Determination of imidazole derivatives by micellar electrokinetic chromatography combined with solid-phase microextraction using activated carbon-polymer monolith as adsorbent

Accepted Manuscript Title: Determination of Imidazole Derivatives by Micellar Electrokinetic Chromatography Combined with Solid-Phase Microextraction using Activated Carbon-Polymer Monolith as Adsorbent Author: Yung-Han Shih Stephen Lirio Chih-Keng Li Wan-Ling Liu Hsi-Ya Huang PII: DOI: Reference:

S0021-9673(15)01264-9 http://dx.doi.org/doi:10.1016/j.chroma.2015.08.067 CHROMA 356818

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

27-5-2015 20-8-2015 31-8-2015

Please cite this article as: Y.-H. Shih, S. Lirio, C.-K. Li, W.-L. Liu, H.Y. Huang, Determination of Imidazole Derivatives by Micellar Electrokinetic Chromatography Combined with Solid-Phase Microextraction using Activated Carbon-Polymer Monolith as Adsorbent, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.08.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Determination of Imidazole Derivatives by Micellar Electrokinetic Chromatography

2

Combined with Solid-Phase Microextraction using Activated Carbon-Polymer

3

Monolith as Adsorbent

ip t

4

Yung-Han Shih‡, Stephen Lirio‡, Chih-Keng Li‡, Wan-Ling Liu, Hsi-Ya Huang*

6

Department of Chemistry, Chung Yuan Christian University, 200 Chung Pei Road,

7

Chung Li District, Taoyuan City, 320, Taiwan.

us

cr

5

8 ‡

These authors contributed equally to this work.

an

9

12

M

10

13

Dr. Hsi-Ya Huang, Department of Chemistry, Chung Yuan Christian University, 200

14

Chung Pei Road, Chung Li District, Taoyuan City, 320, Taiwan.

15

E-mail: [email protected]

16

Tel: ++886-3-2653319

17

Fax: ++886-3-2653399

18

te

d

Corresponding author:

Ac ce p

11

19

Keywords:

20

monolith, caramel colors, imidazole derivatives, micellar electrokinetic chromatography

21

(MEKC)

Solid-phase microextraction (SPME), activated carbon (AC), polymer

22

Page 1 of 33

22

Abstract

23

In this study, an effective method for the separation of imidazole derivatives 2-

24

methylimidazole

25

tetrahydroxybutylimidazole (THI)) in caramel colors using cation-selective exhaustive

26

injection and sweeping micellar electrokinetic chromatography (CSEI-sweeping-MEKC)

27

was developed. The limits of detection (LOD) and quantitation (LOQ) for the CSEI-

28

sweeping-MEKC method exhibited were in the range of 4.3 to 80 µgL-1 and 14 to 270

29

µgL-1, respectively. Meanwhile, a rapid fabrication activated carbon-polymer (AC-

30

polymer) monolithic column as adsorbent in solid-phase microextraction (SPME) of

31

imidazole colors was developed. Under the optimized SPME condition, the extraction

32

recoveries for intra-day, inter-day and column-to-column were in the range of 84.5 to

33

95.1% (< 6.3% RSDs), 85.6 to 96.1% (< 4.9% RSDs), and 81.3 to 96.1% (< 7.1% RSDs),

34

respectively. The LODs and LOQs of AC-polymer monolithic column combined with

35

CSEI-sweeping-MEKC method were in the range of 33.4 to 60.4 µg L-1 and 111.7 to

36

201.2 µg L-1, respectively. The use of AC-polymer as SPME adsorbent demonstrated the

37

reduction of matrix effect in food samples such as soft drink and alcoholic beverage

38

thereby benefiting successful determination of trace-level caramel colors residues with

39

CSEI-sweeping-MEKC method. The developed AC-polymer monolithic column can be

40

reused for more than 30 times without any significant loss in the extraction recovery for

41

imidazole derivatives.

4-

methylimidazole

(4-MEI)

and

2-acetyl-4-

Ac ce p

te

d

M

an

us

cr

ip t

(2-MEI),

42

Page 2 of 33

42

1. Introduction Over the past decades, carbonaceous materials such as activated carbon (AC) has

44

been considered as one of the first materials applied as sorbent in the removal of

45

contaminants in biological, environmental, and food samples due to its high surface area

46

(500 to 1000 m2g-1), nanoporosity, and low cost [1-7]. AC comprises of six-membered

47

rings with sp2-hybridized carbon together with five- and seven-membered non-aromatic

48

carbon rings with varying sizes of micropores that provide sites for adsorption [8,9].

49

Despite the success of AC as adsorbent, several problems associated to it has been

50

encountered such as high cost of reusability, stability in nature, and inability to adsorb

51

inorganic compounds at trace or ultra-trace levels [10]. To improve the stability and

52

adsorbing capacity of AC, modification on its functional surface have been proposed

53

such as oxidative and non-oxidative approach [1]; however, functionalization on its

54

surface requires additional steps and also time consuming. In previous studies, deposition

55

of polypyrrole (Ppy) into the pores and surface of AC with via in situ chemical oxidative

56

polymerization in the presence of FeCl3 [11] or electrochemical polymerization [12] have

57

been used as alternative method for surface functionalization of AC. The incorporation of

58

PPy into the AC (PPy-AC) has facilitated in the removal of sulfates in aqueous solution

59

[11,12].

cr

us

an

M

d

te

Ac ce p

60

ip t

43

Solid-phase microextraction (SPME), first introduced by Arthur and Pawlizyn

61

[13], is a miniaturized form of solid-phase extraction (SPE) that offers several advantages

62

such as short extraction time, less consumption of organic solvents, ease of operation, and

63

can be automated to reduce the cost and time consuming sample preparation [14]. Among

64

the proposed SPME techniques, porous monolithic materials for in-tube SPME (IT-

Page 3 of 33

SPME) has attracted considerable attention due to its chemical and mechanical stability,

66

high surface area and rapid mass transfer under dynamic condition [14]. Typically, IT-

67

SPME utilizes organic polymer [9,15], silica [16] and organic-silica hybrid monoliths [17]

68

as adsorbent. However, drawbacks on their lack of interaction sites may lead to low

69

adsorption efficiency [18]. Recently, several reports showed that by embedding

70

nanoparticles [19], such as metal organic framework (MOF) [20-22] or graphene [23],

71

into the monolithic polymer may increase the interaction sites and surface area. In

72

particular, a rapid and novel synthetic method of incorporating MOF nanoparticles into

73

polymer-based monolith was introduced using ionic liquid (IL) as reaction medium via

74

microwave-assisted polymerization. These MOF-polymer hybrid monoliths were

75

successfully applied as stationary phases in capillary electrochromatography (CEC)

76

system [24-26] and SPME [20].

d

M

an

us

cr

ip t

65

Food colorants like caramel colors are normally associated with food taste and to

78

improve the visual perception of a product. Caramel colors are commonly present in food

79

and beverages under Millard reaction system where carbohydrates like glucose, sucrose

80

and inverted sugars are reacted in the presence of ammonium salts under controlled heat

81

treatment [27]. Previous reports have suggested that during caramelization, undesired

82

compounds such as 2-methylimidazole (2-MEI), 4-methylimidazole (4-MEI) and 2-

83

acetyl-4-tetrahydroxybutyl imidazole (THI) are formed. These side products are deemed

84

as carcinogenic compounds, which can inhibit enzyme (cytochrome P450) [28] at high

85

levels of 2-MEI and 4- MEI; and proven to cause acute hyperexcitation in animals basis

86

[29]. Meanwhile, THI was reported as an immunosuppressive compound [30,31].

87

Consequently, the European commission and 2 Taiwan set a monitoring standard to 250

Ac ce p

te

77

Page 4 of 33

mg kg-1 for 2-MEI and 4-MEI while 25 mg kg-1 for THI to protect humans from these

89

hazardous chemicals [32]. Several analytical methods such as gas chromatography (GC)

90

and high performance liquid chromatography (HPLC) coupled to mass spectrometry (MS)

91

detection have been used successfully in the determination of caramel colors in food

92

samples with good limit of detections (LODs) ranging from 0.10 to 5.0 µgL-1 or µgkg-1

93

[33-41]. Similarly, capillary electrophoresis (CE) has been reported as alternative tool in

94

the separation of imidazole derivatives due to its good separation efficiency, low solvent

95

consumption and low cost. However, the CE methods, capillary isotachophoresis [42]

96

and capillary zone electrophoresis [43,44], used to analyze the 2-MEI and 4-MEI have

97

LODs as low as 0.16 mg L-1, which was poorer than the LC-MS or GC-MS method. To

98

improve the sensitivity of CE, on-line pre-concentration methods such as cation-selective

99

exhaustive injection (CSEI) sweeping (CSEI-sweeping) micellar electrokinetic

100

chromatography (MEKC) [45-47] and anion-selective exhaustive injection (ASEI-

101

sweeping) MEKC [48,49] have been developed to capable of further increasing its

102

sensitivity up to a thousand- to million-folds [45].

cr

us

an

M

d

te

Ac ce p

103

ip t

88

Herein, we propose a new method of separating the imidazole derivatives using

104

CSEI-sweeping-MEKC technique to further improve its detection limit. Despite the early

105

studies about the utilization of AC as adsorbent for the removal of contaminants in

106

environmental, biological and food samples, however, no attempt has been reported using

107

AC as absorbent for in-tube SPME without surface modification. In this work, AC was

108

incorporated into the poly(butyl methacrylate-co-ethylene dimethacrylate) (poly(BMA-

109

EDMA) monolithic column (referred to AC-polymer) via room temperature ionic liquids

110

(RTIL) as reaction media coupled with microwave-assisted heating and then was used as

Page 5 of 33

SPME adsorbent to extract caramel colors in food samples. Using the optimized

112

condition, the AC-polymer monolithic column was applied in SPME of caramel colors in

113

soft drink and alcoholic beverage and extracted imidazole residues were analyzed using

114

the proposed CSEI-MEKC method.

ip t

111

Ac ce p

te

d

M

an

us

cr

115

Page 6 of 33

115

2. Experimental

116

2.1 Chemicals and reagents All chemicals and reagents were at least of analytical grade. 2-Methylimidazole

118

(2-MEI), 4-methylimidazole (4-MEI), 2-acetyl-4-tetrahydroxybutylimidazole (THI) and

119

activated carbon (AC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-

120

Acrylamido-2-methylpropane sulfonic acid (AMPS), butyl methacrylate (BMA)

121

(Lancaster, Ward Hill, MA, USA), ethylene dimethacrylate (EDMA) (ACROS, New

122

Jersey, USA) and azobisisobutyronitrile (AIBN) (Showa, Tokyo, Japan) were used for

123

the polymerization while 3-trimethoxysilylpropyl methacrylate (MSMA, 98%) were

124

purchased from ACROS (Geel, Belgium) for pretreatment of column. Methanol (MeOH),

125

phosphoric acid (H3PO4) and sodium dodecyl sulfate (SDS) were purchased from Merk

126

(Darmstadt, Germany). The acetonitrile (ACN) and sodium hydroxide (NaOH) were

127

purchased from J.T Baker (Phillipsburg, NJ, USA). The ionic liquid, 1-hexyl-3-

128

methylimidazolium tetrafluoroborate ([C6 mim][BF4]), was synthesized in our laboratory

129

and the identity was confirmed by 1H NMR spectroscopy. The uncoated fused-silica

130

capillary with 0.8 mm I.D. and 1.1 mm O.D. was purchased from Kimble Chase

131

(Mexico). The 2- and 4-MEI were dissolved in methanol (MeOH) while THI was in

132

dimethyl sulfoxide (DMSO) at a stock concentration of 1000 µgL-1.

134

cr

us

an

M

d

te

Ac ce p

133

ip t

117

2.2 Instrumentation

135

An LC pump (Model 260D, ISCO, Lincoln, NE, USA) was used for washing the

136

monolithic columns. A scanning electron microscope model JSM-7600F (JEOL, Japan)

137

was used for morphology observation. A FTIR spectroscopy model vertex 70v (Bruker,

Page 7 of 33

Billerica, MA, USA) was used for infrared spectrum measurement. A nitrogen

139

adsorption/desorption equipment model Micromeretics Tri-star 3000 (Norcross, GA,

140

USA) was employed for surface area and pore size measurement. Raman spectrometer

141

model iHR 320 (Horiba Jobin Yvon, France) was used to probe the polymer, AC and

142

AC-polymer.

ip t

138

2.3 Apparatus and operating conditions for CE

us

144

cr

143

All experiments were performed using Beckman Coulter MDQ capillary

146

electrophoresis system equipped with a photo diode array detector (Fullerton, CA, USA).

147

Beckman Coulter MDQ 32 Karat software was used for instrumental control and data

148

analysis. Separations were performed in uncoated fused-silica capillaries of 50.2 cm total

149

length (40 cm to detector) and 50 µm I.D. (Polymicro Technologies, Phoenix, AZ, USA).

150

The carrier electrolyte composed of pH 2 phosphoric acid (20 mM) and 140 mM SDS

151

was used as the running buffer for (normal mode) MEKC and CSEI-sweeping MEKC

152

separation.

154 155

M

d

te

Ac ce p

153

an

145

2.3.1 Normal mode MEKC

The capillaries were conditioned prior to separation by washing with 0.1 M

156

sodium hydroxide (NaOH) solution (10 min, 20 psi), deionized water (10 min, 20 psi)

157

and carrier electrolyte (i.e. running buffer) (5 min, 20 psi). Samples or standard solutions

158

were pressure-injected into the capillary for conventional MEKC (0.5 psi, 3 s).

159

Subsequently, a -20kV voltage was applied for MEKC separation. The temperature of the

Page 8 of 33

160

capillary was maintained at 30 °C and the detection wavelengths were set at 214 nm and

161

280 nm for 2-(4)-MEI and THI, respectively.

162 2.3.2 On-line concentration mode MEKC

ip t

163

The on-line concentration procedure of CSEI-sweeping MEKC used in this study

165

(Scheme S1) was described briefly as below. The capillaries were conditioned prior to

166

separation by washing with 0.1 M NaOH solution (10 min, 20 psi), deionized water (10

167

min, 20 psi) and carrier electrolyte (5 min, 20 psi). A high conductivity zone was

168

pressured-injected into the capillary (acid plug: pH 2, 50 mM H3PO4, 0.5 psi, 420 s)

169

followed by water plug (D.I. water, 0.5 psi, 20s). The sample or standard solutions were

170

mixed with 0.05 mM H3PO4 to convert the imidazole derivatives into cationic form and

171

were then electrokinetically injected into the capillary (10 kV, 120 s). After the sample

172

introduction, the separations were carried out using an electrical voltage of -20 kV. The

173

temperature of the capillary was maintained at 30 °C and the detection wavelengths were

174

set at 214 nm and 280 nm for 2-(4)-MEI and THI, respectively.

176 177

us

an

M

d

te

Ac ce p

175

cr

164

2.4 Synthesis of AC-polymer monolithic column Prior to the preparation of the AC-polymer monolithic column, the inner walls of

178

a 0.8 mm I.D. capillary tube (Kimble Chase, Mexico) were pre-treated according to the

179

procedure described in our previous paper with some modification [20,24-26]. Pre-

180

treatment used NaOH (0.1M, 0.5 mL min-1 for 20 min), H2O (2 mL min-1 for 20 min),

181

methanol (MeOH) (2 mL min-1 for 5 min) and dried in oven for 20 min at 70°C. The

182

column was filled with a mixture of MSMA/MeOH (1:1) then sealed with silicon and

Page 9 of 33

incubated for 17 h at 35°C. Afterwards, the column was washed with MeOH (2 mL min-1

184

for 13 min), H2O (2 mL min-1 for 13 min) and dried with a stream of nitrogen gas. For the

185

preparation of AC-polymer monolith, AC (2 to 8 mg) was suspended in a mixture

186

containing the BMA and EDMA monomers (BMA: 3.6 µL, EDMA: 5.4 µL), porogenic

187

solvent (ionic liquid, [C6mim][BF4], 38 µL), water (3 µL), AMPS (0.5 mg) and AIBN

188

(0.5 mg), while the same composition but no AC addition was used for the neat polymer.

189

The pre-AC-polymer solution was mixed using vortex, sonication, and degas until

190

homogenous and then the pretreated column was filled with this pre-AC-polymer

191

solution. The column containing the pre-AC-polymer mixture was sealed with silicon and

192

submerged in water (350 mL), followed by in situ polymerization via microwave-assisted

193

heating (microwave oven: 900 W for 5 min). An LC pump was used to remove the

194

unreacted starting materials in the capillaries using MeOH.

d

M

an

us

cr

ip t

183

te

195

2.5 SPME procedure and analysis

197

2.5.1 Optimization procedure

198

Ac ce p

196

Prior to the SPME of imidazole derivatives, the monolithic column was pre-

199

conditioned by using 0.5 mL H3PO4 (5 mM, pH2) and 0.5 mL ACN using a controlled

200

syringe pump. Afterwards, 1 mL of 10 mg L-1 caramel standard solution in ACN (sample

201

matrix) was loaded to the SPME column, then washed with 0.5 mL ACN and eluted

202

using 1.5 mL H3PO4 (5 mM, pH2). The eluted sample was dried using oven at 70 °C and

203

re-dissolved in 200 µL carrier electrolyte (see section 2.3) for CE analysis.

204 205

2.5.2 Pre-treatment of real sample

Page 10 of 33

206

For direct injection analysis, the soft drink or alcoholic beverage was diluted with

207

0.05 mM H3PO4 (1:99). The sample solution was electrokinetically injected and analyzed

208

according to the proposed on-line CSEI-sweeping-MEKC method in section 2.3.2. For SPME of soft drink or alcoholic beverage, the samples were diluted with

210

ACN (1:99). The diluted samples were loaded into the pre-conditioned AC-polymer. The

211

same procedure was conducted according to the procedure described in section 2.5.1.

cr

ip t

209

us

212

2.6 Evaluation of the proposed MEKC method and AC-polymer monolithic column for

214

SPME

an

213

Linearity of the method was determined based on the calibration curves obtained

216

by using the optimized condition for a) on-line concentration mode MEKC and b) AC-

217

polymer monolithic column in SPME of imidazole derivatives. For on-line mode MEKC,

218

a fixed amount of imidazole derivatives ranging from 10 to 2500 µg L-1 (10 to 250 µg L-1

219

for MEIs and 250 to 2500 µg L-1 for THI, respectively) were analyzed according to the

220

procedure in Section 2.3.2. Meanwhile, using the optimized condition for AC-polymer

221

monolithic column, a fixed amount of imidazole derivative ranging from 50 to 2500 µg L-

222

1

223

the fabricated SPME column (see Section 2.5.1). The eluted standards (1.5 mL) were

224

dried using oven at 70 °C and re-dissolved in 1.0 mL of 0.05 mM H3PO4 (sample matrix).

225

The sample solution was electrokinetically injected and analyzed according to the

226

proposed on-line CSEI-sweeping-MEKC method in section 2.3.2. The limit of detections

227

(LODs) and quantifications (LOQs) were determined based on the signal-to-noise ratio

228

(S/N) of 3 and 10, respectively.

Ac ce p

te

d

M

215

(50 to 500 µg L-1 for MEIs and 250 to 2500 µg L-1 for THI, respectively) were loaded to

229

Page 11 of 33

229

3. Results and discussion Prior to the extraction of caramel colors, a MEKC method of separating imidazole

231

derivatives was established. Considering their hydrophilic structure and positively

232

charged nature at lower pH (pKa ~ 7.68-8.15 for 2-MEI and 4-MEI) and (pKa ~1.71 and

233

10.63 for THI), the suitable buffer for the electrolyte system in MEKC would be between

234

pH 2 to 7. Within these pH ranges, the imidazole derivatives are protonated to enhance

235

their interaction with the negatively charged SDS micelles at the same time benefits the

236

sweeping ability of SDS micelle to the imidazole analytes for on-line CSEI-sweeping

237

step.

an

us

cr

ip t

230

238

3.1. Separation condition of caramel colors using normal mode MEKC

M

239

In order to make all analytes in cationic form, an acidic carrier electrolyte (H3PO4,

241

20 mM)) was used as running buffer for the separation of imidazole derivatives. To

242

optimize the MEKC separation, the effect of surfactant concentration for the analyte

243

separation was first investigated by varying the SDS concentrations at 120, 140 and 160

244

mM, respectively. As shown in Figure 1a, good baseline separations were achieved in all

245

the concentrations of SDS, and increasing the SDS concentration has no significant

246

impact in either the retention time or resolution in the separation of the analytes. Since

247

the imidazole derivatives carry positive charges in the presence of pH 2 phosphate buffer

248

and -20 kV electric voltage was applied for the separation, a strong electrostatic

249

interaction of the cationic analytes with the anionic surfactant micelle (SDS) had

250

occurred, thus, all analytes migrated toward the detection end (the MEKC separation was

251

performed in the reverse-polarity mode). Considering the migration order of the analytes

252

in Fig. 1a, where 4-MEI first migrated followed by 2-MEI and then THI, the interaction

Ac ce p

te

d

240

Page 12 of 33

of SDS micelles with 4-MEI should be the strongest while the THI is the weakest. Since

254

4-MEI is more hydrophobic and THI is more hydrophilic than the rest of the analytes

255

(octanol-water partition coefficient (LogP) ~0.314, 0.031 and -1.87 for 4-MEI, 2-MEI

256

and THI, respectively), the migration orders of imidazole derivatives were determined

257

based on their hydrophobic characteristics. Further increase in the SDS concentration

258

resulted to high joule heating (i.e. high current obtained in 160 mM SDS) without any

259

improvement in the resolution for the imidazole derivatives. Therefore, 140 mM SDS

260

concentration was utilized as the optimal condition in the subsequent MEKC separation,

261

in which the LODs for imidazole derivatives was in the range of 4.1 to 24.2 mg L-1.

an

us

cr

ip t

253

262

3.2. On-line concentration CSEI-sweeping MEKC

M

263

On-line concentration mode by CSEI-sweeping-MEKC was conducted to further

265

enhance the detection sensitivity of the analytes. The analyte standards were mixed with

266

the acidic matrix (pH 2 H3PO4, 0.05 mM) to make it cationic and electrokinetically

267

injected at +10 kV into the capillary followed by the sweeping step shown in scheme S1.

268

The results suggest that the imidazole derivatives at the concentration of 50 or 250 µg L-1

269

exhibited a high signal intensity and without any loss in the separation resolution when

270

acidic matrix (pH2, 0.05 mM) and long injection time (+10 kV for 120 s) were employed

271

(Fig. 1b). The LODs and limit of quantifications (LOQs) of the analytes were in the range

272

of 4.3 to 80 µg L-1 and 14 to 270 µg L-1, respectively (Table 1). Compared with the

273

results obtained between the normal and on-line concentration modes, it showed that the

274

detection sensitivity of three caramel colors was enhanced about several hundred times

275

(830-, 950- and 300-fold for 4-MEI, 2-MEI and THI, respectively). So far, this is the first

Ac ce p

te

d

264

Page 13 of 33

report about the separation of imidazole derivatives using CSEI-sweeping-MEKC

277

method. In contrast with other reports concerning about the separation of caramel colors

278

(Table S1), our proposed method is able to separate the isomeric form of 2-MEI, 4-MEI

279

and THI and provided good quantitative capacities. The proposed method was also

280

comparable to the previous LC or GC reports using mass spectrometer as detector, where

281

majority of the quantitative abilities (LODs and LOQs) are in the concentration range of

282

µgL-1 for the imidazole derivatives. Therefore, the proposed CSEI-sweeping-MEKC

283

method coupled to UV-visible detector can be used as alternative method for the routine

284

analysis of 2-MEI, 4-MEI and THI residues in food sample.

an

us

cr

ip t

276

286

3.3 Characterization of AC-polymer

M

285

The fabrication of AC-polymer was conducted via RTIL, as porogenic solvent,

288

coupled with microwave-assisted heating. Different parameters were examined to

289

characterize the successful fabrication of AC-polymer. Figure 2a shows the FT-IR spectra

290

of AC-polymer, neat polymer and AC. For AC, the absorption peaks of carbonyl (C=O)

291

and aromatic C=C bending were observed at 1760 and ~1600 cm-1, respectively. For neat

292

polymer, carbonyl (C=O) and alkyl (C-H) stretch for BMA and EDMA were observed at

293

~1700 cm-1 and ~2800-2900 cm-1, respectively. After the incorporation of AC into the

294

polymer system, an infrared absorption peak of aromatic C=C bending at ~1600 cm-1

295

from pristine AC was observed, even if a slight intensity decrease (possibly due to the

296

deposited poly(BMA-EDMA-AMPS) on AC particles). In addition, the C-H stretch from

297

the BMA and EDMA monomers were also preserved in the AC-polymer system. Further

298

characterization was conducted using Raman spectroscopy to probe the existence of AC

Ac ce p

te

d

287

Page 14 of 33

299

into the polymer system. As depicted in Figure 2b, the D and G bands were observed for

300

AC-polymer at 1590 and 1340 cm-1, respectively, which is similar to pristine AC. The

301

results obtained using FT-IR and Raman spectroscopy suggest the successful embedding

302

of

303

polymerization. The BET surface area of the AC-polymer was determined at 15 m2g-1,

304

which was much lower than the pure AC (1061 m2g-1) but almost doubled than the neat

305

polymer (7 m2g-1) (Figure S1). In contrast to the pristine AC, a decrease in the pore

306

volume distribution in AC-polymer was observed (Figure 2c). This observation was also

307

in agreement with the previous reports about the fabrication of PPy-AC polymer using

308

electrochemical or chemical method [11,12]. The decrease in surface area and the pore

309

volume occurred in AC-polymer indicates the possible penetration of partial polymer

310

chain (i.e. poly(BMA-EDMA-AMPS) into the large pores of AC. Scanning electron

311

microscopy (SEM) images also revealed that some of the poly(BMA-EDMA-AMPS) are

312

accumulated onto the surface of AC while a textured structure was observed in pristine

313

AC (Fig. 2d, Fig. S2). Finally, SEM-energy dispersive spectroscopy (SEM-EDS) of AC-

314

polymer showed an increase of carbon amount (81.5%) with corresponding decrease of

315

oxygen (8.5%), while the percentage of carbon and oxygen elements in polymer was

316

about 68.6% and 11.8%, respectively (Fig. S2). Owing to AC’s carbon and polymer’s

317

oxygen and carbon, the above results (i.e. increasing the carbon to oxygen ratio) showed

318

that some AC has been incorporated into the polymer monolith.

319

3.4 Optimization of extraction conditions

the

poly(BMA-EDMA-AMPS)

monolith

via

microwave-assisted

ip t

into

Ac ce p

te

d

M

an

us

cr

AC

320

Next, to demonstrate the feasibility of using AC-polymer as adsorbent for SPME,

321

several parameters affecting the extraction recoveries for the imidazole derivatives such

Page 15 of 33

as amount of charged monomer (AMPS), column length, amount of AC in polymer

323

system, loading solvent and volume of desorption solvent were investigated. Comparison

324

between the neat polymer and AC-polymer was also conducted based on their respective

325

extraction recoveries.

ip t

322

The composition of charged monomer was varied from 0 to 2 mg (i.e. 0 to 4

327

wt.% of pre-polymer solution) of AMPS to determine the effect on the adsorption

328

efficiency of poly(BMA-EDMA-AMPS) (Figure S3). As shown in Figure S3, without the

329

presence of AMPS into the polymer, only 0.3 to 6.9% adsorption efficiency, which is the

330

amount of the adsorbed analyte into the AC-polymer, with less than 7.2 relative standard

331

deviations (RSDs) was observed. Meanwhile, an increase in the adsorption efficiency for

332

imidazole derivatives when the 0.5 mg AMPS (17.8 to 36.1%) was added into the

333

polymer but leveled off at 2 mg (18.2 to 35.1%). The addition of charged monomer

334

(AMPS) improves the hydrophilicity and the degree of negatively charged adsorbent,

335

which contributes to the formation of hydrogen bonding or electrostatic interaction

336

between anionic polymer and cationic analytes. Further increase in the amount of AMPS

337

had no significant effect in the adsorption of the analytes, suggesting that maximum

338

interaction between the poly(BMA-EDMA-AMPS) and analytes has been reached at 0.5

339

mg AMPS.

us

an

M

d

te

Ac ce p

340

cr

326

The extraction recovery of imidazole derivatives using neat polymer (i.e.

341

poly(BMA-EDMA-AMPS)) and AC-polymer were compared at fixed amount of charged

342

monomer (0.5 mg AMPS). The addition of 2 mg AC (20%, wt.% of monomer solution)

343

enhances the extraction recoveries for 2-MEI and 4-MEI for almost two-fold than the

344

neat polymer (from 28.9 to 58.3% and 36.1 to 66.9% for 4- and 2-MEI, respectively)

Page 16 of 33

(Figure S4). The result suggests that the presence of AC improves the extraction recovery

346

for imidazole derivatives, which possibly due to the π-π interaction between the aromatic

347

rings of AC and imidazole derivatives. Also, the existence of -COOH and –OH group in

348

AC, confirmed by FT-IR spectra (Fig. 2a), provided another hydrogen bonding with the –

349

NH and –OH group of imidazole derivatives.

ip t

345

Increasing the column length of AC-polymer would also increase the extraction

351

recovery for imidazole derivatives due to the high amount of adsorbent in the SPME

352

column. The effect of column length of AC-polymer on the extraction efficiencies for the

353

analytes by varying from 3 to 5 cm was investigated. The extraction recoveries of

354

imidazole derivatives were in the range of 18.6 to 66.9% (< 6.7% RSDs) for 3-cm

355

column while 20.3% to 81.93% (< 11.0% RSDs) for 5-cm column (Figure S5). The result

356

showed that the recoveries for all of the analytes were improved when the length of the

357

monolithic column was further increased to 5 cm.

te

d

M

an

us

cr

350

The effect on the extraction efficiency for imidazole derivatives by varying the

359

amount of AC into the polymer system between 2 to 10 mg was conducted. An increase

360

in the amount of AC led to increase in the extraction recoveries for imidazole derivatives

361

(Fig. 3). A recovery range of 51.4 to 83.5% (< 5.8% RSDs) and 61.8 to 92.9% (< 4.8

362

RSDs) were observed when 4 mg (33 wt.%) and 6 mg (43 wt.%) of AC were utilized,

363

respectively. Meanwhile, the extraction recovery for imidazole was further enhanced for

364

8 mg (50%, wt.%) AC with recovery range of 85.6% to 96.1% (< 4.9% RSDs). Attempt

365

in determining the extraction recovery of the analytes using 10 mg of AC was not carried

366

out because of the increase in the backpressure of the monolithic column. This was

367

corroborated based on the obtained backpressures (in psi) of different AC-polymer

Ac ce p

358

Page 17 of 33

monolithic columns using a controlled pump. The backpressure of AC-polymer was

369

found to be stable up to 6 mg AC with < 5 psi while slight increase of 5-7 psi for 8 mg

370

AC. Similarly, more than 20 psi of backpressure was observed when 10 mg AC was

371

utilized into the polymer system. As a result, 8 mg AC was utilized with reasonable

372

permeability for sample loading or solvent elution and good extraction recoveries for the

373

three analytes.

cr

ip t

368

Different solvents for the analytes loading such as methanol (MeOH), acetonitrile

375

(ACN) and 5 mM phosphoric acid (H3PO4, pH 2) (Fig. S6) were investigated. A low

376

adsorption efficiency ranging from 28.1 to 35.9 % (< 7.2% RSDs) was observed when 5

377

mM H3PO4 (pH 2) was used to mix with the analytes while 32.1 to 35.9% (< 8.0% RSDs)

378

for MeOH. A remarkable increase in the adsorption efficiency ranging from 90.0 to

379

94.4% (< 4.9% RSDs) for imidazole derivatives was observed when ACN was utilized as

380

loading solvent. The adsorption ability for each solvent used in mixing with the analytes

381

could be due to their different selective type of polarity. The imidazole derivatives are

382

regarded as hydrophilic compounds with LogP values ranging from 0.031 to -1.87,

383

whereas the LogP for ACN, MeOH and H3PO4 are -0.33, -0.77 and -2.15, respectively.

384

The higher partition coefficient values of the loading solvent (i.e. ACN), the more

385

favorable in forcing the analytes to be adsorbed into the AC-polymer, which is due to the

386

hydrophilic nature of the stationary phase. Meanwhile, a decrease in the adsorption

387

efficiency for MeOH or H3PO4 was observed that could be attributed to their hydrophilic

388

nature than the AC-polymer. As a result, ACN was used as loading solvent to mix with

389

the three analytes in the subsequent study.

Ac ce p

te

d

M

an

us

374

Page 18 of 33

The amount of desorption solvent (5mM pH 2 H3PO4) was also investigated by

391

increasing its volume from 0.5 to 1.5 mL to determine the best extraction recovery for

392

imidazole derivatives. An increase in the extraction recoveries for analytes ranging from

393

85.6 to 96.1% (< 4.9% RSDs) was observed when desorption solvent was further

394

increased to 1.5 mL. This result suggests that an inadequate elution was obtained when

395

low volume of solvent was utilized. Based on the obtained results, the optimized

396

condition of AC-polymer in SPME of imidazole derivatives are as follows: 0.5 mg

397

AMPS, 5-cm column length, 8 mg AC, ACN as loading solvent and 1.5 mL desorption

398

volume using 5 mM pH 2 H3PO4.

an

us

cr

ip t

390

399 3.5 Analytical figure of merit

M

400

The AC-polymer monolithic column was utilized in SPME of imidazole

402

derivatives (10 mg L-1). Using the optimized experimental condition of AC-polymer, the

403

linear ranges for the imidazole derivatives were in the range of 50 to 2500 µg L-1 with

404

correlation coefficient (R2) between 0.997 and 0.998. The LODs (S/N = 3) and LOQs

405

(S/N = 10) of the AC-polymer monolithic column for imidazole derivatives combined

406

with CSEI-sweeping-MEKC method were in the range of 33.4 to 60.4 and 111.7 to 201.2

407

µg L-1, respectively (Table 1). The obtained extraction recoveries for analytes for intra-

408

and inter-day (n=3) were in the range of 84.5 to 95.1% (< 6.3% RSDs) and 85.6 to 96.1%

409

(< 4.9% RSDs), respectively (Table 1). The column-to-column reproducibility has a

410

recovery range of 81.3 to 96.1% (< 7.1% RSDs) (Table 1). Based on the above results, it

411

shows that AC-polymer monolithic column has good repeatability and reproducibility.

412

Based on the long time stability of the AC-polymer monolithic column can be used for at

Ac ce p

te

d

401

Page 19 of 33

413

least 30 times presenting a good extraction recoveries ranging from 80% to 90% for

414

imidazole derivatives without any significant change in the morphology (Figure S7 and

415

S8).

417

ip t

416 3.6. Application to real sample

The fabricated AC-polymer monolithic column was applied in SPME of caramel

419

colors in soft drink and alcoholic beverages (Fig. 4-5 and Table 2, respectively). Direct

420

injection analysis, without AC-polymer pre-treatment, was also conducted using the

421

proposed CSEI-sweeping-MEKC and compared with sample pre-treated using the

422

fabricated AC-polymer. For direct injection analysis of soft drink, the result showed the

423

presence of 2-MEI and 4-MEI (214 nm) while no trace of THI was observed (280 nm)

424

(Fig. 4a). To further demonstrate the advantage of using SPME, the fabricated AC-

425

polymer was used to extract the target analytes in soft drinks and revealed the presence of

426

three imidazole derivatives (Fig. 4b). Based on the above results, the AC-polymer, as

427

sorbent in SPME, could also be useful as sample clean-up to reduce the matrix effect of

428

the real sample. Similarly, alcoholic beverage was also investigated and Figure 5a shows

429

that serious matrix interference was obtained for direct injection analysis. For example,

430

overlapping peaks (i.e. interferences) were observed in determining the presence of 2-

431

MEI and 4-MEI while false peak (*) was seen in THI. Meanwhile, when the alcoholic

432

beverage was subjected in SPME (Fig. 5b), the matrix effect was reduced significantly

433

and showed only the presence of 4-MEI. It also showed that no trace of THI was present

434

in the alcoholic beverage in contrast to the false positive peak (i.e. THI) observed in

435

direct injection analysis. To summarize, the presence of 2-MEI, 4-MEI and THI were

Ac ce p

te

d

M

an

us

cr

418

Page 20 of 33

found 2.6, 3.3 and 1.8 mg L-1, respectively in soft drink; whereas, only 4-MEI with 3.2

437

mg L-1 in alcoholic beverage (Table 2). Furthermore, the fabricated AC-polymer

438

monolithic column was applied for the SPME of caramel colors in soft drink and

439

alcoholic beverage by spiking with 1 mg L-1 of MEIs and THI. The extraction recoveries

440

for soft drink and alcoholic beverage were in the range of 90.5 to 100.3% (< 6.7% RSDs)

441

and 93.6 to 96.6% (< 7.4% RSDs) respectively.

cr

ip t

436

The performance of AC-polymer was compared with other literatures using

443

different stationary phases in SPE of imidazole derivatives. As shown in Table S2, the

444

AC-polymer utilizes small amount of organic solvents (i.e. 2 mL) compared to other

445

stationary phases (i.e. 9 to 27 mL). It also exhibited higher extraction recoveries for the

446

imidazole derivatives ranging from 84.5 to 95.1% than the alkaline diatomite adsorbent.

447

Despite of the obtained high recoveries from the commercially available stationary

448

phases, however, only 2-MEI, 4-MEI, THI or combination of two has been reported.

449

Comparing the other adsorbents for SPME of caramel colors in real sample, the prepared

450

AC-polymer monolithic column afforded higher reusability with reasonable extraction

451

time (~ 40 min), which reduces the laborious work in SPE.

453 454

an

M

d

te

Ac ce p

452

us

442

4. Conclusion

For the first time, we have demonstrated a rapid chromatographic method in the

455

separation of imidazole derivatives (2-MEI, 4-MEI and THI) using CSEI-sweeping-

456

MEKC with high sensitivity and satisfactory detection limits. Similarly, a simple strategy

457

of incorporating activated carbon into the polymer system without any surface

458

modification was developed. The AC-polymer was successfully applied as adsorbent in

Page 21 of 33

SPME of caramel colors in food samples with remarkable extraction recovery,

460

reusability, low detection limits and good mechanical stability. The presence of AC in the

461

polymer monolith exhibited π-π and hydrogen bond interactions between the aromatic

462

rings of the AC and imidazole derivatives. Collating all of the above results, the

463

fabricated AC-polymer and the proposed CSEI-sweeping-MEKC method could be used

464

as adsorbent in SPME and separation of imidazole derivatives in food sample,

465

respectively.

us

cr

ip t

459

466

Ac ce p

te

d

M

an

467

Page 22 of 33

467

References

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510

[1]

ip t

[2]

J. Rivera-Utrilla, M. Sánchez-Polo, V. Gómez-Serrano, P.M. Álvarez, M.C.M. Alvim-Ferraz, J.M. Dias, Activated carbon modifications to enhance its water treatment applications. An overview, J. Hazard. Mater. 187 (2011) 1-23. M. Sevilla, R. Mokaya, Energy storage applications of activated carbons: supercapacitors and hydrogen storage, Energy Environ. Sci. 7 (2014) 12501280. H. Braus, F. Middleton, G. Walton, Orangic Chemical Compounds in Raw and Filtered Surface Waters, Anal. Chem. 23 (1951) 1160-1164. L. Clouzot, B. Marrot, P. Doumenq, N. Roche, 17α-Ethinylestradiol: An endocrine disrupter of great concern. Analytical methods and removal processes applied to water purification. A review, Environ. Prog. 27 (2008) 383-396. S.-H. Lin, R.-S. Juang, Adsorption of phenol and its derivatives from water using synthetic resins and low-cost natural adsorbents: A review, J. Environ. Manage. 90 (2009) 1336-1349. A.A. Rosen, F.M. Middleton, Chlorinated Insecticides in Surface Waters, Anal. Chem. 31 (1959) 1729-1732. Y. Yoon, P. Westerhoff, S.A. Snyder, M. Esparza, HPLC-fluorescence detection and adsorption of bisphenol A, 17β-estradiol, and 17α-ethynyl estradiol on powdered activated carbon, Water Res. 37 (2003) 3530-3537. X. Ren, J. Li, X. Tan, X. Wang, Comparative study of graphene oxide, activated carbon and carbon nanotubes as adsorbents for copper decontamination, Dalton Trans. 42 (2013) 5266-5274. K. Yang, B. Xing, Adsorption of Organic Compounds by Carbon Nanomaterials in Aqueous Phase: Polanyi Theory and Its Application, Chem. Rev. 110 (2010) 5989-6008. Z. Li, X. Chang, X. Zou, X. Zhu, R. Nie, Z. Hu, R. Li, Chemically-modified activated carbon with ethylenediamine for selective solid-phase extraction and preconcentration of metal ions, Anal. Chim. Acta 632 (2009) 272-277. S. Hong, F.S. Cannon, P. Hou, T. Byrne, C. Nieto-Delgado, Sulfate removal from acid mine drainage using polypyrrole-grafted granular activated carbon, Carbon 73 (2014) 51-60. P. Hou, T. Byrne, F.S. Cannon, B.P. Chaplin, S. Hong, C. Nieto-Delgado, Electrochemical regeneration of polypyrrole-tailored activated carbons that have removed sulfate, Carbon 79 (2014) 46-57. C.L. Arthur, J. Pawliszyn, Solid phase microextraction with thermal desorption using fused silica optical fibers, Anal. Chem. 62 (1990) 21452148. A. Namera, T. Saito, Advances in monolithic materials for sample preparation in drug and pharmaceutical analysis, TrAC - Trends Anal. Chem. 45 (2013) 182-196. S. Hjertén, J.-L. Liao, R. Zhang, High-performance liquid chromatography on continuous polymer beds, J. Chromatogr. A 473 (1989) 273-275.

[3]

us

cr

[4]

an

[5] [6]

M

[7]

d

[8]

Ac ce p

te

[9] [10]

[11]

[12] [13] [14] [15]

Page 23 of 33

[21]

[22]

[23]

ip t

te

[24]

cr

[20]

us

[19]

an

[18]

M

[17]

K. Nakanishi, Pore Structure Control of Silica Gels Based on Phase Separation, J. Porous Mater. 4 (1997) 67-112. J. Ou, H. Lin, Z. Zhang, G. Huang, J. Dong, H. Zou, Recent advances in preparation and application of hybrid organic-silica monolithic capillary columns, Electrophoresis 34 (2013) 126-140. H. Kataoka, Current Developments and Future Trends in Solid-phase Microextraction Techniques for Pharmaceutical and Biomedical Analyses, Anal. Sci. 27 (2011) 893-893. F. Svec, Y. Lv, Advances and Recent Trends in the Field of Monolithic Columns for Chromatography, Anal. Chem. 87 (2015) 250-273. C.-L. Lin, S. Lirio, Y.-T. Chen, C.-H. Lin, H.-Y. Huang, A novel hybrid metalorganic framework. Polymeric monolith for solid-phase microextraction, Chem. Eur. J. 20 (2014) 3317-3321. D.-Y. Lyu, C.-X. Yang, X.-P. Yan, Fabrication of aluminum terephthalate metalorganic framework incorporated polymer monolith for the microextraction of non-steroidal anti-inflammatory drugs in water and urine samples, J. Chromatogr. A 1393 (2015) 1-7. A. Saeed, F. Maya, D.J. Xiao, M. Najam-ul-Haq, F. Svec, D.K. Britt, Growth of a Highly Porous Coordination Polymer on a Macroporous Polymer Monolith Support for Enhanced Immobilized Metal Ion Affinity Chromatographic Enrichment of Phosphopeptides, Adv. Funct. Mater. 24 (2014) 5790-5797. S. Tong, X. Zhou, C. Zhou, Y. Li, W. Li, W. Zhou, Q. Jia, A strategy to decorate porous polymer monoliths with graphene oxide and graphene nanosheets, Analyst 138 (2013) 1549-1557. H.-Y. Huang, C.-L. Lin, C.-Y. Wu, Y.-J. Cheng, C.-H. Lin, Metal organic framework–organic polymer monolith stationary phases for capillary electrochromatography and nano-liquid chromatography, Anal. Chim. Acta 779 (2013) 96-103. Y.H. Shih, B. Singco, W.L. Liu, C.H. Hsu, H.Y. Huang, A rapid synthetic method for organic polymer-based monoliths in a room temperature ionic liquid medium via microwave-assisted vinylization and polymerization, Green Chem. 13 (2011) 296-299. B. Singco, C.L. Lin, Y.J. Cheng, Y.H. Shih, H.Y. Huang, Ionic liquids as porogens in the microwave-assisted synthesis of methacrylate monoliths for chromatographic application, Anal. Chim. Acta 746 (2012) 123-133. J.-K. Moon, T. Shibamoto, Formation of Carcinogenic 4(5)-Methylimidazole in Maillard Reaction Systems, J. Agric. Food Chem. 59 (2010) 615-618. M.B. Hargreaves, B.C. Jones, D.A. Smith, A. Gescher, Inhibition of pnitrophenol hydroxylase in rat liver microsomes by small aromatic and heterocyclic molecules, Drug Metab. Dispos. 22 (1994) 806-810. K. Nishie, A.C. Waiss Jr, A.C. Keyl, Toxicity of methylimidazoles, Toxicol. Appl. Pharmacol. 14 (1969) 301-307. R. Gugasyan, C. Losinno, T. Mandel, The effect of 2-acetyl-4tetrahydroxybutylimidazole on lymphocyte subsets during a contact hypersensitivity response in the NOD mouse, Immunol. Lett. 46 (1995) 221227.

d

[16]

Ac ce p

511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556

[25]

[26] [27] [28] [29] [30]

Page 24 of 33

[34] [35]

ip t

te

[37]

d

M

[36]

cr

[33]

us

[32]

G.F. Houben, H.v.d. Berg, M.H.M. Kuijpers, B.W. Lam, H.v. Loveren, W. Seinen, A. Penninks, Effects of the color additive caramel color III and 2-acetyl-4(5)tetrahydroxybutylimidazole (THI) on the immune system of rats, Toxicol. Appl. Pharmacol. 113 (1992) 43-54. EFSA Panel on Food Additives and Nutrient Sources added to Food Scientific Opinion on the reevaluation of caramel colours (E 150a,b,c,d) as food additives, EFSA Journal 9 (2011) 1-103. S. Casal, J.O. Fernandes, M.B.P.P. Oliveira, M.A. Ferreira, Gas chromatographic–mass spectrometric quantification of 4-(5)methylimidazole in roasted coffee after ion-pair extraction, J. Chromatogr. A 976 (2002) 285-291. S.C. Cunha, A.I. Barrado, M.A. Faria, J.O. Fernandes, Assessment of 4-(5)methylimidazole in soft drinks and dark beer, J. Food Compost. Anal. 24 (2011) 609-614. B. Klejdus, J. Moravcová, V. Kubáň, Reversed-phase high-performance liquid chromatographic/mass spectrometric method for separation of 4methylimidazole and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole at pg levels, Anal. Chim. Acta 477 (2003) 49-58. B. Klejdus, J. Moravcová, L. Lojková, J. Vacek, V. Kubáň, Solid-phase extraction of 4(5)-methylimidazole (4MeI) and 2-acetyl-4(5)-(1,2,3,4tetrahydroxybutyl)-imidazole (THI) from foods and beverages with subsequent liquid chromatographic-electrospray mass spectrometric quantification, J. Sep. Sci. 29 (2006) 378-384. L. Lojková, B. Klejdus, J. Moravcová, V. Kubáň, Supercritical fluid extraction (SFE) of 4(5)-methylimidazole (4-MeI) and 2-acetyl-4(5)-(1,2,3,4)tetrahydroxybutyl-imidazole (THI) from ground-coffee with highperformance liquid chromatographic-electrospray mass spectrometric quantification (HPLC/ESI-MS), Food Addit. Contam. 23 (2006) 963-973. C. Moretton, G. Crétier, H. Nigay, J.-L. Rocca, Quantification of 4Methylimidazole in Class III and IV Caramel Colors: Validation of a New Method Based on Heart-Cutting Two-Dimensional Liquid Chromatography (LC-LC), J. Agri. Food Chem. 59 (2011) 3544-3550. C. Schlee, M. Markova, J. Schrank, F. Laplagne, R. Schneider, D.W. Lachenmeier, Determination of 2-methylimidazole, 4-methylimidazole and 2acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole in caramel colours and cola using LC/MS/MS, J. Chromatogr. B 927 (2013) 223-226. P. Wu, L. Zhang, L. Wang, J. Zhang, Y. Tan, J. Tang, B. Ma, X. Pan, W. Jiang, Simultaneous determination of ethyl carbamate and 4-(5-)methylimidazole in yellow rice wine and soy sauce by gas chromatography with mass spectrometry, J. Sep. Sci. 37 (2014) 2172-2176. H. Yamaguchi, T. Masuda, Determination of 4(5)-Methylimidazole in Soy Sauce and Other Foods by LC-MS/MS after Solid-Phase Extraction, J. Agri. Food Chem. 59 (2011) 9770-9775. F. Kvasnička, Determination of 4-methylimidazole in caramel color by capillary isotachophoresis, Electrophoresis 10 (1989) 801-802.

an

[31]

Ac ce p

557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601

[38]

[39]

[40]

[41] [42]

Page 25 of 33

[43]

625

Figure captions

626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644

Figure 1: Electropherograms of imidazole derivatives obtained from a) normal injection and b) CSEI-sweeping-MEKC mode. Carrier electrolytes were composed of (a) 120-160 mM SDS + 20 mM H3PO4 and (b) 140 mM SDS + 20 mM H3PO4. Normal injection (50 mg L-1 imidazole derivative standards) was performed at 0.5 psi, 3 s. For CSEI-sweeping-MEKC procedure, acid plug was pressure-injected (0.5 psi, 420 s) into the capillary then followed by water plug (0.5 psi, 20 s), and thereafter the sample (2-(4)-MEI and THI at 50 µgL-1 and 250 µgL-1, respectively, in 0.05 mM H3PO4), were then electrokinetically injected into the capillary (10 kV, 120 s). Capillary column: 40 cm x 50 µm ID. Separation: -20 kV at 30°C. Peaks: 1) 4MEI, 2) 2-MEI and 3) THI.

[48]

cr

Ac ce p

te

[49]

us

[47]

an

[46]

M

[45]

d

[44]

C.P. Ong, C.L. Ng, H.K. Lee, S.F.Y. Li, Separation of imidazole and its derivatives by capillary electrophoresis, J. Chromatogr. A 686 (1994) 319-324. J.F.d.S. Petruci, E.A. Pereira, A.A. Cardoso, Determination of 2Methylimidazole and 4-Methylimidazole in Caramel Colors by Capillary Electrophoresis, J. Agri. Food Chem. 61 (2013) 2263-2267. J.P. Quirino, S. Terabe, Approaching a Million-Fold Sensitivity Increase in Capillary Electrophoresis with Direct Ultraviolet Detection:  Cation-Selective Exhaustive Injection and Sweeping, Anal. Chem. 72 (2000) 1023-1030. J.P. Quirino, Y. Iwai, K. Otsuka, S. Terabe, Determination of environmentally relevant aromatic amines in the ppt levels by cation selective exhaustive injection-sweeping-micellar electrokinetic chromatography, Electrophoresis 21 (2000) 2899-2903. O. Núñez, J.-B. Kim, E. Moyano, M.T. Galceran, S. Terabe, Analysis of the herbicides paraquat, diquat and difenzoquat in drinking water by micellar electrokinetic chromatography using sweeping and cation selective exhaustive injection, J. Chromatogr. A 961 (2002) 65-75. J.-B. Kim, K. Otsuka, S. Terabe, Anion selective exhaustive injection-sweep– micellar electrokinetic chromatography, J. Chromatogr. A 932 (2001) 129137. L. Zhu, C. Tu, H.K. Lee, On-Line Concentration of Acidic Compounds by AnionSelective Exhaustive Injection-Sweeping-Micellar Electrokinetic Chromatography, Anal. Chem. 74 (2002) 5820-5825.

ip t

602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624

Figure 2: Characterization of AC-polymer obtained from a) FTIR spectroscopy, b) Raman spectroscopy, c) pore size distribution and d) scanning electron microscopy (SEM). For (d), SEM images of AC (upper left), neat polymer (upper right) and ACpolymer (bottom). Figure 3: Extraction efficiencies for imidazole derivatives in the fabricated ACpolymer using different amount of AC.

Page 26 of 33

ip t

Extraction condition: 1) pre-condition: 5 mM H3PO4 (pH2, 0.5 mL); ACN (0.5 mL); 2) sampling: 10 µgL-1 standard in ACN (1 mL); 3) washing: ACN (0.5 mL); 4) desorption: 5 mM H3PO4 (pH2, 1.5 mL) Analysis condition: Sample: eluates was dried in oven at 70°C and re-dissolved in 200 µL MEKC solution (140 mM SDS in 20 mM H3PO4 (pH 2) buffer). Operational conditions are the same as in Figure 1a (normal mode). Table 1: Analytical performance of the proposed CSEI-sweeping MEKC method and fabricated AC-polymer monolithic column for SPME of imidazole derivatives a

cr

Extraction and analysis conditions are the same as in Figure 3 (8 mg of AC). LOD (S/N=3) and LOQ (S/N=10) were calculated based on the obtained S/N ratio d Percent recovery (% RSD); n = 3

us

b,c

an

Figure 4: Electropherograms of soft drink obtained from a) direct injection analysis and b) after SPME. Sample matrix: samples obtained from (a) and (b) were diluted using 0.5 mM H3PO4 (pH 2) (1:99) and ACN (1:99), respectively. CE conditions were the same as in Fig. 1b.

d

M

Extraction conditions for (b) are the same as in Figure 3, except in sampling, where the sample was diluted with ACN (1:99). Analysis condition for (a) and (b) were the same as in Figure 1B (CSEI-sweeping-MEKC) except for samples, which were diluted with ACN (1:99).

te

Figure 5: Electropherograms of alcoholic beverage obtained from a) direct injection analysis and b) after SPME. Extraction conditions were the same as in Fig. 4. Sample matrix and CE conditions were the same as in Fig. 4 and Fig. 1b, respectively.

Ac ce p

645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681

Table 2: Extraction recoveries for imidazole derivatives in SPME of food samples using AC-polymer monolithic column. a

Recovery spiked with 1 mg L-1 of imidazole derivative standards; N.D = not detected

Table 1:

CSEI-MEKC Method

Linear Range (μg L-1)

R2

LOD (μg L-1)b

LOQ (μg L-1)c

Linear Range (μg L-1)

4-MEI

10-250

0.991

6.1

20

50-500

2-MEI

10-250

0.991

4.3

14

50-500

Analytes

AC-polymer monolithic LOD LOQ In (μg LR2 -1 c (μg L ) d 1 )b 9 0.998 37.8 125.9 (3 9 0.997 33.4 111.7 (2

Page 27 of 33

THI

0.995

80

250-5000

0.997

60.4

201.2

aExtraction

and analysis conditions are the same as in Figure 3 (8 mg of AC). (S/N=3) and LOQ (S/N=10) were calculated based on the obtained S/N ratio dPercent recovery (% RSD); n = 3

ip t

b,c LOD

Alcoholic Beverage Concentration Recovery (mg L-1) (%, n=3)a 3.2 96.6 N.D 93.6 N.D 94.3

RSD (%) 2.3 3.0 7.4

an

aRecovery

RSD (%) 2.6 4.1 5.7

cr

Table 2 Cola Sample Analytes Concentration Recovery (mg L-1) (%, n=3)a 4-MEI 3.30 100.3 2-MEI 2.30 90.5 THI 1.80 93.1

spiked with 1 mg L-1 of imidazole derivative standards; N.D = not

te

d

M

detected

Ac ce p

690 691 692 693 694

270

us

682 683 684 685 686 687 688 689

250-5000

Page 28 of 33

8 (6

ip t cr us an M d te Ac ce p 695 696 697 698 699

Figure 1

Page 29 of 33

Ac ce p

706 707 708 709 710 711 712 713

te

d

M

an

us

cr

ip t

700 701 702 703 704 705

Figure 2

Page 30 of 33

ip t cr us an M d te

Figure 3

Ac ce p

714 715 716 717 718

Page 31 of 33

ip t cr us an M d te

Figure 4

Ac ce p

719 720 721

Page 32 of 33

ip t cr us an M Figure 5

Highlights

728 729 730 731 732 733

A new method in the separation of imidazole derivatives using CSEI-

Ac ce p

727

te

726

d

722 723 724 725

sweeping-MEKC

A new method of incorporating polymer into activated carbon (AC-polymer) for in-tube SPME

The AC-polymer exhibited higher extraction efficiency for imidazole derivatives

The AC-polymer monolithic column has potential in SPME application

734

Page 33 of 33