Separation of Ellagitannin-Rich Phenolics from U.S. Pecans and

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Separation of Ellagitannin-Rich Phenolics from U.S. Pecans and Chinese Hickory Nuts Using Fused-Core HPLC Columns and Their Characterization Yi Gong, and Ronald B. Pegg J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01597 • Publication Date (Web): 25 Jun 2017 Downloaded from http://pubs.acs.org on June 25, 2017

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Journal of Agricultural and Food Chemistry

1

Separation of Ellagitannin-Rich Phenolics from U.S. Pecans and Chinese Hickory Nuts

2

Using Fused-Core HPLC Columns and Their Characterization

3 Yi Gong† and Ronald B. Pegg*,†

4 5 6 7

†

Department of Food Science & Technology, College of Agricultural and Environmental Sciences, The University of Georgia, 100 Cedar Street, Athens, GA, 30602, USA

8 9 10 11 12 13 14 15 16 17 18 19 20 21

RUNNING TITLE: Separation of phenolic compounds from pecans and hickory nuts

22 23

*Corresponding author. Tel: (706) 542-1099. Fax: (706) 542-1050. E-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

25

U.S. pecans and Chinese hickory nuts possess a wide array of phenolic constituents with

26

potential health benefits, including phenolic acids and proanthocyanidins. Only limited

27

information is available, however, on their compositions. The present study optimized the

28

separation performance and characterized the low-molecular-weight phenolic fractions of these

29

nuts with C18 and pentafluorophenyl (PFP) fused-core LC columns employing a kinetic

30

approach. Although both types of reversed-phase columns demonstrated similar performance in

31

general, the PFP column furnished greater plate numbers and superior peak shapes for the low-

32

molecular-weight fractions as well as overall separations of ellagic acid derivatives. Analyzed by

33

a 3-µm HILIC column, the high-molecular-weight fraction of pecans possessed more

34

proanthocyanidins than the Chinese hickory nuts, with dimers and trimers (31.4 and 18.34 mg/g

35

crude extract, respectively) being present at the greatest levels. Chinese hickory nuts had lower

36

proanthocyanidins contents, but possessed tetramers and pentamers at 4.46 and 4.01 mg/g crude

37

extract, respectively.

38 39

KEYWORDS: Fused-core columns; Phenolic compounds; Proanthocyanidins; Pecans;

40

Chinese hickory nuts; HILIC


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INTRODUCTION

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Pecans [Carya illinoinensis (Wangenh.) K. Koch] are one of the few tree nuts that are native to

43

North America. They are exported to China and are being sought after there as an alternative to

44

the historically recognized Chinese hickory nut (Carya cathayensis Sarg.). In fact, ca. 50% of the

45

U.S. pecan production has been destined for China over the past three years. The rising demand

46

for tree nuts in general has made China the biggest market and an important trading partner for

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U.S.-grown tree nuts. Furthermore, there is no sign that demand is abating.1–3

48

Both U.S. pecan and Chinese hickory nuts possess a wide array of phenolic constituents with

49

potential health benefits,4–8 but only limited information is available on their composition.9 To

50

facilitate compound identification, Polles et al.10 acid hydrolyzed pecan crude phenolic extracts

51

and then identified and quantitated five phenolics, with ellagic and gallic acids being the

52

predominant compounds. The majority of the phytochemicals present; that is, the free and

53

esterified phenolics were left unmeasured. Robbins et al.11 profiled the phenolics of U.S. pecans

54

after extracting the hydrophilic bioactives from defatted raw pecan nutmeats using an

55

(CH3)2CO:H2O:CH3COOH solvent system. The crude extract obtained was subsequently

56

separated into five fractions via Sephadex LH-20 column chromatography. HPLC-ESI-MSn

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results indicated that ellagic acid and (+)-catechin were the most abundant compounds.

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An early investigation on pecan tannin constituents was published by Polles et al.10, who

59

concluded that tannins were present at 0.70-1.71% levels, depending on the cultivar analyzed.

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Gu et al.12 reported that tree nuts, including pecans, contain exclusively B-type

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proanthocyanidins. A decade later, Robbins et al.11 characterized the proanthocyanidins of

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pecans by hydrophilic interaction liquid chromatography (HILIC), and determined that dimers

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and trimers were the major contributors of the proanthocyanidins profile. In contrast, Gu et al.13,



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who employed a different type of column, concluded that pecans are composed of a considerable

65

percentage of proanthocyanidins with higher degrees of polymerization. The lengthy analytical

66

time and potential co-elution of critical pairs by this method indicates that improved

67

chromatographic separation is needed.

68

Pentafluorophenyl (PFP) and C18 reversed-phases are two of the most utilized stationary

69

phases for separation, identification, and quantitation of phenolic compounds in a variety of

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matrices.14–17 Both stationary phases are now available as sub 3-µm fused-core options. Until

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now, limited information has been available on comparisons of the phenolic separation

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efficiency between PFP and C18 fused-core columns. Mirali et al.18 separated phenolics isolated

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from lentil seed coats. The mobile phase concentration, gradient, and flow rate were optimized

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on both the C18 and PFP fused-core columns. The authors reported that the PFP column

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demonstrated longer interaction and much better resolution of isomeric compounds compared to

76

that of the C18 column. Gómez-Caravaca et al.19 also investigated the efficacy of these two

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stationary phases for phenolic separations from waxy and non-waxy barley samples comprising

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18 different varieties. Analysis revealed that a majority of the barley phenolics (12) are present in

79

their free form. Based on the different gradient elution programs employed for each column type,

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the authors determined that the C18 column yielded superior separations of the free phenolic

81

compounds, whereas the PFP column gave slightly better resolution for the 8 bound compounds.

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However, hierarchical cluster analysis revealed that only separations from the C18 column could

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discriminate the waxy from the non-waxy genotypes. To that end, the authors concluded that the

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fused-core C18 column was more appropriate for analysis of phenolics in barley. These two

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contradicting recommendations on the employment of a fused-core stationary phase clearly

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indicate that the choice of the type of stationary phase should be analyte- or sample-specific. To


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date, no such investigation has been carried out on the phenolics isolated from tree nut species.

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Moreover, the comparisons and recommendations were made only based on the efficacy of

89

separated compounds, and no kinetic data were provided for these two types of stationary phases.

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In the current investigation, C18 and PFP fused-core columns were compared against one

91

another for their separation efficiencies of low-molecular-weight phenolic compounds from U.S.

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pecan [Carya illinoinensis (Wangenh.) K. Koch] and Chinese hickory nuts (Carya cathayensis

93

Sarg.) using kinetic plots. Their high-molecular-weight tannin fractions were also profiled and

94

quantitated. The aim of this research was to establish an optimized method for the separation of

95

tree nut phenolic extracts and their low-molecular-weight fractions utilizing fused-core columns.

96

Furthermore, the similarities and discrepancies of the phenolic compounds isolated from these

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two most-economically important species of the Carya family were compared and summarized.

98 99 100

MATERIALS AND METHODS Chemicals. ACS-grade acetone, methanol, hexanes, ethanol (95%), HPLC-grade water,

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HPLC-grade acetonitrile, cellulose thimbles, and Whatman No. 1 filter paper were purchased

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from the Fisher Scientific Co., LLC (Suwanee, GA). Glacial acetic acid was acquired from VWR

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International, LLC (Suwanee, GA). Sephadex LH-20 and uracil were obtained from the Sigma-

104

Aldrich Chemical Company (St. Louis, MO).

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Extraction and Fractionation of Phenolic Compounds. The U.S. pecans were raw

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'Desirables' supplied by Dr. M.L. Wells of the University of Georgia Department of Horticulture

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(Tifton, GA), while the raw Chinese hickory nuts were acquired by Dr. Randy D. Hudson

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(Ocilla, GA) from a major tree nut processor in Lin’an (Zhejiang Province, PRC). Shelled U.S.

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pecan and Chinese hickory kernel nutmeats were frozen in liquid nitrogen, ground, ca. 20 g were



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defatted with hexanes for roughly 18 h using a Soxhlet apparatus, the phenolic constituents were

111

extracted with (CH3)2CO:H2O:CH3COOH, and then the resultant crude phenolic extracts were

112

fractionated by Sephadex LH-20 column chromatography according to Robbins et al.20 The

113

aqueous residues were then lyophilized and the mass of each dried extract was weighed,

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transferred into an amber vial, capped, and stored at -20 °C until analyzed.

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High-performance Liquid Chromatography. Chromatographic conditions were

116

established to identify and quantitate pecan and hickory nut phenolic compounds by HPLC-ESI-

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QToF-MS (time-of-flight) and HPLC-DAD. Two superficially porous reversed-phase HPLC

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columns with different surface modifications, namely a 150 mm × 4.6 mm i.d., 2.6 µm, Kinetex

119

XB-C18 column with a pore size of 100 Å (Phenomenex, Torrance, CA) and a SecurityGuard

120

cartridge of the same material as well as a 150 mm × 4.6 mm i.d., 2.6 µm, Kinetex PFP column

121

with a pore size of 100 Å (Phenomenex) and a SecurityGuard cartridge of the same material,

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were employed in this investigation. A 1200 series HPLC system (Agilent Technologies, Inc.,

123

Wilmington, DE) was used for developing the chromatographic conditions. Twenty microliters

124

were injected for each pecan or hickory nut low-molecular-weight fraction (10.0 mg/mL in

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mobile phase A followed by a 1:1 dilution) after being filtered through a 0.45-µm PTFE

126

membrane. Detection wavelengths were 255 nm (i.e., ellagic acid and its derivatives), 280 nm

127

(phenolic acids, catechin, epicatechin), 320 nm (phenolic acids notably of the trans-cinnamic

128

acid family), and 360 nm (flavonols). Tentative identification of separated components was

129

achieved by matching UV/Vis spectra and retention times (tR) with standard compounds.

130

The high-molecular-weight phenolics, chiefly the proanthocyanidins, were separated using

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the same Agilent chromatograph but with a 150 mm × 4.6 mm i.d., 3 µm, Luna HILIC column

132

with a pore size of 200 Å (Phenomenex), and SecurityGuard cartridge of the same material. The


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gradient elution employed was based on Kelm et al.21 with modifications. Mobile phases

134

consisted of CH3OH/CH3COOH (98:2, v/v) (solvent A) and CH3OH/H2O/CH3COOH (95:3:2,

135

v/v/v) (solvent B). A linear gradient elution at a flow rate of 1.0 mL/min was run as follows:

136

0−25 min, 0−45% B; 25−30 min, 45−0% B; and then held for an additional 2 min to allow the

137

system to equilibrate. High-molecular-weight extracts from U.S. pecan and Chinese hickory nuts

138

were dissolved in CH3OH and then diluted at a 1:1 (v/v) ratio with the mobile phase A to a final

139

concentration of 5 mg/mL. An aliquot of 10 µL was injected for each sample after filtering

140

through a 0.45-µm PTFE membrane. Fluorescence detection was set at 276/316 nm for

141

excitation/emission wavelengths, respectively. Commercial cocoa proanthocyanidin standards

142

with degrees of polymerization ranging from 2-10 (Planta Analytica LLC, Danbury, CT) were

143

employed to map the tR values. Proanthocyanidins isolated from pecan and hickory nutmeats

144

were quantitated with the calibration curves from individual standards.

145

High-performance Liquid Chromatography-Electrospray Ionization-Mass

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Spectrometry. An 1100 HPLC system (Agilent) coupled to a QToF micro mass spectrometer

147

equipped with an electrospray ionization (ESI) interface (Waters Corporation, Milford, MA) was

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used for identification. The MS conditions were according to Robbins et al.20 The mass

149

spectrometer was operated in the negative-ion mode using capillary voltages of +3.5 kV and -2.5

150

kV, respectively. The microchannel plate detector voltage was set at +2.35 kV. Nitrogen was

151

employed as the desolvation gas at a temperature of 100 °C and flow rate of 150 L/h. Argon was

152

used as the collision gas. For normal MS, the collision voltage was set at 5 V; however for

153

MS/MS, the collision voltage was increased to 30 V. Detection was achieved within a mass

154

range of 50 to 1,100 m/z for low-molecular-weight compounds and 300 to 3,000 m/z for high-

155

molecular-weight species. The MS/MS analyses were acquired by automatic fragmentation



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where the three most intense mass peaks where fragmented. This instrument was calibrated using

157

a Glu−Fibrinogen peptide (Waters) in the MS/MS mode, and MassLynx 4.1 software (Waters)

158

was employed for control and analysis. Comparison of parent molecular ions [M–H]− with

159

known standards was utilized to assist with elucidation of the identities of the phenolic

160

compounds. When necessary, comparisons of tR and [M–H]− values, and fragmentation patterns

161

of phenolic compounds to those reported in the literature were carried out.

162

Van Deemter−Knox Plot. The kinetic characteristics of chromatographic columns are

163

usually assessed by the Van−Deemter equation, describing the height equivalent to a theoretical

164

plate (HETP or H) as the sum of three contributors affected by the linear velocity (u) of the

165

mobile phase.22 The A−C constants in Equation 1 represent eddy diffusion (A term), longitudinal

166

diffusion (B term), and resistance to mass transfer (C term), respectively. =+

167

The H term is related directly to the theoretical plate numbers (N) and column length (L). =

168

+ (1)

(2)

The number of effective theoretical plates was calculated by the following equation: − (3)

= 5.54 .

169

where, tR is the retention time of the compounds-of-interest; t0 is the time for the column dead

170

volume marked by the elution of uracil; and W0.5 is the peak width at half height.

171

To permit a comparison of efficiencies between columns of identical dimensions but packed

172

with materials of different physical or chemical characteristics, the Van−Deemter parameters

173

were fitted and plotted using the Knox equation with reduced parameters:


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ℎ = + 174

+ (4)

In the Knox equation, HETP (H) and linear velocity (u) were transformed into the

175

dimensionless units (h and v), where the reduced plate height h was calculated by dividing the

176

plate height H against the particle size of the column packing material dp, and the liner velocity v

177

was determined according to the following equation:23,24 =

178

! (5) "#

The molecular diffusivity Dm for each standard was estimated by the Wilke-Chang equation25: "# (

%$⁄

&) = 7.4 × 10

+,

(-.)⁄ / (6) 01 .2

179

where, x is the association coefficient introduced to define the effective molecular weight of the

180

mobile phase with respect to the diffusion process. The x value used in this calculation for a

181

CH3CN:H2O mixture was proposed by Miyabe26 as 1.37. The M term represents the average

182

molecular weight of the mobile phase (g/mol); T is the absolute temperature (K); η is the mobile

183

phase viscosity (cP); and V is the molar volume (mL/mol) of the solute at the normal boiling

184

point as estimated by the LeBas method27 based on the summation of atomic contributions.

185

Theoretical Poppe Plot under Isocratic Conditions. The construction of the Van

186

Deemter−Knox plot is based solely on the plate count and linear velocity, while the important

187

factor of column permeability is left out of consideration. The Poppe plot28 is proposed to

188

address this issue, because it is capable of demonstrating the relationship between the plate

189

number (N) and the time equivalent to a theoretical plate (t0/N). The Van Deemter data (u0, H)

190

were rearranged into a new data set (t0, N), calculated using the following formula with column

191

permeability being factored in.



=

∆5%6- 89 7 : (7) 0

=

∆5%6- 89 7 : (8) 0

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192

where, ∆Pmax is the maximum column inlet pressure recommended by the manufacturer. For C18

193

and PFP stationary phases prepared with 2.6-µm superficially porous particles, the maximum

194

back pressure allowed is 60 MPa. However, both columns were coupled with a conventional

195

HPLC system; therefore, the maximum allowable working pressure ∆Pmax was set at 40 MPa.

196

The η term is the viscosity of the mobile phase, which was determined based on its composition

197

and temperature. Kv is the column permeability term and was calculated as follows: 89 =

198 199

0 (9) ∆5

where, ∆P is the pressure drop at the linear velocity over the column length L. Method Optimization. To optimize the chromatographic conditions on the selected fused-

200

core column, mobile phase compositions, starting solvent percentage and gradients, were

201

adjusted and compared until all co-eluting critical pairs were well resolved. The peak resolution

202

(Rs) was calculated for the critical pairs under the selected working chromatographic conditions,

203

which were used as criteria to choose the most effective protocol. The Rs was determined as

204

follows: => =

2( − ) (10) (? − ? )

205

where, tR1 and tR2 are the retention times of the two analytes; t0 is the retention time of the void

206

volume; and WB2 and WB1 are the peak widths of the resolved critical peaks measured at the base.

207

Validation of the Analytical Method. The precision and accuracy of the two fused-core

208

columns studied were assessed by spiking the sample with five representative phenolic 10 ACS Paragon Plus Environment

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standards, including gallic acid, (+)-catechin, ellagic acid, caffeic acid, and proanthocyanidin B2

210

using the proposed chromatographic method. Calibration curves for each standard were prepared

211

with a minimum of ten points comprising concentrations between 0.05 and 1000 µg/mL for each

212

standard. Replicates were injected at each concentration. Calibration curves were constructed by

213

plotting the peak area versus the concentration of each individual standard. Regression analysis

214

was applied to test the linearity of the concentration ranges. Accuracy (% REC) was determined

215

by the closeness of the predicted concentration calculated based on the standard curve to the

216

nominal value of the standard concentration. Precision (% RSD) was measured by calculating the

217

relative standard deviation of the repeated injections using the following formula: @ABCDBEFGHI 9HIJB + KLHIMEFGHI 9HIJB

× 100

218

% REC =

219

% RSD = Standard deviation × 100 / mean

@ABCDBEFGHI 9HIJB

(11) (12)

220

The limit of detection and limit of quantitation were estimated from a series of further

221

dilutions of the aforementioned standards with the mobile phase. The limits of detection were

222

accepted for each standard if the lowest concentration provided a signal-to-noise ratio equal to 3.

223

The limits of quantitation were calculated for peak areas that had RSDs less than 10% for both

224

intraday and interday with a signal-to-noise ratio greater than 10.

225 226

RESULTS AND DISCUSSION

227

Flow Study. The performance and separation efficiency of the columns packed with C18 and

228

PFP fused-core particles on representative phenolic standards including ellagic acid,

229

proanthocyanidin B2, and (+)-catechin were studied and compared using both Van

230

Deemter−Knox and Poppe plots.



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In order to obtain experimental data to construct kinetic plots, the aforementioned phenolic

232

standards (50 µg/mL) were dissolved in the mobile phase and eluted isocratically at a flow rate

233

between 0.05 and 1.0 mL/min at a fixed temperature of 25 °C. The mobile phase composition

234

employed for elution of ellagic acid was CH3CN:H2O:CH3COOH (25:74:1, v/v/v), but for

235

proanthocyanidin B2 and (+)-catechin it was CH3CN:H2O:CH3COOH (15:84:1, v/v/v). The

236

linear velocity, u, and column void time, t0, for both columns were determined using uracil as an

237

unretained marker.

238

The results obtained were used to estimate the constants (A, B, C) by fitting the calculated

239

h−v data to Equation 4 using the least square optimization method. A comparison between the

240

A, B, and C terms, minimum plate height, reduced linear velocity, and plate height for each

241

standard on both the XB-C18 and PFP columns is presented in Table 1. Both Kinetex XB-C18

242

and PFP fused-core columns demonstrated relatively comparable performance when analyzing

243

proanthocyanidin B2 and (+)-catechin. However, an exception was noted for ellagic acid. The

244

two columns employed showed distinct behaviors when this phenolic acid was analyzed. The

245

XB-C18 column demonstrated a much greater minimum plate height at lower mobile phase

246

linear velocities when compared to the PFP packed column, even though the performance of the

247

XB-C18 column was approaching the latter at higher flow rates. When one closely examines the

248

fitted constants, it is evident that the XB-C18 column has an elevated B value of 9.51, almost

249

double of that for the PFP column. Therefore, the PFP column has a distinct advantage over the

250

C18 column with regard to ellagic acid and its derivatives.

251

Kinetic Plots for Separation Speed. To compare the permeability and separation efficiency

252

characteristics of the columns, theoretical Poppe plots for both columns were constructed.

253

Figure 1 plainly shows that the Kinetex PFP column provided more favorable plate time values


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compared to those of the Kinetex XB-C18 column, if the separation was targeted for 10,000

255

plate counts. The points a and b in Figure 1 represent the condition when the vopt and hmin was

256

achieved for ellagic acid on the XB-C18 and PFP columns, respectively. At its optimum, the PFP

257

column (N=16,558) attained almost 2× more plate counts compared to the XB-C18 column

258

(N=8,511), while their required plate times were almost identical. In other words, the PFP

259

column was able to achieve the same plate counts but in a much shorter time relative to that of

260

the XB-X18 column. Nevertheless, both columns demonstrated comparable and satisfactory

261

overall performance when proanthocyanidin B2 and (+)-catechin were analyzed under identical

262

chromatographic conditions; that is, both yielding over 10,000 theoretical plates in less than 2

263

min.

264

Concerning the chemical composition of the phenolics in U.S. pecan and Chinese hickory

265

nutmeats, previous research showed the dominant species to be ellagic acid and a valoneic acid

266

dilactone.11,20 Representative phenolic compounds of U.S. pecans and Chinese hickory nuts are

267

depicted in Figure 2. Based on the separation performance of ellagic acid, the PFP column,

268

therefore, seems better suited for analyzing the phenolic constituents of Carya species.

269

Method Development and Optimization. A number of gradients were tested with the

270

intention of adequately resolving the co-elution of critical pairs of compounds. The

271

chromatographic conditions employed are listed in Table 2. Varying the flow rate greatly

272

affected the separation efficiency of all species, especially for critical pairs containing ellagic

273

acid and its derivatives.

274

As a result of comparing the resolutions achieved using different arrangements of

275

chromatographic conditions, the best linear gradient was selected. The proposed gradient method

276

gave practically baseline separation for all compounds, along with improved peak width and



Page 14 of 39

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shape, including those outlined critical pairs. Juxtaposed to the previous method employing a 5-

278

µm fully porous column, the total run time was shortened from 60 to 35 min at a moderate flow

279

rate of 0.8 mL/min. The volume of mobile phase required for this analysis was also reduced by

280

ca. 50%. The proposed gradient could be tailored to yield acceptable results for ellagitannin-rich

281

phenolic extracts from other food matrices; for instance, nuts and seeds, medicinal plants or

282

fruits, by modifying the linear gradient.

283

Method Validation. Quantitative analysis was performed based on the established

284

calibration curves of commercial standards. These compounds were injected using the optimized

285

gradient across a range of concentrations. The chromatographic parameters, including limits of

286

detection, limits of quantitation, accuracy, and precision, are summarized in Table 3. The

287

calibration curves generated with the proposed method revealed excellent linearity over a wide

288

range on both types of fused-core columns, with regression coefficients close or >0.995,

289

indicating excellent correlation between the analyte concentration and peak area. As expected,

290

the limits of detection and limits of quantitation determined on the PFP and C18 columns were

291

very similar, with the only exception being that of ellagic acid. For the PFP column, the limit of

292

detection of ellagic acid was 9.2 ppb and the limit of quantitation was 22.3 ppb, whereas for the

293

C18 column, the limit of detection was 63.4 ppb and the limit of quantitation was nearly 10 times

294

greater than the value obtained from the PFP column, reaching 210 ppb. Clearly, the limit of

295

detection and limit of quantitation values on the PFP column were much lower when analyzing

296

ellagic acid, which again proves that when operating under the same chromatographic conditions,

297

the PFP column allows for greater sensitivity for ellagic acid and its derivatives compared to the

298

XB-C18 column.


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299


Low-Molecular-Weight Phenolic Compounds in Carya Species. Ninety-nine percent of

300

the crude phenolic extracts applied to the Sephadex LH-20 column were recovered, of which

301

77.6 and 63.2% represented the low-molecular weight fractions of the Chinese hickory nuts and

302

U.S. pecans, respectively. Identification of resolved peaks was achieved either by matching the

303

tR values and MS fragmentation patterns to those of authentic standards (when available), or by

304

assigning tentative identifications based on the interpretation of patterns and comparisons with

305

those reported in the literature. A summary of tentative identifications of the phenolics for the

306

low-molecular-weight and high-molecular-weight fractions in both U.S. pecans and Chinese

307

hickory nuts is available in Table 4 and Table 5, respectively.

308

Phenolic Acids and their Derivatives. Figure 3 depicts the chromatographic separation of

309

the low-molecular-weight phenolic fractions isolated using Sephadex LH-20 from U.S. pecan

310

and Chinese hickory nutmeat phenolic extracts on the Kinetex PFP column. Peak 1 exhibited a

311

molecular ion [M−H]− at m/z 331 and a product ion at m/z 169. The presence of a hexoside was

312

speculated with a neutral loss of 162 Da from the parent ion. Hence, this compound was

313

proposed as gallic acid hexoside. Peak 4 showed a deprotonated parent ion [M−H]− at m/z 169

314

with a maximum UV absorption band at 255 nm. The neutral loss of CO2 [M−H−44]− from the

315

carboxylic acid group was also observed, resulting in a characteristic fragment ion at m/z 125;

316

this compound was confirmed as gallic acid via tR and UV spectrum matching. Applying the

317

same approach, peak 5 with a m/z at 137 and peak 19 with a m/z at 179 were correspondingly

318

identified as p-hydroxybenzoic acid and caffeic acid. Additionally, peak 9 eluted at a tR of 7.3

319

min and was identified as protocatechuic acid hexoside, as revealed by the neutral loss of 162

320

[M−H−hexoside]− and a fragment ion at m/z 153 corresponding to a protocatechuic acid moiety.



321

Page 16 of 39

Hydrolyzable Tannins and Related Compounds. A significant proportion of phenolics

322

found in pecan and hickory nut extracts belong to the ellagitannin family, which eluted during

323

the latter half of the chromatography. The predominant peak, 27, was identified as ellagic acid

324

based on the molecular ion [M−H]− at a m/z 301, along with the UV spectrum and tR values of

325

the authentic standard. In addition to free ellagic acid, several glycosylated ellagic acid

326

derivatives were found in the Carya species, such as peaks 17 and 22. These were identified as

327

ellagic acid hexoside (m/z 463, fragment m/z 301, [M−H−162]−) and ellagic acid pentoside (m/z

328

433, fragment m/z 301, [M−H−132] −). The fragmentation patterns of monoglycosylated ellagic

329

acids were identical to the findings described in pomegranate.29 Peak 25 exhibited a molecular

330

ion with a water adduct at m/z 487 [M−H+18 (H2O)]−. Fragment ions were noted at m/z 425 (loss

331

of CO2) and 301 (ellagic acid). These characteristics indicated that peak 25 was valoneic acid

332

dilactone, a hydrolyzable tannin that is usually present in parallel with ellagic acid. This

333

fragmentation pattern matches closely with that found in walnut phenolics.30 Methylated and

334

glycosylated ellagic acid derivatives were also observed in the samples. Peaks 24 and 26 were

335

identified as methyl ellagic acid hexosides ([M−H]− at m/z 477). The fragment ion registered a

336

loss of hexose (m/z 162) from the parent molecule, and further loss of a methyl group from the

337

aglycone (m/z 315). Noteworthy is that no free methyl ellagic acid was detected in the analyzed

338

samples. Dimethyl ellagic acid hexoside and pentoside were also found in the analyzed samples,

339

as peaks 30 and 31, respectively. They were recognized and elucidated by the two sequential

340

losses of –CH3 moieties from the parent ion, and cleavages of m/z 162 and m/z 132, which are

341

commonly associated with hexosides and pentosides, respectively. Similarly, peak 35 was

342

identified as dimethyl ellagic acid by its consecutive loss of a methyl group. The MS2 mass

343

spectrum of m/z 505 showed strong fragmentation ions at m/z 463 [M−H−42]− and m/z 301,


Page 17 of 39


344

which correspond to the loss of an acetyl group and ellagic acid as an aglycone. This suggested

345

that the compound could be an acetyl derivative of ellagic acid hexoside. Similar compound

346

fragmentations have also been previously reported in camu-camu fruit by Fracassetti et al.31 The

347

ellagic acid fragment ion at m/z 301 and cleavage of the galloyl group (152 Da) also helped in

348

identifying peaks 20, 33, and 36 as digalloyl ellagic acid, ellagic acid galloyl pentoside, and

349

methyl ellagic acid galloyl pentoside, respectively.

350

Another important indication of ellagitannin compounds is the presence of

351

hexahydroxydiphenoyl groups (m/z 302).32 Hexahydroxydiphenoyl moieties can be released

352

through hydrolysis and subsequently lactonized to form ellagic acid.31 Two peaks eluted around

353

a tR of 3.5 min (peaks 2 and 3), and both showed a sizeable [M−H]− at m/z 481 and

354

fragmentation ion at m/z 301 [M−H−180 (glucose)]−. Based on the fragmentation pattern and

355

deprotonated ion mass, these two compounds are likely to be hexahydroxydiphenoyl−glucose

356

isomers. Peak 6 was assigned as galloyl−hexahydroxydiphenoyl−glucose. The molecular ion

357

[M−H]− at m/z 633 generated fragment ions at m/z 481 [M−H−152]−, resulting from the cleavage

358

of a galloyl group. Further fragmentation revealed a product ion at m/z 301

359

[M−H−152−glucose]−; this is likely associated with lactonized hexahydroxydiphenoyl after

360

being hydrolyzed from an ellagitannin. Peak 11, which demonstrated a strong [M−H]− at m/z

361

785, was identified as digalloyl− hexahydroxydiphenoyl−glucose, according to the fragment ion

362

at m/z 633 (loss of galloyl group), m/z 483 (M−H−302, loss of a hexahydroxydiphenoyl moiety),

363

and m/z 301 (loss of galloyl−glucose residue). This compound has not previously been reported

364

for the Carya family. Its presence was miniscule in U.S. pecans but sizable in Chinese hickory

365

nuts.

366

Galloylated glucose esters were also detected in the low-molecular-weight fractions of pecan



Page 18 of 39

367

and hickory nut phenolics, but to a lesser extent. The characteristic fragmentation patterns of

368

gallotannins involve a successive cleavage of galloyl groups from the parent molecule and the

369

presence of cross-ring fragmentation of glucose. Peak 7 was assigned as galloyl glucose with a

370

[M−H]− at m/z 331. MS/MS fragmentation revealed product ions at m/z 169 after glycosidic

371

cleavage (M−H−162, loss of glucoside) and m/z 271 (M−H−60)− as a result of cross-ring

372

fragmentation. Peaks 8 and 12 were recognized as digalloylglucose isomers with molecular ions

373

at m/z 483, giving fragmentation ions at m/z 331 ([M−H−152], loss of galloyl moiety), and m/z

374

313 ([M−H−170], gallic acid neutral loss). A molecular ion for peak 16 [M−H]− at m/z 197

375

yielded two fragment ions at m/z 169 [M−H−28]− and m/z 125 [M−H−28−CO2]−, which

376

corresponded to the loss of an ethyl moiety and CO2. Hence, peak 16 was assigned as ethyl

377

gallate. This compound has previously been reported in U.S. pecan phenolics, but at a much

378

lower level compared to that in Chinese hickory nuts from the current investigation.11

379

Catechins and Derivatives. Flavan-3-ol monomers, namely (+)-catechin and (-)-epicatechin

380

and their galloyl derivatives, are the building blocks of proanthocyanidins. Peaks 13 and 18 were

381

identified as (+)-catechin and (-)-epicatechin ([M−H]− at m/z 289) by their signature mass

382

fragments, and further confirmed by matching tR values and UV spectra with authentic standards.

383

Characteristic fragments included a MS2 fragment at m/z 245 [M−H−CO2]−, m/z 205 [M−H−84]−

384

by cleavage of the A ring, and m/z 179 [M−H−]− by loss of the B-ring structure. Catechin

385

hexoside was proposed for peak 10 by the fragment ion evident at m/z 289 and neutral loss of

386

162 Da. The MS2 spectrum of peak 14 generated ions at m/z 289 and 169, representing the

387

product ions of (epi)catechin and gallic acid, respectively. Further fragmentation showed an ion

388

at m/z 245 as a result of decarboxylation. Other fragments at m/z 205 and 203 match with the

389

characteristic pattern of (epi)catechin. Therefore, peak 14 was assigned as (epi)catechin gallate.


Page 19 of 39


390

The mass spectrum of peak 32 presented a molecular ion [M−H]− at m/z 457. A second order

391

fragmentation of the molecular ion generated fragment ions at m/z 305 ([M−H−152 (C7H4O4)]−)

392

and m/z 169 ([M−H−288]−), corresponding to the loss of one galloyl moiety and an (epi)catechin

393

unit via cleavage of the ester bond. Additionally, a fragment ion at m/z 331 [M−H−126]− was

394

observed. The loss of 162 Da suggests the possibility of a trihydroxybenzene structure, which

395

further was indicated by the presence of an (epi)gallocatechin unit. Therefore, peak 32 was

396

assigned as (epi)gallocatechin gallate. These fragmentations are in accordance to the patterns for

397

catechin derivatives reported in the literature.33

398

Peaks 15 and 23 with [M−H]− at m/z 577 were tentatively identified as a proanthocyanidin B-

399

type dimer, while the potential candidates for peaks 21 and 29 (m/z 575) could be A-type dimers.

400

The molecular ion of B-type dimers [M−H]− at m/z 577 yielded a MS2 ion at m/z 425

401

([M−H−152 (C8H8O3]−) via a retro Diels-Alder rearrangement. A product ion at m/z 451

402

([M−H−126 (C6H6O3)]−) resulted from heterocyclic ring fission between C4−C5 and O−C2 of

403

the pyran ring. Quinone methide ions for B-type dimers were also presented at m/z 289 (cleavage

404

at the terminal unit) and m/z 285 (cleavage at the extension unit). An approximate tR for

405

proanthocyanidin B2 was also confirmed by employing the B-type dimer standard.

406

Correspondingly A-type dimers, which are 2 Da less than the B-type, yielded product ions at m/z

407

423 and m/z 449, as well as quinone methide ions at m/z 289 and m/z 285. These findings and the

408

observed fragmentation patterns are consistent with previously published data.11,12,34,35

409

High-Molecular-Weight Phenolic Compounds in Carya Species. Ninety-nine percent of

410

the crude phenolic extracts applied to the Sephadex LH-20 column were recovered, of which

411

22.4 and 36.8% represented the high-molecular weight fractions of the Chinese hickory nuts and

412

U.S. pecans, respectively. To facilitate identification and to verify the efficacy of the analytical



Page 20 of 39

413

method, proanthocyanidin standards with degrees of polymerization ranging from 2-10 were

414

separated first by HILIC coupled to a fluorescence detector. The chromatographic separation is

415

depicted in Figure 4A. The method provided good resolution for degrees of polymerization up to

416

8, and even degrees of polymerization of 9-10 were also detectable. The resultant high-

417

molecular-weight phenolic fractions from 50% aqueous acetonic elution were then analyzed

418

using this verified method and the results are illustrated in Figure 4B. Because gallic acid ester-

419

and gallocatechin-containing proanthocyanidins are insensitive to fluorescence,36 UV detection

420

at 280 nm was carried out simultaneously. No new signals within the detection limits of the

421

system were revealed, thereby indicating that only (+)-catechin and (-)-epicatechin were the

422

monomeric units of the pecan and hickory nut proanthocyanidins. Proanthocyanidins up to

423

heptamers were discovered in fractionated pecan and hickory nut phenolic extracts by matching

424

the range of tR values, and further confirmation was provided by MS. One can confidently state

425

that based on the separation of the proanthocyanidin standards, any proanthocyanidins with

426

degrees of polymerization larger than seven would have been detected with the current protocol.

427

Robbins et al.11 reported up to hexamers for a pecan high-molecular-weight fraction with

428

exclusively B-type linkages, and the presence of no heptamers. Though proanthocyanidins with

429

degrees of polymerization of 6 and 7 were discernable, their identifications become very difficult

430

due to unavoidable baseline rise, as evident in the chromatogram (Figure 4B). It should be

431

pointed out that the fluorescence response was at the lower range: the existence of heptamers

432

might have been suppressed by the rising chromatographic baseline and noise-to-signal ratio

433

when the degrees of polymerization increased.

434 435

Quantitation of the low-molecular-weight phenolic compounds isolated from U.S. pecan and Chinese hickory nutmeats is summarized in Table 4. It is noteworthy that pecans and hickory


Page 21 of 39


436

nuts shared very similar profiles of their phenolics. To some extent this is not surprising because

437

both tree nut species belong to the Carya family; however, the Chinese hickory possesses twice

438

the total low-molecular-weight phenolic compounds than that detected in U.S. pecans. Compared

439

with the high-molecular-weight fraction, the low-molecular-weight phenolic fraction contains a

440

much greater quantity of hydrolyzable tannins, especially compounds from the ellagitannins

441

family. Specifically, the concentrations of ellagic acid and valoneic acid dilactone in Chinese

442

hickory nuts were 211.7 and 78.3 µg/g crude phenolic extract, respectively. While the

443

corresponding numbers in U.S. pecans were only 86.4 and 48.2 µg/g crude phenolic extract. The

444

concentrations of the proanthocyanidins content were quantitated based on standard curves

445

generated with corresponding proanthocyanidin standards and are reported in Table 5. The

446

predominate proanthocyanidin species of the high-molecular-weight U.S. pecan phenolics were

447

dimers and trimers, accounting for 31.42 and 18.34 mg/g crude extract. Proanthocyanidins with

448

higher degrees of polymerization, namely tetramers, pentamers, hexamers, and heptamers, were

449

present but only in limited quantities. On the contrary when compared with U.S. pecans, the

450

high-molecular-weight fraction from Chinese hickory nuts possesses, in general, a much lower

451

level of proanthocyanidins. Trimers, tetramers, and pentamers outweighed other

452

proanthocyanidin constituents, whereas the dimer content in Chinese hickory nuts was

453

practically negligible.

454

In conclusion, the separation performances of two different reversed-phase fused-core

455

columns, the Kinetex XB-C18 and PFP, were compared using kinetic curves. The Kintex PFP

456

column yielded higher plate numbers and superior peak shapes when analyzing ellagic acid-

457

related species, while the performance of other phenolic compounds was similar to that on a C18

458

column. Based on the preliminary results of pecan phenolic classes, the PFP column was selected



Page 22 of 39

459

for further study: a chromatographic method was optimized to characterize the low-molecular-

460

weight phenolics fractionated from U.S. pecan and Chinese hickory nut crude phenolic extracts.

461

Furthermore, the high-molecular-weight constituents were analyzed and quantitated using

462

HILIC. The identification of phenolic compounds was achieved based on individual commercial

463

standards or tentatively assigned based on MS fragmentation patterns when standards were not

464

available. Overall, U.S. pecans contain much higher proanthocyanidin concentrations, with

465

dimers and trimers being the dominant proanthocyanidin species. In comparison, Chinese

466

hickory nuts possessed much lower proanthocyanidin concentrations, but with higher degrees of

467

polymerization as noted by the predominance of tetramers and pentamers. Conversely, Chinese

468

hickory nuts surpassed pecans in terms of the amount of low-molecular-weight phenolic

469

compounds present, notably the gallotannin species.

470 471

ACKNOWLEDGEMENTS

472

The authors would like to acknowledge the United States Department of Agriculture-National

473

Institute of Food and Agriculture-Specialty Crop Research Initiative (USDA-NIFA-SCRI)

474

Award No. 2011-51181-30674 and the Georgia Agricultural Commodity Commission for Pecans

475

(GACCP) for funding this research. Thanks are extended to Dr. Randy D. Hudson, CEO of the

476

Hudson Pecan Company in Ocilla, GA, for helping to secure representative samples of Chinese

477

hickory nuts. Use of the Proteomics and Mass Spectrometry (PAMS) core facility under the

478

direction of Dr. Dennis Phillips of the University of Georgia Department of Chemistry is greatly

479

appreciated; in particular, thanks are extended to Dr. Kevin D. Clark for assistance with the LC-

480

MS/MS analyses.

481


Page 23 of 39


482

Supporting Information

483

This material is available free of charge via the Internet at http://pubs.acs.org.

484

C18 and PFP fused-core column Van Deemter plots, kinetic plots, and optimized method for the

485

Kinetex PFP column.

486 487

REFERENCES

488

(1) Wessel, D. Shell shock: Chinese demand reshapes U.S. pecan business, 2011.

489

http://www.wsj.com/articles/SB1000142405274870407680457618077424823

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7738, (Accessed: 22 June 2017).

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(2) USDA Economic Research Service. China’s potential as an export market for tree nuts,

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characterization of phenolic compounds from U.S. pecans by liquid chromatography–

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tandem mass spectrometry. J. Agric. Food Chem. 2014, 62, 4332–4341.

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L. Screening of foods containing proanthocyanidins and their structural characterization

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using LC-MS/MS and thiolytic degradation. J. Agric. Food Chem. 2003, 51, 7513–7521.

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(14) Manns, D. C.; Mansfield, A. K. A core–shell column approach to a comprehensive high-

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performance liquid chromatography phenolic analysis of Vitis vinifera L. and interspecific

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hybrid grape juices, wines, and other matrices following either solid phase extraction or

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direct injection. J. Chromatogr. A 2012, 1251, 111–121.

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(15) Olszewska, M. A. New validated high-performance liquid chromatographic method for

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simultaneous analysis of ten flavonoid aglycones in plant extracts using a C18 fused-core

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column and acetonitrile–tetrahydrofuran gradient. J. Sep. Sci. 2012, 35, 2174–2183.

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(16) Wu, J.; Qian, Y.; Mao, P.; Chen, L.; Lu, Y.; Wang, H. Separation and identification of

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phenolic compounds in canned artichoke by LC/DAD/ESI-MS using core–shell C18

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column: A comparative study. J. Chromatogr. B 2013, 927, 173–180.

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(17) Martí, R.; Valcárcel, M.; Herrero-Martínez, J. M.; Cebolla-Cornejo, J.; Roselló, S. Fast

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simultaneous determination of prominent polyphenols in vegetables and fruits by reversed

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phase liquid chromatography using a fused-core column. Food Chem. 2015, 169, 169–179.

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(18) Mirali, M.; Ambrose, S. J.; Wood, S. A.; Vandenberg, A.; Purves, R. W. Development of a

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fast extraction method and optimization of liquid chromatography–mass spectrometry for

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the analysis of phenolic compounds in lentil seed coats. J. Chromatogr. B 2014, 969, 149–

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(19) Gómez-Caravaca, A. M.; Verardo, V.; Berardinelli, A.; Marconi, E.; Caboni, M. F. A

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chemometric approach to determine the phenolic compounds in different barley samples by

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two different stationary phases: A comparison between C18 and pentafluorophenyl core

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antioxidant capacity and phenolic constituents of U.S. pecans. J. Funct. Foods 2015, 15,

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to mass transfer as causes of nonideality in chromatography. Chem. Eng. Sci. 1956, 5, 271–

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compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently

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(30) Regueiro, J.; Sánchez-González, C.; Vallverdú-Queralt, A.; Simal-Gándara, J.; Lamuela-

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ellagitannins, proanthocyanidins and other phenolics, vitamin C and antioxidant capacity of

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two powder products from camu-camu fruit (Myrciaria dubia). Food Chem. 2013, 139,

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578–588.

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liquid chromatography coupled to tandem mass spectrometry. J. Chromatogr. A 2013,

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MS. J. Mass Spectrom. 2012, 47, 502–515.

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Separation and characterization of phenolic compounds from dry-blanched peanut skins by

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(35 ) Li, H-J.; Deinzer, M. L. The mass spectral analysis of isolated hops A-type

590

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593

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Page 29 of 39

595


Figure Captions

596 597

Figure 1. Isocratic Poppe plot for ellagic acid on the Kinetex C18 and PFP fused-cored

598

columns. The curves were fitted according to the Knox equation. Three replications

599

were obtained for each data point.

600 601 602

Figure 2. Representative phenolic compounds isolated from U.S. pecans and Chinese hickory nuts.

603 604

Figure 3. Chromatographic separation of the low-molecular-weight (LMW) phenolic fractions

605

isolated using Sephadex LH-20 from U.S. pecan and Chinese hickory nutmeat

606

phenolic extracts on a Kinetex PFP column at 255 nm.

607 608

Figure 4. Chromatographic separation of (A) proanthocyanidin standards (degrees of

609

polymerization, DPs 2 thru 10) and (B) high-molecular-weight (HMW) phenolic

610

fractions isolated using Sephadex LH-20 from U.S. pecan and Chinese hickory

611

nutmeat phenolic extracts on a hydrophilic interaction liquid chromatography (HILIC)

612

column using fluorescence detection (excitation/emission of 276/316 nm).



Page 30 of 39

Table 1. Summary of Fitted Constants (A, B, C), Optimal Reduced Linear Velocity (v) and Minimum Reduced Plate Height (h) in Knox plots Constructed for the Kinetex C18 and PFP Columns Column

A

B

C

Hmin (µm)

vopt

hmin

C18

1.191

9.511

0.313

14.01

4.67

5.39

PFP

0.427

4.732

0.453

9.17

3.04

3.53

C18

0.135

3.657

0.195

4.94

4.17

1.90

PFP

0.133

3.308

0.209

4.83

3.84

1.86

0.021

3.027

0.293

4.97

3.20

1.91

Analyte: ellagic acid (MW=302)

Analyte: PDB2 (MW=578)

Analyte: catechin (MW=290) C18

PFP 0.294 2.550 0.224 5.01 3.11 1.93 Abbreviations are as follows: MW = molecular weight; and PDB2 = proanthocyanidin dimer B2.


Page 31 of 39


Table 2. Effect of Selected Chromatographic Conditions on the Development and Optimization for Low-Molecular-Weight Phenolic Compounds Isolated from U.S. Pecan and Chinese Hickory Nutmeat Phenolic Extracts on the Kinetex PFP Column Chromatographic conditions Resolution (Rs) for critical peak pairs % Gradient Gradient Time Total Run CH3COOH Flow rate Temp. °C 6/7 10/11 21/22 25/26 (mL/min) Range %B (min) Time (min) 2

0.6

25

5-50

20

25

1.49

0.96

co-elution

co-elution

2

0.6

25

5-50

25

30

2.02

1.01

co-elution

co-elution

2

0.6

25

0-50

25

30

2.12

1.89

0.69

0.44

2

0.6

30

0-60

30

35

1.79

1.55

co-elution

0.70

2

0.7

25

0-50

25

30

1.31

1.39

0.63

0.92

2

0.7

25

0-50

30

35

1.64

1.67

0.87

1.08

2

0.7

30

0-60

30

35

1.42

1.44

0.89

1.13

2

0.8

25

0-60

25

30

1.22

0.89

0.45

1.05

2

0.8

25

0-60

30

35

1.26

1.34

1.21

1.11

1

0.8

25

0-60

30

35

1.32

1.61

1.42

1.33

1

0.8

25

5-60

30

35

0.87

0.91

1.27

1.35

1

0.8

30

0-60

30

35

1.01

0.85

1.13

1.27

1

0.8

30

0-50

30

35

1.16

1.19

1.02

1.15

1

0.9

25

0-60

30

35

0.86

1.02

co-elution co-elution

1 0.9 25 0-50 30 35 co-elution 0.76 1.09 1.17 The gray line highlighted in bold represents the optimal condition for resolving the co-elution of critical pairs of compounds.



Page 32 of 39

Table 3. Chromatographic Parameters of the Proposed Methods on Both Kinetex C18 and PFP Fused-core Columns Accuracy (%REC) Precision (%RSD) Column LOD LOQ Upper limit of Standards Calibration equations r2 type (ppb) (ppb) linearity (ppm) 2 ppm 20 ppm 200 ppm 2 ppm 20 ppm 200 ppm caffeic acid

10.3

34.1

500

y = 121.07x + 11.865

0.9989 100.2

98.3

99.0

1.6

3.0

0.6

gallic acid

14.1

42.0

1000

y = 65.628x – 20.342

0.9999

97.9

100.5

100.3

1.2

3.6

0.7

(+)-catechin

78.3

236.5

1000

y = 16.411x + 0.6211

0.9998

96.6

97.3

101.2

2.5

4.3

1.6

PDB2

51.1

161.3

1000

y = 15.817x – 15.377

0.9994

98.7

96.4

98.4

3.5

0.3

1.0

ellagic acid

63.4

210.2

200

y = 125.41x – 7.3013

0.9994

92.9

93.6

101.6

3.4

1.1

1.8

caffeic acid

13.0

43.2

500

y = 130.69x + 10.404

0.9998

97.4

100.4

99.9

0.9

4.7

0.7

gallic acid

21.3

80.5

1000

y = 68.296x – 8.3698

0.9998

97.8

99.9

100.3

4.8

1.4

0.6

(+)-catechin

83.2

231.1

1000

y = 16.402x + 10.734

0.9999

98.2

99.8

100.4

3.2

1.5

0.6

PDB2

35.9

119.0

1000

y = 15.123x + 3.4259

0.9995

98.6

95.6

101.3

4.6

5.7

1.1

98.2 99.3 2.0 1.0 ellagic acid 9.1 22.3 200 y = 262.38x + 8.2024 0.9998 97.7 2 Abbreviations are as follows: LOD = limit of detection; LOQ = limit of quantitation; r = coefficient of determination; %REC = percent recovery; %RSD = percent relative standard deviation; and PDB2 = proanthocyanidin dimer B2.

0.2

C18

PFP


Page 33 of 39


Table 4. Tentative Identification and Quantitation of Phenolic Compounds Isolated from the Low-MolecularWeight Fractions of U.S. Pecan and Chinese Hickory Nutmeat Phenolic Extracts Concentration of Identified Compounds (µg/g acetonic crude extract)c

Compounds Identification Peak No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

tRa [M-H]- (m/z) (min) 3.15 331 3.42 481 3.71 481 4.47 169 4.82 137 5.17 633 5.49 331 6.72 483 7.33 315 7.68 451 8.14 785 8.81 483 9.57 289 10.89 441 11.21 577 11.70 197 12.64 463 12.90 289 13.28 179 14.44 605 15.14 575 15.46 433 18.12 577 18.96 477 19.37 487 19.87 477 21.16 301 22.11 505 22.66 441 23.81 491 24.30 447 25.26 575 25.82 585 26.27 475 27.60 329 28.06 599

MS2 (m/z)

Tentative Identificationb

Pecan

Hickory nut

169 421-301-275 421-301-275 125 122-111-107 463-421-301 313-169 331-313-169 153 289 633-483-301 331-313-169 245-205-179 289-245-169 451-425-407-289 169-125 301 245-205-179 163-135 453-301 449-423-407-289 301 451-425-407-289 315-300 469-425-301 315-300 217 463-301 289-245-203 328-315-300 315-300 449-423-407-289 433-301 460-329-299 314 447-315

gallic acid hexoside HHDP-glucose isomer HHDP-glucose isomer gallic acid p-hydroxybenzoic acid galloyl-HHDP-glucose monogalloyl-glucose digalloyl-glucose protocatechuic acid hexoside catechin hexoside digalloyl-HHDP-glucose digalloyl-glucose (+)-catechin (epi)catechin gallate PD2B ethyl gallate ellagic acid hexoside (-)-epicatechin caffeic acid digalloyl ellagic acid PD2A ellagic acid pentoside PD2B methyl ellagic acid hexoside valoneic acid dilactone hydrate methyl ellagic acid hexoside ellagic acid ellagic acid acetyl hexoside (epi)catechin gallate dimethyl ellagic acid hexoside methyl ellagic acid pentoside PD2A ellagic acid galloyl pentoside dimethyl ellagic acid pentoside dimethyl ellagic acid methyl ellagic acid galloyl pentoside

10.4 ± 0.8 7.0 ± 0.4 2.4 ± 0.1 7.6 ± 0.2 3.0 ± 0.0 9.6 ± 0.5 4.6 ± 0.1 0.3 ± 0.0 3.3 ± 0.1 10.7 ± 0.2 0.3 ± 0.0 0.3 ± 0.0 9.9 ± 0.9 2.0 ± 0.1 4.1 ± 0.3 1.5 ± 0.1 2.9 ± 0.1 2.8 ± 0.3 2.1 ± 0.1 2.1 ± 0.1 0.7 ± 0.0 8.3 ± 0.7 1.3 ± 0.2 0.8 ± 0.0 48.2 ± 3.1 12.3 ± 0.7 86.4 ± 6.2 7.1 ± 0.6 4.2 ± 0.2 17.7 ± 1.1 7.4 ± 0.8 3.0 ± 0.3 8.3 ± 1.0 6.3 ± 0.7 4.8 ± 0.2 12.0 ± 1.2

11.0 ± 0.63 10.6 ± 0.6 3.5 ± 0.1 12.2 ± 0.7 7.2 ± 0.2 22.3 ± 2.1 12.4 ± 0.8 16.6 ± 1.3 6.3 ± 0.6 17.5 ± 1.0 14.9 ± 1.2 13.4 ± 1.4 4.0 ± 0.3 4.7 ± 0.5 2.9 ± 0.8 33.4 ± 2.3 18.7 ± 1.1 10.6 ± 0.5 5.3 ± 0.2 7.1 ± 0.4 19.9 ±1.0 15.8 ± 1.2 1.1 ± 0.1 0.6 ± 0.0 78.3 ± 4.3 20.2 ± 1.2 211.7 ± 9.7 trace trace 13.0 ± 1.0 24.7 ± 2.1 7.8 ± 0.7 15.5 ± 1.2 4.8 ± 0.4 4.6 ± 0.7 13.1 ± 0.9

a

Retention time (tR) from RP-HPLC analysis performed on the Kinetex PFP column. Tentative identifications were achieved using tR mapping and fragmentation-pattern comparison to those of commercial standards as well as compounds previously reported in pecans by Robbins et al.11 and Regueiro et al.30 Abbreviations are as follows: PD2A = proanthocyanidin A-type dimer; PD2B = proanthocyanidin B-type dimer; HHDP glucose = bis(hexahydroxydiphenoyl) glucose. c All quantitation is based on RP-HPLC analysis; all samples were analyzed in triplicate. Commercial standards were used for caffeic acid, gallic acid, (+)-catechin, ellagic acid, protocatechuic acid, p-hydroxybenzoic acid, and PD2B. All other compounds were quantitated using the most comparable standard. Means were analyzed using the unpaired student t-test: higher values with statistical significance (p

Separation of Ellagitannin-Rich Phenolics from U.S. Pecans and

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