Novel Electrosorption-Enhanced Solid-Phase Microextraction Device

Dec 4, 2017 - Herein, for the first time, we report an ultrafast in vivo solid-phase microextraction (SPME) device based on electrosorption enhancemen...
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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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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

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ABSTRACT

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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

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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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INTRODUCTION

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Ionized pharmaceuticals have been regarded as an important group of emerging

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

43

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

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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

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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

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physiological and environmental influence of ionized pharmaceuticals, experimental

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studies such as monitoring the concentrations in aquatic organisms and measuring

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bioconcentration factor (BCF) values are inevitable.

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

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

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

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

56

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

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the time, labor and expenses. 8

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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

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been

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

well

demonstrated

in

various

studies

involving

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

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

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

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

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

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

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

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

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

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

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significantly shortened because the sampling process is physicochemically

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

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are quite slow.23

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

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

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

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

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by an oxidation or reduction procedure. Although successful applications of

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

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

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

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

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

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

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

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

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

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move towards the coating via electrophoresis and complementary charge attraction,

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

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

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

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

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

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

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

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

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

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

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

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conductivity of the extraction phase. To increase the hydrophilicity and

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

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

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

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

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

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

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

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

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the targeted ionized pharmaceuticals were significantly accelerated, which resulted in

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

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

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values of the pharmaceuticals in tilapia were derived.

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EXPERIMENTAL SECTION

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

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

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

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

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Methanol and acetone were purchased from the Guangzhou Reagent Company

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

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

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

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

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

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

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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

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

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

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

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

15,39-41

The

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

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

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

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

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

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

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

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

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

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

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

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

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

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between the opposite counter electrode needles was 5 mm.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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Instrument and Data Process. The electrochemical experiments were carried out

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

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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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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as higher voltage could provide stronger electrophoresis and complementary charge

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

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

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

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

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

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

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

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

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

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

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

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demonstrated that the electrophoresis and complementary charge attraction forces

233

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

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

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the applied electrosorption enhancement also improved the extraction capacities of the

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

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

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

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

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

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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.

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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

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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

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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

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the sensitivities were excellent and satisfactory for further study.

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

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

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

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

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

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

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

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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

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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

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longitudinal studies without animal sacrifice at each sampling point. The

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

291

were sampled (Figure S6, Table S2).

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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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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

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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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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

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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

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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

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Figure 1. 558x502mm (300 x 300 DPI)

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Figure 2. 973x281mm (300 x 300 DPI)

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Figure 3. 635x440mm (300 x 300 DPI)

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Figure 4. 660x432mm (300 x 300 DPI)

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Figure 5. 609x469mm (300 x 300 DPI)

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For TOC only 38x30mm (300 x 300 DPI)

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