Approach for Downscaling of Electromembrane Extraction as a Lab on

A design of electromembrane extraction (EME) as a lab on-a-chip device was proposed for the extraction and determination of phenazopyridine as the mod...
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A new approach for downscaling of electromembrane extraction as a lab on-a-chip device followed by sensitive Red-Green-Blue detection Mahroo Baharfar, Yadollah Yamini, Shahram Seidi, and Muhammad Balal Arain Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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Analytical Chemistry

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A New Approach for Downscaling of Electromembrane Extraction as a Lab on-a-Chip

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Device Followed by Sensitive Red-Green-Blue Detection

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Mahroo Baharfara, Yadollah Yaminia,*, Shahram Seidib, Muhammad Balal Arainc a

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Department of Chemistry, Faculty of Sciences, Tarbiat Modares University, P.O. Box: 14115-175, Tehran, Iran

b

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Department of Analytical Chemistry, Faculty of Chemistry, K.N. Toosi University of Technology, Tehran, Iran c

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Department of Chemistry, Abdul Wali Khan University, Mardan, K.P.K, Pakistan, 23200

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Corresponding Author:

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*

Email: [email protected], Phone: +98-21-82883449, Fax: +98-21-82883460.

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ABSTRACT

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A new design of electromembrane extraction (EME) as a lab on-a-chip device was

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proposed for the extraction and determination of phenazopyridine as the model analyte. The

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extraction procedure was accomplished by coupling EME and packing a sorbent. The analyte

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was extracted under the applied electrical field across a membrane sheet impregnated by

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nitrophenyl octylether (NPOE) into an acceptor phase. It was followed by the absorption of the

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analyte on strong cation exchanger as a sorbent. The designed chip contained separate spiral

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channels for donor and acceptor phases featuring embedded platinum electrodes to enhance

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extraction efficiency. The selected donor and acceptor phases were 0 mM HCl and 100 mM HCl,

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respectively. The on-chip electromembrane extraction was carried out under the voltage level of

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70 V for 50 min. The analysis was carried out by two modes of a simple Red-Green-Blue (RGB)

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image analysis tool and a conventional HPLC-UV system. After the absorption of the analyte on

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the solid phase, its color changed and a digital picture of the sorbent was taken for the RGB

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analysis. The effective parameters on the performance of the chip device, comprising the EME

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and solid phase microextraction steps, were distinguished and optimized. The accumulation of

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the analyte on the solid phase showed excellent sensitivity and a limit of detection (LOD) lower

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than 1.0 µg L-1 achieved by an image analysis using a smartphone. This device also offered

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acceptable intra- and inter-assay RSD% ( 0.9969) for HPLC-UV and RGB analysis,

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respectively. To investigate the applicability of the method in complicated matrices, urine

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samples of patients being treated with phenazopyridine were analyzed.

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Keywords: Electromembrane extraction; Lab on-a-chip; Phenazopyridine; RGB analysis; Smartphone. 2 ACS Paragon Plus Environment

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Analytical Chemistry

INTRODUCTION

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Design and application of the new generation of miniaturized chemical devices is a current,

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interesting topic in a broad range of research fields from biology and chemistry to engineering1,2.

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The concept behind these microscale lab on-a-chip (LOC) devices is integrating one or several

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processes into a single chip3. LOC systems have undergone extensive promotions since their

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advent, thanks to the numerous advantages associated with these systems, including higher

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efficiency and sample throughput, low consumption of reagents and solvents, portability,

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feasibility of parallelization and automation, and shortening the process time4-6. Utilization of

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LOC devices is also a superior strategy to benefit chemical analysis and integration of, for

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instance, sample preparation, separation, and detection processes over a small platform7. Among

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these steps, sample preparation is a critical step before a chemical analysis. This helps to reduce

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the matrix effect, provide a suitable form of sample for instrumental analysis, concentrate

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analytes, eliminate interferences, and warrant reliable measurements. For this purpose, plenty of

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conventional sample preparation techniques have been proposed during the several past decades,

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entailing excessive amounts of organic solvents and chemicals, time-consuming steps, and

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difficult handling8-10.

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Therefore, the necessity of developing more environmentally and user-friendly techniques

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as well as improving sensitivity, selectivity, and efficiency has eclipsed many research incentives

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in the field of analytical chemistry. Owing to these extensive efforts, several innovative sample

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preparation methods have been established. Among them, membrane-based techniques are well-

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known sample preparation methods because of their advantages over other similar techniques,

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benefiting from high sample clean-up, favorable extraction efficiency, and low required amounts

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of organic solvents11.

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Electromembrane extraction (EME) is one of the relatively new membrane-based

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extraction methods which was introduced at first by Pedersen Bjergaard et al12. The basis of this

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electrically-driven method is related to the fact that the mass transfer of charged species is

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enhanced by the application of an electrical field. Therefore, in this method, two platinum

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electrodes connected to a power supply are responsible for providing an electrical field across a

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thin layer of an organic solvent immobilized in the pores of a porous polypropylene sheet or a

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hollow fiber. Analytes are extracted by the electrical force through the supported liquid

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membrane into the acceptor phase in their ionized form13-15. Since the initiation of EME, much

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advancement has been accomplished to make this technique more efficacious16-18. Due to the

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fascinating and advantageous features of LOC devices, one of the most promising strategies for

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further development of EME is the exploitation of this technique on chip platforms. The early

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studies on this issue go back to 2010 and since then diverse designs of EME as downscaled LOC

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systems have been proposed19,20. However, designing an integrated, sensitive, and portable

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system without any special instrumental analysis still remains a challenge and few works on this

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topic have been reported in the literature21.

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In the present study, a new design of EME as a LOC device followed by a microextraction

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step accomplished by a packed solid phase was proposed for extraction and preconcentration of

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phenazopyridine (PP) as a model analyte. The chip device consisted of spiral channels for both

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donor and acceptor phase compartments to obtain higher extraction efficiency. It is expected that

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the higher amount of the acceptor solution prevents the early saturation of the acceptor phase and

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improves the efficiency of the process. The desired electrical field was applied homogeneously

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throughout the length of the spiral extraction channels. This homogeneity is achieved by the

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constant distance between the electrodes and their fixed position.

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Analytical Chemistry

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In order to integrate the whole analysis process on a single platform, the EME procedure

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was followed by the adsorption of the target analyte on a solid phase. Then, an RGB technique

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was used as the detection method. The accumulation of the analyte on the solid phase caused the

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color change of the sorbent due to the orange color of the target compound. To assess the

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reliability of the RGB method, the HPLC-UV analysis was additionally carried out and the

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obtained results were compared.

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To do the RGB analysis, a digital image of the solid phase was taken by a smartphone and

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its three main color components (red, green, and blue) were analyzed by an image analysis

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software. The relations between variation in concentration and different color components were

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determined, and the best concentration-dependent one was selected as the analytical signal.

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Finally, the applicability of the method was evaluated by the analysis of the real urine samples of

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patients being treated with PP, and then the results of both analysis methods, HPLC-UV system

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and RGB analysis, were compared.

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EXPERIMENTAL

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Chemicals and reagents. Phenazopyridine (PP) was kindly donated by Tehran Shimi

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Pharmaceutical Co (Tehran, Iran). The structure and physicochemical properties of PP is shown

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in Table 1. All chemicals were of analytical grade. Nitrophenyl octylether (NPOE), tris-(2-

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ethylhexyl) phosphate (TEHP), and di-(2-ethylhexyl) phosphate (DEHP) were supplied from

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Fluka (Buchs, Switzerland). 1-Octanol was obtained from Merck (Darmstadt, Germany). The

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Accurel 2E HF (R/P) polypropylene membrane sheet with a thickness of 100 µm and a pore size

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of 0.2 µm was purchased from Membrana (Wup- pertal, Germany). The ultrapure water used in

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this work was prepared by a Younglin 370 series aqua MAX purification instrument (Kyounggi-

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do, Korea). The utilized sorbent was strong cation exchange (SCX) with the particle diameter of

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50 µm and pore size of 60 Å, provided by Analytica International (New York, NY, United

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State). The stock solution of PP was prepared at the concentration of 1.0 mg mL-1 in methanol.

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Standard solutions were freshly prepared from the stock solution by dilution with ultrapure

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water. All of the standard solutions were stored at 4 ºC and protected from light.

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Real samples. To plot the calibration curves and assess figures of merit, all experiments

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were conducted in a drug-free urine sample of a 24-year-old healthy female volunteer. For this

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purpose, two milliliters of the urine sample was spiked with proper amounts of the standard

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solution to obtain the desirable concentration without any pretreatment or dilution. The real urine

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samples were collected from volunteer patients being treated with PP. One of the patients was a

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53-year-old male volunteer, taking a regular dose of 100 mg three times a day, and the other one

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was a 43-year-old female volunteer, taking a single dose of 100 mg. All samplings were carried

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out based on the guidelines for research ethics, and the protocol was approved by an internal

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review board.

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Chromatographic apparatus. Separation and detection of PP was accomplished by an

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Agilent 1260 HPLC system equipped with a quaternary pump, degasser, a 20 µL sample loop

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and UV-Vis detector (Waldbornn, Germany). Elution of PP was carried out by an isocratic

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program at the flow rate of 1.0 mL min-1. The mobile phase consisted of acetonitrile and a 10

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mmol L-1 acetate buffer with the pH of 5.5 (60:40, v/v). For detection and quantification, the

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detector wavelength was set at 395 nm. The results were recorded and analyzed using

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ChemStation for the LC system software (version B.04.03). The elution was performed on an

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ODS-3 column (150 mm × 4.6 mm, with 5 µm particle size) provided by MZ-Analysenteknik

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(Mainz, Germany).

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Analytical Chemistry

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Preparation of the chip device. The chip device consisted of two polymethylmethacrylate

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(PMMA) plates as substrates. In each part, the same pattern of spiral channels was carved with

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the width of 2.0 mm and the depth of 500 µm. The chip device for electromembrane extraction is

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shown in Figure 1A. The upper plate was a compartment for the stagnant acceptor phase and the

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lower plate was dedicated to the flow pass of the sample solution. In each spiral pattern, three

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individual holes were drilled (I.D. of 0.5 mm) to provide inlet and outlet tubes and insert the

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platinum electrodes. As shown in Figure 1B, holes a and b were exploited to provide the inlet

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and outlet tubes, and hole c was used to mount the platinum electrodes. These platinum

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electrodes with the diameter of 0.2 mm were purchased from Pars Platin (Tehran, Iran). The

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electrodes were fixed by bending and embedding along the length of the channels. To mile all

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channels and holes, a SMG-302 CNC micromilling machine from Sadrafan Gostar Industries

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(Tehran, Iran) was used.

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PMMA plates were separated by a piece of NPOE impregnated polypropylene sheet, which

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was located between the two parts. The whole structure was fixed by the aid of bolts and nuts,

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and the membrane sheet was renewed after each extraction. The sample solution was pumped

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into the corresponding channels by a syringe pump obtained from Fanavaran Nano-Meghyas

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(Tehran, Iran), and the acceptor solution was injected into the dedicated channel and withdrawn

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from that by a microsyringe. A transparent silicon tube was packed with 2 mg of SCX and both

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ends of the tube were manually fitted by two frit pieces. Then, it was connected to the outlet tube

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of the acceptor phase.

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On-chip EME coupled with the solid phase microextraction step. A piece of

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polypropylene sheet, with the dimension of 3.5 mm × 3.5 cm, was cut and impregnated by

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NPOE. The desired organic solvent was immobilized in the pores of the porous propylene sheet,

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and the excess amount of that was removed by a small piece of Kleenex. The resulted sheet was

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located between the two parts of the chip. After fixing the whole device, the sample solution was

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introduced to the dedicated channel by a syringe pump. 500 µL of 100 mM HCl was injected into

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the upper channel by a microsyringe as the acceptor phase. After the fulfillment of the EME, the

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acceptor phase was conducted into the solid phase by conducting the excess air at a certain flow

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rate with the same syringe pump. Then, the digital image, captured from the solid phase, was

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decomposed to three main color components (RGB analysis). Through another approach, the

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tube containing the solid phase was mounted and fixed on the needle of a microsyringe, and the

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target analyte was desorbed by the ejection and withdrawal of a 20 µL methanol solution

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containing 40% of 0.2 mol L-1 NaOH. These steps were repeated certain times to ensure the

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complete desorption of the analyte. The final solution was injected to the HPLC-UV system for

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further analysis. A diagram of the whole procedure is illustrated in Figure 1. After each

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extraction, the chip device was carefully washed and dried by ultrapure water and methanol, and

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the packed silicon tube was replaced with a new one.

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RGB analysis. To design a portable device capable of an in-situ analysis, an on-line RGB

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analysis was performed by a smart phone (iPhone SE 2016, USA). For this purpose, the obtained

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solid phase was placed in a certain position and the smart phone was mounted at a fixed place,

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12 cm above the solid phase. A digital photo of the solid phase was taken and was analyzed via

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an iDropper tool, which is an image analysis application available for iOS operating systems.

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The correlation between concentration and intensity of color components, as a concentration-

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dependent signal, was investigated by subtraction of the RGB intensities from white. All

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experiments were carried out with respect to a blank analysis, and optical conditions were the

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same in all cases.

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Analytical Chemistry

RESULTS AND DESCUSSION

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Composition of the supported liquid membrane. Composition of the supported liquid

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membrane (SLM) has a crucial effect on the EME efficiency. Non-volatile organic solvents that

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have definite conductivity, compatibility with polypropylene, less toxicity, immiscibility in

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water, and certain viscosity are worthy of investigation as the SLM in the EME. Considering

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these criteria and based on previous studies, NPOE and 1-octanol are among the widely utilized

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solvents in the EME. In addition, numerous reports in the literature have revealed that

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modification of SLM with DEHP and TEHP as carries can benefit the extraction recovery of the

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EME, especially in the case of polar compounds11. Therefore, 1-octanol and NPOE containing a

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known amount of DEHP and TEHP were evaluated. This effect is shown in Figure 2, according

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to which, NPOE had the best performance and thus was selected as the SLM for further studies.

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Effect of acceptor and donor phase composition. In EME, extraction mechanism is

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mainly based on electrokinetic migration, and thus it is of great importance for the donor phase

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composition to keep the target analytes in their ionized form. In addition, the high concentration

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of HCl in the acceptor phase facilitates the release of the analytes at the interface of the SLM and

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the acceptor phase. The ratio of HCl concentration in the donor phase to that in the acceptor

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phase is defined as ion balance. In line with the reported studies, analyte mass transfer increases

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with the reduction of ion balance22. This fact is attributed to the competition between proton ions

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and target ionized compounds, feasibility of electrolysis reactions, bubble formation, Joule

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heating phenomenon, and other instability problems23. These effects were investigated, and

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according to Figures 3A and 3B, 0 mM (ultrapure water) and 100 mM HCl were chosen as the

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composition of the donor and acceptor phases, respectively.

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Effect of applied voltage. In EME, the electrical field accounts for the main driving force

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to transfer the target analyte across the SLM into the acceptor phase. Basically, the higher the

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applied voltage, the more the extraction efficiency in the EME. Nevertheless, at high voltage

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values, extraction efficiency deteriorates as a result of increasing the current level across the

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SLM. To reach the optimum level of the applied voltage, this parameter was studied within the

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range of 50 to 90 V. The results are shown in Figure 3C. At the voltage level of 70 V, the best

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performance was achieved and was selected as the optimum value.

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Effect of sample solution flow rate. The main characteristic of the non-exhaustive

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extraction methods, such as EME, is that extraction recoveries are limited by the time in which

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the system reaches equilibrium. However, in EME, the extraction time should be adequately

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short due to the unsustainability of the SLM when the voltage is applied. This parameter was

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studied, and as can be seen in Figure 3D, at the optimum flow rate of 40 µL min-1, the system

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reached the desired equilibrium. This flow rate value, regarding 2000 µL of the sample solution,

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corresponds to an extraction time of 50 min.

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Solid phase microextraction parameters. The effective parameters on the extraction

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efficiency of the solid phase microextraction step, including the adsorption flow rate, the type of

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desorption solvent, the number of draw-eject cycles for desorption, and the desorption volume,

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were taken into account. The effect of these parameters on the extraction efficiency of the solid

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phase microextraction step can be found in Figures S1 to S4 in the Supporting Information

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section. Studies were carried out at the flow rate of 120 µL min-1, at which adsorption

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equilibrium was acquired. After the RGB detection for the subsequent analysis by the HPLC-UV

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system, the desorption step is necessary and the related parameters should be investigated. Due

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to the ionized form of the analyte in the EME and the nature of the target drug, SCX was chosen

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Analytical Chemistry

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as the solid phase material. For desorption of the analyte, a basic desorption solvent was

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required, and 20 µL of methanol containing 40% NaOH of 0.2 mol L-1 showed the best

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performance. After 11 cycles of draw-eject, the analyte was successfully desorbed.

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Method evaluation. To assess the analytical performance of the proposed method, figures

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of merit were determined in the blank urine samples by the addition of known amounts of the

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standard solution. In terms of the RGB analysis, the calibration curves were established against

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different color components. Considering the obtained results shown in Table 2, the sum of green

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and blue components showed the best linearity and was thus selected for further studies.

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Linearity, repeatability, and LODs were studied under the optimal conditions. The results are

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summarized in Table 3. In accordance with the obtained results, the level of concentration as low

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as 1.0 µg L-1 can be detected by a simple smartphone image analysis, while the corresponding

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LOD by HPLC-UV was 0.2 µg L-1. In comparison with the instrumental analysis, RGB detection

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by smartphones normally lacks sensitivity, but in this work, the concentrated accumulation of the

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analyte in the solid phase caused outstanding sensitivity. The variation in the color of the solid

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phase at different concentration levels of the case is illustrated in Figure 4A. In all experiments,

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the middle region, just above the first frit, in which the analyte was accumulated more

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homogeneously, was exploited. In this region, there is the least variation in the RGB components

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against the location of the analysis. Corresponding utilized areas for the RGB analysis are also

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shown in Figure 4B. Calibration curves were linear within the range of 30-1000 µg L-1 (r2 =

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0.9969) and 10-1000 µg L-1 (r2 = 0.9980) for RGB detection and HPLC-VU analysis,

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respectively. Relative standard deviations (RSDs), indicative of method precision, were

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evaluated as inter- and intra-assay values based on four replicate measurements. The method

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showed acceptable repeatability with inter- and intra-assay RSD% values less than 6.3 and 7.5,

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respectively.

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Table 4 is provided to compare the characteristics of the proposed method with other

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reported studies in the literature. Compared with the conventional LLE methods, which are

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associated with large amounts of organic solvents and samples and difficult handling, the chip

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device is much more user- and environmental-friendly. The chip-based device also needs lower

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amounts of samples and enjoys less power consumption in comparison with conventional EME.

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Moreover, as opposed to the conventional extraction methods, in this procedure samples can be

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used without any pretreatment owing to the remarkable clean-up of the first membrane-based

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step, which can be a prominent strategy to eliminate pretreatments in the case of solid phase

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methods. More importantly, the main advantage of the proposed system is the simplicity of the

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instrumentation and detection. Detection by the image analysis tool is capable of direct and

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sensitive analyte quantification in complicated matrices. Additionally, elimination of the liquid

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chromatography decreases the cost of analysis and organic solvents used for the elution. It goes

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without saying that detection via smartphone is an available approach which allows in-field

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analysis.

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It is noteworthy that in the case of detecting several colorful species, the analytes can be

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accumulated in different distinct zones of the solid phase by further elution similar to what we

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have in the chromatographic columns. Another fascinating approach for eliminating

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interferences or multi detection is benefiting from the curve resolution methods. This device is a

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simple prototype, paving a new way for sensitive portable detection by LOC devices.

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Additionally, in the case of colorless analytes, derivtization and tagging by a chromophore would

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be helpful, and the interaction between a fluorophore and desired analytes can be even used as an

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Analytical Chemistry

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analytical signal for quantification. Further studies are also being conducted on this issue and the

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results will be reported in not too distant future.

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Analysis of real samples. The LOC system was used for the analysis of the target drug in

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urine samples of patients being treated with PP to assess the applicability of the method. The

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results are presented in Table 5. Accuracy was evaluated as the parameter of error%. The

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obtained values were less than 8.2% for both analysis modes. Furthermore, RSD% values are

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indicative of acceptable precision related to the proposed method. Figures 5A and 6A are typical

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chromatograms, resulted from extraction of PP from patients’ urine samples before and after the

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addition of certain concentration levels. As can be seen, excellent clean-up was acquired

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considering the fact that the samples were used without any pretreatment. In addition, the

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corresponding image analysis of the blank and spiked samples is illustrated below each

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chromatogram (Figures 5B and 6B).

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To validate the RGB method and compare the obtained results by both detection modes, the

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extraction of each real urine sample was repeated three times. The acquired average

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concentration level in each urine sample by the two analysis modes were assessed by a t-test at

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the confidence level of 95%. The related results are shown in Table 6. According to the

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performed t-test, there was no significant difference between the calculated concentration values

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by the RGB method and the HPLC-UV system and both confirmed each other. Therefore, the

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RGB method can be utilized as a reliable, accurate detection mode for sensitive and in-situ

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analysis of the model analyte with the minimum cost.

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CONCLUSIONS

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In the present research, a new LOC device based on the combination of EME with a solid

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phase as the medium for RGB analysis was proposed. Although RGB analysis via smartphones

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normally suffers from poor sensitivity, in this method utilization of the solid phase as a medium

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for concentrated accumulation of the model analyte caused remarkable sensitivity as compared

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with the conventional chromatographic methods. It was shown that RGB intensities can be used

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as a reliable concentration-dependent signal to quantify analytes from the complicated matrices.

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The proposed device can be considered as an encouraging strategy for further developments in

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smartphone-based LOC systems for in-field and inexpensive detections.

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ACKNOWLEDGMENTS

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This work was supported by a grant from the Program of Cooperation (PoC) in Science and

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Technology between the Ministry of Science and Technology, Government of the Islamic

307

Republic of Pakistan and the Ministry of Science, Research, and Technology, Government of the

308

Islamic Republic of Iran for the years 2017-2019 (Project No. 962.201.19503). The authors also

309

gratefully acknowledge the support of University of Sistan and Baluchestan.

310

REFERENCES

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(1) Zhang, Y.; Timperman, A. T. Analyst 2003, 128, 537-542.

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(2) Maruyama, T.; Kaji, T.; Ohkawa, T.; Sotowa, K.-i.; Matsushita, H.; Kubota, F.; Kamiya, N.;

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(9) Ebrahimpour, B.; Yamini, Y.; Seidi, S.; Tajik, M. Anal, Chim. Acta 2015, 885, 98-105.

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(11) Yamini, Y.; Seidi, S.; Rezazadeh, M. Anal. Chim. Acta 2014, 814, 1-22.

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(12) Pedersen-Bjergaard, S.; Rasmussen, K. E. J. Chromatogr. A 2006, 1109, 183-190.

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(13) Kjelsen, I. J. Ø.; Gjelstad, A.; Rasmussen, K. E.; Pedersen-Bjergaard, S. J. Chromatogr. A 2008,

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(15) Gjelstad, A.; Pedersen-Bjergaard, S. Anal. Methods 2013, 5, 4549-4557.

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(16) Rezazadeh, M.; Yamini, Y.; Seidi, S.; Esrafili, A. J. Chromatogr. A 2012, 1262, 214-218.

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Figures captions Figure 1. (A) Equipment used for EME chip device. (B) Schematic illustration of the whole extraction and detection procedure. Figure 2. Effect of SLM composition on extraction efficiency. PP was extracted by the voltage of 60 V, flow rate of 50 µL min-1, 0 and 100 mM HCl for the donor and acceptor phases, respectively. Figure 3. Optimization of (A) donor phase composition, (B) acceptor phase composition, (C) voltage, and (D) flow rate. Figure 4. (A) Final digital images of the solid phase after the analyte absorption as a medium for the RGB analysis at different concentration levels of the case. (B) Utilized zones for the RGB analysis. Figure 5. (A) Typical chromatogram of PP extraction from urine sample of a patient after and before the addition of 200 µg L-1. (B) Corresponding images used for the RGB analysis. Figure 6. (A) Obtained chromatograms after the LOC procedure ((a) non-spiked, (b) spiked at the concentration level of 40 µg L-1. (B) Corresponding images for the RGB analysis.

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Analytical Chemistry

Table 1 Chemical structure and corresponding pKa and Log KO/W values of PP. Name

Chemical structure

Phenazopyridine

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pka

Log KO/W

6.86

2.69

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Table 2 Evaluation of calibration curves against different RGB components Analysis component

Linear dynamic range (µg L-1)

R2

Green

50.0-1000.0

0.9686

Blue

50.0-1000.0

0.9700

Green + Blue

30.0-1000.0

0.9969

Green + Red

50.0-1000.0

0.9657

Blue + Red

50.0-1000.0

0.9649

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Analytical Chemistry

Table 3 Analytical performance of LOC device for determination of PP from urine samples by both the detection approach of HPLC-UV and the RGB analysis Linearity R2 PFa LOQ RSD%b Analysis LOD method (µg L-1) (µg L-1) (µg L-1)

HPLC/UV 0.2 RGB a

1.0

Inter-assay

Intra-assay

5.0

5.0-1000.0

0.9980 25

5.2

7.1

30.0

30.0-1000.0

0.9969 ---

6.3

7.5

Preconcentration based on four-replicate measurements at 250 µg L-1 for HPLC. Based on four-replicated measurements.

b

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Table 4 Comparison of analytical performance of the proposed method with other reported methods for PP determination Method Matrix LOD RSD% LDR R2 (µg L-1)

(µg L-1)

Ref.

LLE/GC -MSa

Plasma

0.3

5.0-500.0

0.9992

2.7

24

LLE/HPLC-UVb

Plasma

10.0

50.0-1000.0

0.9986

8.3

25

Tablets

10.0

2000.0-20000.0

0.9999

-

26

Urine

0.5

1.0-1000.0

0.9960

4.6

27

Plasma

1.0

5.0-1000.0

0.9985

5.9

27

OC-EME/HPLC-UVd

Urine

0.2

5.0-1000.0

0.998

5.2

This work

OC-EME/RGB analysise

Urine

1.0

50.0-1000.0

0.9934

6.3

This work

EME/HPLC-UVc

a

Liquid-liquid extraction-gas chromatography mass spectrometry detection. Liquid-liquid extraction-liquid chromatography ultraviolet detection. c Electromembrane extraction. d On-chip electromembrane extraction. e On-chip electromembrane extraction-RGB image analysis detection. b

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Analytical Chemistry

Table 5 Determination of PP in real samples Sample

Urine 1a

Urine 2b

a b

Analysis method

Creal

Cadded

Cfound

RSD%

(µg L-1)

(µg L-1)

(µg L-1)

(n=4)

HPLC/UV

237.3

200.0

433.7

5.8

-1.8

RGB

234.4

200.0

440.1

7.2

2.8

HPLC/UV

33.3

40.0

72.8

6.6

-1.2

RGB

39.2

40.0

75.9

7.6

-8.2

After 9 h. After 12 h.

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Error%

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Table 6 Validation of the RGB method by t-test Urine 1 -1

PP conc. (µg L )

Average Standard deviation Spooled ttest tTable(4,0.05) = 2.776

HPLC 252.6 234.1 225.2 237.3 13.9

Urine 2 RGB 252.9 230.1 220.2 234.4 16.7

HPLC 36.2 30.6 33.1 33.3 2.8

15.4 2.9 0.23 2.44 ttets˂ tTable(4,0.05) , Not significant

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RGB 35.6 40.9 41.1 39.2 3.1

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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Analytical Chemistry

For TOC only

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