Efficient Enrichment and Analysis of Vicinal Diol-Containing Flavonoid

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

New Analytical Methods

Efficient Enrichment and Analysis of Vicinal Diol-Containing Flavonoid Molecules Using Boronic Acid-Functionalized Particles and Matrixassisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Eunjin Kim, Hyunook Kang, Inseong Choi, Jihyeon Song, Hyejung Mok, Woong Jung, and Woon-Seok Yeo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00832 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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

Page 1 of 31

Journal of Agricultural and Food Chemistry

Efficient Enrichment and Analysis of Vicinal Diol-Containing Flavonoid Molecules Using Boronic Acid-Functionalized Particles and Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Eunjin Kim1, Hyunook Kang1, Insung Choi1, Jihyeon Song2, Hyejung Mok2, Woong Jung3, and Woon-Seok Yeo1,*

1. Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 05029 (Korea) 2. Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029 (Korea) 3. Department of Emergency Medicine, Kyung Hee University Hospital at Kangdong, Seoul 05278 (Korea)

*Corresponding author: W.-S. Yeo Phone: +82-2-2049-6054 E-mail: [email protected]

1

ACS Paragon Plus Environment


1

Page 2 of 31

Abstract

2

Detection and quantitation of flavonoids are relatively difficult compared with those of

3

other small molecule analytes because flavonoids undergo rapid metabolic processes

4

resulting in their elimination from the body. Here, we report an efficient enrichment method

5

for facilitating the analysis of vicinal diol-containing flavonoid molecules using matrix-

6

assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). In

7

our strategy, boronic acid-functionalized polyacrylamide particles were utilized, where

8

boronic acids bound to vicinal diols to form boronate monoesters at basic pH. This complex

9

remained intact during the enrichment processes, and the vicinal diol-containing flavonoids

10

were easily separated by centrifugation and subsequent acidic treatments. The selectivity and

11

the limit of detection of our strategy were confirmed by MS analysis, and the validity was

12

assessed by performing the detection and quantitation of quercetin in mouse organs.

13

Keywords:

14

desorption/ionization time-of-flight mass spectrometry, Vicinal diols

Boronic

acid,

Enrichment,

Flavonoids,

2


Matrix-assisted

laser

Page 3 of 31


15

Introduction

16

Flavonoids, a class of plants and fungus secondary metabolites and abundantly present

17

in the human diet mainly as glycoside conjugates which are transformed to a complex

18

mixture of conjugates in human body.1,2 Flavonoids are a large group of polyphenolic

19

compounds with the skeleton of two benzene rings linked via an oxygen-containing

20

heterocyclic ring. Flavonoids have broad biological and pharmacological activity including

21

anti-allergic, anti-inflammatory, and antioxidant activities with little toxicity,1-3 and therefore,

22

are known to have preventing effects against various diseases such as cancers,4 diabetes,5

23

inflammations,6 and Alzheimer’s disease,7 which have been investigated via in vitro or in

24

animal studies. Conventionally, the analysis of flavonoid aglycones and their conjugates

25

mainly relies on high performance liquid chromatography (HPLC)8-11 and liquid

26

chromatography/mass spectrometry (LC/MS)12-14 which affords straightforward and reliable

27

information. Thin layer chromatography (TLC) is also useful for rapid screening,15,16 but it

28

has disadvantages of low resolution and poor quantitative ability. Electrochemical methods

29

also have been efficiently used,17,18 of which the general use is hampered due to its

30

insufficient selectivity in the presence of compounds with structural similarity. Particularly, it

31

has been known to be relatively difficult to detect and quantitate flavonoid aglycones

32

compared with other small molecules because of poor solubility and rapid metabolic

33

elimination from the human body.19-23 In this regard, reliable analytical methods for the

34

detection and quantitation of flavonoid aglycones via selective and efficient procedures are

35

urgently needed.

36

Here, we report an efficient enrichment method for facilitating the analysis of vicinal

37

diol-containing flavonoid molecules using matrix-assisted laser desorption/ionization time of 3



38

flight mass spectrometry (MALDI-TOF MS). The use of MALDI-TOF MS as an analytical

39

tool for small molecules offers many advantages over other conventional methods in terms of

40

easy sample preparation, rapidity, high sensitivity, low background signals, and no false-

41

positive signals.24-26 In addition, MS analysis has excellent multiplexing capability since it

42

provides molecular weights of analytes, and thus, distinctively discriminates multiple

43

analytes despite of structural similarity. In our strategy, boronic acid-functionalized

44

polyacrylamide particles (BAPs) were harnessed for selective isolation of vicinal diol-

45

containing flavonoid molecules, where boronic acids dominantly form boronate monoester

46

complexes with vicinal diols at basic pH, while decomplexation occurs in the acidic

47

environment resulting in the recovery of boronic acid and vicinal diol functionality.27-30 By

48

using this characteristic, boronic acid-functionalized materials have been widely used for the

49

analysis of vicinal diol-containing hydrophilic biomolecules, mostly saccharides,31-33

50

nucleosides,34 glycopeptides and glycoproteins,35-37 and for various applications including

51

drug delivery systems, enzymatic inhibitions, and cell-capture/release systems.28,38-41

52

However, very few examples have been reported for the study of hydrophobic molecules

53

such as flavonoids because of its low water-solubility and interference of other vicinal diol-

54

containing biomolecules existing in biological systems in high abundance.42,43 As a typical

55

example, Zhang et al. introduced boronate affinity adsorption of cis-diol-containing

56

hydrophobic molecules in nonaqueous solvents such as methanol, acetonitrile, and ethyl

57

acetate.43

58

In the current study, the selectivity of BAPs towards vicinal diol-containing flavonoids

59

was evaluated in the presence of other flavonoids in highly excess amount which have

60

multiple hydroxyl groups and carbonyl groups that potentially interact with boronic acids. 4


Page 4 of 31

Page 5 of 31


61

Next, the practical applicability was assessed by quantitating quercetin spiked in fetal bovine

62

serum. Furthermore, we showed that our method can effectively enrich vicinal diol-

63

containing flavonoids and facilitate the quantitation of the targets present in animal tissues in

64

low abundance. Finally, recyclability of the BAPs was confirmed for validating the practical

65

usability and cost saving of our method.

66

67

Materials and Methods

68

Materials

69

Boronic acid-functionalized particles (100 µmol boronate/mL of settled resin) were

70

purchased from Thermo Fisher Scientific (Mississauga, ON, Canada). Quercetin hydrate,

71

sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), ammonium acetate, and

72

trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethyl

73

acetate (EA) was purchased from Samchun Chemical Co., Ltd. (Seoul, Korea). Naringenin,

74

apigenin, kaempferol, and luteolin were purchased from Tokyo Chemical Industry (Tokyo,

75

Japan). Acetonitrile (ACN) was purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan).

76

Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from

77

WELGENE (Daegu, Korea). Gold nanoparticles (AuNPs, 40 nm diameter) were prepared

78

using the method previously reported by Schwartzberg et al..44

79

80

Methods

81

General 5



82

Quercetin and luteolin were used as target molecules containing a vicinal diol, while

83

naringenin, apigenin, and kaempferol were used as control molecules without a vicinal diol.

84

Quercetin and naringenin were dissolved in 40% ammonium acetate buffer (0.2 M at pH 8.65)

85

in DMSO at various concentrations. Apigenin, kaempferol, and luteolin were dissolved in 30%

86

ammonium acetate buffer (0.2 M at pH 8.65) in DMSO at various concentrations. Boronic

87

acid-functionalized particles containing 0.25 µmol boronates were washed three times with 1

88

mL of ammonium acetate buffer (0.2 M at pH 8.65) by centrifugation at 10,770×g for 30 s,

89

and were resuspended in 200 µL of various solutions of the target molecules and the control

90

molecules. The capture procedure was conducted using a horizontal microtube holder under

91

vortex at room temperature for 2 h. The particles were then washed 3 times with a binding

92

buffer (30% or 40% ammonium acetate buffer in DMSO) by centrifugation at 10,770×g for

93

30 s, and incubated with 100 µL of releasing buffer (50% acetonitrile, 1% TFA in distilled

94

water) under vortex at room temperature for 10 min. Finally, the supernatant (2 µL) was

95

analyzed by MALDI-TOF MS with AuNPs (1.1 nM) as a matrix.

96

97

MALDI-TOF MS analysis

98

MALDI-TOF MS analysis was performed using an Autoflex III MALDI-TOF mass

99

spectrometer (Bruker Daltonics, Germany) using Smartbeam laser as an ionization source.

100

All the spectra were acquired in a positive mode using 19 kV accelerating voltage and a 50

101

Hz repetition rate with the average of 500 shots.

102 6


Page 6 of 31

Page 7 of 31


103

Calibration curve

104

A calibration curve for quantitating quercetin was constructed using three different

105

amounts of the boronic acid-functionalized particles containing boronates of 0.125 µmol,

106

0.25 µmol, or 0.5 µmol. The concentration of apigenin, as an internal standard, was adjusted

107

to 10 µΜ, 40 µΜ, or 100 µΜ. Each experiment was repeated at least 3 times. All quantitation

108

was performed using the intensity sum of adduct ion peaks.

109 110

Quantitation of quercetin in FBS

111

Quercetin (0.8 or 1.2 µmol) was added in FBS (2%, 5%, or 10%). The spiked FBS (200

112

µL) was extracted with EA (500 µL) three times by centrifugation at 10,770×g for 1 min. The

113

combined organic layer was dried in a fume hood at room temperature, and the residue was

114

subjected to the above-mentioned enrichment process and quantitated using the constructed

115

calibration curve.

116

117

Quantitation of quercetin in tissues

118

To quantitate the quercetin contents in the liver, quercetin (3 mg/mouse, human

119

equivalent dose: 12 mg/kg)45 was intravenously injected into mice (n=3), and tissues were

120

extracted with EA using the protocol previously reported by Kang et al..46 In brief, tissues

121

were weighed and homogenized in PBS (tissue:PBS = 1:3, w/v). The homogenates (500 µL)

122

were mixed with 10% SDS (200 µL) by sonication and then mixed with EA (500 µL) by 7



123

sonication for 10 min. The mixture was centrifuged at 10,770×g for 1 min, and the

124

supernatant was collected. The extraction process was repeated 3 times and the combined

125

organic layer was dried in a fume hood at room temperature. The residue was subjected to the

126

above-mentioned enrichment process and was then quantitated using the constructed

127

calibration curve. All animal care and experimental procedures were approved by the Animal

128

Care Committee of Konkuk University.

129

130

Recyclability

131

Quercetin at different amounts of 0.4, 0.8, and 1.2 µmol was incubated with boronic

132

acid-functionalized particles containing 0.25 µmol boronates. After enrichment and release

133

processes, the MALDI-TOF MS analysis was performed. The identical boronic acid-

134

functionalized particles were then reused 2 more times for enrichment and release of

135

quercetin.

136

137

Results & Discussion

138

Selective isolation of vicinal diol-containing flavonoid molecules

139

As shown in the schematic diagram of our method in Figure 1, tissues containing the

140

vicinal diol-containing target molecules were extracted with organic solvents, where other

141

vicinal diol-containing hydrophilic biomolecules (mostly water-soluble) were excluded from

142

the analysis. The tissue extracts were then mixed with BAPs and incubated at basic pH 8


Page 8 of 31

Page 9 of 31


143

leading to selective boronate monoester formation between boronic acid and vicinal diol-

144

containing flavonoids. This complex remained intact during isolation by centrifugation. The

145

subsequent acidic treatment induced the decomplexation and release of the vicinal diol-

146

containing flavonoids, after which the MALDI-TOF MS analysis was performed.

147

Because flavonoids, in general, contain multiple hydroxyl groups and carbonyl groups

148

that potentially interact with boronic acids, we first verified the binding site of BAPs with

149

flavonoid molecules. Four flavonoid molecules—apigenin, kaempferol, luteolin, and

150

quercetin—were individually mixed with BAPs in 30–40% ammonium acetate buffer at pH

151

8.65 (for their structures, see Figure S1). Apigenin and kaempferol do not have a vicinal diol

152

at 3’- and 4’-carbons but possess a potential recognition site (α-hydroxy carbonyl group at 3-

153

and 4-carbons or β-hydroxy carbonyl group at 4- and 5-carbons). Luteolin and quercetin

154

contain a vicinal diol at 3’- and 4’-carbons as well as the potential recognition site. After

155

incubation for 2 h, the mixture was washed, and subsequently treated with a releasing buffer

156

(50% acetonitrile, 1% TFA in distilled water). Finally, the supernatant was analyzed by

157

MALDI-TOF MS with gold nanoparticles as a matrix. As shown in Figure S1, after

158

enrichment, apigenin and kaempferol without a vicinal diol were not observed (Figure S1a

159

and S1b), whereas MS analysis clearly afforded peaks corresponding to luteolin and

160

quercetin (Figure S1c and S1d). This result implies that the vicinal diol at 3’- and 4’-carbons

161

was the recognition site of boronic acid, and vicinal diol-containing flavonoid molecules can

162

be bound to and released from BAPs.

9



163

Next, the selectivity of BAPs towards vicinal diol-containing flavonoids was evaluated

164

in the presence of other flavonoids. We prepared three mixed solutions of flavonoids:

165

quercetin/naringenin at a molar ratio of 1:1 (Figure 2a), quercetin/naringenin/apigenin at a

166

molar ratio of 1:1:1 (Figure 2b), and quercetin/naringenin/apigenin/luteolin at a molar ratio of

167

1:1:1:1 (Figure 2c). Quercetin and luteolin were used as vicinal diol-containing target

168

molecules, while naringenin and apigenin were used as control molecules without a vicinal

169

diol. The MS analysis of the mixtures afforded each peak for flavonoids in the solutions:

170

quercetin (●, m/z 303.04 [M+H+], m/z 325.04 [M+Na+], m/z 341.04 [M+K+]), naringenin (□,

171

m/z 273.07 [M+H+], m/z 295.07 [M+Na+], m/z 311.07 [M+K+]), apigenin (△, m/z 271.05

172

[M+H+], m/z 293.05 [M+Na+], m/z 309.05 [M+K+]), or luteolin (★, m/z 287.05 [M+H+], m/z

173

309.05 [M+Na+], m/z 325.05 [M+K+]) (Figure 2, top panels). However, MS analysis after the

174

isolation process described above gave only peaks for vicinal diol-containing molecules,

175

quercetin and luteolin (Figure 2, bottom panels). This result implies that vicinal diol-

176

containing flavonoid molecules can specifically be isolated by BAPs with excellent

177

selectivity.

178

179

Enrichment of vicinal diol-containing flavonoid molecules using BAPs

180

The feasibility study of our method was performed by assessing the enrichment

181

capability of BAPs in the presence of other flavonoids in highly excess amount. Quercetin

182

was mixed with naringenin at molar ratios of 1:10 (Figure 3a) and 1:100 (Figure 3b), and

183

with naringenin/apigenin at a molar ratio of 1:10:10 (Figure 3c). MS analysis of the mixtures 10


Page 10 of 31

Page 11 of 31


184

displayed major peaks for naringenin and apigenin with a trace of quercetin peaks (Figure 3a,

185

3b, and 3c, top panels). However, the MS spectra after enrichment showed only distinct

186

quercetin peaks (Figure 3a, 3b, and 3c, bottom panels), indicating the excellent enrichment

187

efficiency of BAPs towards vicinal diol-containing quercetin despite the low abundances.

188

189

Quantitation of quercetin in complex samples

190

We next verified the fidelity of our strategy by examining the limit-of-detection (LOD)

191

of quercetin and by confirming the selective enrichment and quantitation of quercetin in

192

highly complex samples. For quantitation, a calibration curve was constructed using three

193

different amounts of the BAPs containing boronates of 0.125 µmol (Figure 4a), 0.25 µmol

194

(Figure 4b), or 0.5 µmol (Figure 4c) with various amounts of quercetin ranging from 0.075 to

195

60 nmol in the presence of apigenin as an internal standard, and the experiment was repeated

196

at least 3 times. We found that the LOD for quercetin was 75 pmol where the signal-to-noise

197

(S/N) ratio was larger than 3. Quercetin/internal standard (Q/IS) ratios at various amounts of

198

quercetin and boronates are listed in Table S1. We next measured Q/IS ratios against

199

quercetin spiked in fetal bovine serum (FBS, 2%, 5%, or 10% diluted with PBS [v/v]). The

200

quercetin spiked in FBS (200 µL) was extracted with EA (500 µL) three times, and the

201

combined organic layer was dried at room temperature. The residue underwent the above-

202

mentioned enrichment process and the quantitation was performed at linear dynamic ranges

203

by a linear regression of the Q/IS ratio against the amount of quercetin (Figure 4, insets). As

204

shown in Table 1, recoveries of the known amount of quercetin added in FBS were obtained

205

from 94% to 102%, and furthermore, we observed a coefficient of variance (CV) of 0.5% to 11



206

5%, which are comparable to or even more efficient than those of previously reported

207

methods.17,47,48 This result clearly indicates that our method is not only consistent but also

208

accurate, and therefore, is feasible and applicable to complex samples.

209

210

Quantitation of quercetin in animal tissues

211

The practical applicability of our method was further evaluated by applying our system

212

to quantitate quercetin contents in animal tissues. Quercetin or PBS as a control was

213

intravenously injected into mice, and the liver from the sacrificed mice was homogenized and

214

extracted with EA using the protocol previously reported by Kang et al..46 The extraction

215

process was repeated 3 times, and the combined organic layer was dried and analyzed with

216

MALDI-TOF MS. As shown in Figure 5, without the enrichment process, only a trace of

217

quercetin peak (●) was observed for the quercetin-injected mice (insets: spectra on an

218

intensity scale of 0–50) (Figure 5a and 5b, top panels). The tissue extracts were then

219

subjected to enrichment and analyzed by MALDI-TOF MS in the presence of IS (△). MS

220

spectra showed clear quercetin peaks for quercetin-injected mice with high densities, while

221

no quercetin peak was observed in the case of the PBS-injected mice (Figure 5a and 5b,

222

bottom panels). The comparison of the intensity between peaks for quercetin and IS allowed

223

quantitation using the constructed calibration curve, resulting that the content of quercetin in

224

the liver from the quercetin-injected mice was 0.475 mg/g of organ. These results strongly

225

imply that our method described in Figure 1 can effectively enrich vicinal diol-containing

226

flavonoids and facilitate the quantitation of the targets present in animal tissues in low 12


Page 12 of 31

Page 13 of 31

227


abundance.

228

229

Recyclability

230

For practical usability and cost savings, BAPs must maintain good reproducibility with

231

repeatable enrichment experiments. Quercetin at different amounts of 0.4, 0.8, and 1.2 µmol

232

was incubated with BAPs containing 0.25 µmol boronates. After enrichment and release

233

processes, the Q/IS ratio was measured by MALDI-TOF MS analysis. The identical BAPs

234

were then reused 2 more times, which showed almost same values of Q/IS ratio for each

235

cycle with a CV of 1.09, 2.17 and 1.81%, indicating the excellent recyclability of the BAPs

236

(Figure S2).

237

238

In summary, we describe herein a strategy for the efficient enrichment and quantitation

239

of vicinal diol-containing flavonoid molecules using boronic acid-functionalized particles

240

(BAPs). We validated the selectivity and efficiency of BAPs towards vicinal diol-containing

241

flavonoid molecules and constructed a calibration curve for quantitating quercetin. The

242

feasibility and applicability of our method were assessed via the quantitation of quercetin

243

spiked in FBS and animal tissues. Furthermore, the recyclability of BAPs was confirmed.

244

Thus, this method is promising from the viewpoint of cost saving and practical applications.

245

A major technical concern of the method is the use of basic pH for selective boronate

246

monoester formation between boronic acid and vicinal diol-containing flavonoids which are

247

known to undergo a chemical modification in alkaline aqueous solution at pH above 10~11.49

248

Although the boronate affinity extraction at pH 8.65 of our method does not seem to induce 13



Page 14 of 31

249

chemical degradation, the use of affinity materials reported by Zhen Liu and coworkers

250

which maintain high binding capacities under neutral or acidic conditions would effectively

251

avoid this possibility.50,51

252

253

Acknowledgments

254

This research was supported by the Priority Research Centers Program (2009-0093824) and

255

Basic Science Research Program (NRF-2016R1D1A1A09918111) through the National

256

Research Foundation (NRF) of Korea funded by the Ministry of Education.

257

258

Abbreviations Used

259

MALDI-TOF

260

spectrometry; HPLC, high performance liquid chromatography; TLC, thin layer

261

chromatography; BAPs, boronic acid-functionalized polyacrylamide particles; SDS, sodium

262

dodecyl sulfate; DMSO, dimethyl sulfoxide; TFA, trifluoroacetic acid; EA, ethyl acetate;

263

ACN, acetonitrile; FBS, fetal bovine serum; PBS, phosphate-buffered saline; AuNPs, gold

264

nanoparticles; LOD, limit-of-detection; S/N, signal-to-noise; Q/IS ratio, quercetin/internal

265

standard ratios; CV, coefficient of variance.

MS,

matrix-assisted

laser

desorption/ionization

266

267

Notes

268

The authors declare no competing financial interest. 14


time-of-flight

mass

Page 15 of 31


269

Supporting Information description

270

Supplementary Table 1. Q/IS ratios at various boronate amounts

271

Supplementary Figure 1. Verification of the recognition site of boronic acid-functionalized

272

particles.

273

Supplementary Figure 2. Verification of the recyclability of the boronic acid-functionalized

274

particles.

15



275

References

276

1.

277

Pharm. Res. 2008, 7, 1089—1099.

278

2.

279

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

280

Chem. 2008, 56, 6185—6205.

281

3.

282

Flavonoids as anti-inflammatory agents: implications in cancer and cardiovascular disease.

283

Inflamm. Res. 2009, 58, 537—552.

284

4.

285

Pharmacother. 2002, 56, 296—301.

286

5.

287

induced diabetic rats. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2003, 135, 357—364.

288

6.

289

Bartlett, J.; Shanmugam, K.; Münch, G.; Wu, M. J. Antioxidant and anti-inflammatory

290

activities of selected medicinal plants containing phenolic and flavonoid compounds. J. Agric.

291

Food Chem. 2011, 59, 12361—12367.

292

7.

293

tea polyphenols in Alzheimer's and Parkinson's diseases. J. Nutr. Biochem. 2004, 15, 506—

294

516.

295

8.

296

A.; Xu, Y. HPLC analyses of flavanols and phenolic acids in the fresh young shoots of tea

297

(Camellia sinensis) grown in Australia. Food Chem. 2004, 84, 253—263.

Tapas, A. R.; Sakarkar, D.; Kakde, R. Flavonoids as nutraceuticals: a review. Trop. J.

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

García-Lafuente, A.; Guillamón, E.; Villares, A.; Rostagno, M. A.; Martínez, J. A.

Le Marchand, L. Cancer preventive effects of flavonoids—a review. Biomed.

Vessal, M.; Hemmati, M.; Vasei, M. Antidiabetic effects of quercetin in streptozocin-

Zhang, L.; Ravipati, A. S.; Koyyalamudi, S. R.; Jeong, S. C.; Reddy, N.; Smith, P. T.;

Weinreb, O.; Mandel, S.; Amit, T.; Youdim, M. B. Neurological mechanisms of green

Yao, L.; Jiang, Y.; Datta, N.; Singanusong, R.; Liu, X.; Duan, J.; Raymont, K.; Lisle,

16


Page 16 of 31

Page 17 of 31


298

9.

299

the phenolic compounds of plant extracts. Investigation of their antioxidant capacity and

300

antimicrobial activity. J. Agric. Food Chem. 2005, 53, 1190—1195.

301

10.

302

performance liquid chromatography and ultra performance liquid chromatography. Talanta

303

2008, 76, 189—199.

304

11.

305

for the analysis of galloylquinic acid derivatives and flavonoids in Copaifera langsdorffii

306

leaves. J. Chromatogr. B 2017, 1061, 240—247.

307

12.

308

Determination of flavonoids in a citrus fruit extract by LC–DAD and LC–MS. Food Chem.

309

2007, 101, 1742—1747.

310

13.

311

Q. Determination of flavonoids by LC/MS and anti-inflammatory activity in Moringa

312

oleifera. J. Funct. Food. 2013, 5, 1892—1899.

313

14.

314

Sindona, G. Comprehensive assay of flavanones in citrus juices and beverages by UHPLC–

315

ESI-MS/MS and derivatization chemistry. Food Chem. 2013, 141, 2328—2333.

316

15.

317

flavonoid aglycones in plant exudates. Acta Chromatogr. 2004, 14, 110—114.

318

16.

319

fingerprint of flavonoids and saponins from Passiflora species. J. Liq. Chromatogr. Relat.

320

Technol. 2005, 28, 2285—2291.

Proestos, C.; Chorianopoulos, N.; Nychas, G.-J.; Komaitis, M. RP-HPLC analysis of

Spáčil, Z.; Nováková, L.; Solich, P. Analysis of phenolic compounds by high

da Silva Motta, E. V.; da Costa, J. d. C.; Bastos, J. K. A validated HPLC-UV method

Bilbao, M. d. L. M.; Andres-Lacueva, C.; Jauregui, O.; Lamuela-Raventos, R. M.

Coppin, J. P.; Xu, Y.; Chen, H.; Pan, M.-H.; Ho, C.-T.; Juliani, R.; Simon, J. E.; Wu,

Di Donna, L.; Taverna, D.; Mazzotti, F.; Benabdelkamel, H.; Attya, M.; Napoli, A.;

Nikolova, M.; Berkov, S.; Ivancheva, S. A rapid TLC method for analysis of external

Birk, C. D.; Provensi, G.; Gosmann, G.; Reginatto, F. H.; Schenkel, E. P. TLC

17



321

17.

322

amino-2-mercapto-1, 3, 4-thiadiazole modified single use sensors for detection of quercetin.

323

Colloids Surf. B Biointerfaces 2013, 106, 181—186.

324

18.

325

on Pd/MoS2-ionic liquid functionalized ordered mesoporous carbon. Electrochim. Acta 2017,

326

247, 657—665.

327

19.

328

and permeability of novel quercetin–amino acid conjugates. Bioorg. Med. Chem. 2009, 17,

329

1164—1171.

330

20.

331

Y.; Crozier, A. Bioavailability of black tea theaflavins: absorption, metabolism, and colonic

332

catabolism. J. Agric. Food Chem. 2017, 65, 5365—5374.

333

21.

334

Anthocyanins and flavanones are more bioavailable than previously perceived: a review of

335

recent evidence. Annu. Rev. Food Sci. Technol. 2017, 8, 155—180.

336

22.

337

Schroeter, H. The metabolome of [2-14 C](−)-epicatechin in humans: Implications for the

338

assessment of efficacy, safety, and mechanisms of action of polyphenolic bioactives. Sci. Rep.

339

2016, 6, 29034.

340

23.

341

Verrall, S.; Stewart, D.; Moffet, T.; Ibars, M.; Lawther, R. Tracking (poly) phenol components

342

from raspberries in ileal fluid. J. Agric. Food Chem. 2014, 62, 7631—7641.

343

24.

Muti, M.; Gençdağ, K.; Nacak, F. M.; Aslan, A. Electrochemical polymerized 5-

Xu, B.; Yang, L.; Zhao, F.; Zeng, B. A novel electrochemical quercetin sensor based

Kim, M. K.; Park, K.-S.; Yeo, W.-S.; Choo, H.; Chong, Y. In vitro solubility, stability

Pereira-Caro, G.; Moreno-Rojas, J. M.; Brindani, N.; Del Rio, D.; Lean, M. E.; Hara,

Kay, C. D.; Pereira-Caro, G.; Ludwig, I. A.; Clifford, M. N.; Crozier, A.

Ottaviani, J. I.; Borges, G.; Momma, T. Y.; Spencer, J. P.; Keen, C. L.; Crozier, A.;

McDougall, G. J.; Conner, S.; Pereira-Caro, G.; Gonzalez-Barrio, R.; Brown, E. M.;

Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T. Protein

18


Page 18 of 31

Page 19 of 31


344

and polymer analyses up to m/z 100 000 by laser ionization time‐of‐flight mass spectrometry.

345

Rapid Commun. Mass Spectrom. 1988, 2, 151—153.

346

25.

347

masses exceeding 10,000 daltons. Anal. Chem. 1988, 60, 2299—2301.

348

26.

349

spectrometry: avoidance of artifacts and analysis of caffeine-precipitated SII thearubigins

350

from 15 commercial black teas. J. Agric. Food Chem. 2012, 60, 4514—4525.

351

27.

352

Overview of their reactions and applications. Boronic Acids: Preparation and Applications in

353

Organic Synthesis and Medicine, 2006; 1., pp. 1—99.

354

28.

355

polymers: from materials to medicine. Chem. Rev. 2015, 116, 1375—1397.

356

29.

357

recognition: structure, properties and applications. Chem. Soc. Rev. 2015, 44, 8097—8123.

358

30.

359

Class Selectivity to Biomimetic Specificity. Acc. Chem. Res. 2017, 50, 2185—2193.

360

31.

361

probe set for electrochemical detection of saccharides. Chin. Chem. Lett. 2013, 24, 291—294.

362

32.

363

Saccharides within Boronic Acid Functionalized Mesoporous Silica Nanoparticles. Angew.

364

Chem. 2015, 127, 6271—6274.

365

33.

366

polymers specific to glycoproteins, glycans and monosaccharides via boronate affinity

Karas, M.; Hillenkamp, F. Laser desorption ionization of proteins with molecular

Drynan, J. W.; Clifford, M. N.; Obuchowicz, J.; Kuhnert, N. MALDI-TOF mass

Hall, D. G. Structure, properties, and preparation of boronic acid derivatives.

Brooks, W. L.; Sumerlin, B. S. Synthesis and applications of boronic acid-containing

Li, D.; Chen, Y.; Liu, Z. Boronate affinity materials for separation and molecular

Liu, Z.; He, H. Synthesis and Applications of Boronate Affinity Materials: From

Li, J.; Sun, Y.-Q.; Wei, Y.-M.; Zheng, J.-B. Phenylboronic acid and dopamine as

Pan, X.; Chen, Y.; Zhao, P.; Li, D.; Liu, Z. Highly Efficient Solid‐Phase Labeling of

Xing, R.; Wang, S.; Bie, Z.; He, H.; Liu, Z. Preparation of molecularly imprinted

19



367

controllable–oriented surface imprinting. Nat. Protoc. 2017, 12, 964.

368

34.

369

specific capture, separation and immobilization of cis-diol biomolecules. Chem. Comm. 2011,

370

47, 5067—5069.

371

35.

372

enzyme-linked immunosorbent assay by boronate affinity-based oriented surface imprinting.

373

Anal. Chem. 2013, 86, 959—966.

374

36.

375

boronate avidity for the selective enrichment of trace glycoproteins. Chem. Sci. 2013, 4,

376

4298—4303.

377

37.

378

monolith-based extraction with matrix-assisted laser desorption/ionization time-of-flight

379

mass spectrometry for efficient analysis of glycoproteins/glycopeptides. Anal. Chim. Acta

380

2014, 834, 1—8.

381

38.

382

hydrogels undergo self-healing at neutral and acidic pH. ACS Macro Lett. 2015, 4, 220—224.

383

39.

384

defined, reversible boronate crosslinked nanocarriers for targeted drug delivery in response to

385

acidic pH values and cis‐diols. Angew. Chem. 2012, 124, 2918—2923.

386

40.

387

phospholipid polymer having phenylboronic acid moiety. Biomed. Mater. 2010, 5, 054101.

388

41.

389

triggered cell capture and release. Bioconjugate Chem. 2017, 28, 2235—2240.

Liu, Y.; Ren, L.; Liu, Z. A unique boronic acid functionalized monolithic capillary for

Bi, X.; Liu, Z. Facile preparation of glycoprotein-imprinted 96-well microplates for

Wang, H.; Bie, Z.; Lü, C.; Liu, Z. Magnetic nanoparticles with dendrimer-assisted

Bie, Z.; Chen, Y.; Li, H.; Wu, R.; Liu, Z. Off-line hyphenation of boronate affinity

Deng, C. C.; Brooks, W. L.; Abboud, K. A.; Sumerlin, B. S. Boronic acid-based

Li, Y.; Xiao, W.; Xiao, K.; Berti, L.; Luo, J.; Tseng, H. P.; Fung, G.; Lam, K. S. Well‐

Saito, A.; Konno, T.; Ikake, H.; Kurita, K.; Ishihara, K. Control of cell function on a

Karimi, F.; Collins, J.; Heath, D. E.; Connal, L. A. Dynamic covalent hydrogels for

20


Page 20 of 31

Page 21 of 31


390

42.

391

functionalized magnetic nanoparticles with a high capacity and their use in the enrichment of

392

cis‐diol‐containing compounds from plasma. Biomed. Chromatogr. 2015, 29, 312—320.

393

43.

394

biomolecules in nonaqueous solvent. Biomed. Chromatogr. 2015, 29, 1133—1136.

395

44.

396

characterization, and tunable optical properties of hollow gold nanospheres. J. Phys. Chem. B

397

2006, 110, 19935—19944.

398

45.

399

and human. J. basic. Clin. Pharm. 2016, 7, 27.

400

46.

401

curcumin biodistribution using optical fluorescence imaging and mass spectrometry. Appl.

402

Biol. Chem. 2016, 59, 291—295.

403

47.

404

of quercetin and curcumin in polyherbal churna and its validation. Int. J. Pharm.

405

Phytopharmacol. Res. 2014, 4, 8—12.

406

48.

407

Determination of quercetin in human plasma using reversed-phase high-performance liquid

408

chromatography. J. Chromatogr. B Biomed. Sci. Appl. 1995, 666, 149—155.

409

49.

410

the chemical modification of quercetin and structurally related flavonoids characterized by

411

optical (UV-visible and Raman) spectroscopy. Phys. Chem. Chem. Phys. 2014, 16, 12802—

412

12811.

Du, J.; He, M.; Wang, X.; Fan, H.; Wei, Y. Facile preparation of boronic acid‐

Zhang, L.; Wang, C.; Wei, Y. Boronate affinity adsorption of cis‐diol‐containing

Schwartzberg, A. M.; Olson, T. Y.; Talley, C. E.; Zhang, J. Z. Synthesis,

Nair, A. B.; Jacob, S. A simple practice guide for dose conversion between animals

Kang, Y. Y.; Choi, I.; Chong, Y.; Yeo, W.-S.; Mok, H. Complementary analysis of

Salunkhe, V. R.; Patil, S. J. UV Spectrophotometric and HPLC method development

Liu, B.; Anderson, D.; Ferry, D. R.; Seymour, L. W.; de Takats, P. G.; Kerr, D. J.

Jurasekova, Z.; Domingo, C.; Garcia-Ramos, J.; Sanchez-Cortes, S. Effect of pH on

21



413

50.

414

Synergistic Co‐monomers: Access to a Boronate‐Functionalized Polymeric Monolith for the

415

Specific Capture of cis‐Diol‐Containing Biomolecules under Neutral Conditions. Angew.

416

Chem. 2009, 121, 6832—6835.

417

51.

418

cis-diol compounds at medium acidic pH condition. Chem. Commun. 2011, 47, 8169—8171.

Ren, L.; Liu, Z.; Liu, Y.; Dou, P.; Chen, H. Y. Ring‐Opening Polymerization with

Li, H.; Liu, Y.; Liu, J.; Liu, Z. A Wulff-type boronate for boronate affinity capture of

22


Page 22 of 31

Page 23 of 31


419

Captions for Table and Figures

420

Table 1. Quantitation of quercetin in FBS at various concentrations

421

Figure 1. Schematic diagram for the efficient enrichment and analysis of vicinal diol-

422

containing flavonoid molecules using boronic acid-functionalized particles and MALDI-TOF

423

MS.

424

Figure 2. Selectivity of boronic acid-functionalized particles towards vicinal diol-containing

425

flavonoid molecules. Quercetin was mixed with (a) naringenin at a molar ratio of 1:1, or (b)

426

naringenin and apigenin at a molar ratio of 1:1:1, or (c) naringenin, apigenin and luteolin at a

427

molar ratio of 1:1:1:1. Quercetin and luteolin were vicinal diol-containing target molecules,

428

while naringenin and apigenin were control molecules without a vicinal diol. Before

429

enrichment, peaks for quercetin (●, m/z 303.04 [M+H+], m/z 325.04 [M+Na+], m/z 341.04

430

[M+K+]), naringenin (□, m/z 273.07 [M+H+], m/z 295.07 [M+Na+], m/z 311.07 [M+K+]),

431

apigenin (△, m/z 271.05 [M+H+], m/z 293.05 [M+Na+], m/z 309.05 [M+K+]), or luteolin (★,

432

m/z 287.05 [M+H+], m/z 309.05 [M+Na+], m/z 325.05 [M+K+]) were observed. After

433

enrichment, only peaks for vicinal diol-containing molecules, quercetin and luteolin, were

434

obtained, indicating the excellent selectivity of boronic acid-functionalized particles towards

435

vicinal diol-containing molecules. *: background signals from the matrix or plate.

436

Figure 3. Efficiency of boronic acid-functionalized particles towards enrichment of vicinal

437

diol-containing flavonoid molecules. Quercetin (●) was mixed with naringenin (□) at

438

molar ratios of (a) 1:10 and (b) 1:100, or mixed with naringenin and apigenin (△) at a molar

439

ratio of (c) 1:10:10. Before enrichment, peaks for quercetin (●) were hardly observed; 23



440

however, after the enrichment, only distinct quercetin peaks were obtained, indicating the

441

excellent selectivity of boronic acid-functionalized particles towards vicinal diol-containing

442

quercetin despite the low abundance of quercetin. *: background signals from the matrix or

443

plate.

444

Figure 4. Calibration curve for quantitating quercetin at various amounts of boronate: 0.125,

445

0.25 and 0.5 µmol. Quantitation was performed at linear dynamic ranges by a linear

446

regression of the quercetin/internal standard (Q/IS) ratio against the amount of quercetin. The

447

error bar represents standard deviation (n=3).

448

Figure 5. Quantitation of quercetin in animal tissues. Liver from (a) PBS-injected mice or (b)

449

quercetin-injected mice was homogenized and extracted with EA. The MS spectra of the

450

tissue extracts without the enrichment process showed only a trace of quercetin peak (●) for

451

the quercetin-injected mice (insets: spectra on an intensity scale of 0–50) (top panels). The

452

tissue extracts subjected to the enrichment with IS (△) gave clear quercetin peaks for

453

quercetin-injected mice with high densities, while no quercetin peak was observed for the

454

PBS-injected mice (bottom panels). *: background signals from the matrix or plate.

24


Page 24 of 31

Page 25 of 31


Table 1

25



Figure 1

26


Page 26 of 31

Page 27 of 31


Figure 2

27



Figure 3

28


Page 28 of 31

Page 29 of 31


Figure 4

29



Figure 5

30


Page 30 of 31

Page 31 of 31


TOC graphic

31


Efficient Enrichment and Analysis of Vicinal Diol-Containing Flavonoid

Recommend Documents