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 Page 1 of 24 Analyst
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4 A novel approach to consecutive extraction of drugs with different properties via
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6 on chip electromembrane extraction
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Yousef Abdossalami Asla, YadollahYaminia,∗, Shahram Seidib
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a
15 Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
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Department of Analytical Chemistry, Faculty of Chemistry, K.N. Toosi University of Technology, Tehran, Iran

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Corresponding author at: Tarbiat Modares University, Department of Chemistry, P.O. Box 14115-175, Tehran, Iran.
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Tel.: +98 21 82883417; Fax: +98 21 82883455.
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E-mail address: [email protected] (Y. Yamini).
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4 Abstract
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6 In the present research, for the first time, a consecutive on chip electromembrane extraction
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9 coupled with high performance liquid chromatography was introduced for analysis of betaxolol
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11 (Bet), naltrexone (Nalt) and nalmefene (Nalm) as model analytes with different chemical properties
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13 from biological samples. The chip consists of two polymethyl methacrylate (PMMA) parts which
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16 two microfluidic channels are craved in each part. These channels were used as a flow path for

Analyst Accepted Manuscript

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18 sample solution and a thin compartment for acceptor phase. A porous polypropylene sheet
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20 membrane impregnated with an organic solvent was placed between two parts of chip device in
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23 order to separate the channels. Two platinum electrodes were bent at the bottom of these channels
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25 that are connected to a power supply providing the electrical driving force for migration of ionized
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analytes from the sample solution through the porous sheet membrane into the acceptor phase. The
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30 new setup provides effective and reproducible extractions by using low volume of sample solution.
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32 Efficient parameters on consecutive electromembrane extraction of the model analytes were
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35 optimized by using one variable at a time method. Under the optimized conditions, the new setup
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37 offered a good linearity in the range of 10.0–500 µg L-1with coefficient of determination (R2) higher
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39 than 0.9932. The relative standard deviation (RSD %) and LOD values were less than 6.8% based on
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42 four replicate measurements and 10.0 µg L-1for the model analytes, respectively. The
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44 preconcentration factors higher than 15.6-fold were obtained. Finally, the proposed method was
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46 successfully applied for determination and quantification of the model analytes in biological
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49 samples.
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51 Keywords: Consecutive on chip extraction; Electromembrane extraction; Betaxolol;
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Naltrexone; Nalmefene.
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4 Introduction
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6 Sample preparation has unique meaning and special importance in the field of analytical
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9 chemistry. For several decades, liquid–liquid extraction (LLE) has been employed for sample
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11 preparation prior to analysis by high performance liquid chromatography and gas chromatography 1-
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. Recent developments of sample preparation are directed toward down scaling and automation of
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16 conventional extraction methods by decreasing acceptor-to-donor ratio in order to reduce organic

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18 solvent, sample solution and chemical reagents consumption 6. Liquid phase micro extraction
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20 (LPME) is a miniaturization of the traditional LLE that performed in several modes such as single
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23 drop microextraction, dispersive liquid-liquid microextraction, porous membrane based LPME and
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25 7-13
solidification of floating organic droplet . Electromembrane extraction (EME), as one of the
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27 14
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LPME methods that was introduced by Pedersen-Bjergaard and coworkers in 2006 . In this
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30 method, the mass transfer is carried out through an organic solvent immobilized within the pores of a
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32 porous membrane by applying an electrical potential difference, as the driving force, between two
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35 platinum electrodes placed on both sides of the porous membrane in donor and acceptor phases.
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37 EME has numerous advantages in comparison with other liquid phase microextraction and provides
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39 more efficient, fast, very selective extractions and excellent sample cleanup 15.
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42 In addition to miniaturization of extraction techniques, downscaling of extraction devices has
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44 important role in decreasing of organic solvent, sample solution and chemical reagents consumption.
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46 Miniaturization of analytical methods and instruments for extraction researches is an area of
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49 burgeoning interest. In many cases, the reduction in size of analytical procedure or technique often
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51 translates to a reduction in analysis time and costs. Miniaturized devices integrate sample
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preparation, separation and detection on a single small chip 16-18. A miniaturized and portable device
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56 is more preferable for the on-site and rapid analysis application.
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The combination of microfluidic technologies and EME would be suitable for such
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6 applications. For the first time, on chip electromembrane extraction (EME)as one type of
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8 miniaturized membrane based LPME techniques was introduced by Pedersen-Bjergaard and
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Rasmussen in 2010 . In this technique, a piece of porous membrane is used as the support for
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13 water–immiscible organic solvent. Afterwards, several reports have been emerged on

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15 implementation of EME as chip based devices to make more developments in this field of area 20-23.
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18 In all of the reported papers using chip devices, one extraction chamber has been used for
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20 extraction. Therefore, analytes with different properties including hydrophilicity, polarity and
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22 functional groups cannot be extracted with these devices. By considering the polarity of various
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25 analytes, different supported liquid membrane (SLM) compositions are required to extract the target
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27 analytes. Adding of a carrier into SLM composition has different effects on the extraction of
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analytes. It has been demonstrated that the use of carriers in SLM composition has strong negative
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32 effect on extraction of nonpolar drugs such as betaxolol (Bet) 24, 25. However, the presence of di-(2-
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34 ethylhexyl) phosphate (DEHP) as an anionic ion pairing reagent is necessary for the extraction of
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37 compounds such as naltrexone (Nalt) and nalmefene (Nalm) 26. Therefore, Bet could be extracted by
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39 use of only pure 2-nitrophenyl octyl ether (NPOE) as the SLM, whereas for the extraction of Nalm
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41 and Nalt a mixture of NPOE and DEHP is required. Consequently, the simultaneous extraction of
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44 these analytes is impossible.
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46 In the current paper, consecutive electromembrane extraction chip as a new on chip EME setup
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48 was introduced for extraction and determination of Nalt, Nalm and Bet from biological samples. The
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51 used analytes were selected as the model compounds with different physicochemical properties to
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53 show the ability of the proposed technique for simultaneous extraction of analytes with different
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chemical properties. In fact, the physicochemical properties of analytes are the important keys for
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the selection of membrane organic solvents so that with selection of a specific composition of
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6 membrane organic solvent selective extraction can be achieved in EME . The proposed method
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8 provides the using possibility of different compositions of membrane organic solvent and thus
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simultaneous extraction of different analytes with different chemical properties. As well known,
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13 todays many combination drugs, which are a mixture of two or more drugs, are used by the patients.
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15 On the other hand a patient may use several drugs, simultaneously. The proposed method can be
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Analyst Accepted Manuscript

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18 used an efficient sample preparation method because it provides the simultaneous analysis
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20 possibility of all drugs by a little volume of a biological sample whereas by other techniques
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22 separated samples, and consequently larger volumes of biological samples, should be used for
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25 analysis of analytes with different physicochemical properties. Here, two extraction chambers were
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27 employed in which anode and cathode electrodes embedded at the bottom of microfluidic extraction
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channels, and SLMs were separately optimized for each class of analytes. As a result, the proposed
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32 system offers suitable conditions for the consecutive extraction of Bet, Nalt and Nalm as model
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34 analytes and can be applied for the extraction of other compounds with similar properties. Effects of
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37 different variables on the extraction efficiency of the analytes were studied and optimized. After
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39 optimization, the method followed by high-performance liquid chromatography–ultraviolet detection
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41 (HPLC–UV) was applied for the extraction and determination of Bet, Nalt and Nalm in human urine
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44 and plasma samples.
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47 Experimental
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50 Chemicals and reagents
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52 Betaxolol was kindly donated by Sina Darou (Tehran, Iran). Nalmefene and naltrexone were
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54 obtained from Parand Darou (Tehran, Iran). Chemical structures, pKa and log P, which P is octanol-
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water partition coefficient, of the analytes were reported in Table 1. 2-Nitrophenyloctyl ether
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6 (NPOE), 2-nitrophenyl phenylether (NPPE), tris-(2-ethylhexyl) phosphate (TEHP) and di-(2-
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8 ethylhexyl) phosphate (DEHP) were purchased from Fluka (Buchs, Switzerland). All of the
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chemicals used were analytical-reagent grades. The Accurel 2E HF (R/P) polypropylene membrane
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13 sheet was supplied by Membrana (Wuppertal, Germany) with the thickness of 150 µm, and pore size
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15 of 0.2 µm. Ultrapure water was prepared by a Younglin 370 series aqua MAX purification
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Analyst Accepted Manuscript

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18 instrument (Kyounggi-do, Korea).
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20 A stock solution containing 2.0 mg mL−1 of Bet, Nalt and Nalm was prepared in methanol. All
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22 standard solutions were stored at 4°C which is protected from light. Working standard solutions
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25 were prepared daily by dilution of the stock solutions with ultrapure water.
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27 Real samples
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Urine sample: To plot the calibration curves and to obtain figures of merit, a human urine
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32 sample was collected from a 29-year-old healthy adult male volunteer. The sampling procedure was
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34 performed according to the guidelines for research ethics. The protocol was approved by an Internal
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37 Review Board. Moreover, written consent was obtained from volunteers prior to the experiment. The
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39 sample was filtered through 0.45-µm pore size cellulose acetate filter from Millipore (Madrid,
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41 Spain). The filtrate was collected in a glass vial, which was carefully cleaned with hydrochloric acid
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44 and washed with deionized water and stored at 4°C to prevent bacterial growth and proteolysis. The
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46 urine sample was diluted 1:4 with ultra-pure water and the pH value was adjusted to 7.00 by adding
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48 proper amounts of hydrochloric acid (100 mM) and/or sodium hydroxide (100 mM) solutions. Then,
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51 the urine sample was spiked with a mixed standard solution to obtain the desired concentration. One
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53 milliliter of the sample solution was subsequently submitted to on chip EME procedure. Further
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urine sample was obtained from a healthy volunteer (29-year-old).
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Plasma sample: Frozen drug-free human plasma sample (blood group B+) was obtained from
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6 the Iranian Blood Transfusion Organization (Tehran, Iran). Prior to use, the plasma sample was
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8 allowed to thaw at room temperature. According to literature, trifluoroacetic acid (TFA) is a suitable
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reagent for protein precipitation and decreasing the matrix effect of real samples such as human
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13 plasma 27. To this end, four drops of concentrated TFA (99 w/w%) were added into 10 mL of each
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15 plasma sample and centrifuged for 5 minutes at 3000 rpm, afterwards. Finally, the supernatant
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18 sample solutions were transferred to a glass container and stored in 4 °C for subsequent experiments.
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20 The pH of 5-fold diluted human plasma samples with ultra-pure water was adjusted at 7.0 using the
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22 proper amounts of hydrochloric acid (100 mM) and/or sodium hydroxide (100 mM) solutions. Then,
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25 the plasma sample was spiked with a mixed standard solution to obtain the desired concentration.
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27 One milliliter of the sample solution was subsequently submitted to on chip EME procedure.
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Chromatographic apparatus
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32 The chromatographic equipment consisted of an Agilent 1260 HPLC system, including a
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34 quaternary pump, degasser, and UV-Vis detector (Waldbronn, Germany). Chromatographic data
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37 were recorded and analyzed by using Chem Station for LC systems software (version B.04.03). The
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39 separations were performed on an ODS-3 column (150 mm × 4.6 mm, with particle size of 5 µm)
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41 from MZ-Analysentechnik (Mainz, Germany) via an isocratic elution at the flow rate of 1.0 mL
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44 min−1. For Bet, Nalt and Nalm, the mobile phase consisted of 10 mM phosphate buffer (pH = 8.0)
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46 and acetonitrile (55:45) respectively for 15 minutes. The detector wavelength was set at 215 nm.
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Manufacture of on chip EME device
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52 The structure of the consecutive chip device for electromembrane extraction is shown in Fig. 1.
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54 The chip consists of two polymethyl methacrylate (PMMA) parts. In each part two long channels
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with a length of 30 mm, depth of 200 µm and width of 1.0 mm was craved. As shown in Fig. 1, the
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channels, which were craved in upper part, acted as acceptor phase channels, whereas the other two
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6 channels locating in lower part are a flow path for donor solution. Moreover, in each part of the
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8 consecutive chip device, six individual holes (with I.D. of 0.5 mm) were drilled. The holes of “a”
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and “f” were connected to inlet and outlet tubes whereas the holes of “b” and “d” were used for the
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13 entrance of platinum electrodes. Holes of “c” and “e” were connected together to deliver extraction
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15 solutions from one extraction chamber to the other one. In each part of the consecutive chip device,
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18 two platinum electrodes were bent and mounted at the bottom of each channel along with its length
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20 after passing through the holes of “b” and “d”. All channels and holes were milled using a CNC
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22 micromilling machine model SMG-302 CNC milling from Sadrafan Gostar Industries (Tehran,
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25 Iran). The channels of sample solution and acceptor phase were separated with two separated small
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27 pieces of porous polypropylene sheet membrane during extraction which was impregnated with
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suitable organic solvents regarding the physicochemical properties of the model analytes. The sheet
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32 membranes were located between the two parts along with lengths of the channels and fixed using
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34 four bolts and nuts during the extraction procedure. After each extraction step, the porous sheet
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37 membrane was replaced with a new one. The sample solution was flowed toward inlet of the sample
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39 channel by a syringe pump. In addition, a micro syringe was used for introducing and withdrawing
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41 of a microliter volume of acceptor solution into the acceptor phase channel.
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44 Consecutive on chip EME procedure
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46 One milliliter of the sample solution containing the target analytes was filled into a Hamilton
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48 syringe and then the syringe mounted on a syringe pump. To impregnate the organic solvent into the
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51 pores of the sheet membrane, two 3 mm × 4 cm pieces of the porous membrane were cut and dipped
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53 into the suitable organic solvents for 5 s and then the excess of organic solutions were gently wiped
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away by using a piece of Kleenex. Then, the membranes were carefully placed between two parts of
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the chip device. The upper and lower parts of the chip were fixed together using bolts and nuts.
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6 Thirty microliters of 100 mM HCl (acceptor solution) was introduced into the acceptor channel by a
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8 microsyringe. Donor phase pumped through the donor channel using a syringe pump. When the
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extraction was completed, the acceptor solution was collected by a microsyringe and injected into
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13 HPLC injection loop. After each extraction, the channels of sample solution and acceptor phase were
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15 washed by ultrapure water and a new porous membrane impregnated with suitable organic solvent
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18 was located and fixed between two parts of extraction chip device for subsequent experiment. Fig. 2
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20 illustrates the extraction process, schematically.
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23 Calculation of preconcentration factor, extraction recovery, and relative recovery
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26 The preconcentration factor (PF) was defined as the ratio of the final analyte concentration in
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28 the acceptor phase (Cf,a) and the initial concentration of analyte (Ci,s) in the sample solution:
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,

 =
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32 ,
(1)
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35 where Cf,a was calculated from a calibration graph obtained from direct injection of standard
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38 solutions of the analytes. The extraction recovery (ER%) was defined as the percentage of the
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40 number of moles of the analyte extracted into the acceptor phase (nf,a) respected to the number of
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42 moles originally present into the sample solution (ni,s).
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45 , ×,
%= × 100 = × 100
,
46 (2)
, , ×,
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50 % =  ,  ×
 × 100 (3)
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54 where Vf,a and Vi,s are the volumes of the acceptor phase and sample solution, respectively. The
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56 relative recovery (RR%) was calculated by the following equation:
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× 100 (4)
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! %= % − 100 (5)
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11 where Cfound, Creal, and Cadded are the concentrations (µg L-1) of analyte after adding a known amount
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13 of standard into the real sample, the concentration of analyte in real sample, and the concentration of
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15 a known amount of standard, which was spiked into the real sample, respectively.
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19 Results and discussion
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22 Selection of supported liquid membrane
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24 Composition of the organic solvent which was used as SLM has an important role in EME. A
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26 suitable liquid membrane enhances the extraction efficiency and selectivity. Composition of
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29 membrane affects the diffusion coefficient of analyte. Furthermore, the range of applied voltage is
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31 determined by nature of SLM composition. Considering the above points, some organic solvents
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33 such as 2-nitrophenyl octyl ether (NPOE), NPOE containing 5, 10 and 15% (v/v) di-(2-ethylhexyl)
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36 phosphate (DEHP), NPOE containing 5, 10 and 15% (v/v) tris-(2-ethylhexyl) phosphate (TEHP) and
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38 NPOE containing 5% TEHP and 5% DEHP was investigated as SLM for the consecutive on chip
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EME. As shown in Fig. 3A, NPOE containing 10% DEHP represented higher extraction efficiency
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43 for Nalt and Nalm where suitable extraction efficiency for Bet was provided when pure NPOE was
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45 used as SLM. Thus, NPOE containing 10% DEHP and NPOE were selected as the optimum SLM
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48 compositions for the subsequent experiments.
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51 Effect of sample solution and acceptor phase compositions
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53 The pHs of sample solution and acceptor phase are critical parameters in extraction efficiency
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of consecutive on chip EME. In EME, the electrokinetic migration of the analytes across the SLM
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into the acceptor solution is affected by ion balance which is defined as the ratio of the total ionic
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6 concentration in the sample solution to that in the acceptor solution . In EME, for basic analytes,
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8 ion balance is mainly considered as the ratio of HCl concentration in sample solution to acceptor
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phase 29. Based on the previous studies on the role of ion balance, it was anticipated that decreasing
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13 of ion balance leads to an increase in the flux of ions through SLM. Therefore, for a basic drug, the
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15 extraction efficiency is increased by increasing the HCl concentration into the acceptor phase and its
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18 decreasing into the sample solution. It can be attributed to this fact that increasing of the HCl
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20 concentration into the acceptor phase not only avoids from partial deprotonation of the analyte and
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22 its back-diffusion into SLM but also, accelerate the analyte release from SLM into the acceptor
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25 solution. On the other hand, for extraction of the basic analyses into the acceptor phase, acidification
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27 of the sample solution is performed to keep the targeted compounds in their ionized form. However,
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there are some limitations for increasing of HCl concentration in both donor and acceptor phase. By
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32 applying an electrical field in EME, cations migrate toward cathode and anions migrate toward
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34 anode through the SLM. Increasing content of ions in each of donor and acceptor phase results to
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37 increasing number of ions migrate through SLM at a given moment, it causes to increasing of the
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39 current level, increasing of Joule heating therefore instability of SLM. Increasing of the current level
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41 between electrodes increase electrolysis reactions on the surfaces of electrodes and therefore bubble
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44 formation risk. Bubble formation increases uncertainties in obtained data for EME. According to the
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46 previous EME works that were carried out for extraction of these model analytes 24-26, 10.0 mM and
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100.0 mM of HCl were selected as donor and acceptor solutions, respectively.
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52 Effect of applied voltage
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54 In EME, the electrical field, which is provided by applying an electrical potential between two
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56 platinum electrodes, stimulates the transfer of analytes through the SLM into the acceptor phase.
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Therefore, mass transfer is greatly depended on the electrical field so that it is expected that
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6 extraction efficiency can be increased by increasing the applied voltage. However, at the high
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8 applied voltages, several problems such as bubble formation due to electrolysis process, SLM
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punctuation, and sparking may be occurred . Therefore, selecting an optimum applied voltage is
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13 necessary to succeed electromembrane extraction. Hence, a series of experiments with various

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15 extraction voltages in the range of 0-140 V were examined to determine the most suitable voltage
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18 while 10 mM HCl and 100 mM HCl was used as donor and acceptor solutions, respectively, for 30
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20 min. As shown in Fig. 3B, relatively high peak areas were obtained at the voltage of 100 V.
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22 Therefore, electrical potential of 100 V was applied for future experiments.
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26 Effect of sample volume
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28 Sampling is one of the main problems related to the analysis of biological real samples. Except
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30 urine, preparing sample with large volumes for other biological samples is difficult. The main
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33 purpose for design and construction of a consecutive on chip EME was low consumption of sample
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35 solution. In the current research, by considering the above reasons, 1.0 mL of sample solution was
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selected for the subsequent experiments.
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41 Effect of sample solution flow rate and extraction time
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43 A syringe pump was used to pump the sample solution into the on chip EME device. The flow
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45 rate of the sample solution plays an essential role in increasing the kinetics and efficiency of
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48 extraction by delivering a sample solution into extraction chambers, increasing the mass transfer and
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30, 31
50 decreasing the thickness of the double layer around SLM . Various sample flow rates in the
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range of 10–60 µL min-1 were examined. As shown in Fig. 3C, extraction efficiency increased by
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55 increasing sample flow rate from 10 to 30 µL min-1, whereas a decreasing was observed by further
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increasing of the flow rate. When the sample flow rate is increased considerably, target analysts do
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6 not have enough time to pass through the SLM into acceptor phase. Also, perforation in the porous
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8 membrane as well as losing of impregnated organic solvents into the pores of porous membrane
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sheets were observed at high sample flow rates.
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13 Fig. 3C also illustrates the effect of extraction time on the extraction efficiency. Extraction
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15 time depends on the sample flow rate, inversely. Therefore, extraction time and sample flow rate was
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18 investigated together. As can be seen, extraction time is increased by decreasing of sample flow rate.
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20 But, stability of SLM is decreased at long extraction durations. When the sample solution is pumped
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22 at low flow rates, accumulation of the ions around SLM is increased which leads to increasing of the
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25 charge density of the double layer; increasing of electrokinetic migration of ions through SLM;
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27 increasing of Joule heating and consequently instability of SLM. Therefore, 30 µL min-1 was
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selected as the optimum sample flow rate.
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33 Method evaluation
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35 To verify the practical applicability of the proposed technique, figures of merit of the proposed
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37 method were studied by using standard solutions of the analytes in drug-free plasma and urine
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40 samples. Optimal conditions were applied to find out linearity, repeatability, and LODs of this
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42 method that is summarized in Table 2. The calibration curves were linear in the range of 10.0–500
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µg L-1 with a coefficient of determination (R2) more than 0.9932. The relative standard deviations
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47 (RSD%) for extraction and determination of the model analytes were less than 6.8% based on four
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49 replicate measurements. LODs less than 10.0 µg L-1 were observed for the target analytes. PF values
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52 higher than 15.6-fold were obtained for the extraction of Bet, Nalt and Nalm at the concentration
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54 level of 100 µg L-1. According to Clarke's analysis of drugs and poisons 32
, the serum therapeutic
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56 concentration range for Bet is 5 to 50 µg L-1. After a single oral dose of 100 mg to four subjects,
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peak plasma concentrations of 10 to 60 (mean 40) µg L-1 of naltrexone were attained in 1 to 2 h. The
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6 peak plasma concentration of 24 healthy males administrated with Nal, aged between 19 and 46
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8 years, were in the range of 12-110 µg L-1. Consequently, the proposed method can be applied for
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extraction and determination of low levels of Nalm, Nalt and Bet from biological fluids.
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14 Application of the proposed method for analysis of real samples
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16 In order to illustrate the performance of the consecutive on chip EME, the procedure was used

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for extraction and determination of Bet, Nalt and Nalm in urine and plasma samples. The obtained
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21 results are presented in Table 3. As it can be seen, model analytes were not found in the investigated
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23 urine and plasma samples. Therefore, urine and plasma samples were spiked with 100 µg L-1 of Bet,
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26 Nalt and Nalm. The relative error for extraction and determination of the model analytes in urine and
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28 plasma samples were less than 5.2%. According to literature, the mean recovery will be within 90 to
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30 110% (or relative Error% between -10% to +10%) 33. Therefore, the results showed that this method
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33 is applicable for the real samples. Fig. 4 shows the typical chromatograms of the extracted model
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35 analytes from urine and plasma samples before and after spiking with 100 µg L-1 of Bet, Nalt and
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Nalm.
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41 Conclusions
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43 In the present paper, for the first time, a consecutive on the chip EME extraction device was
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45 introduced for the analysis of trace amounts of Bet, Nalt and Nalm as model analytes from low
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48 volumes of urine and plasma samples. The effect of various variables affects the extraction
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50 efficiency of the on chip EME procedure were investigated and optimized. Making the extraction
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possibility from low volumes of biological fluids, providing considerable sample cleanup and
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55 acceptable LODs are the advantages that can be accounted for the proposed method. The results
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indicated that the consecutive on chip EME may be a good alternative for drug analysis from
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6 biological samples.
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9 References
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12 1. H. Ebrahimzadeh, Y. Yamini, F. Kamarei and S. Shariati, Anal. Chim. Acta, 2007, 594, 93-
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14 100.
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2. M. Rezaee, Y. Yamini, M. Moradi, A. Saleh, M. Faraji and M. H. Naeeni, J. Supercrit.

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19 Fluids, 2010, 55, 161-168.
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21 3. M. H. Naeeni, Y. Yamini and M. Rezaee, J. Supercrit. Fluids, 2011, 57, 219-226.
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24 4. S. Seidi, Y. Yamini and M. Rezazadeh, J. Chromatogr. B, 2013, 913–914, 138-146.
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26 5. H. Chen, Q. Fang, X.-F. Yin and Z.-L. Fang, Lab Chip, 2005, 5, 719-725.
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28 6. A. Sarafraz-Yazdi and A. Amiri, TrAC Trends Anal. Chem., 2010, 29, 1-14.
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31 7. Y. Ke, W. Li, Y. Wang, X. Hao, R. Jiang, F. Zhu and G. Ouyang, Microchem. J., 2014, 117,
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33 187-193.
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8. H. Ebrahimzadeh, Y. Yamini, A. Gholizade, A. Sedighi and S. Kasraee, Anal. Chim. Acta,
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38 2008, 626, 193-199.
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40 9. Y. Assadi, M. A. Farajzadeh and A. Bidari, in Comprehensive Sampling and Sample
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Preparation, ed. J. Pawliszyn, Academic Press, Oxford, 2012, pp. 181-212.
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45 10. Y.-H. Jian, Y. Hu, T. Wang, J.-L. Liu, C. Zhang and Y. Li, Chin. J. Anal. Chem., 2010, 38,
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47 62-66.
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50 11. M.-I. Leong and S.-D. Huang, J. Chromatogr. A, 2009, 1216, 7645-7650.
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52 12. M. R. Khalili Zanjani, Y. Yamini, S. Shariati and J. Å. Jönsson, Anal. Chim. Acta, 2007, 585,
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54 286-293.
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13. T. Honda, M. Miyazaki, Y. Yamaguchi, H. Nakamura and H. Maeda, Lab Chip, 2007, 7,
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6 366-372.
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8 14. S. Pedersen-Bjergaard and K. E. Rasmussen, J. Chromatogr. A, 2006, 1109, 183-190.
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15. Y. A. Asl, Y. Yamini, M. Rezazadeh and S. Seidi, Anal. Methods, 2015, 7, 197-204.
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13 16. Z.-X. Cai, Q. Fang, H.-W. Chen and Z.-L. Fang, Anal. Chim. Acta, 2006, 556, 151-156.
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15 17. A. Berduque, J. O’Brien, J. Alderman and D. W. M. Arrigan, Electrochem. Commun., 2008,
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18 10, 20-24.
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20 18. H. Shen and Q. Fang, Talanta, 2008, 77, 269-272.
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22 19. N. Petersen, H. Jensen, S. Hansen, S. Foss, D. Snakenborg and S. Pedersen-Bjergaard,
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25 Microfluid Nanofluidics, 2010, 9, 881-888.
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27 20. N. J. Petersen, J. S. Pedersen, N. N. Poulsen, H. Jensen, C. Skonberg, S. H. Hansen and S.
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Pedersen-Bjergaard, Analyst, 2012, 137, 3321-3327.
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32 21. S. Seidi, M. Rezazadeh, Y. Yamini, N. Zamani and S. Esmaili, Analyst, 2014, 139, 5531-
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34 5537.
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37 22. B. Li, N. J. Petersen, M. D. R. Payán, S. H. Hansen and S. Pedersen-Bjergaard, Talanta,
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39 2014, 120, 224-229.
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41 23. M. D. Ramos Payán, H. Jensen, N. J. Petersen, S. H. Hansen and S. Pedersen-Bjergaard,
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44 Anal. Chim. Acta, 2012, 735, 46-53.
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46 24. S. Seidi, Y. Yamini and M. Rezazadeh, J. Pharm. Biomed. Anal., 2011, 56, 859-866.
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48 25. L. Arjomandi-Behzad, Y. Yamini and M. Rezazadeh, Anal. Biochem., 2013, 438, 136-143.
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51 26. M. Rezazadeh, Y. Yamini and S. Seidi, J. Chromatogr. B, 2011, 879, 1143-1148.
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53 27. M. Ghambarian, Y. Yamini and A. Esrafili, J. Chromatogr. A, 2012, 1222, 5-12.
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28. T. M. Middelthon-Bruer, A. Gjelstad, K. E. Rasmussen and S. Pedersen-Bjergaard, J. Sep.
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6 Sci., 2008, 31, 753-759.
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8 29. Y. Yamini, S. Seidi and M. Rezazadeh, Anal. Chim. Acta, 2014, 814, 1-22.
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30. M. Rezazadeh, Y. Yamini and S. Seidi, J. Sep. Sci., 2012, 35, 571-579.
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13 31. A. Gjelstad, K. E. Rasmussen and S. Pedersen-Bjergaard, J. Chromatogr. A, 2007, 1174,

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15 104-111.
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18 32. A. C. Moffat, M. D. Osselton and B. Widdop, Clarke's analysis of drugs and poisons,
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20 Pharmaceutical press London, 2011.
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22 33. G. A. Shabir, JVT, 2005, 10, 314-325.
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26 34. Guidance for Industry; Bioanalytical Method Validation, U.S. Department of Health and
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28 Human Services, Food and Drug Administration, Rockville, 2001.
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35 Figures captions
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38 Fig. 1. Schematic illustration of consecutive on chip EME extraction cell with two extraction
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40 chambers.
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Fig. 2. Schematic illustration of consecutive on chip EME procedure.
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45 Fig. 3. Optimization of (A) SLM composition, (B) applied voltage, (C) sample solution flow rate
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47 and extraction time for Bet, Nalt and Nalm using consecutive on chip EME.
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50 Fig. 4. Typical HPLC chromatograms obtained using consecutive on chip EME procedure for (A) a
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52 urine sample and (B) a plasma sample after (a) and before (b) spiked with Bet, Nalt and Nalm at a
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54 concentration level of 100 µg L-1.
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4 Table 1
5 Chemical structures, pKa and log P of the model analytes.
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7 Name Chemical structure pKa Log P
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12 Betaxolol 9.67 2.54
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19 Naltrexone 8.88 1.36
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29 Nalmefene 9.57 1.95
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4 Table 2
5 Figures of merit of consecutive on chip EME for extraction of Bet, Nalt and Nalm.
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LOD LOQ Linearity
8 Sample Analytes R2 PFa RSD%b
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11 Urine Bet 3.0 10.0 10.0-500 0.9958 17.1 4.2
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13 Nalt 3.0 10.0 10.0-500 0.9997 18.9 5.4

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15 Nalm 2.0 5.0 10.0-500 0.9991 19.5 4.8
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19 Plasma Bet 5.0 10.0 10.0-500 0.9932 15.6 5.4
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21 Nalt 5.0 10.0 10.0-500 0.9974 17.5 5.6
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23 Nalm 3.0 10.0 10.0-500 0.9942 18.9 6.8
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a
25 Preconcentration factor at 100 µg L-1.
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4 Table 3
5 Determination of Bet, Nalt and Nalm in real samples using consecutive on chip EME.
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Sample Analyte Creal Cadded Cfound RSD% Error%
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9 (µg L-1) (µg L-1) (µg L-1) (n=4)
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11 Urine Bet nda 100.0 96.8 4.6 -3.2
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13 Nalt nd 100.0 96.1 5.1 -3.9

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15 Nalm nd 100.0 103.9 4.9 +3. 9
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Plasma Bet nd 100.0 95.1 5.3 -4.9
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22 Nalt nd 100.0 94.8 6.2 -5.2
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