Proanthocyanidins in Wild Sea Buckthorn (HippophaÃ«

Article

Proanthocyanidins in Wild Sea Buckthorn (Hippophaë rhamnoides) Berries Analyzed by RP-, NP- and Hydrophilic Interaction Liquid Chromatography with UV and MS Detection Heikki Kallio, Wei Yang, Pengzhan Liu, and Baoru Yang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jul 2014 Downloaded from http://pubs.acs.org on July 16, 2014

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Journal of Agricultural and Food Chemistry

Proanthocyanidins in Wild Sea Buckthorn (Hippophaë rhamnoides) Berries Analyzed by RP-, NP- and Hydrophilic Interaction Liquid Chromatography with UV and MS Detection

†

†

†

Heikki Kallio *, , Wei Yang , Pengzhan Liu , Baoru Yang

†

†

Food Chemistry and Food Development, Department of Biochemistry, University of Turku, FI20014, Turku, Finland

*Corresponding author (Tel:+35823336870; Fax:+35822317666; E-mail heikki.kallio @utu.fi).

1

ACS Paragon Plus Environment


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ABSTRACT

2

A rapid and sensitive method for profiling of proanthocyanidins (PAs) of sea buckthorn

3

(Hippophaë rhamnoides) berries was established based on aqueous, acidified acetone extraction.

4

The extract was purified by Sephadex column chromatography and analyzed using RP-, NP- and

5

hydrophilic interaction liquid chromatography (HILIC). Negative ion electrospray ionization

6

mass spectrometry (ESI-MS) in single ion recording (SIR) and full scan modes combined with

7

UV detection were used to define the combinations and ratios of PA oligomer classes. PAs with

8

degree of polymerization from 2 to 11 were detected by HILIC-ESI-MS. Quantification of

9

dimeric, trimeric and tetrameric PAs was carried out with ESI-MS-SIR and their molar

10

proportions were 40, 40 and 20%, respectively. Only B-type PAs were found and

11

(epi)gallocatechins were the main monomeric units. More than 60 combinations of

12

(epi)catechins and (epi)gallocatechins of proanthocyanidin dimers and trimers were found.

13

Majority of the PAs were shown to be higher polymers based on the HILIC-UV analysis.

14

15

Keywords: HILIC; Mass spectrometry; Normal phase liquid chromatography;

16

Proanthocyanidins; Reversed phase liquid chromatography; Sea buckthorn; SIR

2


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INTRODUCTION

18

The genus Hippophaë L. in the family Eleagnaceae consists of hardy, thorny, deciduous trees

19

and shrubs with nitrogen-fixing root nodules. The genus is currently recognized to comprise of

20

seven species

21

produces yellow-orange berries by the end of summer. Sea buckthorn is widely distributed in

22

Asia and Europe, and is recently also introduced in North America and in South Africa.

23

1,2

, of which Hippophaë rhamnoides, most commonly known as sea buckthorn,

Sea buckthorn is a rich source of natural antioxidants such as ascorbic acid, tocopherols, 3

24

carotenoids, and flavonoids, as well as of many health-beneficial fatty acids

. Many

25

investigations have demonstrated the berry to present anti-oxidative, anti-bacterial and anti-viral

26

properties and to be effective in treating stomach ulcer and skin diseases 4-7. As early as 900 AD,

27

sea buckthorn was used for nutritional and medicinal purposes, at least in Asia 8. In recent years,

28

sea buckthorn has attracted a great deal of attention in many countries also due to its economic

29

potential and environmental value 9.

30

Proanthocyanidins (PAs), also known as condensed tannins, are oligomeric or polymeric

31

compounds composed of flavan-3-ol subunits. Oligomers and polymers of (epi)catechin or

32

(epi)gallocatechin subunits called procyanidins or prodelphinidins, respectively, are reported to

33

exist in sea buckthorn berries in sufficient amounts, up to 250 mg/100g

34

proanthocyanidins of sea buckthorn berries may have potential health effects on humans because

35

evidence is accumulating that the compounds provide benefits by acting as natural antioxidative,

36

antiviral and antimicrobial substances, and as vasodilators 13-16. Polymeric proanthocyanidins are

37

not absorbed in the gastrointestinal tract

38

(DP) < 4 are at least partially absorbed in the gut. Dimers B1 and B2 are detected in human

10-12

. The

17

, but the PA oligomers of degree of polymerization

3



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plasma after consumption of flavanol-rich food. The PA oligomers have been found to have

40

direct effects on the intestinal mucosa protecting it against oxidative stress or the actions of

41

carcinogens 18,19.

42

Proanthocyandins are commonly isolated into fractions by Sephadex LH-20 column 20

43

chromatography for more detailed investigation

. Both normal-phase liquid chromatography

44

(NP-HPLC) and reversed-phase liquid chromatography (RP-HPLC) have been applied for

45

analysis using UV (280 nm) or fluorescence detectors, or coupled to mass spectrometer

46

These methods are efficient in resolving proanthocyanidins by size on normal-phase columns

47

and by isomers on the reversed-phase ones 23. Procyanidins in pine bark have been analyzed by

48

NP-HPLC and RP-HPLC with electrospray ionization mass spectrometry (ESI-MS)

49

Hydrophilic interaction liquid chromatography (HILIC) as an evolution of NP-HPLC method has

50

been successfully applied for separation of procyanidins by degree of polymerization, and using

51

solvents compatible with RP-HPLC 25-28. Two-dimensional analysis of procyanidins in apple and

52

cocoa with HILIC and RP-HPLC has also been performed to obtain better separation

53

Further, tandem mass spectrometry method by optimizing for the range of cone voltages was

54

used for analyses of a wide range of proanthocyanidins 32.

21-24

.

22

.

29-31

.

55

Selected ion recording (SIR) mode with specific m/z value enhances further the separation and

56

the detection limits. It is possible to reduce overlapping between masses by performing SIR at

57

high resolution, thus increasing further the specificity 33,34.

58

The separation and determination of proanthocyanidins of sea buckthorn is problematic

59

because they occur as highly complex mixtures. The aim of this work was to establish a method

60

for identification of the key PA oligomers in the berries. The crude extract was fractionated by 4


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Sephadex LH-20 column chromatography, and PAs of the fractions were separated according to

62

molecular weight by NP-UPLC-ESI-SIR and HILIC-ESI-SIR, and the isomers were analyzed by

63

RP-HPLC-ESI-SIR. Quantification of the dimers, trimers and tetramers was carried out using a

64

HILIC-ESI-SIR method.

65

66

MATERIALS AND METHODS

67

Plant Materials. Wild sea buckthorn berries (Hippophae rhamnoides ssp. rhamnoides L.)

68

were harvested on September 28th 2011 at Pyhämaa, southern Finland on the coast of Gulf of

69

Bothnia and stored at -18 °C.

70

Chemicals and Reagents. Methanol, dichloromethane and formic acid (all HPLC grade) were

71

purchased from J. T. Baker (Deventer, Holland), acetone and acetonitrile (HPLC grade) from

72

VWR International Oy (Espoo, Finland). Water was obtained from a Millipore system (Billerica,

73

MA). The reference compound procyanidin B2 was purchased from from Extrasynthese (Genay,

74

France).

75

Sample Preparation. Fifty grams of sea buckthorn berries were crushed and extracted three

76

consecutive times with 200 mL of a mixture of acetone, water and acetic acid (80:19.5:0.5, v/v)

77

by sonicating each time for 15 min. After centrifugation and combining of the supernatants,

78

acetone was removed by rotary evaporator. The final volume of c.a. 100 mL was used for

79

Sephadex LH-20 column chromatography (CC).

80

Isolation and Purification of Proanthocyanidin. A 15 mm i.d. × 250 mm glass column

81

packed with five grams of Sephadex LH-20 (Pharmacia, Uppsala, Sweden) was used for column 5



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chromatography. The column was activated and rinsed with 100 mL water. The crude extract was

83

loaded in the column and washed with 60 mL water. Three fractions were collected with 250 mL

84

methanol and water (20:80, v/v, Fraction I), 200 mL acetone and water (70:30, v/v, Fraction II)

85

and 250 mL acetone and water (70:30, v/v, Fraction III) in sequence. The flow of elution was

86

maintained at 1 mL/min by Alitea-XV peristaltic pump (Bioengineering, Wald, Switzerland). All

87

the fractions were dried by vacuum rotary evaporator, re-dissolved in 1 mL methanol and

88

clarified through polytetrafluoroethylene (PTFE) filter (13 mm i.d., 0.45 µm, VWR International,

89

West Chester, PA) before analyses.

90

LC-DAD-MS Apparatus. A Waters Acquity Ultra High Performance LC system (Waters

91

Corp., Milford, MA) which consisted of a sample manager, binary solvent delivery system and

92

Waters 2996 PDA Detector was used. In addition, Waters Quattro Premier tandem quadrupole

93

mass spectrometer (Waters Corp., Milford, MA) equipped with an electrospray-ionization (ESI)

94

source was combined to the system. The chromatograph was operated using MassLynx 4.1

95

software. The system was controlled with MassLynx 4.1 software.

96

Reversed-phase HPLC analyses. The crude extract and its fractions I to III were analyzed by

97

reversed-phase HPLC-DAD to define the content of proanthocyanidins. A Phenomenex Luna

98

RP-C18 column (5 µm, 250 × 4.60 mm, Torrance, CA) combined with a Phenomenex Prodigy

99

guard column (5 µm, 30 × 4.60 mm, Torrance, CA) was used. The mobile phase was run as

100

gradient elution of formic acid/water (0.5:99.5, v/v) as solvent A and acetonitrile/methanol

101

(80:20, v/v) as solvent B at a flow rate of 1 mL/min. The gradient program of solvent B in A

102

(v/v) was 0–5 min 10% B, 5–15 min 10–18% B, 15–25 min 18% B, 25–30 min 18–25% B, 30–

103

35 min 25% B, 35–40 min 25–35% B, 40–45 min 35–60% B, 45–50 min with 60–10% B, and 6


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50–55 min with 10% B 35. The injection volume was 20 µL. The peaks were monitored at two

105

different wavelengths, 280 nm for all the phenolic compounds and 360 nm for the flavonol

106

glycosides. UV spectra of the compounds were recorded. A split joint was used after the UV

107

detector, directing a flow of 0.3 mL/min to the mass spectrometer and the rest to a waste bottle.

108

Normal-phase UPLC analyses. The proanthocyanidin-rich Fraction II was analyzed by

109

normal-phase ultra-high performance liquid chromatography (NP-UPLC). Restek Pinnacle DB

110

Silica UPLC column (1.9 µm, 100 × 2.1 mm, Bellefonte, PA) was used for resolution of sea

111

buckthorn proanthocyanidin clusters for mass spectrometric analysis. The analyses were carried

112

out by a gradient elution of dichloromethane, methanol, water, and acetic acid (82:14:2:2 v/v) as

113

solvent A and methanol, water, and acetic acid (96:2:2 v/v) as solvent B at the mobile phase flow

114

rate of 0.4 mL/min. The gradient program of solvent B in A (v/v) was 0–15 min 0–15% B, 15–20

115

min 15–30% B, 20–25 min 30–80% B, 25–30 min 80–0% B, and 30–35 min with 0% B. The

116

injection volume was 10 µL. The parameter of the UV detector was set as used in the reversed-

117

phase HPLC analyses. The whole flow of 0.4 mL/min was lead to the mass spectrometer after

118

the UV detector.

119

HILIC analyses. A Phenomenex Luna HILIC 200A column (3 µm, 150 × 3.00 mm, Torrance,

120

CA) combined with a Phenomenex Security Guard Cartridge Kit (Torrance, CA) was used for

121

the analysis of Fraction II. Acetonitrile was used as solvent A and formic acid/water (0.5:99.5,

122

v/v) as solvent B with gradient elution. The gradient program of solvent B in A (v/v) was 0–5

123

min 5–20% B, 5–10 min 20–30% B, 10–12 min 30–40% B, 12–15 min 40–65% B, 15–17 min

124

with 65–5% B and 17–30 min with 5% B. The injection volume was 10 µL. The total flow rate

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was 0.6 mL/min and the whole flow was lead to the mass spectrometer after the UV detector.

126

The LC-UV chromatograms were recorded at 280 nm and 360 nm.

127

Mass spectrometry. The electrospray ionization (ESI) source was operated in negative ion

128

mode. The ESI inlet conditions were as follows: capillary voltage, 3.5 kV; cone voltage, 15 V;

129

extractor voltage, 8 V; source temperature, 120 °C and the desolvation temperature, 300 °C.

130

Both full scan and SIR functions were used. The total ion ESI-MS analysis of

131

proanthocyanidins was carried out by scanning the ions from m/z 500 to 3000 for oligomer size

132

screening. The total ion spectra were recorded to elucidate the PA groups with different

133

molecular weights. A smoothing process with a window of m/z ±3 was operated in the

134

MassLynx 4.1 software to combine the peaks of various isotope combinations of the

135

proanthocyanidins in the spectrum. The m/z signals chosen for the selected ion recording (SIR)

136

function were those of the most abundant deprotonated proanthocyanidin ions calculated based

137

on the nominal masses. According to the isotope distribution, the most abundant deprotonated

138

proanthocyanidin ions only consist of

139

were m/z ±0.5 out of the range were discarded. Therefore, the PA molecular ions containing 13C

140

and 14C, were not recorded. The actual molar proportions were estimated by correcting the SIR

141

peak areas by taking the isotopic abundances of 13C, 14C, 2H, 3H, 17O and 18O into account.

12

C, 1H and

16

O. In the SIR model, the m/z ions which

142

Quantitative analysis. The quantive analysis of proanthocyanidin dimers was carried out

143

using HILIC-ESI-SIR method and an external standard. The calibration curves were constructed

144

by analysing standard solutions of procyanidin B2 in methanol in the concentration range of

145

0.01-0.05 mg/mL by plotting the peak areas against the concentrations. The contents of

146

proanthocyanidin dimers and trimers were calculated with the calibration curve of procyanidin 8


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B2 and correction factors 36.

148

RESULTS AND DISCUSSION

149

Purificaction by Column Chromatography. For the extraction of proanthocyanidins from

150

sea buckthorn berries, a mixture of acetone, water and acetic acid (80:19.5:0.5, v/v) was found to

151

serve as an optimal solvent system. A purification step was needed due to the presence of

152

flavonols and other impurities in the extract disturbing the chromatographic and mass

153

spectrometric analyses. Thus, the crude extract and the Sephadex Fractions I - III were analyzed

154

by RP-HPLC at 280 nm and 360 nm, and the UV-spectra were obtained by DAD.

155

Chromatograms of the crude extract and the Fraction II recorded at 280 nm are presented in

156

Figures 1A and 1B. It was found that the non-tannin substances were mainly eluted in Fraction I,

157

and proanthocyanidins were recovered in Fraction II. In the Fractions I and III hardly traces of

158

proanthocyanidins were recognized and Fraction II was used for mass spectrometric analyses of

159

the target compounds. The UV spectra of all the proanthocyanidins were highly similar

160

throughout the chromatogram 1B, from 2.5 min to 45 min at 280 nm. The compounds had

161

pronounced and symmetrical maxima at around 240 and 280 nm without band broadening

162

beyond 300 nm.

163 164

Identification of Proanthocyanidins. The PA Fraction II was analyzed by RP-HPLC, NPUPLC and HILIC combined with DAD and electrospray ionization mass spectrometry.

165

Complexity of the extract and fractions of sea buckthorn resulted in unsatisfactory separation

166

of PAs in the chromatograms recorded by both RP-HPLC-DAD and NP-UPLC-DAD at 280 nm.

9



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The peaks overlapped seriously with each other forming only broad humps of PAs instead of

168

proper resolution as seen in the chromatogram of RP-HPLC-DAD in Fig 1B. However, on the

169

HILIC-DAD chromatogram recorded at 280 nm, 14 peaks eluting between 3.5 and 9 minutes

170

were legible (peaks a to n, Figure 1C). The UV spectra revealed all of them to be PAs and they

171

were tentatively identified based on their ESI mass spectra. Each peak displayed may contain

172

several pronthocyanidins as presented in Table 1. No legible chromatographic peaks and thus no

173

proper spectra of individual PAs were obtained by RP-HPLC- or NP-UPLC-ESI-MS.

174

In addition, full scan mode of ESI-MS was applied to obtain the oligomeric profiles of

175

Fraction II. The mass spectra of HILIC and NP chromatography are shown in Figure 2A and 2B,

176

respectively. The best results were obtained with the HILIC method and oligomers up to 11 PA-

177

units were recorded. Dichloromethane commonly used in normal-phase methods is a harmful

178

solvent both for LC instruments and for the sensitivity of the analysis and is recommended to be

179

replaced by other solvents. e.g. such as used in the HILIC analyses.

180

Identification of proanthocyanidins by total ion spectra. Fraction II was analyzed by RP-

181

HPLC, NP-UPLC and HILIC coupled with electrospray ionization mass spectrometry in

182

negative ion mode.

183

The total ion spectra over the whole chromatogram of the isolated fractions were recorded.

184

Scanning from m/z 500 to 3000 in 14 minutes (from 3.6 to 17.6 min) in HILIC, 22 minutes (from

185

3 to 25 min) in NP-UPLC and 37 minutes (from 3 to 40 min) in RP-UPLC were used to

186

recognize the PAs in Fraction II. The total ion spectra obtained from the HILIC- and NP-ESI-MS

187

are shown in Figure 2. The low-molecular weight parts of the total ion spectra were highly

188

similar, however, the HILIC-ESI-MS was the most sensitive and revealed also the existence of 10


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189

higher oligomers up to DP11 (Fig. 2A). This may be due to the lower water content in the HILIC

190

solvents enhancing the ionization. The [M-H]- ions of PAs with DP from 2 to 7 and [M-2H]2-

191

ions of PAs with DP from 7 to 11 with the HILIC method are collected in Table 1.

192

The ions from m/z 577.52 to 1828.03, 2115.64, 2130.49 of the individual compounds as well

193

as the corresponding full-scan spectra are the deprotonated, average molecular weight

194

proanthocyanidins of DP from 2 to 7. All the compounds represent the B-type

195

proanthocyanidins, dimers (MW 578.14, 594.14, and 610.13), trimers (866.20, 882.20, 898.19,

196

and 914.19), tetramers (1154.27, 1170.26, 1186.26, 1202.25, and 1218.25), pentamers (1474.32,

197

1490.32, 1505.31, and 1522.31), hexamers (1793.37, 1809.37, and 1825.36), heptamers (2114.43

198

and 2130.42). The molecular weights are the exact masses with all the isotope abundances taken

199

into account.

200

No single-charged molecular ions of proanthocyanidins with DP higher than 7 were found. As

201

indicated in Table 1, the ions from m/z 1032.84 to 1665.49 were the [M-2H]2- ions of heptamers

202

(MW 2066.44, 2082.43, 2098.43, 2114.43, 2129.42), octamers (2385.49, 2417.48), nonamers

203

(2626.58, 2642.57, 2658.57, 2674.56, 2690.56, 2706.55, 2722.54, 2738.54), decamers (2994.61,

204

3026.60), and undecamers (3314.67, 3330.66).

205 206

No A-type oligomers with molar masses of two units lower than corresponding B-type PA were found. Tentative identification of all the proanthocyanidins is also presented in Table 1.

207

Identification of proanthocyanidins by NP-UPLC- and HILIC-ESI-MS-SIR.

208

Because of the high complexity of the sea buckthorn extract and fractions, neither the UV

209

analysis nor mass spectrometry in full scan mode gave results detailed enough. The molecular 11



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210

weights of proanthocyanidins are relatively close to each other in each group of the low-DP

211

oligomers and the NP-UPLC and HILIC resolutions were not sufficient for quantitative analysis.

212

Instead, the NP-UPLC-, HILIC- and even RP-HPLC-ESI-MS in the SIR mode provided highly

213

selective methods for determination of proanthocyanidins in sea buckthorn and the three

214

methods complemented well each other.

215

All the twelve [M-H]- ions of the nominal mass proanthocyanidin dimers, trimers and

216

tetramers were scanned in twelve channels by HILIC-, NP-UPLC-, and RP-HPLC-ESI-MS-SIR

217

analyses and displayed in Figure 3. The HILIC and NP chromatograms showed rather systematic

218

difference in retention times based on molecular weight within each of the three oligomer groups.

219

The higher the MW the longer the retention time within a group, but the rule did not apply when

220

all peaks in the twelve molecular weight classes were taken into account. All the PA dimers,

221

trimmers and tetramers defined earlier (Table 1) were eluted in 4 to 12 minutes as shown in

222

Figure 3. Combinations of the monomeric units as well as retention times of the major peaks in

223

each MW group from both the HILIC-ESI-MS-SIR and the NP-UPLC-ESI-MS-SIR analyses are

224

shown in Table 2. Both methods showed relatively low chromatographic resolution in each

225

channel and only poorly resolved peaks were recorded. The HILIC method was more sensitive

226

than the NP-method.

227

Identification of proanthocyanidins by RP-HPLC-ESI-MS-SIR. As an effective separation 35,37

228

technique for phenolic compounds, RP-HPLC is widely used in routine analysis

229

HPLC method coupled to ESI-MS-SIR was used to distinguish the major proanthocyanidin

230

isomer groups with different monoisotopic nominal masses in the twelve channels.

231

. The RP-

Fraction II was analyzed by RP-HPLC-DAD by monitoring at 280 nm, and by RP-HPLC12


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ESI-MS-SIR at the twelve channels (m/z 577, 593 and 609 for proanthocyanidin dimers, m/z 865,

233

881, 897 and 913 for trimers, and m/z 1153, 1169, 1185, 1201 and 1217 for tetramers,

234

respectively) as in NP-UPLC and HILIC-SIR methods. The chromatograms obtained by RP-

235

HPLC method are showed in Figures 3C, F and I and the peaks are listed in Table 2. The

236

analysis provided sufficient separation of proanthocyanidin isomers with same molecular

237

weights among the dimeric and trimeric PAs. Resolution was less good in the case of tetrameric

238

isomers and individual peaks were only occasionally visible. Unlike in NP-UPLC and HILIC

239

chromatography the oligomers with higher MW eluted in the RP analyses on average faster than

240

the smaller counterparts within one polymer group.

241

In the chromatogram of RP-HPLC-ESI-MS-SIR at m/z 577, five peaks (from 1 to 5) were

242

found to display at least five characteristic dimeric procyanidin isomers with composition of

243

[(E)C-(E)C]. At m/z 593 the eleven isomers (from 6 to 16) consisted of one (epi)catechin and one

244

(epi)gallocatechin subunit. Based on the UV spectra recorded, the clear peak at 45 min was not a

245

proanthocyanidin. The twelve peaks from 17 to 28 in the chromatogram at m/z 609 represent the

246

dimeric prodelphinidins of two (E)GC units (Fig 3C).

247

Among the trimers, the peaks from 29 to 36 in the SIR-chromatogram at m/z 865 revealed at

248

least eight isomeric structures of [(E)C-(E)C-(E)C]. The m/z 881 suggested at least twelve

249

isomers consisting of two (epi)catechin and one (epi)gallocatechin subunits, and m/z 897 also

250

twelve compounds of one (epi)catechin and two (epi)gallocatechins. The less resolved peaks

251

from 56 to 62 in the chromatograms at m/z 913 were the isomers of trimeric prodelphinidins (Fig

252

3F). It is clear that many of the trimeric isomers overlap in the chromatograms.

253

The numerous isomers of proanthocyanidin tetramers overlapped significantly each other. 13



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254

The ion peaks [M-H]- from m/z 1153 to 1217 are suggested to consist of tetrameric procyanidins,

255

three (epi)catechin and one (epi)gallocatechin subunits, two (epi)catechin and two

256

(epi)gallocatechin subunits, one (epi)catechin and three (epi)gallocatechin subunits and

257

tetrameric prodelphinidins, respectively (Fig 3I).

258

Quantitative Profiling of Oligomeric Proanthocyanidins. Due to the lack of reference

259

compounds, determination of the ratios of different PA classes of sea buckthorn berries was not

260

possible. It has to be highlighted that all the tree mass spectrometric methods are useful for

261

profiling and comparisons between different samples but none of them give correct proportions.

262

The tentative identification of PA oligomers was based on chromatographic and mass spectral

263

data as well as on UV spectra and literature reports

264

elucidation of the bonds between the monomeric units nor the steroisomerism of the B-ring and

265

hydroxyl group at position 3 could be determined.

10,38

. However, neither exact structural

266

The HILIC-ESI-MS in full scan mode and in SIR function methods were used as the two most

267

sensitive methods to study the quantitative profiles of proanthocyanidins in sea buckthorn. Based

268

on MS analyses illustrated in Figures 2 and 3, it was possible to get an overview of distribution

269

of the compounds in different oligomeric classes in wild, Nordic Hippophaë rhamnoides berries

270

summarized in Table 2.

271

The peak areas of at least the dimeric and trimeric proanthocyanidins in the HPLC-ESI-MS-

272

SIR chromatograms show typically close to linear molar responses regardless of the slight

273

differences in the structures

274

subunits ( [M-H]- at m/z 577) were the most abundant. Their content was close to three times that

275

of [(E)GC-(E)GC] ([M-H]- at m/z 609) and almost twice the content of [(E)C-(E)GC] ([M-H]- at

36

. Thus, among the dimers, the compounds of two (epi) catechin

14


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m/z 593) (Table 2). Over all, in the dimeric PAs, (epi)catechin was the main subunit.

277

(Epi)gallocatechins with m/z 913 formed the most abundant combination (> 30%) within the

278

trimeric PAs. The tetrameric (epi)gallocatechins with m/z 1217 comprised analogously 1/3 of the

279

tetrameric PAs.

280

Information of the two HILIC methods applied gave quite consistent results despite the

281

different calibration profiles. The bigger the molecule was the more isotope ions were lost in the

282

SIR model due to the MW window parameters set in the method. Thus, proportions of the peak

283

areas of the trimeric and tetrameric PAs in the full scan mode were higher than those in the SIR

284

analysis. Results after the isotope-correction are shown in Table 2, and they are quite close to the

285

full scan results. Both ESI-MS-SIR analysis and the total ion spectra with the HILIC method

286

showed the molar proportion of the dimeric and trimeric PAs to approach the level of 40% of the

287

three PA classes studied. The higher oligomers than tetramers were found in the MS analyses in

288

low amounts and all the compounds identified were of type-B proanthocyanidin oligomers.

289

Procyanidin B2 reference compound was used as an external standard for quantification of the

290

procyanidin dimers. Their total content was 0.45 mg/100g (Table 2). Our earlier studies showed

291

that the efficiency of ionization of procyanidin dimers and trimers are very similar in the ESI

292

mode calculated on molar bases 36. Based on this principle, we listed in Table 2 the contents of

293

all PA oligomers with DP ≤ 4 and the contents of proanthocyandin dimers, trimers and tetramers

294

were 0.98 mg/100g, 1.41 mg/100g, and 0.84 mg/100g, respectively.

295

It is worth to notice that the proanthocyanidins of DP2 to DP4 quantified in Table 2 represent

296

only a small part of all the PA oligomers and polymers of sea buckthorn. Figure 1C reveals that

15



Page 16 of 27

297

based on the UV recording at 280 nm there is a huge hump of polymeric PAs, which were not

298

recognized in the MS analyses.

299

Proanthocyanidins in wild sea buckthorn of H. r. ssp. rhamnoides berries were analyzed by

300

RP-, NP- and hydrophilic interaction liquid chromatography (HILIC). HILIC provided

301

separation mainly by PA size and provided the highest sensitivity of the methods applied. PAs

302

with degree of polymerization from 2 to 11 were detected by HILIC-ESI-MS. More than 60

303

combinations of (epi)catechins and (epi)gallocatechins of proanthocyanidin dimers and trimers

304

were found. All of them were B-type compounds and (epi)gallocatechins were the main

305

monomeric units. Quantification of dimeric, trimeric and tetrameric PAs was carried out with

306

ESI-MS-SIR by reference compound.

307

308

309 310

Acknowledgement

Ms Inkeri Peltomaa and Mr. Markku Peltomaa are acknowledged for the sea buckthorn berries.

311 312

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313

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314 315 316

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38. Rösch, D.; Mugge, C.; Fogliano, V.; Kroh, L.W. Antioxidant oligomeric proanthocyanidins from sea buckthorn (Hippophae rhamnoides) Pomace. J. Agric. Food Chem. 2004, 52, 67126718.

423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438

This work was financed by the China Scholarship Council (CSC), China. 20


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439

Figure captions

440

Figure 1. RP-HPLC-DAD chromatograms measured at 280 nm of (A) the crude extract of sea

441

buckthorn berries and of (B) the Sephadex-purified fraction II. The HILIC-DAD chromatogram

442

of (C) fraction II measured at 280 nm.

443 444

Figure 2. The total ion spectrum of fraction II (A) from 3.6 to 17.6 min analyzed by HILIC-

445

ESI-MS and (B) from 3 to 25 min analyzed by NP-UPLC-ESI-MS. The PA signals in the

446

spectrum are marked as deprotonated calculated molar masses where the isotope abundances

447

have been taken into account.

448 449

Figure 3. Selected ion recording (SIR) chromatograms of proanthocyanidins analyzed by ESI-

450

MS in fraction II: dimers (A) HILIC, (B) NP-UPLC and (C) RP-HPLC; trimers (D) HILIC, (E)

451

NP-UPLC and (F) RP-HPLC; tetramers (G) HILIC, (H) NP-UPLC and (I) RP-HPLC.

452

21



Page 22 of 27

Table 1. Composition of Oligomeric Proanthocyanidins Pstimated from The Total Ion Spectrum Obtained by HILIC-ESI-MS. Detected Mass Number of subunits b Molecular a DP

2 2 2 3 3 3 3 4 4 4 4 4 5 5 5 5 6 6 6 7 7 7 7 7 8 8 8 8 8 8 9 9 9 9 9 9 9 9 10 10 10 10 10 10 11 11 a

formula C30H26O12 C30H26O13 C30H26O14 C45H38O18 C45H38O19 C45H38O20 C45H38O21 C60H50O24 C60H50O25 C60H50O26 C60H50O27 C60H50O28 C75H62O32 C75H62O33 C75H62O34 C75H62O35 C90H74O40 C90H74O41 C90H74O42 C105H86O45 C105H86O46 C105H86O47 C105H86O48 C105H86O49 C120H98O50 C120H98O52 C120H98O53 C120H98O54 C120H98O55 C120H98O56 C135H110O56 C135H110O57 C135H110O58 C135H110O59 C135H110O60 C135H110O61 C135H110O62 C135H110O63 C150H122O64 C150H122O66 C150H122O67 C150H122O68 C150H122O69 C150H122O70 C165H134O75 C165H134O76

(E)C 3 2 0 3 2 1 0 4 3 2 1 0 3 2 1 0 2 1 0 4 3 2 1 0 6 4 3 2 1 0 7 6 5 4 3 2 1 0 6 4 3 2 1 0 2 0

(E)GC 0 1 3 0 1 2 3 0 1 2 3 4 2 3 4 5 4 5 6 3 4 5 6 7 2 4 5 6 7 8 2 3 4 5 6 7 8 9 4 6 7 8 9 10 9 11

Exact Mass 578.14 594.14 610.13 866.21 882.20 898.20 914.19 1154.27 1170.26 1186.25 1202.25 1218.25 1474.32 1490.32 1505.31 1522.31 1794.38 1810.37 1826.37 2066.44 2082.43 2098.43 2114.43 2130.42 2337.51 2369.49 2386.50 2402.49 2418.49 2434.48 2626.58 2642.57 2658.57 2674.56 2690.56 2706.55 2722.54 2738.54 2945.62 2978.62 2994.61 3010.61 3026.60 3042.60 3314.67 3330.66

[M-H]577.52 593.52 609.52 865.77 881.77 897.77 913.77 1154.97 1170.34 1186.02 1202.06 1217.88 1473.89 1490.04 1505.34 1522.16 1794.07 1811.01 1828.03

2115.64 2130.49

[M-2H]2-

1032.84 1041.91 1049.18 1057.13 1065.00 1168.25c 1184.24c 1193.29 c 1200.24 1209.18 1216.23c 1312.95 1319.95 1328.61 1336.64 1344.59 1353.51 1361.31 1369.33 1472.31d 1488.30d 1496.57 1504.30d 1513.96 1520.29d 1657.09 1665.49

DP=degree of polymerization; b (E)C=(epi)catechin, (E)GC=(epi)gallocatechin; c [M-2H]2- ions of DP8

overlapped with the [M-H]- ions of DP4; d [M-2H]2- ions of DP10 overlapped with the [M-H]- ions of DP5. 22


Page 23 of 27


Table 2. Identification and Quantification of Oligomeric Proanthocyanidins (DP ≤ 4) in Fraction II of Sea Buckthorn Berries by NP-UPLC-, RPHPLC- and HILIC-ESI-MS-SIR. MS-SIR

Total ion spectrum

DP a

[M-H]m/z b

Rt1 c

Rt2 d

Peak e

Peak f

PA

577

3.98

4.19

a

1-5

(E)C-(E)C

65653

23.6

1.42

20.6

22670

16.2

0.45

dimers

593

4.49

5.51

b

6-16

(E)C-(E)GC

38037

13.7

1.43

12.0

14083

10.0

0.35

609

5.21

5.72

c

17-28

(E)GC-(E)GC

24031

8.7

1.43

7.6

17231

12.3

0.18

PA

865

5.31

5.92

d

29-36

(E)C-(E)C-(E)C

19856

7.1

1.70

7.5

11580

8.3

0.25

trimers

881

5.62

7.04

e

37-43

(E)C-(E)C-(E)GC

20937

7.5

1.71

8.0

8609

6.1

0.27

897

6.13

7.76

f

44-55

(E)C-(E)GC-(E)GC

26843

9.7

1.71

10.1

10866

7.7

0.35

913

6.84

8.58

g-i

56-62

(E)GC-(E)GC-(E)GC

41191

14.8

1.72

15.6

16790

12.0

0.54

1153

5.82

7.96

(E)C-(E)C-(E)C-(E)C

5853

2.1

2.04

2.6

6367

4.5

0.11

PA tetramers

a

Structure assignment g

Peak area

h

%

i

CF

j

%

k

Peak area

l

%

m

Contents (mg/100g)

1169

6.94

8.68

(E)C-(E)C-(E)C-(E)GC

7891

2.8

2.04

3.6

4244

3.0

0.16

1185

7.15

10.01

(E)C-(E)C-(E)GC-(E)GC

5487

2.0

2.05

2.5

5727

4.1

0.11

1201

7.76

10.21

j

(E)C-(E)GC-(E)GC-(E)GC

6947

2.5

2.05

3.1

7927

5.6

0.14

1217

8.58

10.92

k-n

(E)GC-(E)GC-(E)GC-(E)GC

14998

5.4

2.06

6.8

14236

10.1

0.32

DP=degree of polymerization; b Deprotonated nominal masses; c Retention times (min) in HILIC-ESI-MS-SIR show in Figure 3A, 3D and 3G; d Retention times (min) in

NP-UPLC-ESI-MS-SIR show in Figure 3B, 3E and 3H; e Peaks in HILIC-DAD as shown in Figure 1C ; f Peaks in RP-HPLC-ESI-MS-SIR show in Figure 3C and 3F ; g Alternative sequences possible for heterogeneous proanthocyanidins, (E)C=(epi)catechin, (E)GC=(epi)gallocatechin; h Peak areas in HILIC-ESI-MS-SIR show in Figure 3A, 3D and 3G; i Proportions of PA oligomer peak areas in SIR mode; j Correction factors for isotopic abundances; k Proportions PA oligomer peak areas corrected by isotopic abundances; l Peak areas calculated in total ion spectrum; m Proportions of PA oligomer peak areas in full scan mode.

23



Page 24 of 27

Figure 1

24


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Figure 2

25



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Figure 3

26


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Table of Contents Graphic

27


Proanthocyanidins in Wild Sea Buckthorn (HippophaÃ«

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