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View Article OnlineView Journal | View Issue

Electrochemicall

Chemistry & Chemical Engineering Resear

Tehran, Iran. E-mail: ahmadi@ccerci.ac.ir;

Cite this: Analyst, 2015, 140, 497

Received 10th September 2014Accepted 28th October 2014

DOI: 10.1039/c4an01664e

www.rsc.org/analyst

This journal is © The Royal Society of C

y controlled in-tube solid phasemicroextraction of naproxen from urine samplesusing an experimental design

Seyyed Hamid Ahmadi,* Ahmad Manbohi and Kourosh Tabar Heydar

A new in-tube solid phase microextraction approach named electrochemically controlled in-tube solid

phase microextraction (EC in-tube SPME) has been reported. In this approach, in which electrochemistry

and in-tube SPME were combined, the total analysis time was decreased and the sensitivity was

increased. After electropolymerization of pyrrole on the inner surface of a stainless steel tube, the

polypyrrole (PPy)-coated in-tube SPME was coupled on-line to high performance liquid chromatography

(HPLC) to achieve automated in-tube SPME-HPLC analysis. After the completion of the EC-in-tube

SPME-HPLC system, the PPy-coated tube was used as a working electrode for the uptake of naproxen.

It was found that the extraction efficiency could be significantly enhanced using the constant potential.

Plackett–Burman design was employed for screening, to determine the variables significantly affecting

the extraction efficiency. The significant factors were then optimized using a Box–Behnken design. The

linear range and detection limit (S/N ¼ 3) were 0.5–1000 and 0.07 mg L�1, respectively. Urine samples

were successfully analyzed by the proposed method.

Introduction

Naproxen is a non-steroidal anti-inammatory drug (NSAID)commonly used for the reduction of moderate to severe pain,fever, inammation and stiffness. Similar to other NSAIDs,naproxen is capable of producing disturbances in the gastro-intestinal tract.1 Naproxen, as other NSAIDs, is excreted in twodifferent forms: free and forming glucoronides. The clinical andpharmaceutical analysis of these drugs require effectiveanalytical procedures for quality control and pharmacody-namics and pharmacokinetic studies. Several chromatographicmethods have been reported for the determination of naproxenin raw material,2 tablets,3–5 plasma,6–8 urine,9 intestinal perfu-sion samples10 and pharmaceutical preparations.11,12

For the analysis of drugs in complex matrices, sampletreatments such as extraction, preconcentration and clean-upsteps are oen required to improve the sensitivity and selec-tivity. However, traditional off-line methods such as solventextraction are time-consuming and labor-intensive, requirelarge volumes of sample and solvent, suffer from huge risks ofcontamination and analyte loss, and need additional instru-mentation to automate.13

SPME, which has obtained widespread acceptance in manyareas,14,15 can overcome the problems of the traditionalmethods by eliminating the use of organic solvents and inte-grating sample extraction, concentration, and introduction into

ch Center of Iran, P.O. Box 14334-186,

Tel: +98-021-44580799

hemistry 2015

a single step. In-tube SPME is a version of SPME, which can beeasily coupled on-line with HPLC for the analysis of less volatileand thermally labile compounds.16 In-tube SPME allows for theconvenient automation of the extraction process, which notonly saves analysis time but also provides better precisionrelative to manual techniques.14

In the development of the SPME technique, it has been achallenge to extract polar and ionic analytes from watersamples. Among the different types of sorbents used for theextraction of analytes, conducting polymers have attractedconsiderable attention.17 Pyrrole can be polymerized withoxidation reactions by either an electrochemical or a chemicalmethod.18–20 In several works, chemical polymerization of PPyon the inner surface of a capillary has been reported.13,14,21–23

Limitations encountered with this method include thefollowing: (1) because there are no interactions between PPyand the capillary, adhesion of PPy to the capillary is not strongand this can affect the mechanical stability of the polymer. (2)Although there are some reports on producing electricalconductive PPy by chemical polymerization,24 these PPy are notused in SPME and especially in in-tube SPME because theelectrical conductivity of PPy produced by chemical methods isusually low. (3) The number of dopants in chemical polymeri-zation methods are very limited. (4) Physical properties of thesynthesized polymer cannot easily be controlled.

Electrochemical synthesis is more convenient, because thepolymer is directly electrodeposited on the surface of metalsfrom an aqueous solution containing pyrrole and electrolyte.Electrochemically controlled solid-phase microextraction

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(EC-SPME), which was rst suggested by Pawliszyn et al.,combines SPME with electrochemistry.25 EC-SPME has anadvantage over regular SPME, because the neutral charge ofcommercially available SPME apparatus leads to poor analyterecoveries and low analyte partition coefficients for some ana-lytes.26 EC-SPME has been successfully used in the matrixseparation of anionic,27–29 cationic,30 and neutral analytes.31

In a previous report, we electrosynthesized molecularlyimprinted PPy lm on the surface of stainless steel wire as aselective sorbent for benzoate anions.27 In the current work, PPylm was elctropolymerized on the inner surface of a stainlesssteel tube. Then, the PPy-coated tube was coupled on-line toHPLC to achieve an automated in-tube SPME and HPLC setup.Aer the completion of the EC-in-tube SPME-HPLC setup, thePPy-coated tube acted as a working electrode for the uptake ofnaproxen. A Plackett–Burman design was employed forscreening to determine the variables signicantly affecting theextraction efficiency. Then, the signicant factors were opti-mized by using a Box–Behnken design. Finally, the optimizedprocedure was employed to determine naproxen in urinesamples.

ExperimentalChemicals and materials

Naproxen and other materials were purchased from Merck(Darmstadt, Germany). All solvents were HPLC grade and werepurchased from Sigma-Aldrich (Steinheium, Germany). Milli-Qwater (Millipore, Billerica, MA, USA) was used to prepare thesamples.

The required amount of naproxen was dissolved in methanolto obtain a stock solution of the analyte with a concentration of1 mg mL�1. Working standard solutions were freshly preparedby diluting the standard solution of the analyte with ultra-purewater to the required concentrations. Pyrrole was obtained from

Fig. 1 Illustration of the setup for electropolymerization of PPy in thesolution, (3) flow pump, (4) waste solution. WE and CE indicate working

498 | Analyst, 2015, 140, 497–505

Merck (Darmstadt, Germany), distilled and kept under nitrogenin the dark at 4 �C before use. pH was adjusted with 0.1 Msodium hydroxide and hydrochloric acid solutions.

Electrosynthesis of PPy on the inner surface of the tube

Before synthesis, the stainless steel tube (10 cm � 0.80 mm i.d.)was cleaned with amixture of acetone and pure water (70 : 30, v/v). This solution was percolated through the tube for 10 min,and then the tube was thoroughly rinsed with water, acetoneand water, respectively. A solution of 0.1 mol L�1 pyrrole and 0.2mol L�1 lithium perchlorate was placed into a container, purgedwith N2 gas for 2 min, and then used as the mother solution forelectrosynthesis of PPy in the tube. A very thin copper wire (15cm� 0.07 mm diameter) was placed into the tube. The tube andthe wire serve as the working and counter electrodes, respec-tively (Fig. 1). Insulating material was used to prevent electricalconnection between these electrodes. By passing the solutionthrough the tube and applying a constant current of 3 mA, theelectrochemical polymerization of pyrrole was carried out. Inorder to have a PPy-coated tube with good mechanical stability,the peristaltic pump (Master Flex, model 7013-20, Chicago, IL,USA) was turned on and turned off for 30 and 20 s, respectively.This process was performed for 300 s with the constant currentof 3 mA. Following electropolymerization, the wire was removedand the stainless steel tube was washed with methanol toremove the unreacted component. Finally, it was xed in theplace of the injection loop of the HPLC system. When the tubewas not in use, it was lled with 0.01 mol L�1 LiClO4 at roomtemperature.

Scanning electron microscopy (SEM)

The PPy-coated tubes were cut into 1 cm long pieces, and thenanalyzed using a VEGA3 TESCAN scanning electron microscope(20 kV accelerating potential). The approximate thickness of the

tube: (1) power supply (electrochemical device), (2) polymerizationand counter electrodes, respectively.

This journal is © The Royal Society of Chemistry 2015

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PPy was estimated using ImageJ soware (National Institutes ofHealth, USA, version 1.48V).

Instrumentation and analytical conditions

Separation and detection of the naproxen was performed by aKnauer HPLC containing a K-1001 WellChrom HPLC pump anda K-2600 UV-vis detector. Chromatographic data were recordedand analyzed using ChromGate soware (Knauer), version 3.1.The separation was carried out on a C18 column (250 mm � 4.0mm, with 5 mm particle size). The mobile phase consisted of 10mM phosphate buffer, pH 3, and methanol (80 : 20). The owrate of the mobile phase was set at 1.0 mL min�1. Detection wasperformed at the wavelength of 230 nm.

Electrochemical experiments were performed using a Beh-pajuh (BHP 2064+) potentiostat (Isfahan, Iran). All experimentswere carried out at ambient temperature. All pH measurementswere performed at 25 � 0.1 �C using a pH-meter Metrohm 780with a standard uncertainty of 0.1 mV (Metrohm, Switzerland).

EC in-tube SPME-HPLC

The stainless steel tube was xed in the place of the injectionloop of the HPLC system. To complete the three-electrode setup,two zero dead volume internal unions were used as counter andreference electrodes (Fig. 2). Insulating materials were used toprevent short-circuit among these three electrodes. The in-tubeSPME-HPLC setup consisted of two segments: chromatographic(HPLC) and in-tube SPME segments, where a six-port injectionvalve was used to join them. The main parts of the in-tube SPMEsegment consisted of a three-electrode system, an

Fig. 2 Schematic of the EC in-tube SPME-HPLC setup.

This journal is © The Royal Society of Chemistry 2015

electrochemical device and a ow pump. 1/16 in. stainless steeland polyether ether ketone (PEEK) nuts and ferrules were usedto complete the connections. The PPy-coated tube was washedand conditioned by ultrapure water before use as shown inFig. 2 (LOAD), the electroextraction was performed by passingthe sample solution (15 mL) through the stainless steel tubingusing the ow pump when the six-port valve was set to the loadposition. At this time, a positive three-electrode constantpotential was applied. In the load position, the mobile phasewas driven by the HPLC pump through the analytical column toobtain a at baseline in preparation for chromatographicseparation. To prevent contamination of the analytical columnwith residual compounds, the PPy-coated tube was washed withwater (1 mL). Then, 1 mL of the 10 mM buffer solution, pH of 3,and acetonitrile (50 : 50, v/v) was percolated into the tube anddesorption of the analyte was carried out in static mode. Aerthat, the valve was switched to the inject position (Fig. 2,INJECT) and the desorbed analyte was introduced into theHPLC system. The detail of the extraction and separationprogram is listed in Table 1.

Sample preparation

Urine samples were collected from drug-free, healthy volunteers.Any precipitatedmaterial was removed by centrifuging the sampleat 4000 rpm (Hettich, Rotox 32A) for 10 min. The supernatant ofurine was directly spiked with naproxen, diluted 5 times withwater, and adjusted to pH 9 with NaOH. Since urine samples arecomplicated, to reduce interferences, dilution is needed. Thismethod was focused on the determination of the free drug.

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Table 1 Program for EC in-tube SPME-HPLC processa

EventConstant owpump Valve

Wash the tube with H2O Run LoadExtraction of analyte into the PPy-tube Run LoadWash the tube with H2O Run LoadDesorption of analyte in static mode Stop LoadSeparation and detection Stop InjectWash the tube with H2O Run Load

a The ow rate for extraction and washing was kept at 1 mL min�1.Other in-tube SPME conditions are outlined in the text.

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Experimental design approach

Experimental design strategies were applied in two stages: (i)screening the signicant factors in microextraction of naproxenusing a Plackett–Burman design and (ii) optimizing the signif-icant factors using a Box–Behnken design. A Plackett–Burmandesign was applied to evaluate the signicance of six variablesand consists of three central points. Design generation andstatistical analyses were performed by means of the sowarepackage Statgraphics Plus version 5.1 and the Minitab version16.2.2.

Results and discussionElectrosynthesis of PPy

A very thin copper wire was placed into the tube. The tube andthe wire served as working and counter electrodes, respectively.The pump was kept on and off for 30 and 20 s, respectivelythroughout the whole electro-polymerization time. By passingthe solution through the tube and applying a constant currentof 3 mA, electrochemical polymerization of pyrrole was carriedout, thus producing homogeneous PPy with good mechanicalstability. When the pump was kept off during the synthesis,pyrrole monomers had enough time to be polymerized to PPy

Fig. 3 The SEM of PPy-coated tube with magnification of (A) 4.17k� an

500 | Analyst, 2015, 140, 497–505

polymer and when the pump was kept on, gas produced in thecounter electrode and other impurities le the tube.

Characterization of the PPy-coated tube

The morphology of the PPy was investigated by scanning elec-tron microscopy (SEM). The morphology and quality of a poly-meric lm are among the critical features required for its abilityto serve as a SPME device. Porosity of the lm surface and thesize of the inclusion sites vary under different polymerizationconditions.27,28 As can be seen from Fig. 3A, the PPy–ClO4 lmshows a lamentous morphology. The SEM image also showsthat the PPy were homogeneously distributed in the stainlesssteel tube (Fig. 3B). The thickness of the polymer was about10 mm.

Conguration of EC in-tube SPME-HPLC

Before performing EC in-tube SPME and to ensure that thesetup worked correctly, two experiments were designed. A 0.01mol L�1 lithium perchlorate solution was passed through thesystem and cyclic voltammetry (CV) with scan rate of 50 mV s�1

was applied. Furthermore and for comparison, a PPy stainlesssteel ber was immersed in an offline three-electrode cell andthe same CV test was applied. In the offline experiment, thecounter and reference electrodes were the same as for theonline experiment. As can be seen in Fig. 4, the cyclic voltam-mogram obtained by the two experiments were approximatelythe same. These results highlighted two important notes: (1) thePPy-coated tube was able to properly uptake and releaseperchlorate anions, and (2) there were electrical connectionsamong the three electrodes.

Aer ensuring the electrical connections, a 100 ng mL�1

solution of naproxen was passed through the system. Theconductivity of the solution was low and therefore, forincreasing the conductivity, LiClO4 salt was added. Addition ofthis salt increased the solution conductivity.

d (B) 173�.

This journal is © The Royal Society of Chemistry 2015

Fig. 4 Cyclic voltammograms of 0.01 mol L�1 LiClO4 solution at scanrate 50 mV s�1 obtained by online (dashed-line) and offline (line)experiments.

Fig. 5 Normal probability plot (A) and Pareto chart (B) of standardizedeffects in Plackett–Burman design.

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Optimization of EC in-tube SPME-HPLC conditions

Screening of signicant factors. The Plackett–Burmandesign was used as a screening method in order to select thefactors that inuence the microextraction of naproxen by EC in-tube SPME. The Plackett–Burman design is practical especiallywhen the investigator faces a large number of factors. Initially,we selected six factors that were potentially affecting themicroextraction of naproxen. Table 2 shows the factors andlevels applied in the Plackett–Burman design where thedelimitation of experimental regions for each factor was deter-mined from the preliminary experiments. The estimates of themain effects of the factors are shown on a normal probabilityplot of effects (Fig. 5A). All effects that are insignicant arenormally distributed with mean zero and variance s2, and tendto fall along a straight line in the plot. In contrast, signicanteffects have non-zero means and are located far away from thestraight line. The larger the signicant effects, the further awaythey are from the straight line. The results obtained from thenormal probability plot of effects (Fig. 5A) are conrmed with aPareto chart as shown in Fig. 5B. The vertical line in the Paretochart indicates the minimum statistically signicant effectmagnitude for 5% signicance level, while the horizontalcolumn lengths are proportional to the degree of signicance

Table 2 Factors and levels used in the experimental Plackett–Burmandesign

Factors

Levels

Low (�1) High (+1)

pH 3 9Uptake potential (V) 0.2 1.2Uptake time (s) 100 600Release time (s) 100 600Concentration of NaCl (M) 5 � 10�3 5 � 10�2

Flow rate (mL min�1) 1.1 1.5

This journal is © The Royal Society of Chemistry 2015

for each effect. Any effect or interaction that exceeds the verticalline is considered signicant. The factors that were signicantat 5% level (P < 0.05) from the regression analysis wereconsidered to have greater impact on the microextraction ofnaproxen by EC in-tube SPME and were further optimized.Fig. 5 shows that the important factors are uptake potential, pHof solution, and uptake time. To reduce the total extractiontime, the release time of 350 s was selected. Furthermore, theconcentration of NaCl and the ow rate were kept constant incenter values.

Optimization using response surface methodology. One ofthe main advantages of the Box–Behnken design matrix is thatit does not contain combinations for which all factors aresimultaneously at their highest or lowest levels. Therefore thisdesign is useful to avoid experiments performed under extremeconditions.32–34 The levels of signicant factors and their inter-action effects, which inuence the microextraction of naproxenwere analyzed and optimized by the Box–Behnken design. Thenumber of experimental points (N) is dened by the expression:

N ¼ 2k(k � 1) + Cp (1)

where k is the number of variables and Cp is the number ofcenter points. In this study, k and Cp were set at 3 and 3,respectively, which indicate that 15 experiments had to be run.The examined levels of the factors are given in Table 3. Themultiple regression analysis on the resulted response led to the

Analyst, 2015, 140, 497–505 | 501

Table 3 Experimental variables, levels and star points of theBox–Behnken design

Factors

Levels

Optimum valueLow (�1) Central (0) High (+1)

pH 3 6 9 3Uptake potential (V) 0.2 0.7 1.2 1.2Uptake time (s) 100 350 600 350

Fig. 6 Pareto charts of the main effects in the Box–Behnken design.AA, BB, and CC are the quadratic effects of the pH of extraction phase,potential of extraction phase, and extraction time, respectively. AB, AC,and BC are the interaction effects between pH and potential ofextraction phase, pH of extraction phase and extraction time, potentialof extraction phase and extraction time, respectively.

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following second-order polynomial equation, which explainsthe relation of peak area of HPLC with signicant factors andinteractions:

Peak area ¼ 420000.0 � 77339.4 pH + 198243.0 uptake potential

+ 67561.6 uptake time + 52493.3 pH2

� 105415.0 pH � uptake potential

+ 13900.5 pH � uptake time

+ 82922.0 uptake potential2

+ 56540.8 uptake potential

� uptake time � 85514.3 uptake time2 (2)

The adequacy of the model was checked using the analysis ofvariances (ANOVA) and the results are shown in Table 4 andFig. 6. The quality of t of the quadratic polynomial model wasexpressed by the coefficient of determination, R2. According toJoglekar and May,35 R2 should be at least 0.80 for a good t of amodel. In this study, the R2 was 96.67%, which means that theobtained equation was adequate for correlating the experi-mental results. Fig. 7 illustrates the interaction among theinvestigated factors. The existence of interaction means that thefactors may affect the response interactively and not indepen-dently. Thus, their combined effect is greater or less than thatexpected for the straight addition of the effects. Furthermore,these response surface graphs allowed the determination of anoptimal zone. The optimum value for microextraction of nap-roxen was obtained when the pH, uptake potential, uptake time,concentration of NaCl, ow rate, and release time were at

Table 4 Results of analysis of variancea

Source Sum of squares DF Mean square F-ratio p-value

pH 4.78 � 1010 1 4.78 � 1010 13.28 0.015E 3.14 � 1011 1 3.14 � 1011 87.25 0.002T 3.65 � 1010 1 3.65 � 1010 10.13 0.024pH*pH 1.04 � 1010 1 1.02 � 1010 2.82 0.154E*E 2.97 � 1010 1 2.54 � 1010 7.05 0.045T*T 2.70 � 1010 1 2.70 � 1010 7.49 0.041pH*E 4.45 � 1010 1 4.44 � 1010 12.34 0.017pH*T 7.73 � 108 1 7.73 � 108 0.21 0.663E*T 1.28 � 1010 1 1.28 � 1010 3.55 0.118Total error 1.80 � 1010 5 3.60 � 109

Total (corr.) 5.42 � 1011 14

a R-squared ¼ 96.6752%, R-squared (adjusted for d.f.) ¼ 90.6907%,standard error of est. ¼ 60028.7.

Fig. 7 Response surfaces for naproxen using the Box–Behnkendesign obtained by plotting (A) pH of extraction phase vs. potential ofextraction phase, (B) extraction time vs. potential of extraction phase,and (C) pH of extraction phase vs. extraction time.

502 | Analyst, 2015, 140, 497–505 This journal is © The Royal Society of Chemistry 2015

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optimum points. Accumulation was achieved by the appliedpositive potentials. During the oxidation of polypyrrole, thepositive charges were formed on the PPy chain and were used toextract the analyte from the solution. In the next step, thecharge of the PPy was neutralized, and therefore the anionswere released. The inuence of the potential used during thenaproxen uptake step is shown in Fig. 7. The uptake amount fornaproxen increased signicantly with increasing appliedpotential. The uptake of the naproxen to PPy-coated tube wasfound to decrease when negative potentials were applied andincrease with increasing positive potentials.

Desorption of naproxen from the PPy-coated tube. The tubewas washed with water (1 mL) aer each extraction to eliminatethe proteins and other weakly adsorbed components. Someanalytes, such as naproxen, had stronger interactions with theextraction phase and could not be desorbed easily by the mobilephase. Therefore, 1 mL of 10 mM buffer solution, pH of 3, andacetonitrile (50 : 50, v/v) was drawn into the PPy-coated tube toassist desorption of naproxen before switching the valve to theinject position (static mode). No carryover was found aer 350 sof desorption, which was conrmed by the blank analysis per-formed aer extraction.

Precision, limit of detection, and linearity

The repeatability of the EC in-tube SPME-HPLC (run-to-runRSD) was calculated over three analyses of naproxen with thePPy-coated tube. The RSD value obtained is 5.1. Furthermore, tostudy the reproducibility of the PPy-coated tube construction(tube-to-tube RSD), two PPy-coated tubes were electro-poly-merized under the same conditions and used as sorbents in theEC in-tube SPME setup. The RSD of 6.3% was obtained (N ¼ 3).The low RSD values bear evidence to the fact that the

Table 5 Figures of merit of the proposed EC in-tube SPME-HPLC meth

Regression equation

Linearity

LDR R2

y ¼ 7106.3x + 1242.85 0.5–1000 0.9990

a All concentrations are in mg L�1.

Table 6 Comparison of the proposed method with other methods appl

Methoda Linearityb

CS-MNPs-spectrouorimetry 40–1000DLPME-HPLC-UV 100–10 000SPMTE-HPLC-UV 10–10 000DPV 1000–25 000SPME-LC-UV 200–20 000MMSPD-HPLC 50–700SPE-HPLC-DAD 1–50EC in-tube SPME-HPLC 0.5–1000

a Chitosan–polypyrrole magnetic nanocomposite (CS–MNPs), dynamic liqphase membrane tip extraction (SPMTE), differential pulse voltammetphase dispersion (MMSPD), diode array detector (DAD). b The linearitminutes. c Initial volume of 200 mL.

This journal is © The Royal Society of Chemistry 2015

application of a constant electric potential to the PPy-coatedtube was a reliable and reproducible approach to achievequantitative extraction capacity in in-tube SPME extraction bymeans of EC in-tube SPME. The concentration level for calcu-lation of the RSD is 50 mg L�1. The PPy-coated tube has a goodmechanical stability and can be used up to 50 times without atangible change in stability and extraction efficiency.

Calibration curve (peak area counts against analyte concen-trations) parameters are shown in Table 5. The EC in-tube SPMEmethod can enhance sensitivity more than 5 and 12 timesrelative to in-tube SPME and direct injection method (25 mLinjection), respectively. The value of the correlation coefficientshowed an acceptable linearity in the dynamic range. Fromthese results, it can be concluded that the PPy-coated tube couldreliably be used for naproxen. A comparison between the guresof merit of the proposed method and some of the publishedmethods for extraction and determination of naproxen issummarized in Table 6. Clearly, the proposed method has agood sensitivity with a wide dynamic linear range and shortextraction time in comparison to the other methods.

Analysis of urine samples

The applicability of the PPy-coated tube for EC in-tube SPME-HPLC was investigated for urine samples. The microextractionof naproxen from urine samples was performed under theoptimum conditions of EC in-tube SPME with the PPy-coatedtube. The recoveries were between 92–99% (Table 7). As shownin Fig. 8A, no interference peaks were observed in the non-spiked urine sample. Fig. 8B shows a typical chromatogram,obtained aer the EC in-tube SPME-HPLC of a spiked urinesample. Furthermore, for comparison, the chromatogram of

oda

LOD

Precision (RSD%, n ¼ 3)

Run-to-run Tube-to-tube

0.07 5.1 6.3

ied for the extraction and determination of naproxen in urine samples

LODb Extraction timeb Ref.

15 16 3670 35 378 30 38

240 <2 3930 40 4010c >10 411 49 420.07 11.67 This work

uid-phase microextraction (DLPME), solid phase extraction (SPE), solidry (DPV), solid phase microextraction (SPME), magnetic matrix solidy and LOD are expressed in mg L�1. Extraction time is expressed in

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Table 7 Performance of the proposed method for extraction ofnaproxen from the urine samples

SampleAdded(mg L�1)

Found(mg L�1)

RSD%(n ¼ 3) Recovery (%)

Urine 1 10 9.2 6.78 9250 47.6 7.31 95.2

Urine 2 10 9.9 5.96 9950 48.4 7.64 96.8

Fig. 8 HPLC chromatograms of naproxen in urine before spiking (A),after spiking with 0.1 mg mL�1 using the EC in-tube SPME methodcombined with HPLC-UV (B), and using the in-tube SPME-HPLCmethod under optimum conditions (C). The peak area of naproxen inthe case of EC in-tube SPME-HPLC (B) is 712021.

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naproxen aer in-tube SPME-HPLC (without the use of elec-trical potential) was inserted in Fig. 8C.

Conclusions

In this paper, the electropolymerization of pyrrole inside astainless steel tube was successfully performed. Then, thisPPy-coated tube was applied in the EC in-tube SPME-HPLCsetup for the determination of naproxen in urine samples. Ithas been demonstrated that the application of a constantelectric potential to the PPy-coated tube signicantlyimproves the extraction efficiencies of naproxen. The longlifetime, fast analysis, easy automation, reduced solventrequirement and simplication of the whole analyticalprocedure are major points in favor of using the PPy-coatedEC in-tube SPME-HPLC for this purpose. The Plackett–Bur-man design was employed for screening to determine thevariables signicantly affecting the extraction efficiency.The signicant factors were then optimized by using aBox–Behnken design.

504 | Analyst, 2015, 140, 497–505

This EC in-tube SPME-HPLC method can be extended tothe analysis of other groups of analytes with littlemodication.

Acknowledgements

The nancial support of the Research Council of Chemistry andChemical Engineering Research Center of Iran (CCERCI) isgratefully acknowledged.

Notes and references

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