Electromembrane surrounded solid phase microextraction using electrochemically synthesized nanostructured polypyrrole fiber

Journal of Chromatography A, 1443 (2016) 75–82

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Electromembrane surrounded solid phase microextraction using electrochemically synthesized nanostructured polypyrrole fiber Elham Mohammadkhani a , Yadollah Yamini a,∗ , Maryam Rezazadeh a , Shahram Seidi b a b

Department of Chemistry, Tarbiat Modares University, P. O. Box 14115-175, Tehran, Iran Department of Analytical Chemistry, K. N. Toosi University of Technology, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 8 December 2015 Received in revised form 28 February 2016 Accepted 22 March 2016 Available online 24 March 2016 Keywords: Electromembrane extraction Solid phase microextraction Gas chromatography Urine Whole blood

a b s t r a c t Electromembrane surrounded solid phase microextraction using conductive polymers as the sorbent is carried out for the first time for extraction of two antidepressants including amitriptyline (AMI) and doxepin (DOX), as model analytes. The polypyrrole coating was prepared and utilized as both cathode and SPME sorbent. Different variables such as the conditions for preparation of polypyrrole fiber, pH of the donor and the acceptor phases, applied voltage, and extraction time were optimized. Under the optimized conditions, figures of merit of the proposed method were investigated in human whole blood and urine samples. Intra- and inter-assay precisions ranged between 3.1–7.5% and 7.6–12.3%, respectively were obtained in different extraction media. Detection limits of 0.15 and 0.05 for AMI and 0.3 and 0.1 ng mL−1 for DOX were achieved in the urine and blood samples, respectively. Linearity of the method was studied up to 50.0 ng mL−1 for both analytes and coefficients of determination better than 0.9966 were achieved. Regardless of the high sample cleanup, which makes the proposed method suitable for analysis of drugs from complicated matrices, clean chromatograms were obtained. Finally, the proposed method was applied for analysis of AMI and DOX in different real samples and reasonable data were obtained. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Sample preparation is an important issue in analytical chemistry which is often a bottleneck for chemical analysis. As a consequence, a series of steps is required to remove interfering substances, preconcentrate the analyte and increase the sensitivity. Traditionally, liquid–liquid extraction (LLE) has been used for pre-treatment of biological samples, but LLE is laborious and requires environmentally toxic solvents. Due to several advantages, solid-phase extraction (SPE) has become more popular [1] compared to LLE, but it also requires an organic solvent for elution of analytes, solvent evaporation step prior to final analysis, relatively high cost of SPE cartridges and their blockage during extraction procedure. In recent years, modern trends in analytical chemistry are towards simplification, miniaturization, automation, and minimization of organic solvent used in sample preparation. Stir-bar sorptive extraction (SBSE) [2], solid-phase microextraction (SPME) [3], and liquid phase microextraction methods (LPME)

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (Y. Yamini). http://dx.doi.org/10.1016/j.chroma.2016.03.067 0021-9673/© 2016 Elsevier B.V. All rights reserved.

are miniaturized techniques [4], which have been developed for sample preparation. SPME and SBSE are simple and solventless methods. However, the major disadvantages of SPME are its high cost, sample carry-over, fiber fragility, and limited lifetime of the fiber [5]. SBSE needs relative long extraction (30–120 min) and desorption time, and also have carry-over problems [6]. According to the literature over different liquid phase microextraction techniques (LPME), dispersive liquid–liquid microextraction (DLLME) and hollow fiber-based liquid phase microextraction (HF-LPME) have attracted more interests among analytical research community around the world [7,8]. DLLME is a simple and fast method and provides high preconcentration factors. HF-LPME provides high preconcentration factors and produces clean extracts without any need for solvent evaporation and re-constitution as required for LLE and SPE [9]. Also, sample carry-over can be avoided in HF-LPME because the hollow fibers are inexpensive enough to be disposed after each use. Because the LPME tolerates a wide pH range, it can be used in applications, which would not be suitable for SPE or SPME. However, these techniques have some drawbacks; DLLME is only efficient for simple matrices, so that it creates crowded chromatograms for extracts from complex matrices, especially biological fluids. This intensifies distinguish among peaks of

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interferences and analytes. Therefore, sample pretreatment is an unavoidable step for this technique. Moreover, automation of this technique is difficult. For HF-LPME, the extraction time needed is usually high and common extraction times of 30–50 min have been reported [10,11]. Recently, Pedersen-Bjergaardet et al. introduced a novel microextraction technique called electromembrane extraction (EME) [12]. In EME, the driving force for the extraction is a DC electrical potential defrayed over the supported liquid membrane (SLM) and the analytes are extracted by electrokinetic migration. Sufficient ionization of the target analytes in both the sample and the acceptor solution are therefore essential. The ionized compounds are migrated in an electrical field from the sample solution across the SLM into the aqueous phase [13–17]. The use of an electrical potential difference as the driving force reduces the extraction time. Other advantages are high sample cleanup and preconcentration factors [18,19]. There are some difficulties in coupling of EME with gas chromatography (GC) since the acceptor phase is an aqueous solution and direct injection of water to GC may cause some damages. This is while GC is faster, simpler, and less expensive than high performance liquid chromatography (HPLC) and it can easily be coupled with different types of sensitive detectors [20]. Solid phase microextraction (SPME) was originally devised by Pawliszyn et al. as a new technique in 1989 [21]. In this technique, a small amount of extraction phase coated on a solid support, utilized to extract analytes from aqueous or gaseous samples [22,23]. However, this method faces some problems in analysis of nonvolatile or ionizable species in complicated matrices due to fiber saturation with interferences. Therefore, combination of EME and SPME was introduced for the first time in 2013 by Yamini et al. and termed electromembrane surrounded solid phase microextraction (EM-SPME). The setup they used was the same as that in the EME technique, except the cathode (the electrode inserted in the lumen of hollow fiber) was substituted by a carbonaceous pencil lead fiber. By applying electrical field, the model analytes migrated from sample solution through the SLM into an aqueous phase, which was located inside the hollow fiber lumen. The analytes were afterward adsorbed on the solid sorbent, which was also the cathode. Finally, the pencil lead was directly introduced into the GC-FID injection port. Pencil lead was chosen due to its conductive nature, thermal stability, and low cost [24]. However, this fiber contains large amounts of modifiers and these interferences make crowded chromatograms at high temperatures in the GC analysis.

In the present study, EM-SPME using conductive polymers as the sorbent was carried out for the first time for analysis of two antidepressant drugs as the model analytes. Here, AMI and DOX were selected as model compounds to present the applicability of the new coating for EM-SPME and the selectivity were not the aim of this research project. Conductive polymers with conjugated double bonds have attracted much attention as advanced materials [25–31]. Polypyrrole (PPy) is one of the most studied conducting polymers owing to its simple preparation procedure, high conductivity, and relative stability. Application of electrochemically synthesized conductive polymers may increase the extraction efficiency due to its porous structure and could result in cleaner chromatograms in comparison with previous pencil lead fiber [24,32]. Also, the fragility problem which was associated with previous pencil lead is eliminated by new fiber due to deposition of the fiber coating on an unbreakable substrate. Moreover, higher voltages could be applied in EM-SPME system, leading to enhanced extraction efficiency due to the low electrical current through the extraction system, which increases the stability of PPy fiber. 2. Experimental 2.1. EM-SPME equipment The equipment used for the extraction procedure is shown in Fig. 1. A 10 mL glass vial was used as the sample compartment. The platinum electrode used in this work, with the diameter of 0.25 mm, was obtained from Pars Pelatine (Tehran, Iran). The electrodes were coupled to a power supply model 8760T3 with a programmable voltage in the range of 0–600 V and with a current output in the range of 0–500 mA from Paya Pajoohesh Pars (Tehran, Iran). During the extraction, the EM-SPME unit was stirred with a stirring speed in the range of 0–1250 rpm by a heater-magnetic stirrer model 3001 from Heidolph (Kelheim, Germany) using a 1.5 × 0.3 cm magnetic bar. 2.2. Chemicals and materials AMI and DOX were purchased from Razi Pharmaceutical Company (Tehran, Iran). The chemical structure and physicochemical properties of the drugs are provided in Table 1. 2-Nitrophenyl octyl ether (NPOE) and Pyrrol (Py) were obtained from Fluka (Buchs, Switzerland). Distilled Py was prepared freshly prior to the syn-

Fig. 1. Equipment used for the EM-SPME method and mechanism of transport across liquid-liquid-liquid-solid boundaries.

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Table 1 Chemical structures, pKa , logP, and therapeutic concentrations (TCs) of AMI and DOX. Chemical structure

a

IUPAC name

Abbreviation

pKa a

log Pa

TC (ng mL−1 )a

3-(10,11-Dihydro-5Hdibenzo[a,d]cyclohepten-5-ylidene)N,N-dimethyl-1-propanamine

AMI

9.4

4.94

100–200

3-dibenz[b,e]oxepin-11(6H)-ylideneN,N-dimethyl-1-propanamine

DOX

9.0

2.40

20–150

Ref. [38].

thesis process. Sodium dodecylbenzenesulfonate (SDBS), sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and oxalic acid were obtained from Sigma-Aldrich (Milwaukee, WI, USA). All the chemicals used were of analytical reagent grades. The porous hollow fiber used for the SLM was a PPQ3/2 polypropylene hollow fiber from Membrana (Wuppertal, Germany) with inner diameter of 0.6 mm, wall thickness of 200 ␮m, and pore size of 0.2 ␮m. Ultrapure water was obtained from a Young Lin 370 series aquaMAX purification instrument (Kyounggi-do, Korea). Drug-free human blood (blood group O+ and B+ ) was obtained from the Iranian Blood Transfusion Organization (Tehran, Iran). Urine samples were collected from two volunteers 1 h and 24 h after taking 25 mg amitriptyline orally, and from one person who did not consume the drugs at all (as the match matrix for plotting the calibration curves). The samples were stored at −4 ◦ C, thawed, and shaken before extraction. A stock solution containing 1 mg mL−1 of each analyte was prepared in methanol and stored at −4 ◦ C protected from light. The working standard solutions were prepared by dilution of the stock solution in methanol.

Fig. 2. The scanning electron micrograph of the dodecylbenzenesulfate-doped polypyrrole fiber at 30,000-fold magnification.

2.3. Gas chromatography conditions Separation and detection of AMI and DOX were performed by an Agilent 7890A gas chromatograph (Palo Alto, CA, USA) equipped with a split-splitless injector and a flame ionization detector (FID). A 30 m HP-5 Agilent fused-silica capillary column (0.32 mm i. d. and 0.25 ␮m film thickness) was applied for separation of the target compounds. Helium (purity 99.999%) was used as the carrier gas at the constant flow rate of 0.6 mL min−1 . The temperature values of the injector and the detector were set at 280 and 300 ◦ C, respectively. The injection port was operated at the splitless mode. Oven temperature program was 160 ◦ C for 3 min, increased to 280 ◦ C with a ramp of 30 ◦ C min−1 , and held at 280 ◦ C for 3 min. 2.4. Preparation of PPy fiber The PPy sorbent including PPy-DBS (SDBS as the electrolyte and DBS− as the counter ion) was coated on the electrode surface by applying a constant DC potential. For electrochemical synthesis of PPy, the stainless steel electrode was the anode while the cathode was a platinum wire (0.25 mm). The PPy was directly synthesized on the electrode surface from a 0.05 mol L−1 electrolyte solution (SDBS) containing 0.1 mol L−1 of pyrrole monomer by application of a 0.9 V DC potential for 20 min. Five min N2 purging was per-

formed prior to electrosynthesis of PPy fiber to remove the oxygen from the medium. Afterward, the stainless steel electrode coated with PPy-DBS was washed by ultrapure water and methanol to eliminate any unwanted chemicals such as monomers and the supporting electrolyte. Finally, the dried PPy fiber was conditioned by heating under N2 atmosphere at 280 ◦ C. Also, the composition of the conductive fiber was investigated via changing the anion dopant. Since the model analytes carrying a net positive charge, it was anticipated that a bulky dopant is beneficial. A bulky dopant could not leave the polymer structure during application of negative potential. Therefore, the fiber charge could be neutralized by entrance of basic analytes into the polymer. Fig. 2 is a scanning electron microscopic image taken of the PPy-DBS fiber extracting phase. It can be seen that the polymer has a regular and granular (porous) structure adhered to the electrode. The investigation of robustness was carried out by extraction of AMI and DOX at concentration levels of 2 ng mL−1 . The results showed that each fiber could be used for extraction more than 40 successive extractions with no significant changes in extraction efficiency. The fiber-to-fiber reproducibility was assessed by calculating the relative standard deviation (RSD%) for AMI and DOX extraction. The intra-day and inter-day RSDs% were in the range of 7.6–9.2% and 9.8–11.3%, respectively.

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2.5. EM-SPME procedure Ten milliliters of the sample solution containing the model analytes in pure water was transferred into the sample vial. To impregnate the organic liquid membrane in the pores of the HF wall, a 3 cm piece of the hollow fiber was cut out and dipped in the NPOE for 5 s and then the excess of the organic solvent was gently wiped away by blowing air with a Hamilton syringe. Also, pure water was introduced into the lumen of the hollow fiber as the acceptor phase by a microsyringe and then the lower end of hollow fiber was mechanically sealed with a small piece of aluminum foil. The PPy fiber (the cathode) was introduced into the lumen of the hollow fiber. The hollow fiber containing the cathode, together with the SLM and the acceptor solution were afterwards directed into the sample solution. The platinum anode was directly led into the sample solution. Human blood and urine samples were diluted 1:19 and 1:1, respectively. The extraction unit was placed on a stirrer with stirring rate of 700 rpm. The electrodes were subsequently coupled to the power supply and an electrical potential of 70 V for 30 min was applied. When the extraction was completed, the PPy fiber was retracted and connected to the needle of a SPME-syringe. Then, the PPy fiber was inserted into the injection port of GC for thermal desorption of the analytes at 280 ◦ C during 2 min. 3. Results and discussion In EM-SPME, effective parameters should be optimized to achieve the best extraction conditions. Variables that can affect extraction efficiency include the composition of donor and acceptor phases, type of anion dopant, extraction time, applied voltage, and the stirring rate. Also, the effect of desorption time on the extraction signal was investigated. Desorption time was studied in the range of 0.5–5.0 min while the desorption temperature was 280 ◦ C. Efficiency of thermal desorption was improved as desorption time increased from 0.5 to 2.0 min. The chromatographic signal slightly increased and was almost constant after 2.0 min. Thus, desorption time of 2.0 min was selected. The selectivity and high clean up in this extraction system is a result of using the SLM around the SPME fiber. In this work, the SLM was not optimized and selected according to literature [24]. Different anion dopants were considered including EDTA, SDS, SDBS, and SDBS in combination with oxalate ion. The results showed that the most massive dopant offers the best stability and extraction recovery, as it was expected. Thus, SDBS was selected as the ion dopant for fiber preparation. Stirring rate could increase the kinetics and efficiency of extraction by increasing the mass transfer and reducing the thickness of double layer around SLM. The effect of stirring rate on extractability was investigated up to 1250 rpm. A stirring rate of 700 rpm was chosen due to formation of intense vortex into the sample solution and bubble formation around the HF at higher rates. Extraction time and the voltage applied affect the extraction efficiency concurrently and their effects will be studied in Section 3.2 [33–35]. 3.1. The pHs of sample solution and acceptor phase Previous EME investigations [36] have proven that the flux of the analytes has a reverse proportion to the ion balance, which is mainly defined by the pH values of the sample solution and the acceptor phase. The sample solution should be acidic enough; so that the basic analytes carry a net positive charge to be enabled to migrate toward the cathode in an electrical field. To investigate the effect of ion balance, pH of the donor phase was changed in the range of 1.0–6.5, while the pH value in the acceptor phase was varied in the range of 1.0–13.0 by adding appropriate amounts of hydrochloric acid and/or sodium hydroxide solutions. It should be

noted that to study the effect of both acidity and ionic strength of the donor and acceptor phases, experiments were design by changing the concentration of HCl and NaOH solutions in the range of 0–100 mmol L−1 . However, for graphical presentation and to obtain a total view of the effect of the H+ or OH− concentration on extractability, the results should be changed into pH. Thus, the results in Fig. 3A are not the exact pH values and they are just a presentation of the H+ or OH− concentration throughout the donor and acceptor solutions. Fig. 3A shows that by decreasing the pH value of the sample solution, the chromatographic signal decreased. Both model analytes are ionized at the neutral pH value and as the H+ concentration increases, the competition between H+ and cationic analytes decreases the extraction efficiency. Moreover, the extraction efficiency was small at low pH values of the acceptor phase due to H+ predomination in the electrostatic migration toward the PPy fiber electrode. At relatively high pH value of the acceptor phase, extraction efficiency was severely reduced because the analytes were mainly present at their neutral form. This phenomenon confirms the extraction mechanism is electrokinetic migration of cationic species. Thus, neutral pH value (∼7) was chosen as the pH of both the acceptor and donor phases for the rest of the work. 3.2. Applied voltage and extraction time The electrical field mainly provides the driving force for electrokinetic migration of the analytes, which depends on the voltage applied. To investigate the effect of applied voltage and extraction time simultaneously, electrical potential differences in the range of 10–90 V were applied for extraction durations of 15–40 min. As demonstrated in Fig. 3B, when electrical potential of 70 V was applied, maximum amounts of the drugs were adsorbed into the PPy fiber. More increase in the voltage results in loosing of anion dopant, which significantly decreases the sorption ability of the fiber. This is while reduction of applied driving force increases the extraction time. Therefore, 70 V was applied for 30 min to obtain the best results. 3.3. Method validation Figures of merit of the proposed method were investigated in two different media including human blood and human urine according to recommendations of the Food and Drug Administration (FDA). Linearity was studied for AMI and DOX by analysis of extracts obtained from each sample in triplicates. The extraction recovery (ER) was defined as the percentage of the number of moles of the analyte adsorbed on the sorbent (nf ) to those originally present in the sample solution (ni ). ER% =

nf ni

× 100

(1)

The coefficient of variation (CV%) was determined by intraand inter-assays and by five- and three-replicate measurements, respectively, at three concentrations (1, 10, and 20 ng mL−1 ). Accuracy was determined by triplicate analysis of samples containing known amounts of the analyte at three concentrations in the range of expected concentrations. The relative recovery (RR%) and accuracy (Error%) were calculated by the following equations: RR% =

Cfound − Creal × 100 Cadded

Error% = 100 − RR%

(2) (3) (ng mL−1 )

of where Cfound , Creal , and Cadded are the concentrations analyte after addition of known amount of standard into the real sample, the concentration of analyte in real sample, and the concen-

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Fig. 3. (A) Contour plot of peak area vs. pH of donor phase; pH of acceptor phase, (B) Contour plot of peak area vs. extraction time; applied voltage.

tration of known amount of standard spiked into the real sample, respectively. The results summarized in Table 2 show that EM-SPME could effectively be employed for analysis of model drugs even in complicated matrices such as biological fluids. To improve mass transfer of the analytes, human blood and urine samples were diluted 1:19 and 1:1, respectively, with pure water and the pH values were

adjusted to 6.5 by addition of proper amounts of hydrochloric acid or sodium hydroxide solutions. The model drugs were effectively extracted with extraction recoveries in the range of 11.5–51.5%. The calibration plots were linear up to 50 ng mL−1 with coefficients of determination (R2 ) greater than 0.9966. Intra- and inter-assay precisions were ranged between 3.1–7.5% and 7.6–12.3%, respectively (Table 3). Also, calculated Error% for the analytes in the

Table 2 Figures of merit of EM-SPME-GC-FID for analysis of AMI and DOX in urine and whole blood samples. Sample

Analyte

LODa (ng mL−1 )

Linearity range (ng mL−1 )

Calibration curve (ng mL−1 , n = 5)

R2

ER%

Urine

AMI DOX

0.15 (0.3) 0.3 (0.6)

0.3–50 0.5–50

Y = 12.80 (±0.704) X + 5.790 (±0.335) Y = 6.86 (±0.291) X + 0.285 (±0.013)

0.9966 0.9997

20.4 11.5

Blood

AMI DOX

0.05 (1.0) 0.1 (2.0)

0.1–50 0.3–50

Y = 30.53 (±3.81) X + 60.48 (±3.75) Y = 18.91 (±2.12) X + 18.67 (±2.37)

0.9976 0.9990

51.5 28.0

a The LOD values, outside the parenthesis, are based on diluted urine (1:1) and blood (1:19) samples whereas those reported in the parenthesis are calculated for undiluted samples.

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Table 3 Accuracy, precision, and relative recovery of the proposed method for determination of AMI and DOX in pure water and drug-free urine and plasma samples. Analyte

Conc. (ng mL-1 )

Accuracy (Error%)

Precision (CV%)

RR%

Intra-assay (n = 5)

Inter-assay (n = 5)

Intra-assay (n = 5)

Inter-assay (n = 5)

U

B

U

B

U

B

U

B

U

B

AMI

1 10 20

1.3 −6.0 8.5

−0.3 −3.5 −0.6

3.1 −4.5 8.2

−2.3 −5.6 6.5

7.5 3.4 5.3

4.4 6.2 4.2

12.3 7.6 11.3

8.2 9.1 10.1

101.3 94 108.5

99.7 96.5 99.4

DOX

1 10 20

−4.3 −7.3 −9.8

−3.2 −1.6 −5.7

−0.8 3.4 −9.4

1.3 −6.9 7.2

3.7 5.2 3.4

4.1 3.1 6.7

11.4 9.3 10.5

8.6 12.1 9.6

95.7 92.7 90.2

96.8 98.4 94.3

U: Urine, B: Blood.

Fig. 4. A typical chromatogram related to extraction of AMI and DOX at LOQ concentration of each analyte from a blood sample.

range of −9.4% to +9.8% in different matrices demonstrates that the proposed method offers acceptable accuracy even in complicated matrices such as biological fluids. The limits of detection(s) were determined by analyzing a series of spiked samples (n = 3) with decreasing analyte concentrations to create signal to noise (S/N) ratio of 3. LOD values were less than 0.3 ng mL−1 and the limits of quantitation were less than 0.5 ng mL−1 . A typical chromatogram related to extraction of AMI and DOX at LOQ concentration of each analyte from a blood sample is shown in Fig. 4. A comparison of the proposed EM-SPME method with other reported methods for the extraction and determination of AMI and DOX is presented in Table 4. It was shown that along with its simplicity, this technique has high sensitivity and an acceptable repeatability with an important emphasis on the extraction recoveries that make this method more efficient for determination of the analytes. One can see that EM-SPME offers excellent recoveries and LODs. No need for extra sample pretreatment steps is one of the most interesting advantages of the proposed method and it is assumed that the electrical field contributes to break of the bonds between proteins and analytes [37]. Moreover, application of electrochemically synthesized conductive polymers may increase the extraction efficiency due to its porous structure and could result in cleaner chromatograms. On the other hand, as reported in Table 4, some other techniques which provided high extraction recoveries and low LODs and LOQs [47–49], require high cost analytical instruments which are not available in routine laboratories. Also, as mentioned before, LLE and SPE have limitations such as consumption of relatively large amount of toxic organic solvents, solvent evaporation requirement etc. Thus, EM-SPME can be introduced as a novel and simple technique for efficient extraction of analytes from complicated matrices.

Fig. 5. Chromatograms obtained after extraction of drugs from: (A) human urine, (B) human blood ((a) non-spiked sample, (b) spiked sample at the concentration levels of 1.0 ng mL−1 proper to each analyte in urine and 2.0 ng mL−1 proper to each analyte in blood samples, respectively).

3.4. Analysis of real samples In order to evaluate the applicability of the introduced EMSPME method for analysis of real samples, different human blood and urine samples were analyzed. All samples were prepared as explained in the method validation section. To this end, blood and urine samples were diluted 1:19 and 1:1, respectively, with pure water and the pH value was adjusted to 6.5 by addition of proper amounts of hydrochloric acid or sodium hydroxide solutions and optimal conditions were applied for extraction and quantitative analysis of the analytes. Chromatograms obtained after extraction from the human blood (Blood 1) and urine (Urine 1) samples are shown in Fig. 5. Thereafter, to determine the method accuracy, blood and urine samples were spiked with the drugs at 2.0 and 1.0 ng mL−1 levels, respectively, and EM-SPME was carried out to extract the analytes. By FDA definition, a matrix effect is the direct or indirect alteration or interference in response due to the pres-

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Table 4 Comparison of figures of merit of EM-SPME with other analytical techniques for determination of AMI and DOX. Analytical methoda

Analyte

Matrix

LOD (ng mL−1 )

LOQb (ng mL−1 )

Extraction time (min)

ER%

CV%

Reference

IT-SPME-LC–MS IT-SPME-LC–MS dSPME-LC–MS IL-DLLME-␮SPE-HPLC SPME-GC–NPD SPME-GC–NPD UPS ␮-SPE HS-SPME-GC–MS SPME-LC–MS SPME-HPLC-UV SPME-MC-LC EME based on flat membrane-LC–MS LLE-UHPLC–MS/MS SPE-GC–MS EM-SPME EM-SPME EM-SPME EM-SPME EM-SPME EM-SPME EM-SPME EM-SPME EM-SPME EM-SPME

AMI AMI AMI AMI AMI DOX AMI AMI AMI AMI AMI AMI AMI AMI AMI AMI AMI DOX DOX DOX AMI AMI DOX DOX

Urine Plasma Water Water Plasma Plasma Water Hair Plasma Plasma Urine Plasma Blood Blood Water Urine Plasma Water Urine Plasma Blood Urine Blood Urine

0.08 0.07 – 1.0 – – 0.66 0.05 (ng mg−1 ) 0.1 – 3 0.2 1.1 0.7 0.5 1.0 1.0 2.0 2.5 5.0 0.05 0.15 0.1 0.3

1 1 0.01 – 12.5 12.5 2.21 – 50 75 5 0.7 5.5 2.0 2.0 2.5 5.0 5.0 5.0 10.0 0.1 0.3 0.3 0.5

30 30 4 – 10 10 60 50 30 50 210 30 >20 >35 20 20 20 20 20 20 30 30 30 30

– – – – 0.46 0.63 – 0.4–2.3 2.15 – – 89 91–94 90.2–96.7 11.5 10.4 5.9 4.2 4.0 3.1 51.5 20.4 28.0 11.5

2.0 3.9 9.9 3.3 11.5 44.6 – – 14.92 <10 <10.1 <10 <7.2 <7.1 8.5c 4.2c 5.3c 5.5c 4.4c 3.9c 6.2d 7.5d 6.7d 5.4d

[39] [39] [40] [41] [42] [42] [43] [44] [44] [45] [46] [47] [48] [49] [24] [24] [24] [24] [24] [24] This work This work This work This work

a In-tube (IT), liquid chromatography (LC), mass spectrometry (MS), dual solid phase microextraction (dSPME), ionic liquid (IL), dispersive liquid–liquid microextraction (DLLME), micro-solid phase extraction (␮-SPE), high performance liquid chromatography (HPLC), nitrogen-phosphorus detector (NPD), non-nucleophilic urea-groups (UPS), ultraviolet detector (UV), microcolumn (MC), electromembrane extraction (EME), liquid–liquid extraction (LLE), ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS), solid phase extraction (SPE), gas chromatography (GC), electromembrane surrounded solid phase microextraction (EM-SPME). b Limit of quantification. c For five-replicate measurements at 25 ng mL. d For five- replicate measurements at 1.0 ng mL.

Table 5 Determination of AMI and DOX in different urine and blood samples. Sample

Analyte

Creal (ng mL−1 )

Cadded (ng mL−1 )

Cfound (ng mL−1 )

RSD% (n = 3)

Error%

Blood 1

AMI DOX

nd nd

2.0 2.0

2.1 1.96

3.7 4.8

+5.0 −2.0

Blood 2

AMI DOX

nd nd

2.0 2.0

1.93 2.08

4.2 5.1

−3.5 +4.0

Urine 1

AMI DOX

1.6 nd

1.0 1.0

2.54 1.05

6.2 6.5

−6.0 +5.0

Urine 2

AMI DOX

nd nd

1.0 1.0

1.01 0.97

8.3 7.4

+1.0 −3.0

nd: not detected.

ence of unintended analytes or other interfering substances in the sample. Table 5 demonstrates that results of three-replicate analyses of each sample obtained by the proposed technique are in satisfactory agreement with the spiking amounts. No significant matrix effect was observed for the real samples studied and the method offers acceptable accuracy.

a simple and inexpensive method for analysis of low volatile or ionizable compound in complicated matrices.

Acknowledgement The authors gratefully acknowledge financial support from Tarbiat Modares University.

4. Conclusions A new EM-SPME approach with electrochemically synthesized polypyrrole fiber has been introduced for the first time for the extraction of analytes from whole blood and urine samples with excellent efficiency. This technique demonstrated several advantages compared to other extraction methods. In comparison with SPME, by selecting an appropriate organic solvent for SLM, not only the selectivity can be enhanced, but also sample cleanup will be highly improved and makes it possible to apply high voltages that increase extraction recoveries. Furthermore, by using PPy-DBS fiber instead of pencil lead, clean chromatograms were obtained and more porosity of conductive polymer led to improved extraction efficiency. Thus, EM-SPME followed by GC-FID was introduced as

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