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In vivo selective capture and rapid identification of luteolin and its metabolites in rat livers by molecularly imprinted solid-phase microextraction Die Gao, Dan-Dan Wang, Qian Zhang, Fengqing Yang, Zhining Xia, Qi-Hui Zhang, and Chun-Su Yuan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05269 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 24, 2017

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Journal of Agricultural and Food Chemistry

In vivo selective capture and rapid identification of luteolin and its metabolites in rat livers by molecularly imprinted solid-phase microextraction Die Gao1, 2, 3, Dan-Dan Wang1, Qian Zhang3, Feng-QingYang3, Zhi-Ning Xia1, 3,*, Qi-Hui Zhang 3,*, Chun-Su Yuan4 1. School of Pharmaceutical Sciences, Chongqing University, Chongqing, 400030, China; 2. Department of Pharmaceutical Sciences, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan 646000, China； 3. School of Chemistry and Chemical Engineering, Chongqing University, Chongqing, 400030, China; 4. Tang Center for Herbal Medicine Research and Department of Anesthesia & Critical Care, University of Chicago, Chicago, IL 60637, USA.

Correspondence: * To whom correspondence should be addressed: Prof. Zhi-Ning Xia or Dr. Qi-Hui Zhang,

Phone/fax:

86-23-6510-6615,

E-mail:

[email protected]

[email protected].

1

ACS Paragon Plus Environment

or


1

ABSTRACT:

2

A method based on molecularly imprinted solid-phase microextraction

3

(MIP-SPME) coupled with liquid chromatography- quadrupole time-of-flight tandem

4

mass spectrometry (QTOF-MS/MS) was developed for the detection of luteolin and

5

its metabolites in vivo. The MIP-SPME fibers were firstly fabricated by dopamine and

6

silane, then luteolin MIPs coated fibers were successfully prepared using luteolin,

7

acrylamide (AM) and ethylene glycol dimethacrylate (EGDMA) as template,

8

functional monomer and cross-linker, respectively. The characterizations of polymers

9

were analyzed by scanning electron microscopy (SEM), Fourier transform infrared

10

spectroscopy (FT-IR) and Brunauer-Emmett-Teller method (BET). The properties

11

involving adsorption and selective experiments were evaluated, these results revealed

12

that MIPs fibers presented high adsorption capacity and selectivity to luteolin.

13

Furthermore, the developed MIP-SPME coupled with LC-QTOF-MS/MS method was

14

adopted to capture and identify luteolin and its metabolites in rat livers in vivo,

15

eventually, apigenin, chrysoeriol and diosmetin were rapidly identified as metabolites.

16 17

KEYWORDS: Molecularly imprinted polymer, solid phase microextraction, in vivo

18

sampling, luteolin, metabolites, HPLC-MS/MS

19 20 21 22 2


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23

INTRODUCTION

24

Luteolin is a well-known flavone found in many herbal medicines, vegetables

25

and food items such as Lonicera japonica, Chrysanthemum morifolium, celery,

26

peppers, carrots, and peppermint 1-3. Luteolin has gained increasing interest due to the

27

beneficial, pharmacological and biological properties of it, such as anti-inflammation,

28

anti-allergy, anti-diabetic, anti-cancer and neuroprotective activities

29

reported that the metabolism of luteolin in the body had a great impact on its

30

biological effects 8. So elucidation of the metabolism of luteolin in vivo is important

31

to understand the roles of luteolin played in pharmacological and biological processes

32

4-7

. It was

9

. Previous researches are focused on clarifying the pharmacokinetics, metabolism

33

processes and metabolites of luteolin in blood, urine, liver microsomes and tissues

34

homogenates in vitro, and two methylated metabolites namely chrysoeriol and

35

diosmetin have been identified

36

only exert a variety of pharmacological activities similar to those of luteolin, but also

37

exhibit significantly extra pharmacological activities, such as osteoporosis 12-13.

38

8, 10-11

. It is noteworthy that the two metabolites not

In recent researches, the research objects of luteolin are limited to blood, urine, 9, 11, 14

39

liver microsomes and liver homogenates

. Although analysis of blood sample

40

can explain some metabolic process of luteolin, they are performed by sacrificing

41

several rats for each data point. At the same time, the experimental animals should

42

withstand serial blood collection 15. Nowadays, much attention has been paid on the

43

metabolism of analytes in liver, which is the main metabolic organ of mammals.

44

Moreover, researches demonstrated that flavonoids are usually enriched and 3



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45

metabolized in liver, and the enzymes in liver, such as catchol-O-methyltransfeas and

46

CYP450s et al. play important roles in the metabolism and biotransformation of

47

flavonoids

48

homogenates to simulate the metabolic process of analytes in liver

49

these methods can be used to explain some changes of analytes, which were brought

50

by liver enzymes in microsomes and homogenates, these methods frequently have

51

serious invasiveness to living system, even resulted in the death of animals. Therefore,

52

more simple and reliable metabolic analysis method in vivo is necessary for metabolic

53

studies.

16-17

. However, most of the researches used liver microsomes and liver 18-19

. Although

54

An ideal in vivo sampling technique should be highly specific, solvent-free, and

55

integration of the sampling, sample preparation and analysis steps. Furthermore, this

56

technique needs a minimized morphology and has slight invasiveness to living

57

systems

58

subsequent analysis in vivo is solid-phase microextraction (SPME)

59

minimal invasiveness to living systems, and only small fractions of analytes are

60

removed from the analyzed samples

61

that SPME is suitable for in vivo sample preparation and subsequent analysis in

62

different fields such as botanicals, food science, pharmaceutical research and

63

development, microorganisms in the body, analysis of environmental pollutants,

64

disease diagnosis et al. 23.

15

. One of the most promising techniques for rapid sample preparation and 20

. SPME brings

21-22

. The ongoing basic studies have elucidated

65

In recent years, a number of commercially available and laboratory-made SPME

66

sorbents have been proved to be useful for in vivo drugs tracking such as 4


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24

, SPME biocompatible C18 fibers

22

67

polydimethylsiloxane (PDMS) fibers

68

bioinspired polydopamine sheathed nanofibers

69

and laboratory-made SPME fibers mostly suffer from insufficient selectivity for

70

analytes. It is noteworthy that there has been a growing interest in the design of

71

various new sorbents based on molecularly imprinted polymers (MIPs) for SPME

72

(MIP-SPME). Compared with commercial SPME fibers, MIPs sorbents based SPME

73

fibers are usually prepared easily with low cost. Besides, owing to MIPs’ imprinting

74

according to the size of cavity and functional groups of the selected template

75

molecule, they perform high selectivity towards the specific analyte in complex

76

system such as biological samples. To date, MIPs as fiber coatings for SPME, MIPs

77

for in-tube SPME, monolithic MIPs fibers for SPME, membrane MIPs for SPME et al.

78

have been successfully prepared

79

coatings SPME fiber is successfully synthesized for the selective removal and

80

extraction of the antiviral drug abacavir in environmental and biological matrices 27.

81

An in-tube MIP-SPME device was used for determination of 4-nitrophenol (a kind of

82

environmental pollutants) in environmental water samples

83

MIP hybrid monolith and applied it in selective recognition and capture of lysozyme

84

from complex biological samples. A MIP-coated hollow fiber membrane for the

85

SPME was successfully used to extract triazines from environmental waters

86

Whereas, the recent studies about MIP-SPME have been limited to pre-concentrating

87

and selectively extracting target analyte from complex system in vitro, no study about

88

MIP-SPME on analyzing analytes such as metabolites in vivo has been reported.

25

,

et al.. However, these commercial

26

. For instances, a new abacavir based MIPs

5


28

. Lin et al

29

prepared a

30

.


89

In the present study, a synthesized MIP material with luteolin as template is

90

coated on stainless steel fiber according to previous literature 31. The new MIP-SPME

91

fibers are designed for the pre-concentrate and selectively extract of luteolin and its

92

metabolites in vivo. The characterizations of polymers on fibers were analyzed by

93

FT-IR, BET and SEM. The adsorption and selectivity of MIPs fibers were evaluated

94

by high-performance liquid chromatography (HPLC). Finally, the proposed

95

MIP-SPME fibers for pre-concentrate and selectively extract of luteolin and its

96

metabolites in rat livers were firstly developed.

97

MATERIALS AND METHODS

98

Reagents. Luteolin, quercetin, rutin and ombuin were purchased from PUSH

99

Bio-technology Co., Ltd. (Sichuan, China). Ethylene glycol dimethacrylate

100

(EGDMA), 3-Methacryloxypropyltrimethoxysilane (3-MPS), azobisisobutyronitrile

101

(AIBN), and acrylamide (AM), dopamine hydrochloride (DA) were purchased from

102

Aladdin Reagent Co., Ltd (Shanghai, China). EGDMA were used after vacuum

103

distillation. AIBN was used after recrystallization. Methanol and acetonitrile were of

104

chromatographic grade and obtained from Adamas Reagent Co., Ltd (Shanghai,

105

China). All solutions used for HPLC were filtered through a 0.22 µm filter before use.

106

Stainless steel fibers (200 µm in diameter, medical grade) were purchased from

107

Yongheng hardware Company (Shenzheng, China). Quartz capillaries (1 mm I.D.)

108

were purchased from Huaxi medical university instrument plant (Chengdu, China).

109

Commercial PDMS and DVB fibers were purchased from Supelco (Sigma-Aldrich，

110

St.Louis, MO, USA). 6


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Animals. Nine conscious male Sprague-Dawley rats (200-250 g, the Animal

112

Centre, Chongqing Medical University) were used as subjects for in vitro (three rats)

113

and in vivo (six rats) experiments. The animals were acclimatized to their new

114

environment for a minimum of 5 days prior to experiments. Before experiments, food

115

was not given for 14 h and water was available at any time. All experimental

116

procedures were approved by the Institutional Animal ethical Committee of

117

Chongqing University, and were conducted according to the Guide forthe Careand

118

Use of Laboratory Animal of National Institute of Health (Publication No. 80-23,

119 120 121

revised 1996). Instrument and Chromatographic Conditions. The details about instrument and chromatographic conditions were described under Supplemental Methods .

122

Surface Treatment of Stainless Steel Fibers. The surface treatment of stainless

123

steel fibers were based on literature 31. The details were described under Supplemental

124

Methods .

125

Preparation of MIPs Coated Stainless Steel Fibers. The MIPs fibers of

126

luteolin were synthesized with the optimal experiment conditions as follows: The

127

template luteolin (0.01 mmol) and functional monomer AM (0.06 mmol) were

128

dissolved in the selected porogen solvent (3 mL acetontrile and 1 mL methanol) in a

129

10 mL glass tube and pre-polymerized by rotating at 150 rpm for 5 h at room

130

temperature. After that, cross-linker EGDMA (0.37 mL) and initiator AIBN (10 mg)

131

were added under ultrasonic. To remove oxygen, the mixed solution was purged with

132

nitrogen gas for 10 min. Each modified fiber was put into a glass capillary (1.0 mm 7



133

I.D) and eight glass capillaries were put into the test tube containing the reaction

134

system. Then, the reaction mixture was refluxed in an oil bath at 60 oC for 24 h. The

135

prepared MIPs fibers were removed from the glass capillary and washed with

136

methanol/acetic acid (8:2, v/v) with slight oscillation until no templates were detected

137

in the eluent by HPLC. For comparison, the non-imprinted polymers were also

138

prepared using an identical procedure without the addition of luteolin.

139

Coating Characterization. The scanning electron micrography of MIPs fibers

140

were obtained with a JSM-7600F Feld emission scanning electron microscope (JEOL,

141

Japan). The FT-IR spectra of MIPs and NIPs fibers were determined by using a

142

Fourier Transform Infrared Spectrometer (Shimadzu, Japan). The samples were

143

ground with anhydrous KBr and the spectra recorded between 4000 and 500 cm-1. The

144

surface areas measurement was based on the Brunauer-Emmett-Teller (BET), and the

145

pore-size distribution was based on the Barrett-Joyner-Halenda (BJH) formula

146

(Autosorb-iQ, America).

147

Adsorption Experiments of MIPs Fibers. Analysis of Luteolin by HPLC.

148

Standard curve of luteolin analyzed by HPLC method were presented in supporting

149

information (Supplemental Methods).

150

Static Adsorption Experiment. To evaluate the adsorption capacity of MIPs fibers,

151

one of the MIPs fibers or NIPs fibers was put into 5 mL of luteolin methanol-water

152

mixture solution (55:45, v/v) with the concentration range of 5-100 µg/mL,

153

respectively. Then, the mixtures were shaken for 12 h at room temperature in an

154

oscillator. Finally, the supernatants were filtrated through 0.22 µm membrane filter 8


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155

and measured by HPLC. The adsorption capacity of Q (µg on each MIPs fiber) was

156

calculated with the following equation 32. QMIP = (Ci - Cf) ×

157 158 159

Here, Ci and Cf are initial and final concentration (µg/mL), respectively, V is the volume of adsorption solution (mL) and n is the number of MIPs fibers used.

160

Adsorption Kinetics Experiment. In order to further evaluate the adsorption

161

capacity of MIPs fibers, one of the MIPs or NIPs fibers was put into 5 mL of 100

162

µg/mL luteolin methanol-water mixture solution (55:45, v/v). Then, the mixtures were

163

shaken for 5, 10, 20, 30, 40, 60, 80 and 100 min, respectively, at room temperature in

164

an oscillator. According to the changes of luteolin concentration in the solution before

165

and after adsorption, the adsorption capacity of the polymers toward luteolin can be

166

calculated.

167

Selectivity of the MIPs Fibers to Luteolin. The selectivity of MIPs fibers to

168

luteolin was compared with four structural analogues (rutin, quercetin, luteolin and

169

ombuin). Firstly, one of the MIPs or NIPs fibers was put into 4 mL of the mixture of

170

rutin, quercetin, luteolin and ombuin (100 µg/mL each). Then, the mixtures were

171

shaken for 12 h at room temperature. The residual concentrations of four compounds

172

after the adsorption were analyzed by HPLC.

173

MIP-SPME Procedures. Preparation of Liver Homogenates. The liver

174

homogenates were prepared for the further determination of the total concentrations

175

of luteolin in homogenates, the details were described under Supplemental Methods.

176

Extraction Time Profile. Extraction time profiles of target luteolin were used to 9



177

determine the optimum extraction time for in vivo MIP-SPME sampling, the details

178

were described under Supplemental Methods .

179

Diffusion-Based Calibration: Pre-Determined Sample In Rates. The pre-

180

equilibrium SPME sampling of living rats was calibrated with the sampling-rate

181

calibration method, the details were described under Supplemental Methods.

182

MIP-SPME Sampling Method Used for the Measurement of Luteolin and Its

183

Metabolites in Livers of Rats In Vivo. Firstly, pentobarbital sodium solution (50 mg/kg)

184

was used to anesthetize rats via intraperitoneal administration. After 10-15min, the

185

rats were subconscious. At this time, luteolin dissolved in 0.5% CMC-Na was orally

186

administered to rats (n= 6) at the dosage of 200 mg/kg. Animals were partially

187

restricted during the time needed to insert the MIPs fibers into the interface for

188

sampling. Then, by using crosscutting technology, it was opened a minilaparotomy at

189

the downside of breastbone of rat. After that, a syringe needle was transverse inserted

190

into the rat liver to a depth of approximately 2.0 cm. Then, the needle was removed,

191

and the fiber was inserted into the hole for approximately 2.0 cm to pierce through the

192

liver and extracted for 10 min. A new fiber was used for each sampling time period at

193

a same rat, and six rats were used for sampling. The time period was set as 25-35 min

194

(30 min), 55-65 min (60 min), 85-95 min (90 min), 115-125 min (120 min),

195

175-185min (180 min), 235-245 min (240min), 295-305 min (300 min) and 355-365

196

min (360 min). The fiber was then rinsed with deionized water and dried. After that,

197

the fiber was desorbed using 500 µL methanol-acetic acid solution (8:2, v/v) and the

198

eluent was condensed to 50 µL. At last, 5 µL of eluent was dissolved in 100 µL 10


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199

methanol for HPLC-MS/MS analysis.

200

Comparison of prepared MIPs-SPME fiber with commercial PDMS and DVB

201

fibers. In order to evaluate the selectivity and extraction efficiency of the prepared

202

MIPs-SPME fiber, the extraction efficiency in liver of rat of commercial PDMS and

203

DVB fibers were also tested. The extraction and analysis methods of PDMS and DVB

204

fibers was the same with the methods of prepared MIPs-SPME fiber.

205

HPLC Analysis. Please refer to the Supporting Information ( Supplemental

206

Methods ) for details.

207

RESULTS AND DISCUSSION

208

Preparation of MIPs Fibers. The suitable modification of stainless steel fiber

209

surface is critical for the further synthesis of MIPs fibers. In the present study, the

210

modifications of fibers were carried out according to the previous literature

211

schematic diagram of MIPs fibers preparation was shown in Figure 1.

31, 33

. The

212

Characterization. SEM Analysis. The morphologies of bare stainless steel fiber

213

and MIPs fiber were characterized by SEM, and the results were shown in Figure 2.

214

As shown in Figure 2A, the bare stainless steel fiber had a smooth surface. After

215

coated by MIPs coatings, the surface became rough (Figure 2 B-D). As shown in

216

Figure 2B and C, uniform morphology, higher crosslink degree polymers were found

217

on the fiber. Moreover, as shown in Figure 2D, highly cross-linked and porous

218

structure was observed remarkably.

219

IR Analysis. The functional groups of MIPs coatings were subjected to

220

characterize by FT-IR spectroscopy, the results were shown in Figure S1，other 11



221

Page 12 of 38

information about IR analysis, please refer to the Supporting Information.

222

BET Analysis. The N2 adsorption isotherm was used to determine the specific

223

surface area, pore size and pore volume of MIPs and NIPs coatings. As shown in

224

Table S1, the surface area, pore size and pore volume of MIPs coatings were all larger

225

than those of NIPs coatings. These results demonstrated that more pores formed in

226

MIPs after the removal of template.

227

Binding Experiments. Static Adsorption. The static adsorption of luteolin on

228

MIPs and NIPs fibers were investigated. As shown in Figure 3A, MIPs fibers had

229

apparently higher adsorption capacity than that of NIPs fibers in the same lueolin

230

concentration condition. With the increase of luteolin concentration, the adsorption

231

capacity of MIPs were increased. Moreover, the adsorption data were fitted with

232

Langmuir and Freundlich equations (Table 1), respectively. The plot Ce/Qe versus Ce

233

was used to validate the linearized Langmuir isotherm. The Langmuir isotherm

234

equation were described as y=0.0227x+0.0446 (R2 =0.9971) and y=0.042x+0.356 ( R2

235

=0.9930) for the MIPs and NIPs fibers, respectively. Moreover, the plot logQe versus

236

logCe was used to validate the linearized Freundlich isotherm. The Freundlich

237

isotherm

238

y=0.507x+0.487(R2 =0.9641) for the MIPs and NIPs fibers, respectively. The results

239

demonstrated that the Langmuir isotherm model was more suitable for the

240

experimental results than the Freundlich isotherm model. According to the Langmuir

241

isotherm equation, Qmax of MIPs and NIPs were separately calculated as 45.45 mg/g

242

and 23.81 mg/g. Furthemore, the imprinting factor was calculated as 1.91 from the

equation

was

described

as

y=0.459x+1.055

12


(R2=0.9196)

and

Page 13 of 38


243

Qmax of MIPs and NIPs fibers according to Langmuir equation.

244

Kinetic Adsorption. The Figure 3B illustrated the kinetic adsorption curves of the

245

MIPs and NIPs fibers to luteolin. In the first 40 min, a rapid increase of the binding

246

capacity of luteolin on MIPs fibers could be detected, then the adsorption rate

247

increased slowly till the adsorption equilibrium was reached at 60 min. However, the

248

NIPs fibers could reached adsorption equilibrium within 40 min. The results indicated

249

that MIPs have more specific binding sites and capacity in comparison with NIPs,

250

resulting in more saturation time was needed for MIPs fibers 36.

251

Selectivity Experiment. To investigate the selectivity of MIPs fibers, four

252

structurally analogues (luteolin rutin, quercetin and ombuin) were selected as the

253

competitive compounds. The molecular structures were displayed in Figure S2. The

254

calibration curves, LOD, LOQ and RSD of the detection method were shown in Table

255

2. As shown in Figure 3C and 3D, the results demonstrated that MIPs fibers had

256

higher bining capacity for luteolin in comparison with rutin, quercetin and ombuin,

257

respectively. However, NIPs fibers had no special selectivity to luteolin. Therefore,

258

compared with the selective adsorption capacity to luteolin, the MIPs fibers presented

259

better selectivity for luteolin in the mixed solution.

260

Application of MIPs Fibers for Solid Phase Microextraction In Vivo.

261

Determination of Sampling Rates of MIPs Fibers in Livers of Living Rats. The

262

sampling-rate calibration method was successfully applied in quantification of

263

analytes in in vivo SPME sampling, such as in living rat blood, living fish muscle,

264

living fish brain and et al.

21-22, 37

. This calibration method assumed that the sampling 13



265

rate remains constant throughout the duration of sampling within the linear regimen of

266

the extraction time profile

267

determined according to the following equation:

38

. The sampling rate of analyte in vivo sample was

268

Rs = (1) ∙

269

Here, Rs is the sampling rate, n was the extracted amount of the analyte in a fiber. Cs

270

is the concentration in sample matrix (determined by traditional extraction method of

271

analytes in liver). t is the extraction duration under optimum conditions.

272

Firstly, extraction time profiles of luteolin were used to determine the optimum

273

extraction time for in vivo MIP-SPME sampling in the present study. As shown in

274

Figure S3A, the amounts extracted of luteolin seemed to be linearly in proportion to

275

the extraction time between 5-15 min. Considering that the extraction amount of

276

luteolin by using MIP-SPME fiber, it was limited and the metabolites of luteolin were

277

constantly changing during the extraction duration, 10 min was selected as the

278

extraction duration.

279

Then a serious of concentrations of luteolin were added into prepared liver

280

homogenates and MIPs fibers were used to extract luteolin from liver homogenates.

281

The total luteolin and extracted luteolin in each group of liver homogenates were

282

determined by HPLC. As shown in Figure S3B. The amounts of extracted luteolin

283

seemed to be linearly in range of 0.98-8.97 µg of luteolin in prepared liver

284

homogenates. The linear regression equation for n-Cs was n=0.1281Cs+0.9118 with

285

R2 equal to 0.9984. The excellent linearity of the n-Cs curves demonstrated that the

286

sampling rates were constants for a certain range of concentrations. According to the 14


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Page 15 of 38


287

equation (1), Rs was calculated as 0.0042 g/min.

288

Time Profile of In Vivo MIP-SPME Sampling of Luteolin in Rat Livers. The

289

developed in vivo MIP-SPME method was the first time to be used to monitor the

290

concentrations of luteolin in rat livers. In this study, the rats were still alive after

291

deploying the custom-made MIP-SPME fibers in their livers, and the in vivo

292

MIP-SPME process of luteolin and its metabolites were shown in Figure 4. After

293

extraction by MIP-SPME fibers at 30, 60, 90, 120, 180, 240, 300 and 360 min,

294

extracts at each time point were detected by using HPLC method. The time profiles of

295

luteolin concentrations determined in rat livers were shown in Figure 5A and the

296

chromatogram of extract in rat liver at 120 min was shown in Figure 5B. The results

297

demonstrated that luteolin and three metabolites could be detected. Moreover, the

298

luteolin concentrations in rat livers were slowly distributed and slowly decayed after

299

oral administration of luteolin in rats. The maximum concentration (Cmax) of luteolin

300

was 2.46 ± 0.29 µg/g, and this concentration was observed at about 120 min after oral

301

administration of luteolin (200 mg/kg).

302

Identification of the Luteolin Metabolites Using HPLC-MS/MS. HPLC-MS/MS

303

method was used to further identify the metabolites of luteolin. As shown in Figure

304

S4 and table S2, luteolin and its three metabolites were detected.

305

Luteolin (M0) was detected as deprotonated molecular ion [M+H]

+

at m/z 287

306

with the retention time at 5.613 min. It gave signals at m/z 258, 241 and 152. As

307

illustrated in Figure 5C, the m/z 287 lost -CO to give m/z 258. The m/z 258 was

308

further fragmented to be m/z 241 by the loss of H2O. The m/z 152 was obtained from 15



309

Page 16 of 38

m/z287 by RDA cleavage.

310

Metabolite M1 was detected as deprotonated molecular ion [M+H] + at m/z 271

311

with the retention time at 6.618 min. As shown in Figure 5D, the m/z 152 was also

312

obtained from m/z 287 by RDA cleavage. According to the previous literatures

313

the dehydroxylation of polyhydroxyl flavonoids often occurred at the place of 3' in B

314

rings (if it had hydroxy at 3’). Therefore, M1 was tentatively identified as apigenin.

39-40

,

315

Metabolites M2 and M3 were detected as deprotonated molecular ion [M+H] + at

316

m/z 301. The retention time of them was at 6.857 and 6.875 min, respectively. As

317

shown in Figure 5E and 5F, the m/z 301 lost H2O to give m/z 286. The m/z 286 was

318

further fragmented to be m/z 258 by the loss of -CO. Them/z152 was obtained from

319

m/z 301 by RDA cleavage. Methylation was a major metabolic pathway of

320

polyhydroxyl flavonoids such as rhamnetin, 7, 4´-dihydroxyflavone, chrysin, et al..

321

Their methylated derivatives were detected highly in tissues of rats 41-42. For luteolin,

322

previous studies revealed that chrysoeriol and diosmetin have been identified as two

323

major metabolites of it in tissues homogenates in vitro 8. Combined with previous

324

study and MS/MS data in our experiment, M2 and M3 were tentatively identified as

325

chrysoeriol and diosmetin.

326

Therefore, three metabolites named apigenin, chrysoeriol and diosmetin were

327

extracted and detected by using MIP-SPME combining with HPLC-MS/MS method.

328

Previous study only found methylation metabolites of luteolin by using in vitro

329

method. However, in vivo MIP-SPME sampling, not only methylation metabolites

330

named chrysoeriol and diosmetin, but also dehydroxylation metabolite named 16


Page 17 of 38


331

apigenin were found and identified. Therefore, in vivo MIP-SPME sampling method

332

had obvious advantages than in vitro method: 1) MIP-SPME sampling could extract

333

more metabolites of luteolin in vivo, for which the metabolic pathway of luteolin was

334

more reliable. 2) In vitro method had serious invasiveness to living systems, even

335

resulted in the massive death of animals, which could be avoided by using in vivo

336

MIP-SPME sampling. 3) In vivo MIP-SPME sampling had good selectivity for

337

luteolin and its metabolites, which could reduce the interference of other impurities in

338

livers. Therefore, the luteolin based MIP-SPME sampling method was effectively to

339

extract luteolin and its dehydroxylation and methylation metabolites in vivo.

340

Moreover, the MIP-SPME sampling method had good selectivity for extraction target

341

molecule from complex living systems based on special structures of MIPs materials.

342

However, the commercial PDMS and DVB fibers could only extraction luteolin

343

in liver. The chromatogram of extracts in rat liver at 120 min was shown in Figure S5.

344

The results showed that only a small amount of luteolin could be exracted by using

345

PDMS and DVB fibers，and the concentrations of luteolin were much lower than that

346

extracted by MIPs-SPME fiber. Moreover, no metabolites could be detected using

347

HPLC method. These results demonstated that the selectivity of commercial PDMS

348

and DVB fibers were much lower than prepared MIPs-SPME fiber, and MIPs-SPME

349

fiber was more suitable for extracting luteolin and its metabolites in complex system.

350

In conclusion, in the present work, based on the stainless steel fibers, luteolin

351

based MIPs coated fibers were successfully prepared for the first time. The results of

352

binding and selectivity experiments demonstrated that the prepared MIPs fibers 17



353

showed high adsorption capacity and good selectivity to luteolin. Moreover, a in vivo

354

MIP-SPME sampling method was proposed for non-lethal extraction of luteolin and

355

its metabolites. The results indicated that apigenin, chrysoeriol and diosmetin which

356

detected by HPLC-MS/MS were identified as metabolites of luteolin in livers. In

357

comparison with the in vitro method in traditional, which depended on the sacrifice of

358

animals, in vivo MIP-SPME sampling method would make it possible to carry out the

359

repeated sampling on the same experiment animal. It is also noteworthy that in vivo

360

MIP-SPME sampling method had good selectivity and pre-concentrated ability for the

361

target molecule and its metabolites from complex living systems.

362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 18


Page 18 of 38

Page 19 of 38


379

ABBREVIATIONS USED

380

MIPs = molecularly imprinted polymers

381

SPME = solid phase microextraction

382

HPLC = high-performance liquid chromatography

383

QTOF-MS/MS = quadrupole time-of-flight tandem mass spectrometry

384

AM = acrylamide

385

EGDMA = ethylene glycol dimethyl acrylate

386

AIBN = azodiisobutyronitrile

387

3-MPS = 3-methacryloxypropyltrimethoxysilane

388

DA = dopamine hydrochloride

389

CMC-Na = carboxymethylcellulose sodium

390

PDMS = polydimethylsiloxane

391

DVB = divinyl benzene

392

SEM = scanning electron microscopy

393

FT-IR = Fourier transform infrared

394

BET = Brunauer-Emmett-Teller

395 396 397 398 399 400 401 402 403 404 405 406 407 19



408

SUPPORTING INFORMATION DESCRIPTION

409

Figure S1: The FT-IR spectra of MIPs (after elution) and NIPs. Figure S2：The

410

structure of luteolin and its structural analogues. Figure S3：n-t (A) and n-Cs (B)

411

curves of in vitro MIP-SPME of rat livers homogenates. Figure S4: The total ion

412

current of extract extracted by using MIP-SPME sampling method. Figure S5: The

413

chromatogram of extract in rat livers at 120 min by using commercial PDMS fiber (A)

414

and DVB fiber (B).

415

Table S1：Specific surface area, pore structure parameters of MIPs and NIPs coatings.

416

Table S2: Luteolin and its metabolites identified in rat livers using UPLC-MS/MS.

417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 20


Page 20 of 38

Page 21 of 38


434

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572 573

Notes.

574

This work was supported by the National Natural Science Foundation of China

575

(No.21275169 and No.81202886); the Fundamental and Frontier Research Fund of

576

Chongqing under Grant cstc2014jcyjA10108; Fundamental Research Funds for the

577

Central Universities under Grant CQDXWL-2014-Z007.

578

The authors declare no competing financial interest.

579

27



FIGURE CAPTIONS Figure 1. The synthesis process of luteolin based MIPs fibers. Figure 2. The SEM images of the MIPs fiber. (A) The SEM image of bare fiber, (B) The SEM image of MIPs fiber at 80x, (C) The SEM image of MIPs fiber at 200x, (D) The SEM image of MIPs fiber at 5000x. Figure 3. Binding and selectivity experiments of MIPs and NIPs fibers. (A) The static adsorption isotherm of MIPs and NIPs fibers. (B) The rebinding dynamic curves of luteolin on MIPs and NIPs fibers. (C) Selectivity study of MIPs fibers towards luteolin. (a) The chromatograms of initial solution of luteolin and its structural analogues. (b) The chromatograms of the residual solution of luteolin and its structural analogues after adsorption with MIPs fibers. (c) The chromatograms of the residual solution of luteolin and its structural analogues after adsorption with NIPs fibers. (B) The binding amounts of rutin, quercetin, luteolin and ombuin on MIPs and NIPs fibers, respectively. Figure 4. The process of in vivo MIP-SPME sampling of luteolin and its metabolites in the livers of rats. (A) Fixed of rat after anesthesia and drug orally administration. (B) Opened a minilaparotomy at the downside of breastbone of rat. (C) A syringe needle was transverse inserted into the rat liver. (D) The fiber was inserted into the hole after the syringe needle was removed for MIP-SPME sampling. (E) The schematic diagram of the prepared MIP-SPME fiber apparatus for the analysis of luteolin and its metabolites in vivo.

28


Page 28 of 38

Page 29 of 38


Figure 5. Uptake tracing of luteolin and its metabolies in livers of rats in vivo using HPLC and HPLC-MS/MS mehod. (A) The time profiles of luteolin concentrations determined in rat livers. (B) The chromatogram of extract in rat livers at 120 min. (C) The HPLC-MS/MS spectra and proposed fragmentation pathway of luteolin (m/z 287 ). (D) The HPLC-MS/MS spectra and proposed fragmentation pathway of apigenin (m/z 271). (E) The HPLC-MS/MS spectra and proposed fragmentation pathway of chrysoeriol (m/z 301). (F) The HPLC-MS/MS spectra and proposed fragmentation pathway of diosmetin (m/z 301).

29



Figure 1

30


Page 30 of 38

Page 31 of 38


Figure 2

31



Figure 3

32


Page 32 of 38

Page 33 of 38


Figure 4

33



Figure 5

34


Page 34 of 38

Page 35 of 38


35



Page 36 of 38

Table 1 Equilibrium Parameters for the Adsorption of Luteoin onto MIPs Coated Fibers and NIPs Coated Fibers Langmuir equation

Freundlich equation

Qm a

KLb

R2

(mg/g)

(L/mg)

MIP-SPME

45.45

0.0005

0.9971

NIP-SPME

23.81

0.00012

0.9935

KF c

nd

R2

11.35

2.174

0.9192

3.074

1.975

0.9643

(mg1-1/nL1/ng-1)

Qm a was the adsorption capacity at the maximum adsorption capacity of materials (mg/g). KLb was c

d

Langmuir adsorption constants. KF was Freundlich adsorption constant and 1/ n was the adsorption index indicating adsorption strength.

36


Page 37 of 38


Table 2 Linear Regression Data and Precision of Four Compounds in Selectivity Study of MIPs Analytes

Linear regression

Precision

Calibrationcurves

Correlation coefficient

Linear range （µg/mL)

LOQ (µg/mL)

LOD (µg/mL)

Intra-day RSD(%)

Inter-day RSD(%)

Rutin

y=103.478x+181.421

0.9994

2.89-200.02

0.18

0.06

0.4

2.1

Quercetin

y=175.665x+210.9767

0.9995

4.02-193.67

0.21

0.05

0.5

1.7

Luteolin

y=270.161x-338.036

0.9993

2.01-100.91

0.05

0.01

0.3

1.1

Ombuin

y=174.359x-633.929

0.9992

4.83-100.98

0.12

0.06

0.4

3.2

y and x stand for the peak area (mAU) and the concentration (μg/mL) of the analytes, respectively.

37



TABLE OF CONTENTS GRAPHIC (TOC)

38


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In Vivo Selective Capture and Rapid Identification ... - ACS Publications

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