Electromembrane surrounded solid phase microextraction: A novel approach for efficient extraction from complicated matrices

Journal of Chromatography A, 1280 (2013) 16–22

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Electromembrane surrounded solid phase microextraction: A novel approach for efficient extraction from complicated matrices Maryam Rezazadeh, Yadollah Yamini ∗ , Shahram Seidi, Behnam Ebrahimpour Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 20 November 2012 Received in revised form 6 January 2013 Accepted 7 January 2013 Available online 16 January 2013 Keywords: Electromembrane extraction Solid phase microextraction Gas chromatography Urine Plasma

a b s t r a c t In the present work, electromembrane surrounded solid phase microextraction (EM-SPME) is introduced for the first time. The organic liquid membrane, which consists of 2-nitrophenyl octyl ether (NPOE), was immobilized in the pores of a hollow fiber (HF) and the basic analytes migrated in an electrical field from aqueous sample solution through the liquid membrane and into aqueous acceptor phase and then they were adsorbed on the solid sorbent, which acts as the cathode. Effective parameters such as composition of organic liquid membrane, pH of donor and acceptor phases, applied voltage and extraction time were optimized for extraction of amitriptyline (AMI) and doxepin (DOX) as model analytes and figures of merit of the method were investigated in pure water, human plasma, and urine samples. To extract the model analytes from 24 mL neutral sample solution across organic liquid membrane and into aqueous acceptor phase, 120 V electrical potential was applied for 20 min and finally the drugs were adsorbed on a carbonaceous cathode. Regardless of high sample cleanup, which make the proposed method suitable for the analysis of drugs from complicated matrices, extraction efficiencies in the range of 3.1–11.5% and good detection limits (less than 5 ng mL−1 ) with admissible repeatability and reproducibility (intra- and inter-assay precisions ranged between 4.0–8.5% and 7.5–12.2%, respectively) were obtained from different extraction media. Linearity of the method was studied in the range of 2.0–500.0 ng mL−1 and 5.0–500.0 ng mL−1 for AMI and DOX, respectively and coefficient of determination higher than 0.9947 were achieved. Finally, the proposed method was applied for the analysis of AMI and DOX in real samples. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Sample preparation is still the most important challenge for the analysis of different compounds from various complex matrices, especially biological fluids. During the last decade, new modern sample preparation methods with respect to simplification, miniaturization, and minimization of the organic solvent usage have been developed. Solid phase microextraction (SPME) and liquid phase microextraction (LPME) are miniaturized techniques; introduced for these purposes [1,2]. SPME requires a small amount of extraction phase coated on a solid support. Selectivity of this technique is determined by composition of the extraction phase. Headspace SPME (HS-SPME) mode is a suitable method for extraction of analytes from dirty matrices. However, only appreciably volatile compounds could be effectively extracted using HS-SPME. Since in direct immersion SPME (DI-SPME), the fiber is directly exposed to the sample, the efficiency is increased especially for nonvolatile or

∗ Corresponding author. Tel.: +98 21 82883417; fax: +98 21 88006544. E-mail address: [email protected] (Y. Yamini). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.01.034

ionizable compounds. However, the use of DI-SPME is limited in complicated matrices due to fiber saturation, which decreases the fiber lifetime and extraction efficiency. Therefore, neither HSSPME nor DI-SPME is useful for extraction of analytes from very dirty samples containing nonvolatile or high molecular interfering compounds. To overcome these drawbacks, micro-solid-phase extraction (␮-SPE), involving the use of a sorbent wrapped in a porous membrane sheet, was introduced [3,4] and SPME using HF membranes has been developed to protect the extraction fiber [5–8]. Also, combination of electrosorption and SPME (ES-SPME) was studied for extraction of charged species from water samples [9–11]. Thus, the selectivity and efficiency of DI-SPME for extraction of ionic analytes could be improved by application of electrical potential to the extraction fiber [12]. However, it still suffers from the problems DI-SPME faced and despite the potentials of the technique to improve the extractability; it is not recommended for extraction from complicated matrices. Instead of common SPME fibers, carbonaceous materials such as pencil lead could act as a suitable sorbent in ES-SPME due to its conductive nature, high thermal stability, availability, and low cost [9]. Hollow fiber-based liquid phase microextraction (HF-LPME) has been employed as a simple sample preparation method since 1999 [2]. High cleanup and preconcentration factor are some advantages

M. Rezazadeh et al. / J. Chromatogr. A 1280 (2013) 16–22

of HF-LPME technique. HF-LPME is an effective method for extraction of compounds from complicated matrices and provides clean chromatograms. One of the advantages of HF-LPME is elimination of possible carry-over problems since the hollow fiber is not expensive and can be discarded after each extraction. The extraction mechanism in HF-LPME is based on passive diffusion, which is a relatively slow process, so that common extraction times of 30–50 min have been reported [13]. Therefore, electromembrane extraction (EME) was introduced using an electrical field as an effective driving force to increase the extraction efficiency and reduce the extraction time of conventional HF-LPME [14] and some improvements have been made on EME so far [15–21]. The basis of EME is migration of ionized compounds in an electrical field, from sample solution across supported liquid membrane (SLM) into aqueous acceptor phase. The attempts to figure out the transportation mechanisms of the ions through the organic phase in EME technique could not explain the exact extraction mechanism, up to now. However, the permeation of a fully ionized analyte across a lipophilic membrane was studied, recently [22]. It was demonstrated that there are two transfer mechanisms, due to naked ions and ion-pairs. Also, results show that transfer via ion-pairs occurs at a rate that is close to 3 orders of magnitude higher than the ionic one [22]. Since the acceptor phase is an aqueous solution and direct injection of water may cause some problems for gas chromatography (GC) instrument, there are some difficulties in coupling EME with GC. However, GC is simpler, faster, and less expensive than high performance liquid chromatography and it can easily be coupled with different types of sensitive detectors. Amitriptyline (AMI) and doxepin (DOX) have been used to treat endogenous depression, phobic states, panic attacks, neuropathic pain states, and pediatric enuresis. Due to their relatively narrow therapeutic/toxic index, monitoring their levels in biological fluids is necessary for effective and appropriate patient treatment [23,24]. To get rid of the inherent complexity of biological samples, which limits the selectivity and sensitivity of the determinations, using some sample preparation techniques is needed prior to analysis. In the present work, combination of electrically enhanced three-phase LPME and SPME is introduced for the first time as a novel and efficient method for extraction of ionizable analytes from

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complicated matrices. A 120 V electrical field was applied to make the model analytes migrate from sample solution through the SLM into an aqueous phase, which was located inside the lumen of HF. The analytes were afterward adsorbed on the solid adsorbent, which was also the cathode. Finally, the sorbent was directly introduced into GC-FID injection port. The analytes were desorbed and transferred into the column for separation and analysis. The proposed method not only offers high sample cleanup, which enables SPME technique to the analysis of ionized compound in complex matrices, but using electrical potential as an effective driving force may increase the extraction efficiency. 2. Experimental 2.1. EM-SPME equipment The equipment used for the extraction procedure is shown in Fig. 1. A 24 mL vial with an internal diameter of 2.5 cm and a height of 5.5 cm was used. The platinum electrode used in this work, with diameters 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 heatermagnetic stirrer model 3001 from Heidolph (Kelheim, Germany) using a 1.5 cm × 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), tris-(2-ethylhexyl) phosphate (TEHP), and di-(2ethylhexyl) phosphate (DEHP) were purchased from Fluka (Buchs, Switzerland). Dihexyl ether, 1-octanol, and octanoic acid were obtained from Merck (Darmstadt, Germany). All the chemicals used were of analytical reagent grades. The porous HF used for the SLM was a PPQ3/2 polypropylene HF from Membrana (Wuppertal,

Fig. 1. Equipment used for the EM-SPME method and mechanism of transport across liquid–liquid–liquid–solid boundaries. The flux of the analytes is presented by “i” and o, aq, and f represent the organic, the aqueous and the carbonaceous fiber, respectively.

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Table 1 Chemical structures, pKa , log P, 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-5H-dibenzo[a,d]cyclohepten-5ylidene)-N,N-dimethyl-1-propanamine

AMI

9.4

4.94

100–200

3–Dibenz[b,e]oxepin-11(6H)-ylidene-N,N-dimethyl-1propanamine

DOX

9.0

2.40

20–150

Ref. [26].

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 (Kyounggido, Korea). The HB pencil lead fibers with the diameter of 0.3 mm (Owner (Seoul, Korea)) were purchased from local market. The fibers were conditioned by heating under N2 atmosphere from the room temperature to 350 ◦ C by a ramp of 3 ◦ C min−1 , held for 120 min at 350 ◦ C, and then were allowed to cool to the ambient temperature. Drug-free human plasma (blood group A+ and O+ ) was obtained from the Iranian Blood Transfusion Organization (Tehran, Iran). Urine samples were collected from two persons who took amitriptyline 25 mg, orally after 1 h and 24 h and one person who had not consumed the drugs at all (as 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. Working standard solutions were prepared by dilution of the stock solution in methanol. 2.3. Gas chromatography conditions Separation and detection of AMI and DOX were performed using 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 temperatures of injector and 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. EM-SPME procedure Twenty-four 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 2.8 cm piece of the HF 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 HF as the acceptor phase by a microsyringe and then the lower end of the HF was mechanically sealed. The pencil lead fiber (the cathode) was introduced into the lumen of the HF. The HF containing the cathode, together with the SLM and the acceptor solution, was afterward directed into the sample solution. The platinum anode was led directly into the sample solution. The electrodes were subsequently coupled to the power supply and the extraction unit was placed on a stirrer with stirring rate of 1250 rpm. When the extraction was completed, the pencil lead was retracted into the SPME syringe needle. The tractable nature of pencil lead facilitated its insertion into the SPME syringe. The carbonaceous fiber was well fitted into the syringe plunger by pushing the pencil lead with a little force. Then, the pencil lead was inserted into the GC injection port for thermal desorption of the analytes at 280 ◦ C for 2 min. 3. Results and discussion Effective parameters should be optimized to achieve the best extraction conditions. Variables that can affect extraction efficiency include composition of the supported liquid membrane (SLM), donor and acceptor phases’ compositions, extraction time, applied voltage, and 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 after 2.0 min, but desorption of some interferences from the pencil lead fiber resulted in crowded chromatograms. Thus, desorption time of 2.0 min was selected. 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 sample solution and bubble formation around the HF at higher rates. The presence of high content of ionic substances causes an increase in the value of the ion balance (), which is defined as the ratio of the total ionic concentration in the sample solution to that in the acceptor solution [25], which in turn decreases the flux of analytes across the SLM. In fact, with increasing concentration of other ions in the sample solution competition between these ions and

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target analyte ions for migration toward electrodes through the SLM increase, resulted to reduction of extraction efficiency. Also, increasing of salt content resulted in increasing the number of ions to migrate through SLM, which subsequently caused increasing of Joule heating and instability of SLM. The effect of  was investigated using solutions containing 2.5% NaCl. In the presence of salt, extraction recoveries of AMI and DOX were decreased 21% and 13% respectively. Since it has a negative effect on extraction efficiency, all the subsequent experiments in EME were performed in the absence of salt. Extraction time and the applied voltage affect the extraction efficiency concurrently [27–29]. Increase in extraction time limits the voltage and vice versa. On the other hand, the total ionic concentration of the donor phase to that of the acceptor phase, which is mainly determined by the pH values of donor and acceptor phases, influences the flux over the membrane [25]. Since there is an antagonistic effect among these parameters, they were simultaneously considered and the interaction of time–voltage and the pH values of the acceptor and donor phases were investigated. 3.1. Selection of organic liquid membrane The SLM should be water-immiscible and its polarity should be similar to that of the polypropylene fiber so that it can be easily immobilized within the pores of the fiber. SLM should have an appropriate electrical resistance to keep the electrical current of the system in its lowest possible level, even when high voltages were applied and charged analytes should have suitable solubility in the SLM to allow their transportation. Experiments with different types of water-immiscible organic solvents, which have suitable chemical properties to enable electrokinetic migration of the model analytes including 1-ocnanol, octanoic acid, dihexyl ether, and NPOE were conducted. As demonstrated in Fig. 2A, drugs were slightly extracted when octanoic acid or dihexyl ether were immobilized in the pores of HF. Since best results were obtained using NPOE as SLM, the effect of addition of some carriers such as DEHP and TEHP to NPOE was examined. Both carriers had negative effects on extractability of the model analytes. Therefore, NPOE was selected as the organic liquid membrane for further experiments. 3.2. The pHs of sample solution and acceptor phase It was found that the flux of the analyte is increased by decreasing the ion balance [25]. Ion balance is mainly determined by the pH values of sample solution and acceptor phase. 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 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 effects of both acidities and ionic strengths of donor and acceptor phases, experiments were designed by changing the concentration of HCl and NaOH solutions in the range of 0–100 mM. However for graphical presentation and to obtain a total view of the effect of the concentration of H+ or OH− on extractability, the results should be changed to pH. Thus, the results in Fig. 2B are not the exact pH values and they are just the representation of the concentrations of H+ or OH− in the donor and acceptor solutions. Normalized peak area for each experimental point was selected as response objective for the study. To normalize the peak areas, the peak area of each analyte was divided by its smallest peak area, which was obtained in all the experiments. Normalized peak areas for the analytes were subsequently added for each experimental point and used in the calculation of total normalized peak area. The contour plots of the results were designed using the software package Minitab 16

Fig. 2. (A) Optimization of membrane composition, (B) contour plot of normalized peak area vs. pH of donor phase; pH of acceptor phase, and (C) contour plot of normalized peak area vs. extraction time; applied voltage.

trial for Windows. Fig. 2B shows that the chromatographic signal decreased by decreasing the pH value of sample solution. Both of the model analytes are ionized at the neutral pH value and as the concentration of H+ increases, competition between H+ and cationic analytes decreases the extraction efficiency. On the other hand, the extraction efficiency was small at low pH values of the acceptor phase due to H+ predomination in the electrostatic migration toward the pencil lead electrode. At a relatively high pH value of the acceptor phase, extraction yield was severely reduced because the analytes were mainly present as their neutral form (this phenomenon confirms that the extraction mechanism is electrokinetic migration of cationic species). Thus, neutral pH value was chosen as the pH of both acceptor and donor phases for the rest of the work. 3.3. Applied voltage and extraction time The driving force for electrokinetic migration of the analytes is mainly provided by the electrical field, which depends on the applied voltage. To investigate the effect of applied voltage and extraction time simultaneously, electrical potential differences in

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

Analyte

LOD (ng mL−1 )

Water

AMI DOX

0.5 2.0

Urine

AMI DOX

Plasma

AMI DOX

Linearity (ng mL−1 )

R2

ER%

2.0–500 5.0–500

0.9947 0.9973

11.5 4.2

1.0 2.5

2.5–500 5.0–500

0.9986 0.9978

10.4 4.0

1.0 5.0

5.0–500 10.0–500

0.9977 0.9968

5.9 3.1

the range of 50–260 V were applied for extraction durations of 5–20 min. As demonstrated in Fig. 2C, maximum amounts of the drugs were adsorbed into the pencil lead fiber when electrical potential of 120 V was applied. It is worthy to note that electrolysis may occur as a result of increasing of the electrical current of the system. Since EME system has relatively high electrical resistance due to presence of the organic solvent between the electrodes, and also the surfaces of electrodes are very small, the current level is low by applying 120 V electrical potential. Therefore, H2 generation on the surface of the cathode is negligible and no bobbles were observed during the extraction process. More increase in the voltage results in decreasing of normalized peak area due to mass transfer resistance and built-up of a boundary layer of ions at the interfaces at both sides of SLM or saturation of the analyte in the acceptor phase. Also, as pH slightly increased in the acceptor solution due to electrolysis, analyte back-extraction into the donor phase leads to reduction of extraction efficiency. Furthermore, a relatively low voltage leads to extraction protraction. Therefore, the extraction efficiency is improved by increasing the extraction time. Finally, 120 V was applied for 20 min to obtain the best results. 3.4. Method validation Figures of merit of the proposed method were investigated in three different media including pure water, human plasma, and human urine, according to recommendations of the Food and Drug Administration (FDA). Linearity was studied for AMI and DOX by the analysis of extracts obtained from the aliquots of each sample in triplicates. The extraction recovery (ER) was defined as the percentage of the number of moles of the analyte adsorbed into 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, at three concentrations (25, 100, and 400 ng mL−1 ), respectively.

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

(2)

Error% = RR% − 100

(3) (ng mL−1 )

where Cfound , Creal , and Cadded are the concentrations of analyte after addition of known amount of standard into the real sample, the concentration of analyte in real sample, and the concentration of known amount of standard which was spiked into the real sample, respectively. The results summarized in Table 2 show that EM-SPME could effectively be employed for the analysis of model drugs even from complicated matrices such as biological fluids. To improve mass transfer of the analytes, human plasma and urine samples were diluted 1:3 and 1:1, respectively, with pure water and the pH value was adjusted to 6.5 by addition of proper amounts of hydrochloric acid and/or sodium hydroxide solutions. The model drugs were effectively extracted with recoveries in the range of 3.1%–11.5%. The calibration plots were linear up to 500 ng mL−1 with coefficient of determination (R2 ) greater than 0.9947. Intra- and inter-assay precisions ranged between 4.0–8.5% and 7.5–12.2%, respectively (Table 3). Also, calculated Error% for the analytes in the range of −8.9% to +10.0% for different matrices demonstrates that the proposed method offers acceptable accuracy even in complicated matrices such as human plasma and urine samples. The limits of detection (LODs) were determined by analyzing a series of spiked samples (n = 3) with decreasing analyte concentrations and were less than 5 ng mL−1 and the limits of quantitation were less than 10 ng mL−1 . Comparison of proposed EM-SPME with other solid phase-based methods for extraction of AMI and DOX is provided in Table 4. One can see that along with simple equipment, EM-SPME offers excellent recoveries and LODs in a relatively short time. Since the organic liquid membrane amplifies the electrical resistance of the system, it is possible to apply high voltages, which reinforce the electrical

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 %) Intra-assay (n = 3) a

b

RRd %

Precision (CV%) Inter-assay (n = 3) c

Intra-assay (n = 5)

Inter-assay (n = 3)

W

W

W

U

P

W

U

P

U

P

U

P

W

U

P

AMI

25 100 400

+0.2 −0.2 +1.7

+3.4 +8.2 −2.0

+3.1 −3.0 −0.1

−1.2 −2.5 −5.4

−0.8 −4.5 +6.9

+0.9 +2.3 −8.6

8.5 6.9 5.2

4.2 5.5 4.3

5.3 4.0 4.3

9.6 8.2 7.5

11.0 8.8 7.9

10.0 10.2 8.1

100.2 99.8 101.7

103.4 108.2 98.0

103.1 97.0 99.9

DOX

25 100 400

−6.3 +6.6 −1.6

−8.1 −6.5 −6.2

−9.5 −2.7 +0.6

+0.8 −2.2 −4.4

−1.6 +6.5 +10.0

+2.6 +5.5 −8.9

5.5 5.8 5.1

4.4 6.0 4.4

3.9 5.7 5.5

9.5 11.7 10.9

9.9 12.2 9.0

10.7 9.5 10.4

93.7 106.6 98.4

92.4 93.5 93.8

90.5 97.3 100.6

a b c d

W: Water. U: Urine. P: Plasma. Relative recovery.

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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 SPME-GC-NPD SPME-GC-NPD HS-SPME-GC-MS SPME-LC-MS SPME-HPLC-UV SPME-MC-LC EME-SPME EME-SPME EME-SPME EME-SPME EME-SPME EME-SPME

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

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

0.08 0.07 – – – 0.05 (ng mg−1 ) 0.1 – 3 0.5 1.0 1.0 2.0 2.5 5.0

1 1 0.01 12.5 12.5 – 50 75 5 2.0 2.5 5.0 5.0 5.0 10.0

30 30 40 10 10 50 30 50 210 20 20 20 20 20 20

– – – 0.46 0.63 0.4–2.3 2.15 – – 11.5 10.4 5.9 4.2 4.0 3.1

2.0 3.9 9.9 11.5 44.6 – 14.92 <10 <10.1 8.5c 4.2c 5.3c 5.5c 4.4c 3.9c

[30] [30] [31] [32] [32] [33] [33] [34] [35] This work This work This work This work This work This work

a In-tube (IT), liquid chromatography (LC), mass spectrometry (MS), dual solid phase microextraction (dSPME), nitrogen–phosphorus detector (NPD), high performance liquid chromatography (HPLC), ultraviolet detector (UV), microcolumn (MC). b Limit of quantification. c For five-replicate measurements at 25 ng mL−1 .

Fig. 3. Chromatograms obtained after extraction of drugs from (A) human urine, (B) human plasma ((a) non-spiked sample, (b) spiked sample). 1; AMI, 2; DOX.

field and extraction recoveries. Also, solitude chromatograms are obtained even by extraction from biological fluids. Therefore, using expensive extraction and analysis methods such as in-tube SPME and liquid chromatography/mass spectrometry is unnecessary. 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 [13]. Thus, EM-SPME could be introduced as a novel and simple technique for efficient extraction of analytes from complicated matrices. 3.5. Analysis of real samples In order to evaluate the applicability of the introduced EM-SPME method to the analysis of real samples, different human plasma

and urine samples were analyzed. All samples were prepared as explained in the method validation section. To this end, plasma and urine samples were diluted 1:3 and 1:1, respectively, with pure water and the pH value was adjusted to 6.5 by addition of proper amounts of hydrochloric acid and/or sodium hydroxide solutions and optimal conditions were applied for quantitative analysis. Chromatograms obtained after extraction from human plasma and urine samples are shown in Fig. 3. Thereafter, to determine the method accuracy, each sample was spiked at 50 ng mL−1 of the drugs and EM-SPME was carried out to calculate extraction error. By FDA’s definition, a matrix effect is the direct or indirect alteration or interference in response due to the presence 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

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

Analyte

Creal (ng mL−1 )

Cadded (ng mL−1 )

RSD% (n = 3)

Error%

Plasma 1

AMI DOX

nda nd

50.0 50.0

49.3 52.5

6.0 4.7

−1.4 +5.0

Plasma 2

AMI DOX

nd nd

50.0 50.0

50.8 48.1

7.6 3.6

+1.6 −3.8

Urine 1

AMI DOX

153.9 nd

50.0 50.0

204.6 52.8

5.5 6.9

+1.4 +5.6

Urine 2

AMI DOX

nd nd

50.0 50.0

49.1 52.3

7.3 8.1

−1.8 +4.6

a

Not detected.

Cfound (ng mL−1 )

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