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A Novel Electrosorption-enhanced Solid-phase Microextraction Device for Ultrafast In Vivo Sampling of Ionized Pharmaceuticals in Fish Junlang Qiu, Fuxin Wang, Tianlang Zhang, Le Chen, Yuan Liu, Fang Zhu, and Gangfeng Ouyang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04883 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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Environmental Science & Technology

1

A

Novel

Electrosorption-enhanced

Solid-phase

2

Microextraction Device for Ultrafast In Vivo Sampling of

3

Ionized Pharmaceuticals in Fish

4 5

Junlang Qiu†, Fuxin Wang†, Tianlang Zhang†, Le Chen‡, Yuan Liu‡, Fang Zhu*,†, and

6

Gangfeng Ouyang*,†

7 8

†

9

Chemistry, Sun Yat-Sen University, Guangzhou 510275, China

10 11

‡

MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of

Department of Food Science and Technology, College of Food Science and

Technology, Shanghai Ocean University, Shanghai 201306, China

12 13

*

Corresponding author. Tel. & Fax: +86-20-84110845

14 15

E-mail: [email protected] (F. Zhu); [email protected] (G. Ouyang).

16 17

ACS Paragon Plus Environment


18

ABSTRACT

19

Decreasing the tedious sample preparation duration is one of the most important

20

concerns for the environmental analytical chemistry especially for in vivo

21

experiments. However, due to the slow mass diffusion paths for most of the

22

conventional methods, ultrafast in vivo sampling remains challenging. Herein, for the

23

first time, we report an ultrafast in vivo solid-phase microextraction (SPME) device

24

based on electrosorption enhancement and a novel custom-made CNT@PPY@pNE

25

fiber for in vivo sampling of ionized acidic pharmaceuticals in fish. This sampling

26

device exhibited an excellent robustness, reproducibility, matrix effect-resistant

27

capacity and quantitative ability. Importantly, the extraction kinetics of the targeted

28

ionized pharmaceuticals were significantly accelerated using the device, which

29

significantly improved the sensitivity of the SPME in vivo sampling method (limits of

30

detection ranged from 0.12 ng·g-1 to 0.25 ng·g-1) and shorten the sampling time (only

31

1 min). The proposed approach was successfully applied to monitor the

32

concentrations of ionized pharmaceuticals in living fish, which demonstrated that the

33

device and fiber were suitable for ultrafast in vivo sampling and continuous

34

monitoring. In addition, the bioconcentration factor (BCF) values of the

35

pharmaceuticals were derived in tilapia (Oreochromis mossambicus) for the first time,

36

based on the data of ultrafast in vivo sampling. Therefore, we developed and validated

37

an effective and ultrafast SPME sampling device for in vivo sampling of ionized

38

analytes in living organisms and this state-of-the-art method provides an alternative

39

technique for future in vivo studies.


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40

INTRODUCTION

41

Ionized pharmaceuticals have been regarded as an important group of emerging

42

contaminants in the environment due to their widespread use and continuous release

43

in aquatic environments.1-3 These pharmaceuticals are primarily released through

44

urban wastewater and can further spread through the water cycle and food chain,

45

making the presence of pharmaceuticals in the aquatic environment important to

46

human health safety.4,5 As previously reported, trace amount of pharmaceuticals may

47

lead to subtle effects on the ecosystem, and the adverse effect on aquatic organisms

48

due to exposure of pharmaceuticals has been demonstrated.6 To assess the

49

physiological and environmental influence of ionized pharmaceuticals, experimental

50

studies such as monitoring the concentrations in aquatic organisms and measuring

51

bioconcentration factor (BCF) values are inevitable.

52

Sample preparation is regarded as the most time consuming step in a typical

53

environmental analytical process.7 For bioanalysis experiments, animal sacrifice and

54

tedious clean up steps with organic solvents are also a problem. In vivo solid-phase

55

microextraction (SPME) is a possible solution because this approach integrates

56

sampling, extraction, and preconcentration into a single step to significantly reduce

57

the time, labor and expenses. 8

58

In recent years, in vivo SPME has been widely employed for sampling and sample

59

preparation in many bioanalysis studies.9-11 The advantages of in vivo SPME have

60

been

61

metabolomics,9 pollutant detection,10 and central nervous system studies.13 In general,

well

demonstrated

in

various

studies

involving


clinical

analysis,12


62

new investigations of in vivo SPME can be divided into two categories including the

63

preparation of novel highly efficient fibers14-17 and the sampling of different

64

compounds in living organisms.9,11,18 Although great progress has been made during

65

the development of in vivo SPME, scientists are still investigating methods to shorten

66

the sampling duration of the in vivo SPME method to promote analytical

67

efficiency19-21 and capture short-term or fast dynamic events that occur in biological

68

systems.22 Some novel high performance SPME fibers for in vivo sampling of

69

exogenous and endogenous analytes of interests have been recently reported.11,14,15

70

However, the synthesis routes for these coatings are typically complicated and tedious,

71

which limit application of these fibers. In addition, the sampling time cannot be

72

significantly shortened because the sampling process is physicochemically

73

spontaneous (passive diffusion) and the diffusion rates of the analytes in bio-tissues

74

are quite slow.23

75

In the past decade, electrochemically controlled SPME (EC-SPME) has been

76

extensively applied to extract analytes of interests from aqueous samples.24-27

77

Conductive polymers were used as stationary phase to incorporate analytes by anion

78

or cation exchange. The charge on the polymers can be electrochemically controlled

79

by an oxidation or reduction procedure. Although successful applications of

80

EC-SPME for direct extraction of analytes from water have been reported, some

81

studies revealed that the lifetime of the fiber in the EC-SPME was short and

82

successive extraction was difficult due to the redox reactions of the stationary

83

phase.28,29 Moreover, the extraction efficiency of EC-SPME is primarily dependent on


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84

the ion-exchange capacity of the stationary phase. However, the small volume of the

85

stationary phase limits the ion exchange capacity, which limits the extraction

86

efficiency.30,31 Electrosorption-enhanced SPME (EE-SPME) is another approach that

87

has been proposed to combined electrosorption and SPME to directly extract analytes

88

from aqueous samples.32,33 An electric field is applied to make the ionized analytes

89

move towards the coating via electrophoresis and complementary charge attraction,

90

which significantly improves the extraction efficiency. In addition, no redox reactions

91

occur during the EE-SPME.32 However, due to the large size and coating material of

92

the sampling device, the reported EE-SPME systems are limited in detecting analytes

93

of interest in aqueous matrices without further application.32,33

94

In the current study, we designed and fabricated an ultrafast in vivo SPME sampling

95

device based on electrosorption enhancement. First, a novel custom-made

96

CNT@PPY@pNE fiber was developed using a simple polymerization and

97

dip-coating method. Carbon nanotubes (CNTs) were used as the main extraction

98

phase because they can easily form a 3D interconnected architecture by stacking and

99

have been extensively used as adsorbent for extraction.11,34,35 Polypyrrole (PPY) is a

100

biocompatible and conductive polymer,36-38 which was used to improve the

101

conductivity of the extraction phase. To increase the hydrophilicity and

102

biocompatibility of the coating material, a uniform thin layer of polynorepinephrine

103

(pNE) was placed on the surface of the fiber coating because pNE can provide

104

bioinspired and bio-interface properties15,39-41 and improve the sorption affinity of the

105

ionized pharmaceuticals due to the positive charges on the surface.14,15 Then, a



106

sampling device with a four-direction electric field was designed and fabricated. The

107

experimental results indicate that the device, coupled with the custom-made

108

CNT@PPY@pNE fiber, exhibited excellent robustness, reproducibility, matrix

109

effect-resistant capacity and quantitative ability. In addition, the extraction kinetics for

110

the targeted ionized pharmaceuticals were significantly accelerated, which resulted in

111

excellent sensitivity with a much shorter sampling time. Finally, the proposed

112

approach was then applied to monitor pharmaceuticals in living tilapia, and the BCF

113

values of the pharmaceuticals in tilapia were derived.

114 115

EXPERIMENTAL SECTION

116

Reagents and Materials. Multi-walled carbon nanotubes (O.D. 8~15 nm; I.D. 3~5

117

nm; length ~50 µm), dimethylformamide (DMF) and pyrrole were purchased from

118

Aladdin Reagent (Shanghai, China). Ketoprofen (KET), norepinephrine, eugenol and

119

polyacrylonitrile (PAN) were purchased from J&K Scientific (Beijing, China).

120

Methanol and acetone were purchased from the Guangzhou Reagent Company

121

(Guangzhou, China). Bovine serum was purchased from Sigma Aldrich (Shanghai,

122

China). Tolfenamic acid (TOL), mefenamic acid (MEF), flufenamic acid (FLU) and

123

gemfibrozil (GEM) were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo,

124

Japan). Stainless steel wires (SSWs, 127µm and 480 µm in diameter, medical grade)

125

were purchased from Small Parts Inc. (Miami Lakes, USA).

126

Preparation of the Custom-made Fibers. The PPY was synthesized onto the CNT

127

from a pyrrole monomer by oxidative polymerization using FeCl3 as an oxidant


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(Figure 1A (i)).42,43 Then, the coating was prepared on the SWW via dip-coating

129

method with the dispersive slurry of PAN and PPY/CNT (Figure 1A (ii)).11,15

130

Subsequently, The pNE modification were performed by immersing the coating in the

131

norepinephrine solution (2.0 mg·mL-1) for 16 h (Figure 1A (iii)).

132

procedures were detailed in the supporting information (Method S1).

15,39-41

The

133

Fabrication of the Proposed Sampling Device. The structure of the proposed

134

sampling device is shown in Figure 1B. The sharpened SPME assembling was placed

135

on the center of a glass tube and fastened with glass sealant. Five needles that were

136

fabricated by sharpened 480 µm SSWs (the diameter of the tip was appropriately 100

137

µm) were fastened on the glass tube using epoxy resin. Then, the epoxy resin layer

138

was wrapped with insulating tapes. Four of these needles were placed on the top,

139

bottom, left and right sides of the SPME assembly, and the last needle was placed

140

between two of the previously mentioned needles. The length of the glass tube was 3

141

cm, and the total length of the needles was 5.5 cm. Therefore, the excess portion of

142

the needles was 2.5 cm. The SPME assembly was connected to the work electrode

143

(WE) of the electrochemical workstation. The four needles were combined and

144

connected to the counter electrode (CE), and the remaining needle was connected to

145

the reference electrode (RE) and acted as a quasi-reference electrode. The distance

146

between the opposite counter electrode needles was 5 mm.

147

Animals and Exposure. The mature tilapia (Oreochromis mossambicus) were

148

purchased from a local fishery, and reared in aerated aquariums containing

149

dechlorinated tap water for 2 weeks prior to the experiment. The fish were separated



150

into two groups and reared in dechlorinated tap water (loading rate was 6.8 g·L-1;

151

weight of fish per liter water) that was spiked with the stock solution (500 µg·mL−1 of

152

each pharmaceuticals, in methanol) at 10.0 µg·L−1 for each pharmaceutical. One

153

group was used to determine the sampling rates (after 3 d of exposure), and the other

154

group was used for monitoring. To maintain a constant concentration of

155

pharmaceuticals, the water was refreshed and respiked with the initial amounts of

156

stock solution every 12 h. The total monitoring period was 360 h. The concentrations

157

of the pharmaceuticals in water were also monitored, and the sampling points for

158

water were the same as those for the fish. Commercial C18 SPME fibers were used

159

for water analysis, and an external calibration method was used for quantification.

160

The water quality was monitored daily during the entire monitoring period (pH 6.7,

161

dissolved oxygen 6.6 ± 0.4 ppm, and temperature 27.3 ± 1.4 °C). The weight and

162

length of the fish were recorded (i.e. length 25.2 to 31.3 cm, median 28.5 cm; weight

163

461 to 653 g, median 568 g). All of the animal experiments were approved by the

164

Animal Ethical and Welfare Committee of Sun Yat-sen University.

165

In Vivo Electrosorption-enhanced Solid-phase Microextraction (EE-SPME). A

166

fish was anaesthetized in dechlorinated tap water that was spiked with eugenol (0.1%,

167

v/v) until loss of vertical equilibrium. The custom-made fiber was inserted into the

168

sharpened SPME assembly, and then the electrode needles were stabbed into the

169

dorsal-epaxial muscle of the fish to a depth of approximately 1.5 cm (Figure 1C (i)).

170

Next, the sharpened SPME assembly was stabbed into the dorsal-epaxial muscle to a

171

depth of approximately 1.5 cm (Figure 1C (ii)). Then, the SPME assembly was


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carefully withdrawn to expose the fiber in the muscle (Figure 1C (iii)). The fish was

173

placed in a small tank to avoid disturbance from swimming and isolate the sampling

174

device from water (Video S1). Then, a constant voltage of 1.2 V was applied to the

175

device. The health status of the fish was ensured during sampling and the entire

176

monitoring period. After 1 min of sampling duration, the SPME assembly was placed

177

back into the muscle, and then, the assembly and fiber were withdrawn from the

178

muscle (Figure 1C (iv&v)). Next, the electrode needles were withdrawn from the

179

muscle, and the fiber was rinsed with deionized water and dried with a Kimwipe

180

tissue. Then, the fiber was desorbed in 90 µL of methanol for 30 min at a vortex rate

181

of 400 rpm, and 10 µL of the KET standard solution (100 ng·mL−1) was added as an

182

internal standard to calibrate the ionization efficiency of the instrument analysis.

183 184

Liquid Extraction (LE). The details of the LE of the fish dorsal-epaxial muscle are described in the Supporting Information (Method S2).

185

Instrument and Data Process. The electrochemical experiments were carried out

186

on a CHI750A electrochemical workstation (CH instrument, Shanghai, China). The

187

details of the HPLC-MS-MS analysis are described in the Supporting Information

188

(Method S3). All of the data were processed with GraphPad Prism 5.

189 190

RESULTS AND DISCUSSION

191

Characterization of the Novel Custom-made Fibers. The coating of the

192

custom-made fiber was prepared based on the 3D interconnected stacking architecture

193

of CNTs. A biocompatible and acid-resistant polymer (i.e. PAN) was used as the glue



194

to attach the PPY/CNT composite to the SSW.11 The oxidative polymerization of the

195

pyrrole monomer on the CNT was confirmed by X-Ray Diffraction (XRD) and

196

Fourier Transform Infrared Spectroscopy (FTIR) analyses (Figure S1-A&B). The

197

element analysis data indicated that PPY was successfully polymerized onto the CNT

198

(Figure S1-C) and the element mapping data further demonstrated that PPY was

199

uniformly wrapped onto the CNT (Figure S1-D). The bioinspired pNE sheath on the

200

coating was confirmed by X-ray photoelectron spectroscopy (XPS) analysis (Figure

201

S1-E). The transmission electron microscope (TEM) images (Figure S2-A&B) also

202

show that the PPY layer was uniformly wrapped on the CNT and the pNE sheath on

203

PPY/CNT was also observed. In the Scanning electron microscope (SEM) images

204

(Figure S2-C, E&F), the coating appeared to possess a homogeneous surface, and the

205

stacking of the CNTs formed 3D interconnected pores which could facilitate mass

206

transfer in the coating. The cross section of the fiber indicated that the coating

207

thickness was approximately 20 µm (Figure S2-D).

208

Evaluation of the Proposed Sampling System. The device was designed and

209

fabricated in the lab and coupled to the custom-made fiber for sampling. The

210

three-electrode system of an electrochemical workstation was used to provide a stable

211

electric field. The deionized water that used for extraction was mixed with bovine

212

serum (Sigma Aldrich, Shanghai) (1%, v/v). To ensure the lifetime of the fiber, the

213

threshold value of the applied voltage required evaluation. To do this, the stability and

214

robustness of the fiber towards the electric field were evaluated by cyclic voltammetry.

215

The result indicated that the pNE modified fiber possessed better electric resistance


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than the unmodified fiber (Figure S3-A). Redox reactions were observed when the

217

voltage was increased to 1.75 V for the modified fiber and 1.0 V for the unmodified

218

fiber. This result indicated that the modified fiber can withstand higher voltage, and it

219

also revealed that the modified fiber had the potential for higher extraction efficiency

220

as higher voltage could provide stronger electrophoresis and complementary charge

221

attraction forces. The applied voltage was also evaluated for the pNE modified and

222

unmodified fibers (Figure S3-B). The extraction efficiency of the unmodified fiber

223

increased as the voltage increased. However, the extraction efficiency remained stable

224

for the pNE modified fiber from 1.2 V to 1.5 V. Because a repeated higher voltage

225

would lead to the redox reaction of the coating materials, the applied voltage for the

226

pNE modified fiber was set to 1.2 V based on fiber lifetime consideration.

227

The extraction kinetics of the three situations (i.e., (A) unmodified coating with

228

electrosorption enhancement (0.75 V), (B) pNE modified coating with electrosorption

229

enhancement (1.2 V) and (C) pNE modified coating without electrosorption

230

enhancement) were evaluated (Figure 2). By comparing situation (B) and (C), the

231

extraction kinetics of (B) were significantly faster than those of (C), which

232

demonstrated that the electrophoresis and complementary charge attraction forces

233

significantly accelerate the diffusion rates of the ionized analytes. In addition, the

234

equilibrium extraction amount in (B) was higher than that in (C), which revealed that

235

the applied electrosorption enhancement also improved the extraction capacities of the

236

coating. The results indicated that the sensitivity of the proposed EE-SPME sampling

237

system with a very short sampling duration (~1 min) may be better than that of the



238

common SPME process with a relatively long sampling duration (~20 min). The

239

extraction kinetics were ultrafast for both (A) and (B) but the equilibrium extraction

240

amounts were higher in (B) than those in (A). These results were due to: (1) the

241

higher voltage that was applied in (B) provided greater extraction capacity

242

improvements and (2) the modified pNE sheath improved the sorption affinity of the

243

ionized pharmaceuticals due to the positive charges on the surfaces.14,15 The extraction efficiency of the proposed sampling system was compared to that

244 245

of

246

fibers, which has been previously used for sampling of pharmaceuticals in fish

247

(coating: length 1.0 cm, thickness 330 µm; custom-made)21,44,45 and polar polyacrylate

248

(PA) fibers (coating: length 1.0 cm, thickness 85µm; Supelco, Bellefonte, PA, USA)

249

(Figure 3). Although the coating thickness and volume of the custom-made fiber were

250

much smaller than those of the commercial fibers, the extraction efficiency of the

251

proposed EE-SPME system was still much higher than those of the commercial fibers.

252

The remarkably high extraction efficiency of the proposed SPME system in a short

253

sampling duration (5 min) indicated that this sampling system possessed excellent

254

sensitivity in an ultrafast sampling process.

255

commercially available SPME fibers including polydimethylsiloxane (PDMS)

In

addition,

the

proposed

sampling system

exhibited excellent matrix

256

effect-resistant capacities because no biomacromolecules were detected in the

257

desorption solutions by MALDI-TOF-MS (Figure S5) and no significant ionization

258

bias were detected between desorption solutions and control solutions in the

259

LC-MS-MS analysis. The ionization efficiencies of the desorption solution of three


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different concentrations ranged from 96.06% to 100.75% compared to the control (the

261

standard solution without desorption of the prepared fiber), which indicates that no

262

significant ionization bias were observed (Table S1). In addition, the pNE surface

263

modification could form a bioinspired interface, and therefore, the fibers would not

264

cause rejection reactions during sampling in living animals.15,39,40

265

In Vivo Sampling and Monitoring in Fish. The proposed sampling system was

266

used for sampling of the targeted ionized pharmaceuticals in living fish. The

267

sampling-rate calibration method was used for quantification,21 and the sampling rates

268

were determined (Table 1). The sampling rates were much faster (approximately

269

20-100 times) than those reported in previous studies (Figure S4),14,15,21,40 which

270

revealed that the extraction kinetics of the proposed sampling system in living fish

271

muscle were very fast, compared to conventional in vivo SPME. Additionally, the

272

accuracy of the proposed method was confirmed by comparison with LE (Figure 4).

273

The in vivo sampling duration was set as 1 min for the subsequent experiments, and

274

the sensitivities were excellent and satisfactory for further study.

275

The limit of detection, limit of quantitation, linear range and regression coefficients

276

in spiked fish dorsal-epaxial muscle homogenate are shown in Table 1. The proposed

277

system possessed satisfactory stability with relative standard deviations (RSD) of

278

intrafiber and interfiber reproducibilities ranging from 2.97% to 8.56% and 3.04% to

279

9.50%, respectively, for six sampling-desorption cycles (Table 1). These results

280

demonstrated that the proposed system was stable for repeated uses in a complex

281

biological matrix. In addition, the sensitivity (limits of detection ranged from 0.12



282

ng·g-1 to 0.25 ng·g-1) was much better compared to those in the previously reported

283

studies14,15,21,39 even though the sampling duration was shortened from approximately

284

10-20 min to only 1 min.

285

Next, the established method was applied to monitor the concentrations of four

286

ionized pharmaceuticals in living fish. All the pharmaceuticals were successfully

287

monitored in a 360 h monitoring period (Figure 5). The monitoring experiment

288

revealed that the established ultrafast sampling method permits repeated temporal and

289

longitudinal studies without animal sacrifice at each sampling point. The

290

concentrations of the pharmaceuticals in the water were also monitored when the fish

291

were sampled (Figure S6, Table S2).

292

The ionized pharmaceuticals have been recognized as emerging contaminants due

293

to their physiological activities, wide usage and constant discharge to the environment.

294

The characterization of the bioaccumulative features is important for physiological

295

and environmental assessments of the ionized pharmaceuticals. Here we derived the

296

BCF values using the proposed ultrafast in vivo SPME sampling system, and the BCF

297

values were calculated based on the ratio of the concentrations in the fish to those in

298

the water. =

concentrations in fish concentrations in water

299

The BCF values became steady after 168 h of exposure, and the BCF values ranged

300

from 1.84 to 16.18 (Table S3). The significant decrease in the sampling duration

301

results in more precise, accurate and faster in vivo analytical data to provide better

302

environmental and biological analyses of ionized pharmaceuticals in pharmacokinetic


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303

and metabolism studies. In addition to laboratory experiments, the established method

304

could be a useful tool for the sampling and analysis of ionized compounds in wild

305

animals.

306

Implication

307

electrosorption-enhanced solid-phase microextraction system for ultrafast and

308

sensitive in vivo sampling of ionized pharmaceuticals in fish. This sampling system

309

exhibited excellent robustness, reproducibility, matrix effect-resistant capacity and

310

quantitative ability. The extraction kinetics for the targeted ionized pharmaceuticals

311

were significantly accelerated, and the sensitivity was excellent with a very short

312

sampling duration of only 1 min. The proposed approach was successfully applied to

313

monitor the concentrations of ionized pharmaceuticals in living fish, which

314

demonstrated that the device and the fiber were suitable for in vivo sampling and

315

long-term continuous monitoring. In addition, the BCF values of the pharmaceuticals

316

were derived based on the data of ultrafast in vivo sampling. Overall, the proposed

317

state-of-the-art approach opens up a new avenue for ultrafast in vivo sampling and

318

advanced the implementation of SPME for future in vivo studies.

for

Application.

In

summary,

we

developed

a

novel

319 320

ASSOCIATED CONTENT

321

Supporting Information

322

HPLC-MS/MS analytical parameters, LE method, detailed preparation and

323

characterization results of the custom-made novel SPME fiber, monitoring of the

324

concentrations of pharmaceuticals in water, ionization efficiencies of the



325

matrix-impacted standard solutions to the control with LC-MS-MS analysis,

326

quantification parameters of the pharmaceuticals in water, the BCF values of different

327

monitoring periods, the video file of the in vivo sampling in fish with the proposed

328

sampling system. This material is available free of charge via the Internet at

329

http://pubs.acs.org.

330 331

AUTHOR INFORMATION

332

Corresponding Author

333

*Tel./Fax: +86-20-84110845. E-mail: [email protected].

334

*Tel./Fax: +86-20-84110845. E-mail: [email protected].

335

Notes

336

The authors declare no competing financial interest.

337 338

Acknowledgements We acknowledge financial support from the projects of National Natural Science

339 340

Foundation of China (21377172, 21477166, 21527813, 21677182).

341 342

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496

Figure Captions

497

Figure 1. (A) Flow diagram for the preparation of the novel SPME fiber. (B)

498

Representation of the proposed sampling device and the electrosorption-enhanced

499

SPME with the device for ionized pharmaceuticals. (C) The in vivo sampling

500

procedure in dorsal-epaxial muscle of fish with proposed sampling system. To better

501

illustrate the procedure, the front and back counter electrode needles as well as the

502

reference electrode needle were omitted in the scheme.

503 504

Figure 2. Extraction kinetic profiles of the three situations including (A) unmodified

505

coating with electrosorption enhancement, (B) modified coating with electrosorption

506

enhancement and (C) modified coating without electrosorption enhancement. The

507

extractions were conducted at room temperature and the concentrations of the

508

pharmaceuticals in deionized water were 10 ng·g-1. The error bars represent the

509

standard deviations (n = 6).

510 511

Figure 3. Comparison of the extraction efficiencies between the proposed sampling

512

system and commercial fibers (polydimethylsiloxane (PDMS) fiber and polyacrylate

513

(PA) fiber). The extraction duration was 5 min for the proposed system and 10 min for

514

the commercial fibers; the concentrations of the pharmaceuticals in deionized water

515

were 10 ng·g-1. The error bars represent the standard deviations (n = 6).

516 517

Figure 4. The mean concentrations of the four ionized pharmaceuticals in



Page 26 of 33

518

dorsal-epaxial muscle of the six fish determined with liquid extraction (LE) and the

519

proposed

520

(EE-SPME-SR). The error bars represent the standard deviations (n = 6).

electrosorption-enhanced

SPME

with

sampling-rate

calibration

521 522

Figure 5. In vivo sampling and continuous monitoring of the ionized pharmaceuticals

523

in living fish using the proposed sampling system. The error bars represent the

524

standard deviations (n=6).

525


Page 27 of 33


526

Table 1. Intra-fiber and inter-fiber reproducibilities (%) of the proposed sampling system (n=6) as well as limits of detection (LOD) (S/N=3,

527

ng·g-1) and limits of quantification (LOQ) (S/N=10, ng·g-1) for the in vivo sampling of fish dorsal-epaxial muscle. A linear range (ng·g-1) was

528

achieved in spiked fish dorsal-epaxial muscle homogenate, and the correlation coefficient (R2) values are presented. The sampling rates

529

(mg·min-1) of the pharmaceuticals in fish muscle with the proposed sampling system are also presented. Analytes

Intrafiber

Interfiber

LOD

LOQ

Linear range

R2

Sampling rates

MEF

2.97

9.50

0.12

0.40

1-500

0.9994

31.81±3.87

FLU

5.20

4.13

0.24

0.82

1-500

0.9988

17.84±3.21

TOL

8.48

6.77

0.25

0.83

1-500

0.9987

8.44±1.52

GEM

8.56

3.04

0.18

0.62

1-500

0.9989

8.81±1.55

530



Figure 1. 558x502mm (300 x 300 DPI)


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Figure 2. 973x281mm (300 x 300 DPI)



Figure 3. 635x440mm (300 x 300 DPI)


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Figure 4. 660x432mm (300 x 300 DPI)



Figure 5. 609x469mm (300 x 300 DPI)


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For TOC only 38x30mm (300 x 300 DPI)


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