Capillary electrophoresis with capacitively coupled contactless conductivity detection: A universal tool for the determination of supported liquid membrane selectivity in electromembrane extraction of complex samples

Journal of Chromatography A, 1267 (2012) 96–101

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Capillary electrophoresis with capacitively coupled contactless conductivity detection: A universal tool for the determination of supported liquid membrane selectivity in electromembrane extraction of complex samples ˇ ∗ , Petr Boˇcek Pavel Kubán Institute of Analytical Chemistry of the Academy of Sciences of the Czech Republic, v.v.i., Veveˇrí 97, CZ-60200 Brno, Czech Republic

a r t i c l e

i n f o

Article history: Available online 13 July 2012 Keywords: Capacitively coupled contactless conductivity detection Capillary electrophoresis Complex samples Electromembrane extraction Membrane selectivity Supported liquid membranes

a b s t r a c t Monitoring the selectivity of supported liquid membranes (SLMs) is of paramount importance since the amount and type of compounds that are transferred across a SLM directly influence the transfer efficiency, reproducibility and accuracy. To apply a correct SLM in particular sample pretreatment, rapid determination of the transfer of analytes and matrix compounds across the SLM is necessary, which requires the use of an analytical method with universal detection technique. Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4 D) has proven to be a useful tool for the determination of SLM selectivity. Background electrolyte solution consisting of 1 M acetic acid (pH 2.4) was used for simultaneous separation and detection of three basic drugs (nortriptyline, haloperidol and loperamide) and major matrix components (inorganic cations, proteins, amino acids, etc.) after electromembrane extraction (EME) of standard solutions and complex samples. The CE-C4 D method has evidenced for the first time that large proteins, such as human serum albumin, are efficiently retained on all examined SLMs and that transfer of other matrix components and the analytes is strongly SLM dependent. Excellent transfer of the analytes was achieved across SLMs impregnated with 2-nitrophenyl octyl ether (NPOE) or 1-ethyl-2-nitrobenzene, however, an increased co-extraction of interfering matrix components, which disabled quantitative determination of haloperidol with the current CE-C4 D setup, was observed for the latter. After addition of a commonly used ion carrier (bis(2-ethylhexyl)phosphate) to NPOE, a wide range of matrix components were transferred across the SLM with no measurable transfer of the analytes. Best selectivity regarding transfer of the basic drugs and elimination of matrix components was obtained using SLM impregnated with NPOE. An optimized EME-CE-C4 D method was used to determine the basic drugs in various samples and satisfactory analytical parameters were obtained. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Liquid phase microextraction (LPME) using supported liquid membranes (SLMs) was first described by Audunsson [1] and has attracted significant attention in subsequent years due to its favorable features compared to conventional liquid–liquid and solid-phase extraction techniques. LPME with SLMs has minimized environmental impact, lowered the volumes of pretreated samples, speeded-up the pretreatment process and reduced the overall analysis costs. Moreover, it has been shown suitable for pretreatment of various complex samples and results into acceptor solutions, which are applicable to virtually any analytical technique

∗ Corresponding author. Tel.: +420 532290140, fax: +420 541212113. ˇ E-mail address: [email protected] (P. Kubán). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.07.010

[2–6]. LPME using SLMs has later developed into hollow fiberLPME (HF-LPME) [3,7] and electromembrane extraction (EME) [4–6]. In HF-LPME and EME, a porous inert supporting material (usually polypropylene sheet or hollow fiber) is impregnated with a water immiscible organic solvent to form a SLM. The SLM separates two compartments usually filled with aqueous donor (complex sample) and acceptor (diluted acid or base) solutions. The transfer of analytes from the donor into the acceptor solution is driven either by diffusion in HF-LPME or by a combination of electromigration and diffusion in EME whereas matrix components, such as biochemical macromolecules, small inorganic ions and solid particles, are retained on the SLM [3,6]. Due to the fact that analytes are transferred across the SLM by different physico-chemical means, equilibrium of the extraction process can be reached after different extraction periods in HF-LPME and EME. Recently, it has been

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shown that extraction times in EME can be significantly reduced compared to HF-LPME [8] and EME has therefore become very popular [4–6]. Although EME has been rapidly established as an emerging sample pretreatment technique, selection of a SLM, which is the most important variable affecting the overall EME performance, is currently based on empirical rules where the organic solvent selection is performed on experimental experience and similarities with already published literature. Selectivity of SLMs (and consequently the EME performance) is usually not investigated comprehensively and is determined solely by measurement of the transfer of target analytes, which is achieved by subsequent capillary electrophoresis (CE) or HPLC analysis of the acceptor solution. These analytical techniques are mostly coupled to selective detection methods (e.g. UV–vis, MS, fluorescence, etc.) with excellent capability of detecting target analytes, however, selective detection only rarely provides any information regarding the transfer of interfering matrix components. In EME, SLMs are mostly impregnated with pure organic solvents but as the number of analytes investigated by EME is growing, the composition of SLMs is often tailored by addition of suitable ion carrier and/or ion exchanger organic ions for efficient transfer of particular analytes [9–12]. Presence of these additives in SLMs usually results in additional transfer of matrix ions as has been shown by increased transfer of inorganic cations after addition of bis(2-ethylhexyl)phosphate (DEHP) [11,12]. Obviously, the ions “invisible” to selective detection methods are also transferred across the SLM, result into higher electric current during EME, contribute to the overall conductivity and may have an effect on the EME performance. A deeper insight into the extraction process, where the EME performance is considered not only by measuring the efficiency of the transfer of analytes across the SLM but also by the cross-SLM transfer of matrix components, might be helpful in rapid determination of most suitable SLMs and in development of new, possibly more efficient SLMs. Conductivity is considered a universal detection technique since all charged species can be visualized conductometrically. In the last decade, conductivity detection has undergone a renaissance by introduction of an axial capacitively coupled contactless conductivity detection (C4 D) [13,14]. Fundamental principles and practical applications of axial C4 D were described in several extensive publications [15–18] and in a series of review articles [19–23], respectively. Although combination of C4 D with liquid chromatography, flow injection analysis and other analytical methods was described, C4 D is mostly coupled to CE, which offers excellent separation efficiency, short separation times, minimal sample volume requirements and simple instrumentation. In addition, simultaneous determination of various analytes (inorganic, organic, biochemical) can be achieved in rather simple background electrolyte (BGE) solutions. The above-mentioned features predetermine CE-C4 D to be a suitable analytical tool for rapid determination of comprehensive SLM selectivity. In this contribution, CE-C4 D was used for the first time for monitoring of the transfer of target analytes and matrix components across SLMs. Three basic drugs and selected inorganic cations, small biomolecules and large biomolecules served as model analytes and matrix components, respectively. Various liquid membranes were examined and effect of the organic solvent type as well as of the ion carrier addition was investigated with respect to transfer of all ionic species across the SLM. The most suitable composition of SLM was selected upon CE-C4 D analyses of various EME pretreated standard solutions and complex samples. The study has proved that optimum SLM composition may provide high extraction efficiency of the target analytes and minimum transfer of interfering matrix components and that CE-C4 D is a universal tool for such an optimization.

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2. Materials and methods 2.1. Instrumentation 2.1.1. Electromembrane extraction system The equipment and procedures used for the EME were described earlier [12]. Briefly, 900 ␮L of a donor solution was filled into a glass vial, which was attached to a MS1 Minishaker (IKA Works Inc., Wilmington, NC, USA). A 3 cm long piece of a polypropylene hollow fiber (Accurel PP 300/1200, Membrana, Wuppertal, Germany; wall thickness of 300 ␮m, internal diameter of 1200 ␮m, and pore size of 0.2 ␮m) was heat-sealed at the bottom and was used as a single use extraction unit. Before EME, each extraction unit was impregnated for 10 s with a solution forming the liquid membrane (pure organic solvent or organic solvent with an additive). Immediately, the HF lumen was filled with 20 ␮L of an acceptor solution and excessive solvent was wiped off using a lint-free medical wipe. The HF extraction unit was then placed into the donor solution. Two platinum electrodes (0.5 mm platinum wires) were connected to a DC power supply (ES 0300-0.45; Delta Elektronika BV, Zierikzee, The Netherlands). Cathode was immersed into an acceptor solution in the HF lumen and anode into a donor solution in the vial. EME was started by switching on the power supply and agitation of the vial with the donor solution and the HF. Electric current in the EME system was continuously monitored using an M-3800 (Metex, Seoul, Korea) digital multimeter. After the extraction was completed the acceptor solution was transferred to a plastic microvial for CE analysis and the HF was discarded. All EME experiments were performed at ambient temperature of 24 ± 2 ◦ C. 2.1.2. Capillary electrophoresis system A purpose-built CE instrument was employed for all CE runs. A high voltage power supply unit (CZE1000R, Spellman, Pulborough, UK) was operated at a potential of +20 kV applied at the injection side of the separation capillary for all runs. Detection was performed with a C4 D assembled at the Department of Physical Chemistry of the Charles University in Prague [24], which was operated at a frequency of 1.25 MHz. Separation capillary used was a fused-silica capillary (25 ␮m ID, 375 ␮m OD, 36 cm total length and 16 cm effective length; Polymicro Technologies, Phoenix, AZ, USA). Before use, new capillary was flushed for 2 min with deionized (DI) water and for 10 min with 1 M acetic acid, which was used as the background electrolyte (BGE) solution. Between two CE runs, separation capillary was rinsed with the BGE solution for 1 min. Injection of standard solutions and pretreated complex samples was carried out hydrodynamically by elevating the capillary injection end to a height of 15 cm for 60 s, which represents less than 1.5% (2.7 nL) of the total capillary volume. All CE-C4 D measurements were performed at ambient temperature of 24 ± 2 ◦ C. The data were acquired by Orca 2800 (Ecom, Prague, Czech Republic) A/D converter and processed in Clarity software (Data Apex, Prague, Czech Republic). 2.2. Reagents, standards, BGE solutions and real samples All chemicals were of reagent grade and DI water with resistivity higher than 18 M cm was used throughout. Stock solutions of inorganic cations (1.5 M Na+ , 100 mM NH4 + , 100 mM K+ , 100 mM Ca2+ and 100 mM Mg2+ , Pliva-Lachema, Brno, Czech Republic) were prepared from their corresponding chloride salts. Stock solutions of creatinine (Crea), lysine (Lys), and histidine (His) (10 mM; Sigma, Steinheim, Germany and Fluka, Buchs, Switzerland) were prepared from pure chemicals. Stock solutions of basic drugs (1000 mg/L; Sigma) were prepared from pure chemical (haloperidol) and from their hydrochloride salts (nortriptyline, loperamide) and were diluted with pure methanol (Sigma). Stock solution of human

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serum albumin (HSA; Sigma, 40 g/L) was prepared in DI water. Standard solutions for CE were prepared from these stock solutions and were diluted with DI water. Standard donor solutions for EMEs consisted of selected matrix ions (75 mM Na+ , 2 mM NH4 + , 2 mM K+ , 1 mM Ca2+ , 0.5 mM Mg2+ , 50 ␮M Crea, Lys, His, and 20 g/L HSA) and various concentrations of the three basic drugs and were prepared in 1 M acetic acid. Concentrations of these ions correspond to 1:1 diluted human plasma (ammonium was included in the donor solutions due to its presence in urine and other complex samples). Organic solvents for EME (2-nitrophenyl octyl ether, NPOE; 1-ethyl2-nitrobenzene, ENB; 1-octanol and bis(2-ethylhexyl)phosphate, DEHP) were obtained from Sigma and were of highest available purity. The solvents were used without any further purification. BGE solutions for CE were prepared from concentrated acetic acid (Fluka), degassed in an ultrasonic bath and kept at 4 ◦ C. BGE solutions were used for about a month. Human plasma sample was purchased as a lyophilized powder from Sigma and was prepared according to supplier’s instructions. Human urine was obtained from volunteers at the Institute of Analytical Chemistry. Bovine milk and red wine were purchased in a local supermarket. Urine, milk and wine were refrigerated at 4 ◦ C, plasma was deep frozen at −20 ◦ C. Before EME, all complex samples were left to equilibrate to ambient temperature and then diluted 1:1 by mixing 450 ␮L of a particular sample with 450 ␮L of 2 M acetic acid and spiked to a given concentration of the three basic drugs.

Fig. 1. CE-C4 D determination of selected matrix components and basic drugs in a standard solution. CE conditions: BGE solution, 1 M acetic acid at pH 2.4, voltage, +20 kV, injection, 15 cm for 60 s. Analyte concentrations: NH4 + , K+ , Ca2+ , Na+ and Mg2+ (n/a due to contamination from HSA), HSA (7.5 ␮M), Crea, Lys, His (15 ␮M), nortriptyline, haloperidol, loperamide (2.5 mg/L).

3. Results and discussion

its ca. 100-fold lower concentration compared to Na+ in most real samples, BGE solution consisting of 1 M acetic acid was used in following experiments. CE-C4 D determination of the selected cations in 1 M acetic acid is depicted in Fig. 1. Concentrations of inorganic cations in the standard solution are not specified since contamination by inorganic cations was observed in pure HSA solution and has therefore affected their final concentrations in the standard solution.

3.1. Selection of model substances and BGE solution

3.2. Electromembrane extraction parameters

First, a set of target analytes (nortriptyline, haloperidol and loperamide) was considered. These basic drugs have pKa values of 9.7, 8.3 and 8.7, respectively, [9] and are positively charged in acidic solutions. They are normally transferred across SLMs as cations and determined in cationic CE mode using acidic BGE solutions [9,10]. Second, potential interfering cations, which are commonly present in most complex samples, were summarized and representative candidates were selected for each group. Na+ , NH4 + , K+ , Ca2+ and Mg2+ were selected as the most abundant inorganic cations, Crea, Lys and His as representative small biomolecules and HSA as the large biomolecule, which is highly abundant in biological samples and may seriously deteriorate subsequent analytical procedures. A standard solution consisting of the three basic drugs and the nine matrix components was prepared in DI water and was used for selection and optimization of the BGE solution for CE. Composition of the BGE solution has to be carefully optimized since species with various electrophoretic properties have to be determined simultaneously. It has been shown recently that small and large biomolecules can be determined by CE-C4 D in BGE solutions consisting of weak organic acids, such as of acetic acid [25]. A stable performance and excellent sensitivity can be obtained for CE-C4 D determination of underivatized amino acids and related compounds in acetic acid based BGE solutions, too [12,26,27]. CE-C4 D determination of several basic drugs and their inorganic counter-cations in acidic solutions was also reported [28]. BGE solutions consisting of various concentrations of acetic acid were therefore examined and separation of the 12 cations was monitored. Best conditions for determination of all species were obtained in 1 M acetic acid with partial comigration of NH4 + and K+ and full comigration of Na+ and Mg2+ . Baseline separation of NH4 + /K+ and Na+ /Mg2+ was achieved by further increase of acetic acid concentration to 3–6 M at the expense of slightly reduced detection sensitivity and longer separation times. However, since the determination of Mg2+ is not of particular importance due to

EME parameters were selected according to previous publications on EME of basic drugs [9,10] and were slightly modified in order to achieve good analytical performance of the CE-C4 D system. Hydrochloric acid, which was previously used to acidify donor and acceptor solutions in EME of basic drugs, was replaced with acetic acid since the latter was used as the BGE solution for CE-C4 D analyses of acceptor solutions. 1 M acetic acid was used in donor solutions and 100 mM acetic acid was used as the acceptor solution. Agitation of the extraction device was performed at 800 rpm and various electric potentials were applied to the two electrodes depending on the SLM used. 3.3. Monitoring of EME transfer of analytes and matrix components NPOE was used as the liquid membrane and EME experiments were performed at 150 V. Standard solution for EME was prepared in 1 M acetic acid (for details on cation concentrations, see Section 2.2) and contained 500 ␮g/L of the three basic drugs. The standard solution was extracted for 3, 5 and 10 min in order to compare the time-dependent transfer of the three basic drugs and selected matrix cations across the SLM and the resulting electropherograms are depicted in Fig. 2. In addition, a blank solution (1 M acetic acid only) was extracted for 5 min and the electropherogram is also included in Fig. 2. It can be seen, that the three drugs were increasingly transferred across the SLM for 3–10 min, which indicates that EME equilibrium has not been reached yet. In contrast, there is no evidence of HSA and amino acid transfer across the SLM as these compounds were not detected in the resulting acceptor solutions. By the comparison of HF pore size (200 nm) and average protein size of HSA (<7 nm), globulins (<10 nm) and fibrinogens (<50 nm) [29], it is obvious that the most abundant body fluid proteins are not retained by the HF itself but by the organic liquid filled in the pores. Inorganic cations were also efficiently retained on the

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Fig. 2. CE-C4 D determination of EME pretreated standard solution at various extraction times. EME conditions: liquid membrane: NPOE, agitation: 800 rpm, extraction voltage: 150 V, acceptor solution: 100 mM acetic acid. Donor solution for EME was prepared in 1 M acetic acid. Concentration of basic drugs is 500 ␮g/L. Blank was a solution of 1 M acetic acid and was extracted for 5 min. CE conditions as for Fig. 1. *Unknown compounds.

SLM as only small peaks of inorganic cations were observed in all traces. The analytical signals of the transferred inorganic cations were constant for the blank and the standard solutions extracted for 3–10 min, which indicates that inorganic cations were transferred to the acceptor solution mainly from the SLM but not from the donor solution. If these cations originated from the donor solution, their concentrations would gradually increase for longer extraction times, similarly to what is observed for the three analytes. It has been shown recently that even analytical grade organic solvents contain inorganic contamination, which can be determined using EME [30]. In addition, several unknown peaks, labeled with asterisks, were detected in acceptor solutions, which show that further contamination from the SLM takes place during the EME. In order to examine origin of the inorganic and unknown contaminants in Fig. 2, additional experiments were performed, which are summarized in Supplementary Data. Nevertheless, a comprehensive study is necessary in order to determine all sources of contamination and possible ways of their elimination. The unknown contaminants were not identified since this is far beyond the scope of this publication. For the same reason, unknown peaks were also not identified in all subsequent sections. Fig. 2 demonstrates the benefits of the EME process monitoring since in this case CE-C4 D proves that the selected matrix ions are efficiently retained on the SLM whereas the basic drugs are gradually transferred from donor to acceptor solution. Fig. 2 also evidences that extraction efficiency for the basic drugs improves for longer EME times.

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Fig. 3. CE-C4 D determination of EME pretreated standard solution using various SLMs. EME conditions: agitation: 800 rpm, extraction time: 10 min, extraction voltage: (a) NPOE (150 V), (b) ENB (10 V), (c) 1-octanol (5 V), (d) NPOE/DEHP 85/15% v/v (25 V), acceptor solution: 100 mM acetic acid. Donor solution for EME was prepared in 1 M acetic acid. Concentration of basic drugs is 500 ␮g/L. CE conditions as for Fig. 1.

cations transferred from standard donor solution across various SLMs to acceptor solution is given in Table 1. The three basic drugs were efficiently transferred across SLMs formed by the pure organic solvents. Slightly higher transfer of nortriptyline and haloperidol was observed for SLM impregnated with ENB and transfer of haloperidol and loperamide was reduced for SLM impregnated with 1-octanol. After addition of the ion carrier to NPOE, no measurable transfer of the three basic drugs across the resulting SLM was observed. A drastic reduction of transfer of basic drugs across SLMs based on NPOE/DEHP has been reported previously [9]. Peak corresponding to HSA was not observed in any electropherogram indicating that HSA was efficiently retained on all four SLMs. Crea, Lys and His were completely retained on the SLMs formed by NPOE and 1-octanol and a small peak of Crea was observed for ENB, showing its partial transfer across this SLM. Crea and partially also Lys and His were transferred across NPOE with addition of DEHP. This is, however, not surprising since SLMs with addition of DEHP have been used for EME of a range of amino acids [12]. A significant difference can be observed for transfer of inorganic cations across various SLMs. NPOE and ENB result in low transfer of all inorganic cations and considering the fact that Na+ concentration in donor solution is 75 mM, only a trace of its original concentration is detected in acceptor solution after EME. An increased transfer of inorganic cations can be observed for 1octanol and NPOE/DEHP, demonstrating that these SLMs are less efficient in elimination of inorganic cations.

3.4. Selectivity of various SLMs examined with standard solutions 3.5. Selectivity of various SLMs examined with human urine Three liquid membranes consisting of pure organic solvents (NPOE, ENB and 1-octanol) and a liquid membrane with addition of an ion carrier (NPOE/DEHP, 85/15%), which were previously used in EME of basic drugs [9,31,32], were examined. Transfer of the analytes across the SLMs and simultaneous retention of matrix components was monitored. EME parameters were kept constant for all liquid membranes except for extraction voltage that was varied between 150 V (NPOE), 10 V (ENB), 5 V (1-octanol) and 25 V (NPOE/DEHP). Various extraction voltages were selected for the four SLMs in order to maintain stable EME performance at comparable electric currents. Standard solution of the three basic drugs and selected matrix components (concentrations were same as in Sections 2.2 and 3.3) was extracted for 10 min at 800 rpm. Resulting electropherograms are depicted in Fig. 3 and an overview of recoveries, calculated according to [9], of selected

To further investigate the selectivity of various SLMs, the same experiments were performed with human urine (prepared according to instructions in Section 2.2) and spiked to a final concentration of 250 ␮g/L of the three basic drugs. Fig. 4 depicts electropherograms for the EME pretreated urine sample using the four different SLMs and Table 1 shows recovery values for selected cations. Note that recovery values are not listed for species, which are naturally present in urine due to their unknown initial concentrations, and for species, which comigrate with matrix components. Similarly as with the standard solution, the three basic drugs were best transferred across NPOE and ENB and a reduced transfer rate was observed for 1-octanol. No peaks, which could be clearly identified as the three analytes, were observed for NPOE with addition of DEHP.

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Table 1 Recoveries (%) of selected analytes extracted from standard solution and urine across various SLMs. EME conditions as for Fig. 3. CE conditions as for Fig. 1. K+ /NH4 +

Ca2+

Na+ /Mg2+

Standard solution (concentrations of basic drugs 500 ␮g/L) NPOE <0.1 <0.1 <0.02 ENB <0.1 <0.1 <0.02 1-octanol <0.2 <0.5 <0.1 <0.2 <0.2 NPOE/DEHP <0.2 Urine (concentrations of basic drugs 250 ␮g/L) n.a. n.a. n.a. NPOE n.a. n.a. n.a. ENB 1-octanol n.a. n.a. n.a. NPOE/DEHP n.a. n.a. n.a.

HSA

Crea

Lys

His

Nortriptyline

Haloperidol

Loperamide

0 0 0 0

0 0.2 0 1

0 0 0 0.5

0 0 0 0.1

48 51 38 0

46 50 22 0

55 57 15 0

n.d. n.d. n.d. n.d.

n.d. n.a. n.d. n.a.

n.d. n.d. n.d. n.a.

n.a.* n.a.* n.a.* n.a.*

44 46 15 0

42 n.a.* 4 0

49 49 1.5 0

n.a., not available due to unknown initial concentration; n.a.*, not available due to comigration with other peak(s); n.d., not detected.

Based on data in Fig. 4, inorganic cations were almost completely eliminated by NPOE and ENB and an increased transfer of inorganic cations was observed for EME across 1-octanol and NPOE/DEHP, which is consistent with the findings in Section 3.4. HSA, which is present in urine in significantly lower concentrations than in plasma, was not detected in any acceptor solution. More interestingly, extractions of the urine sample resulted in an increased transfer of various matrix components across the SLMs and significant differences were observed for the selectivity of the four SLMs. Crea and Lys were efficiently retained by NPOE and 1-octanol, and a decent peak of Crea was observed for ENB. Large peak of Crea and a small peak of Lys were observed for NPOE/DEHP. His identification was not possible in any acceptor solution due to its comigration with a large unknown peak migrating at 1.4 min. A number of unknown matrix compounds were observed in EME treated urine using NPOE/DEHP. Identification of nortriptyline and haloperidol was not possible due to their comigration with the matrix compounds and loperamide was not detected. Since no transfer of the basic drugs across this SLM was observed for standard solution in Section 3.4, we assume that basic drugs were not extracted from the urine matrix, too, and the peaks in the electropherogram represent interfering matrix cations only. Several interfering compounds can be also observed in acceptor solution obtained after EME using ENB. These compounds migrate between the peak of nortriptyline and loperamide and disable correct quantification of haloperidol. Minimum matrix components are observed for the analysis of the EME pretreated urine using NPOE and 1-octanol. Obviously, selectivity of the examined liquid membranes varies significantly and although the target analytes are transferred across SLMs impregnated with NPOE, ENB

Fig. 4. CE-C4 D determination of EME pretreated human urine using various SLMs. EME and CE conditions as for Fig. 3. (a) NPOE, (b) ENB, (c) 1-octanol, (d) NPOE/DEHP 85/15% v/v. Urine was diluted 1:1 with 2 M acetic acid and spiked with 250 ␮g/L of basic drugs.

and 1-octanol, ENB and 1-octanol are less suitable for applications in complex samples due to the increased transfer of matrix components and due to the reduced transfer rate of the analytes, respectively. NPOE with addition of DEHP is not suitable for two major reasons. Firstly, the target analytes are not transferred across the SLM and secondly, matrix components are not efficiently retained on the SLM and interfere with subsequent CE analysis. 3.6. Selectivity of NPOE-impregnated SLM for various complex samples Based on the previous results, NPOE was selected as the most suitable liquid membrane and red wine, bovine milk, human urine and human plasma were pretreated in order to monitor the SLM selectivity for various complex matrices. The complex samples were prepared according to instructions in Section 2.2 and spiked to a final concentration of 250 ␮g/L of the three basic drugs. Fig. 5 depicts resulting electropherograms for the EME pretreated complex samples. The analyses of EME pretreated milk and plasma samples result in clearly defined peaks of the three basic drugs and only limited number of matrix ions can be detected. Moreover, the major unknown peaks, labeled with asterisks, appear to be a permanent contamination from the SLM since the analytical signals are almost identical for all four complex samples and were observed also for the EME treated standard solutions. Several additional peaks can be observed for urine and wine samples, which apparently originate from the sample matrix. Wine and urine contain a range of various organic and biochemical compounds and this example clearly demonstrates the necessity of the SLM

Fig. 5. CE-C4 D determination of EME pretreated complex samples. EME conditions: liquid membrane: NPOE, agitation: 800 rpm, extraction voltage: 150 V, extraction time: 10 min, acceptor solution: 100 mM acetic acid. Complex samples for EME were diluted 1:1 with 2 M acetic acid, (a) bovine milk, (b) human plasma, (c) human urine, (d) red wine. Concentration of basic drugs is 250 ␮g/L. CE conditions as for Fig. 1. *Unknown compounds.

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selectivity monitoring. Although NPOE exhibits an excellent selectivity for EME of basic drugs [9,10], the SLM selectivity is not absolute and other components are simultaneously co-extracted across the SLM from complex matrices. Therefore, a second selective tool, orthogonal to the extraction technique, such as separation using CE or HPLC with selective detection (UV–vis, MS, etc.) is necessary in order to obtain the required selectivity. A possible approach for elimination of the co-extracted components using optimized CE-C4 D method along with analytical parameters of the method is described in Supplementary Data.

Grant Agency of the Czech Republic (Grant No. P206/10/1219) is gratefully acknowledged.

4. Conclusions

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SLMs are progressive tools for pretreatment of complex samples and LPME using SLMs can easily be combined with CE, HPLC and other analytical techniques. Optimization of the SLM selectivity is very important; however, in order to optimize it, monitoring of all types of species present in complex samples is necessary, which is not possible with selective detection methods. The combination of CE with universal C4 D has shown high potential in that respect and a new CE-C4 D method for rapid determination of SLM selectivity is reported on the example of EME of basic drugs from complex samples. Target analytes and major matrix components (e.g. small inorganic cations, small biomolecules, proteins, etc.) can be simultaneously separated and detected in a common BGE solution and the entire process of their transfer across various SLMs can be monitored. The developed CE-C4 D method has evidenced for the first time that large proteins, such as HSA, are not transferred across SLMs prepared from selected organic solvents. The transfer of target analytes, small inorganic cations and small biomolecules is strongly SLM dependent and significant differences in their transfer rates were observed for various SLMs. Since CE-C4 D is suitable for analysis of all charged species, it can be used for rapid determination of selectivity of various SLMs for a wide range of analytes in many complex samples. We believe that the proposed approach might significantly improve the process of the SLM selection and enable rapid introduction of new and possibly more efficient SLMs in all SLM-based sample pretreatment methods. Acknowledgments Financial support from the Academy of Sciences of the Czech Republic (Institute Research Funding AV0Z40310501) and the

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.chroma.2012.07.010. References