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Low-voltage electrically-enhanced microextraction as a novel technique for simultaneous extraction of acidic and basic drugs from biological fluids
Journal of Chromatography A, 1243 (2012) 6–13
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Low-voltage electrically-enhanced microextraction as a novel technique for simultaneous extraction of acidic and basic drugs from biological fluids Shahram Seidi, Yadollah Yamini ∗ , Maryam Rezazadeh, Ali Esrafili 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 17 March 2012 Received in revised form 18 April 2012 Accepted 19 April 2012 Available online 26 April 2012 Keywords: Electromembrane Diclofenac Hollow fiber Nalmefene Simultaneous extraction Low voltage
a b s t r a c t In the present work, for the first time a new set-up was presented for simultaneous extraction of acidic and basic drugs using a recent novel electrically-enhanced microextraction technique, termed electromembrane extraction at low voltages followed by high performance liquid chromatography with ultraviolet detection. Nalmefene (NAL) as a basic drug and diclofenac (DIC) as an acidic drug were extracted from 24 mL aqueous sample solutions at neutral pH into 10 L of each acidified (HCl 50 mM) and basic (NaOH 50 mM) acceptor solution, respectively. Supported liquid membranes including 2-nitrophenyl octyl ether containing 5% di-(2-ethylhexyl) phosphate and 1-octanol were used to ensure efficient extraction of NAL and DIC, respectively. Low voltage of 40 V was applied over the SLMs during 14 min extraction time. The influences of fundamental parameters affecting the transport of target drugs were optimized using experimental design. Under optimal conditions, NAL and DIC were extracted with extraction recoveries of 12.5 and 14.6, respectively, which corresponded to preconcentration factors of 300 and 350, respectively. The proposed technique provided good linearity with correlation coefficient values higher than 0.9956 over a concentration range of 8–500 g L−1 and 12–500 g L−1 for NAL and DIC, respectively. Limits of detection and quantifications, and intra-day precisions (n = 3) were less than 4 g L−1 , 12 g L−1 , and 10.1%, respectively. Extraction and determination of NAL and DIC in human urine samples were successfully performed. In light of the data obtained in the present work, this new set-up for EME with low voltages has a future potential as a simple, selective, and fast sample preparation technique for simultaneous extraction and determination of acidic and basic drugs in different complicated matrices. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, determination of pharmaceutical drugs in biological fluids has found noticeable importance in medical sciences, due to increasing of medicine consuming among people. Along with the mentioned point, determination of medicine contents in different biological fluids helps to get information about mechanism, side effects, and kinetics of these compounds in the body. So, this subject has high degree of importance in pharmaceutical sciences. However, determination of drugs and metabolites in biological fluids such as plasma and urine is still a very challenging task. The area of largest difficulty is often the sample pretreatment step. In the past two decades, a large number of modern sample preparation techniques including solvent free extraction techniques or extraction techniques with a very high sample to solvent ratio, which leads to a high preconcentration factor of analytes have been introduced.
∗ Corresponding author. Tel.: +98 21 82883417; fax: +98 21 88006544. E-mail address: [email protected] (Y. Yamini). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.04.050
Among the emerging techniques, liquid phase microextraction based on hollow fiber (HF-LPME) is one of the most promising developments for preconcentration, separation, and cleanup purposes. Nevertheless, in spite of the potential capabilities of HFLPME, the extraction time needed in this technique is usually high and common extraction times of 30–50 min have been reported [1]. In 2006, Pedersen-Bjergaard et al. introduced a novel microextraction technique called electromembrane extraction (EME) [2], to overcome this limitation of HF-LPME. The present method has a same set-up as HF-LPME, besides the use of a power supply and two electrodes to sustain the voltage across the SLM. EME was performed effectively for extraction of various compounds from different matrices with variable voltages in the range of 9–300 V [2–24]. In EME, only a single class of analytes (basic or acidic analytes) is extracted. In a recent work, Basheer et al. introduced an interesting method for simultaneous extraction of acidic and basic drugs from wastewater samples using four sheets of porous polypropylene membrane, which were combined and heat-sealed at three edges [10]. In the present work, a new EME set-up based on two separated pieces of hollow fibers were used for simultaneous extraction of
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by a heater-magnetic stirrer model 3001 from Heidolph (Kelheim, Germany) using a 1.5 cm × 0.3 cm magnetic bar.
2.2. Chemicals and materials Nalmefene (NAL), morphine (Mor) codeine (Cod), oxymorphone (Oxy), mefenamic acid (Mef) and diclofenac (DIC) were kindly donated by Parand Darou (Tehran, Iran) and the Department of Medical Sciences of Tehran University (Tehran, Iran). 2-Nitrophenyl octyl ether (NPOE) and di-(2-ethylhexyl) phosphate (DEHP) were purchased from Fluka (Buchs, Switzerland). Cetyltrimethylammonium bromide, CTAB (C19 H42 BrN), NaClO4 , pyridines and 1-octanol were purchased from Merck (Darmstadt, Germany). Standards of chlorophenols were purchased 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 aquaMAx purification system 370 series (Seoul, Korea). Fig. 1. A schematic illustration of the equipment for simultaneous extraction of acidic and basic drugs by EME. Different parts of the set-up are shown in the figure.
acidic and basic drugs. It makes possible to employ the best SLM for each class of analytes separately, whereas this possibility is to some extent difficult for hand-made envelope by sheet membranes. Also, the electrical resistance of extraction system increases sowing to the use of two layers of organic solvents that reduces the risk of electrolysis and bubble formation during extraction time. In comparison with hand-made envelope using sheet membranes, the presented set-up in this work is simpler; the volume of acceptor phase is more repeatable, and the risk of membrane pollution during development of the set-up is very lower. Also, in the previous work, the volume of final acceptor phase is relatively high (50 L) due to limitation of sizes of sheet fibers needed for making compartments, whereas reaching low volume for acceptor phase (even lower than 5 L) can easily be achieved using hollow fibers. Using high volume of acceptor phase causes dilution and reduction of determination sensitivity; especially in the case of gas chromatography that only 1–3 L of extract is injected into the instrument. In the previous work, the final step of extraction process is basically controlled by distribution ratios, which may increases the extraction time. This is while in the present work, the most effective transport mechanism is electrokinetic migration, which reduces the extraction time. Finally, high performance liquid chromatography with ultraviolet detection was employed for analysis of acceptor solution, which needs no derivatization steps.
2. Experimental 2.1. Equipment for electromembrane extraction The equipment used for the extraction procedure is shown in Fig. 1. A 24-mL vial with internal diameter of 2.5 cm and height of 5.5 cm was used. The electrodes used in this work were platinum wires with diameters of 0.25 mm, and were 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 EME unit was stirred with a stirring speed in the range of 0–1250 rpm
2.3. Biological and standard solutions Urine samples were collected from four volunteers, two of which were treated with DIC tablets. The samples were stored in sterilized bottles at −4 ◦ C, thawed and shaken before extraction. A stock solution containing 1 mg mL−1 of NAL and DIC were prepared in methanol and stored at −4 ◦ C. Working standard solutions were prepared by dilution of the stock solutions in water.
2.4. HPLC conditions Separations and detections of the target analytes were performed by a Varian HPLC (Walnut Creek, CA, USA) containing a 9012 HPLC pump, a six-port Cheminert HPLC valve from Valco Instruments (Houston, TX, USA) with a 20-L sample loop and a Varian 9050 UV–Vis detector. Chromatographic data were recorded and analyzed using Chromana software (version 3.6.4). The separations were carried out on an ODS-3 column (250 mm × 4.0 mm, with 5 m particle size) from TeknoKroma (Barcelona, Spain). Separation of NAL and DIC was performed using an isocratic elution at the flow rate of 1.0 mL min−1 . The mobile phase consisted of 50 mM NaClO4 and acetonitrile (65:35, v/v). Quantification of both NAL and DIC was accomplished by measuring peak areas at wavelength of 210 nm. Total analysis time was 12 min. Separation and determination of chlorophenoles were carried out according to the literature [25]. Mobile phase of pyridines was a buffer solution containing 50 mM acetic acid and sodium phosphate (pH = 8.0) and acetonitrile (50:50, v/v). The separation program was an isocratic elution at the flow rate of 0.8 mL min−1 . All determinations were performed at 254 nm. The chromatographic separation of morphine, codeine and oxymorphone was performed with a mobile phase consisting of 10 mM phosphate buffer adjusted to pH 2.5 and acetonitrile, and delivered at a flow rate of 0.8 mL min−1 . The gradient program was as follows: starting with 15% acetonitrile, then increasing to 70% in 0–12 min, keeping constant until 13 min, thereafter restored to 15% in 3 min. The detector wavelength was set at 210 nm. The mobile phase for separation of mefenamic acid was 50% acetonitrile and 50% ammonium acetate aqueous solution (50 mM, pH = 5.3). The flow rate was 1.0 mL min−1 . The detection wavelength was set at 285 nm.
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2.5. Procedure for EME Twenty four milliliters of the sample solution containing target analytes in deionized water was transferred into the sample vial. To impregnate the organic solutions in the pores of cathodic and anodic hollow fiber walls, two 3.8-cm pieces of hollow fibers were cut out and dipped in the organic solutions for 5 s and then the excess of organic solutions were gently wiped away by air blowing using a 500-L Hamilton syringe. The upper ends of hollow fibers were connected to two medical needle tips as guiding tubes, which were inserted through the rubber cap of the vial. Ten microliters of 50 mM HCl and NaOH as cathodic and anodic acceptor solutions were introduced into the lumen of the hollow fibers by a microsyringe, respectively. The lower ends of the hollow fibers were sealed with two small pieces of aluminum foil (thickness of 280 m). One of the electrodes, the cathode, was introduced into the lumen of the cathodic fiber and the other was inserted into the lumen of the anodic fiber. The fibers containing the cathode and anode, SLM and the acceptor solutions were afterward directed into the sample solution. The electrodes were subsequently coupled to the power supply and the extraction unit was placed on a stirrer with stirring speed of 700 rpm. The predetermined voltage of 40 V was turned on and extraction was performed for 14 min. Under the applied voltage, the target analytes migrated from aqueous sample into SLM, and then transported into acceptor phases (Fig. 1). After the extraction was completed, the acceptor solutions were collected by a microsyringe, mixed together into a microtube and injected into the HPLC for further analysis. 2.6. Calculation of preconcentration factor, extraction recovery, and relative recovery The preconcentration factor (PF) was defined as the ratio of the final analyte concentration in the acceptor phase (Cf,a ) to the initial concentration of analyte (Ci,s ) in the sample solution: PF =
Cf,a
(1)
Ci,s
where Cf,a was calculated from a calibration graph obtained from direct injection of NAL and DIC standard solutions. The extraction recovery (ER%) was defined as the percentage of the number of moles of analyte originally present in the sample (ni,s ), which was extracted to the acceptor phase (nf,a ). ER% =
nf,a ni,s
ER% =
× 100 =
Va Vf
Cf,a Va Ci,s Vf
× 100
PF × 100
(2)
(3)
where Va and Vf represent the volumes of acceptor phase and sample solution, respectively. Relative recovery (RR%) was acquired from the following equation: RR% =
Cfound − Creal Cadded
× 100
(4)
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. 2.7. Data analysis and statistical methods In order to obtain the optimum conditions for simultaneous extraction of NAL and DIC, a central composite design (CCD) was
Fig. 2. The chromatograms obtained from (I) a spiked water sample at 5 V (a) and no application of electrical potential (b); (II) a urine sample after spiking with NAL and DIC at concentration of 500 g L−1 (a) and before spiking (b) under optimal conditions; (III) chromatogram of a non-spiked urine sample under optimal conditions that was taken from a volunteer who had treated with DIC. For more details see the text.
used. For this purpose, STATISTICA software trial version 8.0 (StatSoft, Tulsa, OK, USA) was employed to generate the experimental matrix and evaluate the results. 3. Results and discussion 3.1. Initial experiments In this work, experimental design method was used for optimization of effective parameters on EME. However, some initial investigations were carried out before optimization process by experimental design method to find some primary information about these parameters. To this aim, 24 mL of deionized water spiked with target analytes at concentration of 500 g L−1 was agitated for 15 min with stirring speed of 700 rpm. According to the literatures, NPOE containing 5% DEHP and 1-octanol were used as the best SLMs for extraction of NAL and DIC, respectively [9,17]. The lumens of the fibers were filled with 100 mM HCl and NaOH as cathodic and anodic acceptor solutions, respectively. Initial experiments at low voltages showed that NAL and DIC can easily be extracted at these voltages. Fig. 2I shows the chromatograms obtained for simultaneous extraction of DIC and NAL at 5 V (a) and
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without application of voltage (b). As can be seen from this figure, no considerable extraction occurs in the absence of voltage that indicates the importance of application of voltage in EME system as the main driving force. Some tests concerning the effects of the stirring rate and ionic strength of the donor phase in the electromigration process were realized. As it is known, stirring speed plays an essential role in increasing the kinetics and efficiency of extraction by increasing the mass transfer and reducing the thickness of double layer around SLM. To study the effect of stirring rate in more details, the effect of stirring speed on extraction efficiency was investigated up to 1250 rpm. A stirring rate of 700 rpm was chosen as the best amount due to formation of intense vortex and bubble formation into sample solution at higher speeds. Sodium chloride was added to the donor phase for studying the effect of ionic strength on the extraction efficiencies of DIC and NAL at concentration levels of 2.5% and 5% (w/v). Presence of salt caused a decrease in the extraction efficiency and obtaining of undesirable results. In 2007, Pedersen-Bjergaard et al. introduced a mathematical model based on the Nernst–Planck equation [11], which investigates EME in more details from a theoretical point of view. According to the model, increasing the ionic strength in sample solution increases the ion balance () that leads to reduction of extraction efficiency. The ion balance is defined as the total concentration of ions in sample solution to that in acceptor phase [11]. The reason of this phenomenon is increasing of the competition among analyte and other ions for entering into the acceptor solution. It is remarkable that presence of high contents of salt in the donor solution increases variation of acceptor phase volume and so decreases extraction repeatability. Also, it may result in puncture of SLM and creation of arc between the electrodes. Similar observations have been reported [17]. 3.2. Variation in the SLM composition (organic solvent) According to earlier findings, the chemical nature of the supported liquid membrane is highly critical for the success of EME. There are specific requirements for a solvent to be used as a SLM in EME [7]. 1-Octanol and NPOE have been the best candidates for acidic and basic drugs in EME, respectively, up to now [1,3]. It has been found that addition of hydrophobic ion-pair reagents to SLM would enable better phase transfer and electrokinetic migration of analytes [2,7,9]. To investigate this effect, 2.5% (w/v) CTAB in 1-octanol and 5% (v/v) DEHP in NPOE were used as the anodic and cathodic SLMs, respectively. Addition of DEHP showed good improvement in extraction efficiency of NAL, while using CTAB gave variable results and thus did not provide any general guidance on the trends of extraction for DIC. Therefore, 1-octanol was chosen as anodic SLM. In a new experiment, DEHP was gradually added to NPOE corresponding to 10% and 15% (v/v). The results are shown in Fig. 3. As seen from the results, there is no noticeable difference between extraction efficiency of NAL at 5% and 10% (v/v) of DEHP, while further addition caused to a decrease in its extractability. So, NPOE containing 5% (v/v) of DEHP was selected as cathodic SLM. Generally, decreasing of an analyte extractability with addition of ion-pair reagents may be due to a decrease in electrical resistance of SLM and an increase in the current level and bubble formation or strong interaction of the ion-pair and analyte complex with the organic solvent [2,7]. 3.3. Results for the central composite design (CCD) Response surface methodology (RSM) is a collection of statistical and mathematical methods that involves experimental designs
9
Fig. 3. Effect of SLM composition on extraction efficiencies of NAL and DIC by EME; spiked concentration: 500 g L−1 , voltage: 30 V, sample solution: 24 mL of urine at pH 6.5, acceptor solutions: 100 mM HCl (cathodic) and 100 mM NaOH (anodic), extraction time: 10 min, and stirring rate: 700 rpm.
to achieve adequate and reliable measurement of the response of interest. RSM usually contains three steps: (1) design and experiments; (2) response surface modeling through regression; and (3) optimization. The main objective of RSM is to determine the optimal operational conditions of the process or to determine a region that meets the operating specifications. Among different RSM experimental designs, face centered central composite design (FCCCD) has been widely used because it requires fewer experimental runs and provides sufficient information. Different variables can affect the extraction efficiency of EME procedure, including type of organic solvent (SLM), volume of sample solution, pH of donor and acceptor phases, stirring rate, salt%, temperature, extraction time, and voltage. Variables were chosen with the aim of reducing the extraction time and process cost. The proposal includes parameters such as extraction time, voltage, and pH of donor and acceptor phases. Separated study of membrane organic solvent can give optimum SLM, simplicity of experimental design method, and reduction of number of runs. Therefore, this parameter was separately optimized at the first. Temperature is another factor, which can affect the flux of ions through SLM. Theoretically, increase of temperature decreases the electrical driving force in EME, while increases diffusion coefficient of ions into SLM [11]. However, according to previous studies and our experience, an increase in temperature led to puncture of SLM, and increase in bubble formation and arc phenomenon probability due to increase in solubility of membrane organic solvent; especially for long extraction time. Furthermore, control of temperature is relatively difficult as it can increase the experiment uncertainties; thus, working at ambient temperature can be a good benefit for a microextraction technique. To pass the analytes through electrical field, it is necessary to change them to their ionizable forms. NAL and DIC have pKa values of 7.6 and 4.2, respectively; so, they are charged (NAL+ and DIC− ) in deionized water solution (donor phase) and easily transferred to cathodic and anodic acceptor solutions under electrical field. Based on the literatures, pH in the sample solution is not highly critical for the EME process, so that the extraction efficiency is found to be maintained constant in an extended pH range [2,10,13]. Based on initial experiments, addition of salt has negative effect on extraction efficiencies of NAL and DIC, and increasing the stirring speed above 700 rpm is not experimentally possible. Therefore,
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these factors were not taken into account in the experimental design. By this strategy for factor selection, 17 permutations of conditions were obtained by varying the three remained variables at the lower, middle, and upper levels (5, 12.5 and 20 min for time, 10, 40 and 70 V for voltage, 1, 50 and 100 mM for concentration of cathodic and anodic acceptor phases). Normalized peak area was used as the experimental response for each permutation [18]. The data obtained were evaluated by analysis of variance (ANOVA) at 95% confidence level. Results indicate that time (X1 ) and voltage (X2 ) have significant effects on extraction recovery, and the X1 , X2 , and the concentration of cathodic and anodic acceptor phases (X3 ) interactions and quadratic terms of all the three factors are significant. In addition, there is no evidence of lack of fit at the 95% confidence level, meaning that the model is explaining the observed differences in the response variable. In the next step of the design, a response surface model was developed by considering all significant interactions in the FCCCD. The R2 value of model was obtained higher than 0.97, so the regression model predicts the experimental responses adequately. Normalized peak area = −29.1570 + 4.8038X1 − 0.1705X12 + 1.2933X2 − 0.0137X22 + 0.0077X3 − 0.0022X32 − 0.0079X1 X2 + 0.0094X1 X3 + 0.0010X2 X3
(5)
Based on the analysis, it was found that the normalized peak area increased with the increase of time, voltage, and concentration of the acceptor phase to a certain level and then decreased beyond that. According to the EME theoretical model [11], the main driving force for migration of the analytes across the liquid membrane is provided by the electrical field. Strength of the electrical field is dependent on the applied voltage. Therefore, applied voltage is one of the most important parameters that should be regarded. Tendency of basic drugs to deprotonate in the artificial liquid membrane was relatively high. Application of high electrical potential differences promotes their efficient migration through the artificial membrane and reduces deprotonation opportunity of these drugs, which resulted in higher extraction recovery [2]. Time is another parameter that can affect the flux of analytes in EME. Voltage and time are two parameters that act in parallel ways. Both time and voltage directly increase the flux of ions, and thus increase the extraction recovery. However, there is an antagonistic effect when they are simultaneously considered. It means that an increase in extraction time limits the voltage and vice versa. Increase in voltage causes an increase in the number of ions crossing through the membrane. This leads to heat generation (Joule heating phenomenon) and loss of membrane organic solvent as a function of time. Therefore, time duration of membrane stability reduces by increasing of voltage [2,8,9,19–21]. As it is known in EME, electrolysis can occur at surfaces of both electrodes according to the following reactions: Anodic acceptor phase : H2 O → 2H+ + 1/2O2 + 2e−
(6)
Cathodic acceptor phase : 2H+ + 2e− → H2
(7)
The probabilities of these reactions increase with as the time and voltage increases, owing to the decrease in membrane stability and resistance and increase in system current flow. Electrolysis on the surface of both electrodes slightly increases as the pH value of the cathodic acceptor solution increases and the pH value of the anodic acceptor solution decreases. This may result from neutralization of analytes and back-extraction of them into the donor phase and decrease of extraction efficiencies [2,22–24].
Also, statistical analysis showed that extraction efficiencies were increased by decrease in the pH value of cathodic acceptor solution (below the pKa of basic analyte) and increase in the pH value of anodic acceptor solution (above the pKa of acidic analyte). These variations occur due to easily releasing of the analytes into the cathodic and anodic acceptor solutions, respectively. However, there are some limitations for the increase of proton and hydroxide ions concentrations of the cathodic and anodic acceptor solutions, respectively. These types of pH adjustments improve the probability of electrolysis reactions and bubble formation on the surfaces of electrodes; especially with the increase in extraction time and voltage. Bubble formation causes intensive variation in the collected volumes of acceptor solutions and increases uncertainties of the data obtained by EME. The final step was to find the optimum conditions of voltage, extraction time, and concentration of HCl and NaOH as cathodic and anodic acceptor phases, respectively. In order to optimize the extraction efficiency of EME, the desirability function approach was applied to analyze the regression model equations using STATISTICA software. Desirability function is an interesting and precise technique to simultaneously determine the optimal amounts of input variables. This technique was first developed by Harrington [26], and was later optimized by Derringer and Suich [27] for specifying the relationship between predicted responses on a dependent variables and the desirability of the responses. The results of FCCCD design matrix represented the maximum of 34.070 and minimum of 2.000 for normalized peak area. According to these values, desirability function settings for each dependent variable of the normalized peak area are depicted at the right hand side of Fig. 4: desirability of 1.0 was assigned for maximum (34.070), 0.0 for minimum (2.000), and 0.5 for middle (18.035). On the basis of these calculations and desirability score of 1, the optimized variables were found to be 14 min, 40 V, 50 mM, and 50 mM for extraction time, voltage, and concentration of HCl and NaOH as cathodic and anodic acceptor solutions, respectively. 3.4. Method validation The optimized conditions were applied to test the applicability of the proposed EME method for extraction of model drugs from human urine samples. Calibration curves were plotted in drug free urine samples and figures of merit of the method were investigated. For this purpose, urine sample was spiked with the analytes and extraction was accomplished after dilution of urine samples (1:1) with pure water and pH adjustment. The pH was adjusted by dropwise addition of 5.0 M NaOH and/or HCl solutions, such that the final pH of samples was adjusted at 6.5. The proposed technique provided good linearity with correlation coefficient values higher than 0.9956 over a concentration range of 8–500 g L−1 and 12–500 g L−1 for NAL and DIC, respectively. As seen from Table 1, this system is capable of simultaneous extraction and determination of acidic and basic analytes with good limits of detection (LODs). Also, 8 g L−1 and 12 g L−1 were determined as limits of quantification (LOQs) for basic and acidic drugs, respectively. Repeatability was evaluated by performing triple analysis of the drugs at concentration level of 50 g L−1 and the relative standard deviations (RSDs%) were less than 10.1%. The preconcentration factors of 300 and 375 were obtained for DIC and NAL, respectively, which illustrate the ability of this new EME set-up for trace analysis of real samples. 3.5. Analysis of real samples The proposed method was applied to determine the concentration of NAL and DIC in urine samples. Firstly, samples were diluted
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Fig. 4. Profiles for predicted values and desirability function of normalized peak area. Dashed lines indicate current values after optimization.
Table 1 Figures of merit of the method in a drug-free urine sample. Analyte
LOD (g L−1 )
LOQ (g L−1 )
Linearity LOQ – 500 (g L−1 )
R2
RSD%a (n = 3)
PF
ER%
NAL DIC
2 4
8 12
y = 0.6102x + 8.8599 y = 0.5435x − 0.6951
0.9981 0.9956
7.4 8.9
350 300
14.6 12.5
a
RSDs% were calculated at concentration of 50 g L−1 for each drug.
1:1 with ultrapure water and their pH values were adjusted at 6.5. The pH was adjusted by dropwise addition of 5.0 M NaOH and/or HCl solutions, such that the final pH of samples was adjusted at 6.5. Then, 24 mL of each solution was transferred into the sample vial and evaluated by the EME process. Fig. 2II shows the chromatograms obtained for a blank urine sample before and after spiking with 500 g L−1 of NAL and DIC. The corresponding relative recoveries (RR%) and RSDs% are summarized in Table 2. As can be seen, the relative recoveries for both drugs in spiked urine samples are between 90% and 98%. These results demonstrated that
Table 2 Relative recoveries and standard deviations of NAL and DIC from spiked blank urine samples and actual urine samples. Sample Urine 1
Urine 2
Urine 3
Urine 4
a b
Initial concentration (g L−1 ) RR%b RSD% (n = 3) Initial concentration (g L−1 ) RR% RSD% (n = 3) Initial concentration (g L−1 ) RR% RSD% (n = 3) Initial concentration (g L−1 ) RR% RSD% (n = 3)
NAL
DIC
nda 98.0 5.1 nd 91.0 6.6 nd 95.0 5.8 nd 96.0 8.7
nd 96.0 6.5 nd 93 7.8
nd, no detection. 50 g L−1 of each drug was added to calculate relative recovery (RR%).
the matrices of the analyzed real urine samples possess negligible effect on the proposed methods and also excellent sample clean-up was obtained. Fig. 2III depicts the resulted chromatogram of a non-spiked urine sample, which was taken from a volunteer who had treated with DIC. 3.6. Investigation applicability of the proposed configuration for simultaneous extraction of other compounds On the basis of the experiments discussed above, the performance of the proposed configuration was investigated for simultaneous extraction of some other basic and acidic compounds. Twelve different compounds including 4-chlorophenol (4-CP, pKa = 9.43), 2,3-dichlorophenol (2,3-DCP, pKa = 7.7), 2,5dichlorophenol (2,5-DCP, pKa = 7.2) and 2,4,6-trichlorophenol (2,4,6-TCP, pKa = 6.23) as acidic environmental pollutants, pyridine (Py, pKa = 5.25), 3-methyl pyridine (3-MPy, pKa = 5.63), 2,4-lutidine (2,4-Lu, pKa = 6.46) and quinoline (Qu, pKa = 4.81) as basic environmental pollutants, morphine (Mor, pKa = 8.0), oxymorphone (Oxy, pKa = 8.5) and codeine (Cod, pKa = 8.2) as basic drugs and mefenamic acid (Mef, pKa = 4.4) as an acidic drug were extracted under optimal conditions which were obtained for DIC and NAL. The acidic and basic environmental pollutants were extracted together (Fig. 5A) and also simultaneous extraction of acidic and basic dugs were investigated (Fig. 5B). Extraction of these compounds was carried out under optimal conditions of NAL and DIC and acceptable results were obtained (Table 3). It should be noted that some of these
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Fig. 5. HPLC chromatograms related to simultaneous extraction of different acidic and basic compounds utilized new configuration of EME. Pyridines, chlorophenols, basic drugs and acidic drug were present at 250, 250, 500 and 500 g L−1 , respectively.
Table 3 The obtained preconcentration factors (PFs) and repeatability (RSDs%) for simultaneous extraction of different acidic and basic environmental and pharmaceutical compounds under optimal conditions of NAL and DIC. Analyte classification
Type of analyte
Analyte
Environmental
Basic
PY 3-MPy 2,4-Lu Qu 4-CP 2,3-DCP 2,5-DCP 2,4,6-TCP Mor Oxy Cod Mef
Acidic
Pharmaceutical
Basic
Acidic
PFa 120 142 174 96 46 43 38 42 66 45 114 32
RSD% (n = 3) 4.6 4.8 5.1 4.5 6.5 7.3 6.2 6.5 5.3 6.8 5.5 7.5
a
Pyridines, chlorophenols, basic drugs and acidic drug were present at 250, 250, 500 and 500 g L−1 , respectively.
compounds such as basic drugs are so polar; however, they were easily extracted under modest conditions. This observation indicates the important role of electrical field in EME as well as applicability of the proposed configuration of EME for simultaneous extraction of various acidic and basic compounds from different matrices. 4. Conclusions In the present work, for the first time, a new EME set-up was introduced for simultaneous extraction and determination of trace amounts of acidic and basic drugs in biological samples. Since the SLMs were optimized for each class of analytes, this system offers more efficient extraction. Low voltage EME with optimized SLM for each class of analytes effectively prevents the extraction of interferences. Despite simplicity, this EME system presents high selectivity and sample cleanup that makes this method suitable
for simultaneous extraction of different classes of analytes from complicated matrices. Acknowledgements The authors gratefully acknowledge financial support from Tarbiat Modares University. Also, the support provided by the National Elite Foundation (Tehran, Iran) is highly appreciated. References [1] K.E. Rasmussen, S. Pedersen-Bjergaard, Trends Anal. Chem. 23 (2004) 1. [2] S. Pedersen-Bjergaard, K.E. Rasmussen, J. Chromatogr. A 1109 (2006) 183. [3] M. Balchen, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1152 (2007) 220. [4] I.J.Ø. Kjelsen, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1180 (2008) 1. [5] L.E. Eibak, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1217 (2010) 5050. [6] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1124 (2006) 29. [7] J. Lee, F. Khalilian, H. Bagheri, H.K. Lee, J. Chromatogr. A 1216 (2009) 7687. [8] M. Balchen, L. Reubsaet, S. Pedersen-Bjergaard, J. Chromatogr. A 1194 (2008) 143. [9] M. Rezazadeh, Y. Yamini, S. Seidi, J. Chromatogr. B 879 (2011) 1143. [10] C. Basheer, J. Lee, S. Pedersen-Bjergaard, K.E. Rasmussen, H.K. Lee, J. Chromatogr. A 1217 (2010) 6661. [11] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1174 (2007) 104. [12] T.M. Middelthon-Bruer, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Sep. Sci. 31 (2008) 753. [13] A. Gjelstad, H. Jensen, K.E. Rasmussen, S. Pedersen-Bjergaard, Anal. Chim. Acta (2012), http://dx.doi.org/10.1016/j.aca.2011.12.039. [14] T. Sikanen, S. Pedersen-Bjergaard, H. Jensen, R. Kostiainen, K.E. Rasmussen, T. Kotiaho, Anal. Chim. Acta 658 (2010) 133. [15] L.E.E. Eibak, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Pharm. Biomed. 57 (2012) 33. [16] M. Balchen, H. Lund, L. Reubsaet, S. Pedersen-Bjergaard, Anal. Chim. Acta 716 (2012) 16–23. [17] M. Ramos-Payán, M.Á. Bello-López, R. Fernádez-Torres, M. Villar-Navarro, M. Callejón-Mochon, Talanta 85 (2011) 394. [18] S. Seidi, Y. Yamini, T. Baheri, R. Feizbakhsh, J. Chromatogr. A 1218 (2011) 3958. ˇ L. Strieglerová, P. Gebauer, P. Boˇcek, Electrophoresis 32 (2011) [19] P. Kubán, 1025.
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[24] A. Gjelstad, T.M. Andersen, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1157 (2007) 38. [25] J. Huang, J. Liu, C. Zhang, J. Wei, L. Mei, S. Yu, G. Li, L. Xu, J. Chromatogr. A 1219 (2012) 66. [26] E.C. Harrington, Ind. Qual. Control 21 (1965) 494. [27] G. Derringer, R. Suich, J. Qual. Technol. 12 (1980) 214.
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Low-voltage electrically-enhanced microextraction as a novel technique for simultaneous extraction of acidic and basic drugs from biological fluids Shahram Seidi, Yadollah Yamini ∗ , Maryam Rezazadeh, Ali Esrafili 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 17 March 2012 Received in revised form 18 April 2012 Accepted 19 April 2012 Available online 26 April 2012 Keywords: Electromembrane Diclofenac Hollow fiber Nalmefene Simultaneous extraction Low voltage
a b s t r a c t In the present work, for the first time a new set-up was presented for simultaneous extraction of acidic and basic drugs using a recent novel electrically-enhanced microextraction technique, termed electromembrane extraction at low voltages followed by high performance liquid chromatography with ultraviolet detection. Nalmefene (NAL) as a basic drug and diclofenac (DIC) as an acidic drug were extracted from 24 mL aqueous sample solutions at neutral pH into 10 L of each acidified (HCl 50 mM) and basic (NaOH 50 mM) acceptor solution, respectively. Supported liquid membranes including 2-nitrophenyl octyl ether containing 5% di-(2-ethylhexyl) phosphate and 1-octanol were used to ensure efficient extraction of NAL and DIC, respectively. Low voltage of 40 V was applied over the SLMs during 14 min extraction time. The influences of fundamental parameters affecting the transport of target drugs were optimized using experimental design. Under optimal conditions, NAL and DIC were extracted with extraction recoveries of 12.5 and 14.6, respectively, which corresponded to preconcentration factors of 300 and 350, respectively. The proposed technique provided good linearity with correlation coefficient values higher than 0.9956 over a concentration range of 8–500 g L−1 and 12–500 g L−1 for NAL and DIC, respectively. Limits of detection and quantifications, and intra-day precisions (n = 3) were less than 4 g L−1 , 12 g L−1 , and 10.1%, respectively. Extraction and determination of NAL and DIC in human urine samples were successfully performed. In light of the data obtained in the present work, this new set-up for EME with low voltages has a future potential as a simple, selective, and fast sample preparation technique for simultaneous extraction and determination of acidic and basic drugs in different complicated matrices. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, determination of pharmaceutical drugs in biological fluids has found noticeable importance in medical sciences, due to increasing of medicine consuming among people. Along with the mentioned point, determination of medicine contents in different biological fluids helps to get information about mechanism, side effects, and kinetics of these compounds in the body. So, this subject has high degree of importance in pharmaceutical sciences. However, determination of drugs and metabolites in biological fluids such as plasma and urine is still a very challenging task. The area of largest difficulty is often the sample pretreatment step. In the past two decades, a large number of modern sample preparation techniques including solvent free extraction techniques or extraction techniques with a very high sample to solvent ratio, which leads to a high preconcentration factor of analytes have been introduced.
∗ Corresponding author. Tel.: +98 21 82883417; fax: +98 21 88006544. E-mail address: [email protected] (Y. Yamini). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.04.050
Among the emerging techniques, liquid phase microextraction based on hollow fiber (HF-LPME) is one of the most promising developments for preconcentration, separation, and cleanup purposes. Nevertheless, in spite of the potential capabilities of HFLPME, the extraction time needed in this technique is usually high and common extraction times of 30–50 min have been reported [1]. In 2006, Pedersen-Bjergaard et al. introduced a novel microextraction technique called electromembrane extraction (EME) [2], to overcome this limitation of HF-LPME. The present method has a same set-up as HF-LPME, besides the use of a power supply and two electrodes to sustain the voltage across the SLM. EME was performed effectively for extraction of various compounds from different matrices with variable voltages in the range of 9–300 V [2–24]. In EME, only a single class of analytes (basic or acidic analytes) is extracted. In a recent work, Basheer et al. introduced an interesting method for simultaneous extraction of acidic and basic drugs from wastewater samples using four sheets of porous polypropylene membrane, which were combined and heat-sealed at three edges [10]. In the present work, a new EME set-up based on two separated pieces of hollow fibers were used for simultaneous extraction of
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by a heater-magnetic stirrer model 3001 from Heidolph (Kelheim, Germany) using a 1.5 cm × 0.3 cm magnetic bar.
2.2. Chemicals and materials Nalmefene (NAL), morphine (Mor) codeine (Cod), oxymorphone (Oxy), mefenamic acid (Mef) and diclofenac (DIC) were kindly donated by Parand Darou (Tehran, Iran) and the Department of Medical Sciences of Tehran University (Tehran, Iran). 2-Nitrophenyl octyl ether (NPOE) and di-(2-ethylhexyl) phosphate (DEHP) were purchased from Fluka (Buchs, Switzerland). Cetyltrimethylammonium bromide, CTAB (C19 H42 BrN), NaClO4 , pyridines and 1-octanol were purchased from Merck (Darmstadt, Germany). Standards of chlorophenols were purchased 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 aquaMAx purification system 370 series (Seoul, Korea). Fig. 1. A schematic illustration of the equipment for simultaneous extraction of acidic and basic drugs by EME. Different parts of the set-up are shown in the figure.
acidic and basic drugs. It makes possible to employ the best SLM for each class of analytes separately, whereas this possibility is to some extent difficult for hand-made envelope by sheet membranes. Also, the electrical resistance of extraction system increases sowing to the use of two layers of organic solvents that reduces the risk of electrolysis and bubble formation during extraction time. In comparison with hand-made envelope using sheet membranes, the presented set-up in this work is simpler; the volume of acceptor phase is more repeatable, and the risk of membrane pollution during development of the set-up is very lower. Also, in the previous work, the volume of final acceptor phase is relatively high (50 L) due to limitation of sizes of sheet fibers needed for making compartments, whereas reaching low volume for acceptor phase (even lower than 5 L) can easily be achieved using hollow fibers. Using high volume of acceptor phase causes dilution and reduction of determination sensitivity; especially in the case of gas chromatography that only 1–3 L of extract is injected into the instrument. In the previous work, the final step of extraction process is basically controlled by distribution ratios, which may increases the extraction time. This is while in the present work, the most effective transport mechanism is electrokinetic migration, which reduces the extraction time. Finally, high performance liquid chromatography with ultraviolet detection was employed for analysis of acceptor solution, which needs no derivatization steps.
2. Experimental 2.1. Equipment for electromembrane extraction The equipment used for the extraction procedure is shown in Fig. 1. A 24-mL vial with internal diameter of 2.5 cm and height of 5.5 cm was used. The electrodes used in this work were platinum wires with diameters of 0.25 mm, and were 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 EME unit was stirred with a stirring speed in the range of 0–1250 rpm
2.3. Biological and standard solutions Urine samples were collected from four volunteers, two of which were treated with DIC tablets. The samples were stored in sterilized bottles at −4 ◦ C, thawed and shaken before extraction. A stock solution containing 1 mg mL−1 of NAL and DIC were prepared in methanol and stored at −4 ◦ C. Working standard solutions were prepared by dilution of the stock solutions in water.
2.4. HPLC conditions Separations and detections of the target analytes were performed by a Varian HPLC (Walnut Creek, CA, USA) containing a 9012 HPLC pump, a six-port Cheminert HPLC valve from Valco Instruments (Houston, TX, USA) with a 20-L sample loop and a Varian 9050 UV–Vis detector. Chromatographic data were recorded and analyzed using Chromana software (version 3.6.4). The separations were carried out on an ODS-3 column (250 mm × 4.0 mm, with 5 m particle size) from TeknoKroma (Barcelona, Spain). Separation of NAL and DIC was performed using an isocratic elution at the flow rate of 1.0 mL min−1 . The mobile phase consisted of 50 mM NaClO4 and acetonitrile (65:35, v/v). Quantification of both NAL and DIC was accomplished by measuring peak areas at wavelength of 210 nm. Total analysis time was 12 min. Separation and determination of chlorophenoles were carried out according to the literature [25]. Mobile phase of pyridines was a buffer solution containing 50 mM acetic acid and sodium phosphate (pH = 8.0) and acetonitrile (50:50, v/v). The separation program was an isocratic elution at the flow rate of 0.8 mL min−1 . All determinations were performed at 254 nm. The chromatographic separation of morphine, codeine and oxymorphone was performed with a mobile phase consisting of 10 mM phosphate buffer adjusted to pH 2.5 and acetonitrile, and delivered at a flow rate of 0.8 mL min−1 . The gradient program was as follows: starting with 15% acetonitrile, then increasing to 70% in 0–12 min, keeping constant until 13 min, thereafter restored to 15% in 3 min. The detector wavelength was set at 210 nm. The mobile phase for separation of mefenamic acid was 50% acetonitrile and 50% ammonium acetate aqueous solution (50 mM, pH = 5.3). The flow rate was 1.0 mL min−1 . The detection wavelength was set at 285 nm.
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2.5. Procedure for EME Twenty four milliliters of the sample solution containing target analytes in deionized water was transferred into the sample vial. To impregnate the organic solutions in the pores of cathodic and anodic hollow fiber walls, two 3.8-cm pieces of hollow fibers were cut out and dipped in the organic solutions for 5 s and then the excess of organic solutions were gently wiped away by air blowing using a 500-L Hamilton syringe. The upper ends of hollow fibers were connected to two medical needle tips as guiding tubes, which were inserted through the rubber cap of the vial. Ten microliters of 50 mM HCl and NaOH as cathodic and anodic acceptor solutions were introduced into the lumen of the hollow fibers by a microsyringe, respectively. The lower ends of the hollow fibers were sealed with two small pieces of aluminum foil (thickness of 280 m). One of the electrodes, the cathode, was introduced into the lumen of the cathodic fiber and the other was inserted into the lumen of the anodic fiber. The fibers containing the cathode and anode, SLM and the acceptor solutions were afterward directed into the sample solution. The electrodes were subsequently coupled to the power supply and the extraction unit was placed on a stirrer with stirring speed of 700 rpm. The predetermined voltage of 40 V was turned on and extraction was performed for 14 min. Under the applied voltage, the target analytes migrated from aqueous sample into SLM, and then transported into acceptor phases (Fig. 1). After the extraction was completed, the acceptor solutions were collected by a microsyringe, mixed together into a microtube and injected into the HPLC for further analysis. 2.6. Calculation of preconcentration factor, extraction recovery, and relative recovery The preconcentration factor (PF) was defined as the ratio of the final analyte concentration in the acceptor phase (Cf,a ) to the initial concentration of analyte (Ci,s ) in the sample solution: PF =
Cf,a
(1)
Ci,s
where Cf,a was calculated from a calibration graph obtained from direct injection of NAL and DIC standard solutions. The extraction recovery (ER%) was defined as the percentage of the number of moles of analyte originally present in the sample (ni,s ), which was extracted to the acceptor phase (nf,a ). ER% =
nf,a ni,s
ER% =
× 100 =
Va Vf
Cf,a Va Ci,s Vf
× 100
PF × 100
(2)
(3)
where Va and Vf represent the volumes of acceptor phase and sample solution, respectively. Relative recovery (RR%) was acquired from the following equation: RR% =
Cfound − Creal Cadded
× 100
(4)
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. 2.7. Data analysis and statistical methods In order to obtain the optimum conditions for simultaneous extraction of NAL and DIC, a central composite design (CCD) was
Fig. 2. The chromatograms obtained from (I) a spiked water sample at 5 V (a) and no application of electrical potential (b); (II) a urine sample after spiking with NAL and DIC at concentration of 500 g L−1 (a) and before spiking (b) under optimal conditions; (III) chromatogram of a non-spiked urine sample under optimal conditions that was taken from a volunteer who had treated with DIC. For more details see the text.
used. For this purpose, STATISTICA software trial version 8.0 (StatSoft, Tulsa, OK, USA) was employed to generate the experimental matrix and evaluate the results. 3. Results and discussion 3.1. Initial experiments In this work, experimental design method was used for optimization of effective parameters on EME. However, some initial investigations were carried out before optimization process by experimental design method to find some primary information about these parameters. To this aim, 24 mL of deionized water spiked with target analytes at concentration of 500 g L−1 was agitated for 15 min with stirring speed of 700 rpm. According to the literatures, NPOE containing 5% DEHP and 1-octanol were used as the best SLMs for extraction of NAL and DIC, respectively [9,17]. The lumens of the fibers were filled with 100 mM HCl and NaOH as cathodic and anodic acceptor solutions, respectively. Initial experiments at low voltages showed that NAL and DIC can easily be extracted at these voltages. Fig. 2I shows the chromatograms obtained for simultaneous extraction of DIC and NAL at 5 V (a) and
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without application of voltage (b). As can be seen from this figure, no considerable extraction occurs in the absence of voltage that indicates the importance of application of voltage in EME system as the main driving force. Some tests concerning the effects of the stirring rate and ionic strength of the donor phase in the electromigration process were realized. As it is known, stirring speed plays an essential role in increasing the kinetics and efficiency of extraction by increasing the mass transfer and reducing the thickness of double layer around SLM. To study the effect of stirring rate in more details, the effect of stirring speed on extraction efficiency was investigated up to 1250 rpm. A stirring rate of 700 rpm was chosen as the best amount due to formation of intense vortex and bubble formation into sample solution at higher speeds. Sodium chloride was added to the donor phase for studying the effect of ionic strength on the extraction efficiencies of DIC and NAL at concentration levels of 2.5% and 5% (w/v). Presence of salt caused a decrease in the extraction efficiency and obtaining of undesirable results. In 2007, Pedersen-Bjergaard et al. introduced a mathematical model based on the Nernst–Planck equation [11], which investigates EME in more details from a theoretical point of view. According to the model, increasing the ionic strength in sample solution increases the ion balance () that leads to reduction of extraction efficiency. The ion balance is defined as the total concentration of ions in sample solution to that in acceptor phase [11]. The reason of this phenomenon is increasing of the competition among analyte and other ions for entering into the acceptor solution. It is remarkable that presence of high contents of salt in the donor solution increases variation of acceptor phase volume and so decreases extraction repeatability. Also, it may result in puncture of SLM and creation of arc between the electrodes. Similar observations have been reported [17]. 3.2. Variation in the SLM composition (organic solvent) According to earlier findings, the chemical nature of the supported liquid membrane is highly critical for the success of EME. There are specific requirements for a solvent to be used as a SLM in EME [7]. 1-Octanol and NPOE have been the best candidates for acidic and basic drugs in EME, respectively, up to now [1,3]. It has been found that addition of hydrophobic ion-pair reagents to SLM would enable better phase transfer and electrokinetic migration of analytes [2,7,9]. To investigate this effect, 2.5% (w/v) CTAB in 1-octanol and 5% (v/v) DEHP in NPOE were used as the anodic and cathodic SLMs, respectively. Addition of DEHP showed good improvement in extraction efficiency of NAL, while using CTAB gave variable results and thus did not provide any general guidance on the trends of extraction for DIC. Therefore, 1-octanol was chosen as anodic SLM. In a new experiment, DEHP was gradually added to NPOE corresponding to 10% and 15% (v/v). The results are shown in Fig. 3. As seen from the results, there is no noticeable difference between extraction efficiency of NAL at 5% and 10% (v/v) of DEHP, while further addition caused to a decrease in its extractability. So, NPOE containing 5% (v/v) of DEHP was selected as cathodic SLM. Generally, decreasing of an analyte extractability with addition of ion-pair reagents may be due to a decrease in electrical resistance of SLM and an increase in the current level and bubble formation or strong interaction of the ion-pair and analyte complex with the organic solvent [2,7]. 3.3. Results for the central composite design (CCD) Response surface methodology (RSM) is a collection of statistical and mathematical methods that involves experimental designs
9
Fig. 3. Effect of SLM composition on extraction efficiencies of NAL and DIC by EME; spiked concentration: 500 g L−1 , voltage: 30 V, sample solution: 24 mL of urine at pH 6.5, acceptor solutions: 100 mM HCl (cathodic) and 100 mM NaOH (anodic), extraction time: 10 min, and stirring rate: 700 rpm.
to achieve adequate and reliable measurement of the response of interest. RSM usually contains three steps: (1) design and experiments; (2) response surface modeling through regression; and (3) optimization. The main objective of RSM is to determine the optimal operational conditions of the process or to determine a region that meets the operating specifications. Among different RSM experimental designs, face centered central composite design (FCCCD) has been widely used because it requires fewer experimental runs and provides sufficient information. Different variables can affect the extraction efficiency of EME procedure, including type of organic solvent (SLM), volume of sample solution, pH of donor and acceptor phases, stirring rate, salt%, temperature, extraction time, and voltage. Variables were chosen with the aim of reducing the extraction time and process cost. The proposal includes parameters such as extraction time, voltage, and pH of donor and acceptor phases. Separated study of membrane organic solvent can give optimum SLM, simplicity of experimental design method, and reduction of number of runs. Therefore, this parameter was separately optimized at the first. Temperature is another factor, which can affect the flux of ions through SLM. Theoretically, increase of temperature decreases the electrical driving force in EME, while increases diffusion coefficient of ions into SLM [11]. However, according to previous studies and our experience, an increase in temperature led to puncture of SLM, and increase in bubble formation and arc phenomenon probability due to increase in solubility of membrane organic solvent; especially for long extraction time. Furthermore, control of temperature is relatively difficult as it can increase the experiment uncertainties; thus, working at ambient temperature can be a good benefit for a microextraction technique. To pass the analytes through electrical field, it is necessary to change them to their ionizable forms. NAL and DIC have pKa values of 7.6 and 4.2, respectively; so, they are charged (NAL+ and DIC− ) in deionized water solution (donor phase) and easily transferred to cathodic and anodic acceptor solutions under electrical field. Based on the literatures, pH in the sample solution is not highly critical for the EME process, so that the extraction efficiency is found to be maintained constant in an extended pH range [2,10,13]. Based on initial experiments, addition of salt has negative effect on extraction efficiencies of NAL and DIC, and increasing the stirring speed above 700 rpm is not experimentally possible. Therefore,
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these factors were not taken into account in the experimental design. By this strategy for factor selection, 17 permutations of conditions were obtained by varying the three remained variables at the lower, middle, and upper levels (5, 12.5 and 20 min for time, 10, 40 and 70 V for voltage, 1, 50 and 100 mM for concentration of cathodic and anodic acceptor phases). Normalized peak area was used as the experimental response for each permutation [18]. The data obtained were evaluated by analysis of variance (ANOVA) at 95% confidence level. Results indicate that time (X1 ) and voltage (X2 ) have significant effects on extraction recovery, and the X1 , X2 , and the concentration of cathodic and anodic acceptor phases (X3 ) interactions and quadratic terms of all the three factors are significant. In addition, there is no evidence of lack of fit at the 95% confidence level, meaning that the model is explaining the observed differences in the response variable. In the next step of the design, a response surface model was developed by considering all significant interactions in the FCCCD. The R2 value of model was obtained higher than 0.97, so the regression model predicts the experimental responses adequately. Normalized peak area = −29.1570 + 4.8038X1 − 0.1705X12 + 1.2933X2 − 0.0137X22 + 0.0077X3 − 0.0022X32 − 0.0079X1 X2 + 0.0094X1 X3 + 0.0010X2 X3
(5)
Based on the analysis, it was found that the normalized peak area increased with the increase of time, voltage, and concentration of the acceptor phase to a certain level and then decreased beyond that. According to the EME theoretical model [11], the main driving force for migration of the analytes across the liquid membrane is provided by the electrical field. Strength of the electrical field is dependent on the applied voltage. Therefore, applied voltage is one of the most important parameters that should be regarded. Tendency of basic drugs to deprotonate in the artificial liquid membrane was relatively high. Application of high electrical potential differences promotes their efficient migration through the artificial membrane and reduces deprotonation opportunity of these drugs, which resulted in higher extraction recovery [2]. Time is another parameter that can affect the flux of analytes in EME. Voltage and time are two parameters that act in parallel ways. Both time and voltage directly increase the flux of ions, and thus increase the extraction recovery. However, there is an antagonistic effect when they are simultaneously considered. It means that an increase in extraction time limits the voltage and vice versa. Increase in voltage causes an increase in the number of ions crossing through the membrane. This leads to heat generation (Joule heating phenomenon) and loss of membrane organic solvent as a function of time. Therefore, time duration of membrane stability reduces by increasing of voltage [2,8,9,19–21]. As it is known in EME, electrolysis can occur at surfaces of both electrodes according to the following reactions: Anodic acceptor phase : H2 O → 2H+ + 1/2O2 + 2e−
(6)
Cathodic acceptor phase : 2H+ + 2e− → H2
(7)
The probabilities of these reactions increase with as the time and voltage increases, owing to the decrease in membrane stability and resistance and increase in system current flow. Electrolysis on the surface of both electrodes slightly increases as the pH value of the cathodic acceptor solution increases and the pH value of the anodic acceptor solution decreases. This may result from neutralization of analytes and back-extraction of them into the donor phase and decrease of extraction efficiencies [2,22–24].
Also, statistical analysis showed that extraction efficiencies were increased by decrease in the pH value of cathodic acceptor solution (below the pKa of basic analyte) and increase in the pH value of anodic acceptor solution (above the pKa of acidic analyte). These variations occur due to easily releasing of the analytes into the cathodic and anodic acceptor solutions, respectively. However, there are some limitations for the increase of proton and hydroxide ions concentrations of the cathodic and anodic acceptor solutions, respectively. These types of pH adjustments improve the probability of electrolysis reactions and bubble formation on the surfaces of electrodes; especially with the increase in extraction time and voltage. Bubble formation causes intensive variation in the collected volumes of acceptor solutions and increases uncertainties of the data obtained by EME. The final step was to find the optimum conditions of voltage, extraction time, and concentration of HCl and NaOH as cathodic and anodic acceptor phases, respectively. In order to optimize the extraction efficiency of EME, the desirability function approach was applied to analyze the regression model equations using STATISTICA software. Desirability function is an interesting and precise technique to simultaneously determine the optimal amounts of input variables. This technique was first developed by Harrington [26], and was later optimized by Derringer and Suich [27] for specifying the relationship between predicted responses on a dependent variables and the desirability of the responses. The results of FCCCD design matrix represented the maximum of 34.070 and minimum of 2.000 for normalized peak area. According to these values, desirability function settings for each dependent variable of the normalized peak area are depicted at the right hand side of Fig. 4: desirability of 1.0 was assigned for maximum (34.070), 0.0 for minimum (2.000), and 0.5 for middle (18.035). On the basis of these calculations and desirability score of 1, the optimized variables were found to be 14 min, 40 V, 50 mM, and 50 mM for extraction time, voltage, and concentration of HCl and NaOH as cathodic and anodic acceptor solutions, respectively. 3.4. Method validation The optimized conditions were applied to test the applicability of the proposed EME method for extraction of model drugs from human urine samples. Calibration curves were plotted in drug free urine samples and figures of merit of the method were investigated. For this purpose, urine sample was spiked with the analytes and extraction was accomplished after dilution of urine samples (1:1) with pure water and pH adjustment. The pH was adjusted by dropwise addition of 5.0 M NaOH and/or HCl solutions, such that the final pH of samples was adjusted at 6.5. The proposed technique provided good linearity with correlation coefficient values higher than 0.9956 over a concentration range of 8–500 g L−1 and 12–500 g L−1 for NAL and DIC, respectively. As seen from Table 1, this system is capable of simultaneous extraction and determination of acidic and basic analytes with good limits of detection (LODs). Also, 8 g L−1 and 12 g L−1 were determined as limits of quantification (LOQs) for basic and acidic drugs, respectively. Repeatability was evaluated by performing triple analysis of the drugs at concentration level of 50 g L−1 and the relative standard deviations (RSDs%) were less than 10.1%. The preconcentration factors of 300 and 375 were obtained for DIC and NAL, respectively, which illustrate the ability of this new EME set-up for trace analysis of real samples. 3.5. Analysis of real samples The proposed method was applied to determine the concentration of NAL and DIC in urine samples. Firstly, samples were diluted
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Fig. 4. Profiles for predicted values and desirability function of normalized peak area. Dashed lines indicate current values after optimization.
Table 1 Figures of merit of the method in a drug-free urine sample. Analyte
LOD (g L−1 )
LOQ (g L−1 )
Linearity LOQ – 500 (g L−1 )
R2
RSD%a (n = 3)
PF
ER%
NAL DIC
2 4
8 12
y = 0.6102x + 8.8599 y = 0.5435x − 0.6951
0.9981 0.9956
7.4 8.9
350 300
14.6 12.5
a
RSDs% were calculated at concentration of 50 g L−1 for each drug.
1:1 with ultrapure water and their pH values were adjusted at 6.5. The pH was adjusted by dropwise addition of 5.0 M NaOH and/or HCl solutions, such that the final pH of samples was adjusted at 6.5. Then, 24 mL of each solution was transferred into the sample vial and evaluated by the EME process. Fig. 2II shows the chromatograms obtained for a blank urine sample before and after spiking with 500 g L−1 of NAL and DIC. The corresponding relative recoveries (RR%) and RSDs% are summarized in Table 2. As can be seen, the relative recoveries for both drugs in spiked urine samples are between 90% and 98%. These results demonstrated that
Table 2 Relative recoveries and standard deviations of NAL and DIC from spiked blank urine samples and actual urine samples. Sample Urine 1
Urine 2
Urine 3
Urine 4
a b
Initial concentration (g L−1 ) RR%b RSD% (n = 3) Initial concentration (g L−1 ) RR% RSD% (n = 3) Initial concentration (g L−1 ) RR% RSD% (n = 3) Initial concentration (g L−1 ) RR% RSD% (n = 3)
NAL
DIC
nda 98.0 5.1 nd 91.0 6.6 nd 95.0 5.8 nd 96.0 8.7
nd 96.0 6.5 nd 93 7.8
nd, no detection. 50 g L−1 of each drug was added to calculate relative recovery (RR%).
the matrices of the analyzed real urine samples possess negligible effect on the proposed methods and also excellent sample clean-up was obtained. Fig. 2III depicts the resulted chromatogram of a non-spiked urine sample, which was taken from a volunteer who had treated with DIC. 3.6. Investigation applicability of the proposed configuration for simultaneous extraction of other compounds On the basis of the experiments discussed above, the performance of the proposed configuration was investigated for simultaneous extraction of some other basic and acidic compounds. Twelve different compounds including 4-chlorophenol (4-CP, pKa = 9.43), 2,3-dichlorophenol (2,3-DCP, pKa = 7.7), 2,5dichlorophenol (2,5-DCP, pKa = 7.2) and 2,4,6-trichlorophenol (2,4,6-TCP, pKa = 6.23) as acidic environmental pollutants, pyridine (Py, pKa = 5.25), 3-methyl pyridine (3-MPy, pKa = 5.63), 2,4-lutidine (2,4-Lu, pKa = 6.46) and quinoline (Qu, pKa = 4.81) as basic environmental pollutants, morphine (Mor, pKa = 8.0), oxymorphone (Oxy, pKa = 8.5) and codeine (Cod, pKa = 8.2) as basic drugs and mefenamic acid (Mef, pKa = 4.4) as an acidic drug were extracted under optimal conditions which were obtained for DIC and NAL. The acidic and basic environmental pollutants were extracted together (Fig. 5A) and also simultaneous extraction of acidic and basic dugs were investigated (Fig. 5B). Extraction of these compounds was carried out under optimal conditions of NAL and DIC and acceptable results were obtained (Table 3). It should be noted that some of these
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S. Seidi et al. / J. Chromatogr. A 1243 (2012) 6–13
Fig. 5. HPLC chromatograms related to simultaneous extraction of different acidic and basic compounds utilized new configuration of EME. Pyridines, chlorophenols, basic drugs and acidic drug were present at 250, 250, 500 and 500 g L−1 , respectively.
Table 3 The obtained preconcentration factors (PFs) and repeatability (RSDs%) for simultaneous extraction of different acidic and basic environmental and pharmaceutical compounds under optimal conditions of NAL and DIC. Analyte classification
Type of analyte
Analyte
Environmental
Basic
PY 3-MPy 2,4-Lu Qu 4-CP 2,3-DCP 2,5-DCP 2,4,6-TCP Mor Oxy Cod Mef
Acidic
Pharmaceutical
Basic
Acidic
PFa 120 142 174 96 46 43 38 42 66 45 114 32
RSD% (n = 3) 4.6 4.8 5.1 4.5 6.5 7.3 6.2 6.5 5.3 6.8 5.5 7.5
a
Pyridines, chlorophenols, basic drugs and acidic drug were present at 250, 250, 500 and 500 g L−1 , respectively.
compounds such as basic drugs are so polar; however, they were easily extracted under modest conditions. This observation indicates the important role of electrical field in EME as well as applicability of the proposed configuration of EME for simultaneous extraction of various acidic and basic compounds from different matrices. 4. Conclusions In the present work, for the first time, a new EME set-up was introduced for simultaneous extraction and determination of trace amounts of acidic and basic drugs in biological samples. Since the SLMs were optimized for each class of analytes, this system offers more efficient extraction. Low voltage EME with optimized SLM for each class of analytes effectively prevents the extraction of interferences. Despite simplicity, this EME system presents high selectivity and sample cleanup that makes this method suitable
for simultaneous extraction of different classes of analytes from complicated matrices. Acknowledgements The authors gratefully acknowledge financial support from Tarbiat Modares University. Also, the support provided by the National Elite Foundation (Tehran, Iran) is highly appreciated. References [1] K.E. Rasmussen, S. Pedersen-Bjergaard, Trends Anal. Chem. 23 (2004) 1. [2] S. Pedersen-Bjergaard, K.E. Rasmussen, J. Chromatogr. A 1109 (2006) 183. [3] M. Balchen, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1152 (2007) 220. [4] I.J.Ø. Kjelsen, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1180 (2008) 1. [5] L.E. Eibak, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1217 (2010) 5050. [6] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1124 (2006) 29. [7] J. Lee, F. Khalilian, H. Bagheri, H.K. Lee, J. Chromatogr. A 1216 (2009) 7687. [8] M. Balchen, L. Reubsaet, S. Pedersen-Bjergaard, J. Chromatogr. A 1194 (2008) 143. [9] M. Rezazadeh, Y. Yamini, S. Seidi, J. Chromatogr. B 879 (2011) 1143. [10] C. Basheer, J. Lee, S. Pedersen-Bjergaard, K.E. Rasmussen, H.K. Lee, J. Chromatogr. A 1217 (2010) 6661. [11] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1174 (2007) 104. [12] T.M. Middelthon-Bruer, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Sep. Sci. 31 (2008) 753. [13] A. Gjelstad, H. Jensen, K.E. Rasmussen, S. Pedersen-Bjergaard, Anal. Chim. Acta (2012), http://dx.doi.org/10.1016/j.aca.2011.12.039. [14] T. Sikanen, S. Pedersen-Bjergaard, H. Jensen, R. Kostiainen, K.E. Rasmussen, T. Kotiaho, Anal. Chim. Acta 658 (2010) 133. [15] L.E.E. Eibak, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Pharm. Biomed. 57 (2012) 33. [16] M. Balchen, H. Lund, L. Reubsaet, S. Pedersen-Bjergaard, Anal. Chim. Acta 716 (2012) 16–23. [17] M. Ramos-Payán, M.Á. Bello-López, R. Fernádez-Torres, M. Villar-Navarro, M. Callejón-Mochon, Talanta 85 (2011) 394. [18] S. Seidi, Y. Yamini, T. Baheri, R. Feizbakhsh, J. Chromatogr. A 1218 (2011) 3958. ˇ L. Strieglerová, P. Gebauer, P. Boˇcek, Electrophoresis 32 (2011) [19] P. Kubán, 1025.
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