanidins before and after thiolysis were

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analyzed using normal phase HPLC to examine the completeness of depolymerization according

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to a published method 2. They were separated on a Luna silica column (250 × 4.6 mm, 5 μm,

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Phenomenex, Torrance, CA) at a flow rate of 1.0 mL/min on an Agilent 1200 HPLC system (Palo

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Alto, CA). The binary gradient consisted of dichloromethane, methanol, water, and acetic acid

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[(A) 82:14:2:2; v/v/v/v] and methanol, water, and acetic acid [(B) 96:2:2; v/v/v]. The gradient was

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as follows: 0‐20 min, 0‐11.7% B; 20‐50 min, 11.7‐25.6% B; 50‐55 min, 25.6‐87.7% B; 55‐65min, 5



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87.7% B; 65‐70 min, 87.7‐0% B, followed by a 5‐min equilibration period. The peaks were

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monitored by fluorescence detection with excitation at 231 nm and emission at 320 nm.

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Thiolytic products of procyanidins were separated on the reversed phase HPLC columns

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using water (2% acetic acid) as solvent A and methanol as solvent B. The column temperature

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was set at 25 °C. The detection wavelength was 280 nm with 8 nm of width. The reference

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wavelength was 400 nm with 10 nm of width. The gradient and flow rate were optimized for the

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best separation with peak resolution Rs ≥ 1 and shortest running time 11. Separation was

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compared between two columns: a StableBond C18 column (5 µm, 250 × 4.6 mm, Agilent

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Technologies, Palo Alto, CA) and a Kinetex C18 column (2.6 µm, 150 × 3 mm, Phenomenex,

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Torrance, CA). The mass spectra of thiolytic products were acquired at positive mode using

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electrospray ionization on Agilent 6220 ESI‐TOF mass spectrometer (Agilent Technologies, Palo

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Alto, CA). The thiolytic reactions and structures of thiolytic products are shown in Figure 1. The

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retention times of each thiolytic product on HPLC is listed in Table 3.

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Impacts of reaction conditions on thiolysis. Impacts of temperature, reaction time,

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acidity, and fold excess of thiolytic reagents on the yields of thiolytic adducts were evaluated

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using single factor experiments. Ten mg of partially purified cranberry procyanidins was dissolved

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in 1.0 mL 95% ethanol to prepare 10 mg/mL cranberry procyanidins solution. Eighty microliters

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of this solution was depolymerized using 80 µL of cysteamine dissolved in 95% ethanol. Acidity

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was adjusted using 12 M HCl. Temperature was tested at three levels (60, 70, 80 oC). Reaction

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time and acidity of solution were tested at four levels (10, 15, 20, 25 min) (0.1, 0.2, 0.3, 0.4 M).

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Fold excess of cysteamine was tested at 2, 5, 10, and 50 folds. Completion of depolymerization

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was monitored using normal phase HPLC. The peak areas of individual thiolytic product were 6


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obtained using reserved phase HPLC under optimized conditions. They were compared between

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different depolymerization conditions to obtain the optimal thiolytic reaction conditions.

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Extraction and purification of procyanidins from cranberry products. Craisin dried

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cranberry and cranberry concentrate dietary supplement were extracted using a published

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method 4. One gram of ground sample was extracted in 10 mL of 70% (v/v) aqueous acetone

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acidified with 0.5% (v/v) of acetic acid in 15‐mL capped test tube. The tube was vortexed for 1

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min followed by sonication at 25 oC for 10 min. Then the tube was remained at room temperature

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in darkness for 20 min. Additional 5 minutes of sonication was conducted before the tube was

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centrifuged at 2265 g for 15 min. Eight mL of extract was pipetted into a 50‐mL tube, and the

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solvent was evaporated under partial vacuum at 25 oC in a SpeedVac concentrator (ISS110‐115,

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Thermo, Marietta, OH). The dried residue was dispersed in 5 mL of 30% (v/v) aqueous methanol

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before loading onto a solid phase extraction (SPE) column. The SPE column was a 12‐mL tube

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containing 3 g of Sephadex LH‐20 which were equilibrated in 30% aqueous methanol for 4 hours.

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Cranberry juice (2.0 mL) was loaded to SPE column without extraction. Samples were loaded to

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SPE columns at a flow rate of 0.25 mL/min. Forty ml of 30% aqueous methanol was used to elute

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the column to remove sugars and other phenols at the flow rate of 1.25 mL/min. Procyanidins

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were recovered from SPE column by eluting with 60 mL of 70% (v/v) aqueous acetone at a flow

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rate of 0.63 mL/min. The eluents were evaporated in a SpeedVac concentrator and residues were

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dissolved in 2 mL of 95% ethanol before thiolysis reaction.

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Standard curves and linearity. Procyanidin A2 and B2 were dissolved in 95% ethanol with

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concentration ranging from 0.01 mg/mL to 10 mg/mL. Standard solutions of different

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concentrations were depolymerized under the optimized thiolysis conditions (20 minutes of 7



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reaction time, 70 °C of reaction temperature, 0.30 M of HCl, and 500 mg/mL of cysteamine).

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Depolymerized samples were analyzed using reversed phased HPLC with the optimized method

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after filtration through 0.45 µm filter unit. Three standards curves were generated for different

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thiolytic products of procyanidins according to Table 2. The calibration curves were weighed

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using least squares method in Minitab (Version 17.1.0, Minitab Inc., State College, PA). Curve

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weighing was applied to adjust the data by a factor related to an inverse function of the

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concentration 12. Under different weighing factors, estimated concentrations calculated based

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on the calibration curve were converted to the recovery. Relative Error (RE) was the difference

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between recovery and 100%. The weighing factor that results in the lowest sum of the relative

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error possesses the highest possibility to be the most appropriate one. Correlation coefficient (R2

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> 0.98) of each calibration curve and relative error (RE0.98. Among them, the least sum of relative errors was observed when using 1/x as a

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weighing factor. The relative error of each data point was lower than 20% except the two lowest

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points 0.01 and 0.025 mg/mL. Therefore, for the sum of catechin and epicatechin, 1/x was

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considered the best weighing factor and the linearity range was determined to be 0.05‐20

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mg/mL. For the thio‐epicatechin, the lowest sum of relative error and acceptable R2 (0.9934)

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indicated the best weighing factor as 1/x2.

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The calibration curve and linearity ranges are summarized in Table 5. The limit of

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detection and quantification were calculated based on the average of slopes and standard

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deviation of intercepts of calibration curves using the best weighing factor. The LOQ of

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procyanidin A2 (0.050 mg/mL) was comparable to that of catechin + epicatechin (0.054 mg/mL)

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and thio‐epicatechin (0.030 mg/mL). The LOQs were all higher or equal to the lower end of linear

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range, which suggested that the data were valid. The LOD of catechin + epicatechin was about 5

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times higher than epicatechin reported in a previous HPLC‐UV method where epicatechin was

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analyzed without a chemical reaction and S/N=3 was used to define LOD 26. LOD in the present

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research was calculated using a more conservative statistical approach 14. The higher LOD was

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also attributed to larger deviation of data caused by the thiolysis reaction of standards. 16


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Accuracy. Accuracy of this method was tested using a spike and recovery study at three

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concentrations (Table 6). In a blank liquid matrix of Gatorade, accuracy was 84.8% and 109% at

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low concentration. The values met the suggested criteria of AOAC International which is 85‐

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110%. However, at low concentration in a blank solid matrix of wheat flour, recoveries were

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76.6% and 82.1%, which were lower than the suggested standard by AOAC International. But at

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medium and high concentration for both matrixes, recovery values were within the range of 84%

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to 108%, which closely matched the AOAC suggested criteria (90‐108%). The solid matrix

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underwent an addition extraction step before purification on SPE compared to liquid matrix. It

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explained the lower recovery rate in flour than in Gatorade beverage. Overall, the recovery values

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were higher than 84%, suggesting that accuracy of the method was appropriate.

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Precision. Precision was tested at three concentrations. At medium and high

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concentrations, the relative standard deviations of analyses were below 7.5% for the intra‐day

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assays and below 9% for the inter‐day assays, except for a high value of 13% for medium

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concentration of procyanidin B2 spiked in wheat flour. At a low concentration of 0.5 mg/g or 0.5

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mg/mL, precision for both intra‐day and inter‐day precision were above 10% with two exceptions

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of 7.9% and 9.2% (Table 6). The precision values at low concentration were higher because

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dilution of sample contributes to large errors and uncertainty. AOAC International suggests the

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repeatability of concentrations of 0.1% and 0.01% as 3% and 4%, respectively. Although the

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obtained results were higher than suggested, these values were at acceptable levels considering

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the complicated sample preparation steps which included extraction, purification using SPE and

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a chemical reaction.

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Method application on cranberry products. This method was applied to quantitate and

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characterize procyanidins in craisins, cranberry juice, and a dietary supplement containing

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cranberry concentrate. The data are shown in Table 7. Cranberry dietary supplement contained

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the highest amount of procyanidins at 0.915 mg/g. However, this value was markedly lower than

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the labeled procyanidin concentration of 187 mg/g which was mostly likely obtained using a

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DMAC colorimetric method. The large variance may also result from the coexisting of fruit matrix

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or impurities in the cranberry concentrate powder. The total procyanidin content in cranberry

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juices was 0.140 mg/mL, which was within a reported range of 0.044‐0.777 mg/mL which was

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measured using DMAC method with procyanidin A2 as a standard (Feliciano and others 2012).

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Craisins contained higher amount of total procyanidins with significantly larger ADP than that in

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cranberry juice. Ratios of A‐type linkage for procyanidins in cranberry juice was significantly

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higher that of craisins. Their average degrees of polymerization (ADP) were similar. It was

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reported that juicing process resulted in loss of procyanidins and cleavage of B‐type interflavan

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bonds 19. Possible changes of procyanidins during processing of Craisins have not been reported.

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A‐type procyanidin equivalents (APE) reflect the absolute amount of flavan‐3‐ols linked by A‐type

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linkages. Table 7 shows that cranberry dietary supplement contained the highest APE whereas

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cranberry juice contained the least. APE is a new concept for A‐type procyanidins and it may

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correlate better with the bioactivity of cranberry procyanidins than total procyanidins because

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only A‐type procyanidins in cranberries have anti‐adhesion activity.

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In conclusion, cysteamine was utilized as a low‐odor substitute of toluene‐α‐thiol for

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thiolysis depolymerization of procyanidins. Reaction temperature of 70 °C and reaction time of

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20 min, with 0.3 M of HCl were determined as an optimum depolymerization condition. A 36‐min 18


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reversed phase HPLC gradient was developed for separation of cranberry procyanidin thiolytic

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products. Procyanidin dimer A and dimer B were used as external standards. Single‐lab

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validations of the method including detection and quantification limits, linearity, precision and

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recovery were conducted per criteria set by the AOAC International. The strength of this method

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is that it can simultaneously quantitate total procyanidins, average degree of polymerization,

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ratio of A‐type linkages, and A‐type procyanidin equivalents in cranberry products. This method

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also has several drawbacks. Sample preparation procedure was lengthy and includes a chemical

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reaction, which may have caused poor recovery at low concentration. Some unique or modified

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procyanidins, such as trimers with two sets of A‐type linkages or aldehyde modified cranberry

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procyanidins 27, could not be quantified using this method. This method measures only

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extractable procyanidins. However, it can be modified to include unextractable procyanidins by

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combining extraction and depolymerization steps in a “one‐pot” approach.

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Chemistry 2004, 87, 465‐470.

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27. Arbenz, A.; Avérous, L., Chemical modification of tannins to elaborate aromatic biobased macromolecular architectures. Green Chemistry 2015, 17, 2626‐2646.

450

451

Funding: This research was funded in part by a grant from Ocean Spray Cranberries, Inc.

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452

FIGURE CAPTIONS

453

Figure 1 Thiolytic products of three representative procyanidin trimers (A, B, C) using cysteamine

454

as a thiolysis reagent. (1): catechin, (2): epicatechin, (3): thio‐epicatechin (3,4‐trans‐epicatechin

455

cysteamine thioether), (4): procyanidin A2, (5): thio‐A2 (procyanidin A2 cysteamine thioether)

456

Figure 2 HPLC chromatograms of thiolyzed partially purified cranberry procyanidins on Kinetex

457

2.6 µm C18 column (A) and StableBond 5 µm C18 column (B). Detection wavelength: 280 nm.

458

Peak 1‐5 match the structure of the same number in Figure 1.

459

Figure 3 Normal phase HPLC chromatograms of partially purified cranberry procyanidins before

460

(A) and after thiolysis (B). Thiolysis was performed at 70 °C in 0.3 M acidity and 5‐fold excess of

461

cysteamine for 20‐minute. Peaks 1‐4 match structure of the same number in Figure 1. 6:

462

procyanidin B2, 7: procyanidin trimers. 8: procyanidin polymers, 9: unknown compounds

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Table 1. HPLC‐MS Analysis of Thiolytic Products of Partially Purified Cranberry Procyanidins a

No (1) (2) (3) (4) (5)

Calculated Thiolytic products monoisotopic [M+H]+ Catechin 12.7 C15H14O6 291.0868 Epicatechin 20.6 C15H14O6 291.0868 Thio‐epicatechin 14.3 C17H19O6SN 366.1011 Procyanidin A2 29.4 C30H24O12 577.1346 Thio‐A2 18.4 C32H29O12SN 652.1488 a Number of thiolytic products (1‐5) matches those in Figure 1. Retention time (min)

Observed monoisotopic [M+H]+

Molecular formula

291.0863 291.0863 366.1006 577.1341 652.1483

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Table 2. Standard Curves for Thiolytic Products Standard curve

X‐axis (independent variable)

#1

Weight or molar amount of Summed peak areas terminal unit in procyanidin of (1) and (2) B2

(3): Thio‐epicatechin

#2

Weight or molar amount of Peak area of (3) extension unit in procyanidin B2

#3

Weight or molar amount of Peak area of (4) procyanidin A2

(4): Procyanidin A2

Y‐axis (dependent variable) *

*Number of thiolytic products (1‐5) matches those in Figure 1.

25


Thiolytic products quantified * (1) + (2): Catechin + Epicatechin

(5): Thio‐A2


Table 3. HPLC Peak Areas of Five Thiolytic Products of Partially Purified Cranberry Procyanidins under Different Thiolysis Conditions Chromatogram Peak Area

(1) Catechin (2) Epicatechin (3)Thio‐epicatechin (4) Procyanidin A2 (5)Thio‐A2 60 37.5±1.5 C 313.5±11.7 AB 1112.5±27.1 B 538.8±39.9 B 133.6±9.9 C Temperature 70 57.1±2.1 B 340.7±15.4 A 1534.1±86.8 A 670.8±30.1 A 349.1±3.4 B (°C)a 80 93.0±1.5 A 295.1±5.4 B 1645.8±48.5 A 671.2±9.5 A 731.7±13.2 A 10 50.1±11.8 B 320.2±8.3 BC 1241±22.8 C 588.2±23.1 B 220.3±11.6 D 15 55.3±1.9 B 343.9±10.2 A 1436±59.7 B 655±4.4 A 342.4±15.6 C Time (minute)b 20 63.7±2.8 AB 332.3±3.8 AB 1497±71.8 AB 678.4±25 A 441.1±3.6 B 25 71.6±1.8 A 310.8±3.5 C 1622.8±7.7 A 687.2±15.9 A 538.9±3 A 0.1 43.4±1.7 C 309.9±8.7 B 1252.9±65 B 586.6±22.5 B 219.4±6.9 D c 0.2 63.8±1.6 B 333.2±11.1 A 1499.3±95 AB 683±15.1 A 448.2±6.2 C Acidity (M) 0.3 79.4±0.9 A 318.7±4.5 AB 1625.4±57 A 690±13.3 A 578±11.5 B 0.4 79.3±7.5 A 288.7±4.9 C 1567.6±172 A 638.7±46.1 AB 676.3±19 A 2 93.4±7.7 A 319.5±11 A 1546.4±86.7 A 679.6±45.6 A 391.2±4.8 B 5 100.5±3.3 A 332.8±3.8 A 1564.7±80.4 A 702.3±21.2 A 530.2±33.3 A Fold excess of d cysteamine 10 98.5±9 A 346.7±17.6 A 1618.9±39.9 A 721.1±16.3 A 567±35.9 A 50 83.2±4.6 A 329.8±24 A 1569.1±42 A 709.4±22.6 A 584.6±19.2 A Data are mean ± standard deviation for 3 determinations; Chemical structures of thiolytic products (1‐5) are shown in Figure 1; Data in the same column with different letter labels are significantly different. a Reactions were conducted for 15 minutes using 0.2 M HCl and 500 mg/mL cysteamine (50 folds) b Reactions were conducted under 70 °C using 0.2 M HCl and 500 mg/mL cysteamine (50 folds) c Reactions were conducted under 70 °C for 20 minutes using 500mg/mL cysteamine (50 folds) d Reactions were conducted under 70 °C for 20 minutes using 0.3 M HCl 26


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Table 4. Relative Errors and Correlation Coefficients of Calibration Curves of Procyanidin A2, Thio‐Epicatechin and Epicatechin Using Different Weighing Factors Weighing

1/x0

1/x0.5

1/x

1/x2

1/x3

Relative error

12.8

3.10

1.23

0.962

2.80

R2

0.9983

0.9976

0.9961

0.9786

0.9578

Relative error

10.6

1.94

0.82

0.46

0.64

R2

0.9975

0.9976

0.9973

0.9934

0.986

Relative error

5.37

2.32

1.45

1.30

2.67

R2

0.9996

0.9994

0.9984

0.9709

0.9646

(1)+(2) Catechin+epiatechina (3) Thio‐epicatechina

(4) Procyanidin A2a a

Molecular structures of thiolytic products (1‐4) are shown in Figure 1.

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Table 5. Linear Range, Limit of Detection (LOD), and Limit of Quantification (LOQ) of Thiolytic Products of Procyanidin A2 and Procyanidin B2 Linear range (mg/mL)

Thiolytic adducts a

Calibration curve

Average slope Intercept*

R2

LOD (mg/mL)

LOQ (mg/mL)

(1)+(2) Catechin+epicatechin 0.05‐20.0

y=376x‐13.5

376

13.5±2.05 0.9961

0.018

0.054

(3) Thio‐epicatechin a

0.01‐10.0

y=372x+2.13

372

2.13±1.15 0.9934

0.010

0.031

(4) Procyanidin A2 a

0.05‐10.0

y=962x‐10.3

962

10.3±4.9 0.9994

0.017

0.051

*Intercept is mean ± standard deviation for 3 determinations a

Molecular structures of thiolytic products (1‐4) are shown in Figure 1.

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Table 6. Recovery and Precision of Spiked Procyanidin A2 and B2 Standards in Wheat Flour and Gatorade Beverage

Concentrations

Procyanidin A2

0.5

76.6 ± 6.5

82.1 ± 9.4

8.4

11

16.1

14.9

1.0

84.3 ± 6.1

85.4 ± 10.8

7.2

13

7.4

6.9

5.0

84.5 ± 5.3

92.5 ± 6.2

6.2

6.7

6.7

6.1

0.5

84.8 ± 14.3

109 ± 13.1

16.9

12.0

10.7

3.3

1.0

84.0 ± 4.0

108 ± 4.3

4.8

4.0

9.6

3.8

5.0

104 ± 7.6

93 ± 6.8

7.3

7.3

6.1

7.3

Wheat Flour (mg/g)

Gatorade (mg/mL)

Precision (RSD, %)

Recovery (%)

Intra‐day

Inter‐day

Procyanidin Procyanidin Procyanidin Procyanidin Procyanidin B2 A2 B2 A2 B2

*Data are mean ± standard deviation for 3 determinations

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Table 7. Quantitation and Characterization of Procyanidins in Three Representative Cranberry Products Cranberry products

TPwa (mg/mL or mg/g)

TPmb (mmole/mL or mmole/g)

Craisins dried cranberries

0.384±0.014

0.92±0.03

4.7±0.3 A 21.8%±0.7% B

83.8±5.9 B

Cranberry juice cocktail

0.140±0.005

0.33±0.01

3.2±0.3 B 26.7%±0.9% A

37.4±2.4 B

Dietary supplement containing cranberry extract

0.915±0.138

2.07±0.32

2.7±0.2 B 29.2%±2.6% A

258±55 A

ADPc

RALd

APEe (µg/mL or µg/ g)

*Data are mean ± standard deviation for 3 determinations; Data in the same column with different letter labels are significantly different. a Total procyanidins in weight b Total procyanidins in mole c Average degree of polymerization d Ratio of A‐type linkages e A‐type procyanidin equivalents

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Figure 1 OH O

HO

OH

OH OH

OH OH HO

O

O

OH

(1) catechin

OH

OH

+

OH

OH

OH

OH

or

OH

OH

OH OH HO

O

HO

OH

N H2

OH

O

HO HO

O

A

OH

OH HS

OH

S

N H2

(2) epicatechin

(3) thio-epicatechin

OH OH OH O

HO

O

OH

O

O

OH

+

OH

S

N H2

OH O

OH

OH

HO

(3) thio-epicatechin

OH

(4) procyanidin A2

OH

HO

OH OH

OH

NH2

O

O

HO

OH

HS

OH OH

B

O

HO OH

OH OH HO

OH

OH

OH

OH

OH OH

O

OH

HS

OH OH

HO

O

OH

O

OH

OH OH

OH

C

OH

NH2

OH OH HO

OH

OH OH

(2) epicatechin

(1) catechin

OH

O

HO

or

OH

O

OH OH

O

HO

O

OH

HO

+

O

OH

O

OH

OH OH OH

S

(5) thio-A2

N H2

OH OH

31



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Figure 2 32


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Figure 3

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Page 34 of 34

TOC Graphic

34


Development of a Thiolysis HPLC Method for the Analysis of

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