Combination of Electromembrane Extraction and Liquid-Phase

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Combination of electromembrane extraction and liquid-phase microextraction in a single step: Simultaneous group separation of acidic and basic drugs Chuixiu Huang, Knut Fredrik Seip, Astrid Gjelstad, Xiantao Shen, and Stig Pedersen-Bjergaard Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01610 • Publication Date (Web): 03 Jun 2015 Downloaded from http://pubs.acs.org on June 8, 2015

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Analytical Chemistry

Combination of electromembrane extraction and liquid-phase microextraction in a single step: Simultaneous group separation of acidic and basic drugs Chuixiu Huang,†, § Knut Fredrik Seip, † Astrid Gjelstad, † Xiantao Shen,*, ‡, § and Stig PedersenBjergaard,*,†, ⊥ †

School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway

‡

Institute of Environmental Medicine, School of Public Health, Tongji Medical College,

Huazhong University of Science and Technology, Hangkong Road #13, Wuhan 430030, China §

G&T Septech AS, PO Box 33, 1917 Ytre Enebakk, Norway

Department of Pharmacy, Faculty of Health and Medical Sciences, Faculty of Pharmaceutical

⊥

Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark Stig Pedersen-Bjergaard: [email protected] Xiantao Shen: [email protected]

ACS Paragon Plus Environment

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ABSTRACT: Electromembrane extraction (EME) and liquid-phase microextraction (LPME) were combined in a single step for the first time to realize simultaneous and clear group separation of basic and acidic drugs. Using 2-nitrophenyl octyl ether as the supported liquid membrane (SLM) for EME and di-hexyl ether as the SLM for LPME, basic and acidic drugs were extracted and separated simultaneously from low pH sample by EME and LPME, respectively. After 15 min extraction, basic drugs (citalopram and sertraline) were exhaustively extracted, whereas the recoveries for acidic drugs (ketoprofen and ibuprofen) were in the range of 76%-86%. Longer extraction time provided higher recoveries for the acidic drugs, but this somewhat deteriorated the group separation. Matrices effects from the coexisting acidic drugs/basic drugs were tested, and we observed that simultaneous EME/LPME was not affected by coexisting drugs at high concentration. This approach was further investigated from human plasma. Extraction recoveries were strongly dependent on dilution of plasma with buffer and on extraction time. Finally, this simultaneous EME/LPME approach was evaluated in combination with LC-MS. The linearity range for the basic and acidic drugs were 10-600 ng/mL and 1-60 µg/mL, respectively, with R2 >0.997 for all analytes. The repeatability at three different levels for all analytes was less than 15%. The limits of quantification (LOQ, S/N = 10) were found to be 4.0-6.3 ng/mL and 0.6-0.9 µg/mL for basic and acidic drugs, respectively. Simultaneous EME/LPME enabled efficient group separation of basic and acidic analytes under optimum experimental conditions for both EME and LPME.

KEYWORDS: Electromembrane extraction (EME); liquid-phase microextraction (LPME); simultaneous extraction; group separation; LC/MS; human plasma


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INTRODUCTION Generally, direct analysis of samples with complex matrices is not possible, and the analysis of pharmaceuticals in biological matrices is therefore still a challenge.1,2 Thus, efficient and green sample preparation methods are necessary for reliable determination of drugs in biological samples.3 Up to date, different sample preparation methods have been proposed to remove major matrix components prior to the instrument analysis. Major matrix components may reduce the sensitivity, and may also interfere with the determination of target analytes. Group separation of target analytes may be preferable prior to the analysis, because compounds with similar chemical or physical properties can be treated in a uniform manner.4 As known, protein precipitation (PP), liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are traditional sample preparation techniques, and in principle all these techniques are suitable for simultaneous determination of basic and acidic drugs in biological samples.5,6 For example, SPE was used for the simultaneous determination of basic and acidic pharmaceuticals from aquatic environmental matrices with recoveries from 27 to 120%. 7 In another work, simultaneous determination of basic and acidic pharmaceuticals from human whole blood was achieved with recoveries ranging from 20 to 114% by combining PP and SPE. 8 However, simultaneous group separation of similar compounds by the above techniques may not be possible.7,8 Microextraction techniques such as solid-phase microextraction (SPME) and liquid-phase microextraction (LPME) have been introduced to meet the demands of green sample preparation method. 9 SPME highly depends on the distribution coefficient of the analytes between the sample matrix and the extraction phase, which is the coating on the solid support.10 Accordingly,


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SPME is a non-exhaustive sample preparation technique, and basic and acidic drugs can be extracted simultaneously, but group separation is not possible.11,12 In 1996, LPME was first introduced as the format of single-drop microextraction (SDME) 13, where the analytes are extracted into a single drop of organic solvent from the sample based on distribution constants.13, 14 , 15 Subsequently, hollow fiber liquid-phase microextraction (HFLPME) was introduced in 1999. 16 The supported liquid membrane (SLM) in the HF-LPME system was an organic solvent impregnated in the pores of a hollow fiber membrane. The extraction is governed by passive diffusion of neutral species from the sample through the SLM, and into the acceptor solution. Definitely, HF-LPME provides excellent clean-up due to the high selectivity of the SLM, and sufficient enrichment factors due to the tuneable ratio between the sample volume and the acceptor volume.17 Nevertheless, LPME is not suitable for simultaneous extraction of acidic and basic drugs.18 Electromembrane extraction was introduced as a miniaturized sample preparation technique in 2006. 19 Low cost, environmental friendly, simple operation and fast extraction are some advantages of EME.20,21 During EME, charged analytes in the sample are extracted selectively through the supported liquid membrane under the influence of an electric field, and finally into an acceptor solution to realize isolation and clean-up. Up to date, EME has been applied for the extraction of basic drugs22,23 and acidic drugs24 individually. Most recently, flat membrane-based EME was developed to provide exhaustive extraction of basic drugs from biological samples.25 In addition, EME has also been used for simultaneous group separation of basic and acidic drugs at a certain sample pH, where the acidic drugs were negatively charged and the basic drugs were positively charged. 26 With an envelope-EME system, the recovery for simultaneous extraction of acidic and basic drugs ranged between 18% and 80%, but special care was needed for the sample


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pH to ionize both the acidic and basic drugs. 27 As reported by Yamini et al., a basic drug (nalmefene) and an acidic drug (diclofenac) were extracted simultaneously using dual EME with a recovery of 12.5% and 14.6%, respectively, where EME was conducted from sample (neutral pH) using 40 V, and with an SLM of NPOE and 1-octanol for basic and acidic drugs, respectively.28 In a subsequent paper, ibuprofen and thebaine were extracted simultaneously and successfully using dual EME from neutral sample with 40 V.29 The idea of simultaneous group extraction of basic and acidic drugs using dual EME is very interesting, though exact control of the distribution of the electrical field across the extraction system may be a challenge under dual EME conditions. However, when the proposed dual EME approach was applied for the simultaneous extraction of some other basic compounds with pKa7, the obtained recoveries were calculated to be in the range of 1.6%-7.2%.28 This low recoveries might due to the fact that for dual EME, the used experimental parameters (sample pH and extraction voltage) were selected as a compromise29, and netrual pH in sample was not optimum to ionize such kind of analytes efficiently.20,21,29,30 Inspired by the above works, together with the fact that low pH in sample is the optimum pH for EME of basic analytes and LPME of acidic analytes theoretically and practically18,25, and combining the advantages of both EME and LPME, we herein proposed a new concept for simultaneous group separation of basic and acidic drugs (Scheme 1), where the principle of EME and LPME were integrated together for the first time. This simultaneous EME/LPME method gave the opportunity to optimize the experimental conditions for EME (basic analytes) and LPME (acidic analytes) independently from sample with optimum pH for EME of basic analytes and for LPME of acidic analytes.18,25 In comparison with dual EME, our combination of EME and LPME also solved the challenge mentioned above about electrical field distribution. In this


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work, major operational parameters were optimized, including the SLM, the acceptor solution, background electrolytes, and the extraction time. Also, the purity of the group separation was studied to verify that the acceptor phase containing basic analytes was not contaminated with acidic compounds and vice versa. Finally, this simultaneous EME/LPME approach was evaluated by LC-MS using two basic drugs and two acidic drugs as model analytes. EXPERIMENTAL SECTION Chemicals. Citalopram hydrobromide (CIT) (20 mg tablet) was produced by Lundbeck (Copenhagen, Denmark), and sertraline hydrochloride (SER) (50 mg tablet) was produced by Pfizer Italiana (Latina, Italy). The internal standard for basic drugs - fluoxetine-d5 solution in methanol (1 mg/mL) was supplied by ISOTEC (Miamisburg, OH, USA). Ketoprofen (KET), ibuprofen (IBU) and ibuprofen-d3 (internal standard for acidic drugs) were all from SigmaAldrich (St. Louis, MO, USA). 2-Nitrophenyl octyl ether (NPOE) was from Fluka (Buchs, Switzerland). 2,4-Dimethyl-1-nitrobenzene (DMNB), 2-undecanone, 1-ethyl-2-nitrobenzene (ENB), 2,2-dimethyl-1-propyl benzene (DMPB), phenyl benzene (PB), 1-nonanol, 1octanol, dihexyl ether (DHE), were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Isopropyl nitrobenzene (IPNB) was produced by Tokyo Chemical Industry (Tokyo, Japan). A Milli-Q water purification system (Millipore, Darmstadt, Germany) was employed for the purification of water. Acetonitrile, formic acid, ammonium acetate, hydrochloric acid, and sodium hydroxide were all supplied from Merck (Darmstadt, Germany). Preparation of solutions. The stock solutions of ketoprofen (5 mg/mL), ibuprofen (5 mg/mL), sertraline (1 mg/mL) and citalopram (1 mg/mL) were obtained by dissolving the compounds in ethanol individually. All these stock solutions were stored at 4 oC after a filtration with a 0.45 µm filter. The standard solution (STD, 10 µg/mL for each analyte) was prepared by diluting the


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stock solutions with 100 mM HCl solution. Drug-free human plasma from Oslo University Hospital (Oslo, Norway) was stored at -32 oC, and was diluted with 100 mM HCl prior to extraction. Simultaneous EME/LPME set-up and procedure. The set-up of simultaneous EME/LPME is shown in Scheme 1. The home-made flat-membrane-based acceptor compartment for both EME and LPME is a wide end-closed 10-200 µl pipette tip (Sartorius Biohit Liquid Handling Oy, Helsinki, Finland) with a piece of Accurel PP 1E (R/P) flat membrane (Membrana, Wuppertal, Germany) with a thickness of 100 µm, an average pore size of 100 nm, a porosity of 69%31 and an area of approximately 28 mm2 sealed on the bottom. The fabrication of this flatmembrane-based acceptor compartment was described elsewhere.25 The donor compartment was a disposable plastic pipette (SARSTEDT, Nümbrecht, Germany) with a cut of the tip. The SLM of EME and LPME were 3 µl of NPOE and DHE, respectively. The acceptor solution of EME and LPME were 100 µl of 100 mM HCl and 100 mM NaOH, respectively. The “L- shaped” cathode and anode, made of platinum rods (d= 0.5 mm, K. A. Rasmussen, Hamar, Norway), were introduced into the sample and EME acceptor solution (100 mM HCl), respectively. Afterwards, two acceptor compartments were inserted into the sample compartment with a gap of approximately 1 mm between the SLMs and the interface of the sample. The electrodes were connected to an ES 0300-0.45 power supply (Delta Elektronika BV, Zierikzee, Netherlands), which was coupled to a home-made current monitor system to record the instantaneous current continuously. After the voltage was set with LabVIEW 8.2 software (National Instruments, Austin, TX, USA), the EME and LPME process was initiated by starting the Vibramax 100 agitator (Heidolph Instruments, Kelheim, Germany) with a speed of 1200 rpm. After the default extraction time, the extractions were terminated by turning off the agitator manually.


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Immediately, the acceptor solutions after EME and LPME were collected individually and subsequently analyzed by HPLC-UV or LC-MS. High Performance Liquid Chromatography (HPLC) -UV Analysis. A Dionex Ultimate 3000 system equipped with an auto-sampler (WPS-3000SL), a VWD-3400 UV/VIS detector (all from Dionex Corporation, Sunnyvale, CA, USA), a degasser (SRD-3200), a column oven (FLM3100), and a pump (HPG-3200M) was used for the HPLC-UV analysis, and the UV/VIS detector was operated at 214 nm. Chromeleon software (v. 6.80 SP2 Build 2212) from Dionex Corporation was used to process the analysis and to collect the data. A Gemini C18 column (150 mm X 2.00 mm, 5 µm) was used for the separation, with an injection volume of 20 µL. Mobile phase A was 20 mM formic acid containing 5% of acetonitrile (v/v), and mobile phase B was acetonitrile containing 5% of 20 mM formic acid (v/v). Mobile phase B was increased from 15 to 90% within 4 min at a flow rate of 0.4 mL/min, afterwards, it was decreased to 15% within 0.1 min, and this condition was kept for 2 min. Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis. LC-MS analysis was carried out using an Ultimate 3000-system (Sunnyvale, CA, USA), including an autosampler (WPS-3000TSL), a degasser (SRD 3300), a micro pump (LPG-3000), and a mass spectrometer detector (LTQ XL linear ion trap). Chromeleon client software (v.6.80 SR8 build 2633) was used for LC control, and Xcalibur software (v. 2.1.0.1139) was used for processing and control of LTQ XL and data acquisition. The separation for both basic and acidic drugs was carried out on a 1 × 50 mm Biobasic-C8 column with a particle size of 5 µm (Thermo Scientific, Waltham, MA, USA). For basic drugs, the ratio of 20 mM formic acid and acetonitrile in mobile phase A and B were 95%/5% and 5%/95% (v/v), respectively. However, for acidic drugs, 20 mM formic acid was replaced by 20 mM ABC (ammonium bicarbonate) in mobile phase A and B. The


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gradient for separation was described in the electronic supporting information (LC-MS). The MS was operated with electrospray ionization in either positive (basic drugs) or negative (acidic drugs) ion mode. Collision energy and product ions for each target ion are presented in Table S1. RESULTS AND DISCUSSION

Proof-of-Concept. The aim of the current work is to show the possibility of combining EME and LPME for group separation of basic and acidic analytes. EME was used to extract basic analytes from low pH sample to low pH acceptor solution. The basic analytes, which were positively charged in the sample at low pH, were extracted by electro-kinetic migration through an SLM of NPOE with a voltage of 300 V. As shown in Scheme 1, the cathode was placed in the acceptor solution (100 µL), while the anode was located in the acidified sample (600 µL). Citalopram (CIT) and sertraline (SER) (Table 1) were selected as model basic analytes, and have previously been extracted by EME.18,25, 32 The operational parameters used for EME in the current work were based on previous experience.25 LPME was used to extract acidic analytes from low pH sample to high pH acceptor solution. The acidic analytes were neutral at low pH, and were extracted by passive diffusion through an SLM of DHE. Ibuprofen (IBU) and ketoprofen (KET) (Table 1) were selected as model acidic analytes.18,28,33,34 The sample and acceptor solution volume were 600 and 100 µL, respectively. Initially, EME was conducted from 6-fold diluted standard solution with 100 mM HCl alone using 300 V for 15 min with an SLM of NPOE. Afterwards, both of the acceptor solution and the sample were analyzed, and mass balance data for all analytes in three phases are reported in Table 2. As shown, CIT and SER were extracted exhaustively, which is in accordance with our previous observations.25 Interestingly, KET and IBU were not detected in the acceptor solution,


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and only a small fraction was found in the sample. This supported that KET and IBU were highly trapped in the hydrophobic SLM of NPOE with single EME.24 Similarly, LPME was performed from 6-fold diluted standard solution with 100 mM HCl alone with an SLM of DHE18 for 15 min, and the mass balance data in three phases for both basic and acidic analytes are summarized in Table 3. As shown, the acidic drugs were extracted exhaustively. As expected, the positively charged basic drugs showed no affinity towards the SLM (DHE), and remained in the sample. This was due to the selectivity of the SLM, where only neutral species can pass through the SLM barrier by passive diffusion.16 In the following step, EME and LPME were combined and performed simultaneously for group separation of basic and acidic analytes from 6-fold diluted standard solution with 100 mM HCl. The mass balance data for all analytes in different phases are summarized in Table 4, and the group separation performance is illustrated with the chromatograms (Figure 1). Again, the basic drugs were extracted exhaustively, and clearly, the EME performance was unaffected by the simultaneous LPME process. The LPME recoveries for the acidic drugs KET and IBU were 86% and 76%, respectively, because small fractions of KET and IBU were trapped in the SLMs of EME and LPME. Clearly, the LPME process was slightly affected by the EME process in the sense that small amounts of the acidic analytes were extracted into the NPOE membrane in the EME part of the system. However, in comparison with single EME process, the amount of acidic drugs trapped in the hydrophobic SLM was reduced significantly, which was due to the competitive extraction by the LPME part of the simultaneous EME/LPME system.

Optimization of major operational parameters. In a series of experiments, different organic solvents were tested as SLM for LPME, including 2-undecanone, 1-nonanol, 1-octanol, DHE, DMPB, PB, ENB, IPNB, DMNB and NPOE. The solvents were selected based on


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previous experiences.18,33 DMPB, PB, ENB, IPNB and DMNB generated background peaks in the chromatogram as observed previously.25 Thus, solvent related impurities leaked to the acceptor solutions during extraction and added undesired complexity to the HPLC separation. The recoveries with the other SLMs are presented in Figure 2. As seen, DHE provided the highest recovery for the target analytes. Therefore, DHE was selected as the SLM for the LPME. For the EME part, NPOE was used without further optimization based on previous experience.25 In LPME, the target must be neutral in the sample to promote extraction, and charged in the acceptor solution to avoid back-extraction. Therefore, the background composition of the sample and the acceptor solution was tested. As known, the ion balance in the EME system plays a role for efficient extraction.35 However, in this series of experiments, the acceptor solution of EME was kept the same as the background of the sample for convenient operation, since under such condition, exhaustive EME of basic drugs has been achieved (Table 2). The tested sample background and EME acceptor solution were 100 mM, 10 mM and 1 mM HCl. The results are summarized in Table S2, which shows that 100 mM HCl in the sample was optimal for the recovery of the acidic analytes. This ensured the pH in the sample to be low enough during the whole extraction process to keep the acidic drugs neutral, although pH might shift slightly due to electrolysis.28,29,36,37 The tested concentrations of HCl in the sample and in the EME acceptor solution showed no effect on the recoveries of the basic analytes, because pH in the sample and in the EME acceptor solution was substantially lower than the pKa values of the target (Table 1), so the basic analytes were always fully charged in all cases. Meanwhile, the tested acceptor solution for LPME included 100 mM, 10 mM and 1 mM NaOH. The recoveries for the acidic analytes were not affected by the NaOH concentration (1-100 mM) in the acceptor solution


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(Table S3), because the acidic analytes were fully charged and prevented from back-extraction into the SLM in all cases. Subsequently, different acceptor solution volumes were investigated for the LPME part of the system. The recoveries for all model substances versus the acceptor solution volume (50, 75, 100 and 150 µL) for LPME are plotted in Figure 3a. In the examined range, the acceptor solution volume for LPME did not affect the EME efficiency, and the recovery for the acidic analytes increased with increasing acceptor solution volume up to 100 µL, which was consistent with previous observations.25 Accordingly, 100 µL was selected as the optimal acceptor solution volume for LPME. The acceptor solution volume for EME was kept at 100 µL, since basic drugs were exhaustively extracted with such conditions. In a final optimization experiment, extractions were carried out for 15, 20 and 30 min, respectively. The LPME recovery versus extraction time is plotted in Figure 3b. As seen from this figure, LPME recoveries increased slightly with time, and a long extraction time was advantageous for high LPME recoveries. For EME, recoveries remained almost unaffected by the investigated extraction time. However, extraction time exceeding 15 min caused a small fraction of the acidic analytes to be extracted by EME (Figure S1). Most probably, this was due to possible competitive extraction and pH shift in the EME acceptor phase.28,29,36,37 Based on the results, we concluded that 15 min was the optimal extraction time to provide a clear group separation and high recovery, but longer time was needed if higher recoveries are preferable.

Interference effects during group separation. Another series of experiments were conducted to investigate the effects of the presence of high level of acidic substances (KET and IBU) on the EME of the basic substances (CIT and SER), and vice versa. The recoveries for all analytes at different concentrations are shown in Table 5 and Table 6, respectively. As seen, the


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presence of acidic drugs with different initial concentrations (0, 2 and 10 µg/mL) did not affect the recoveries of CIT and SER with an initial concentration of 0.5 µg/mL by EME (Table 5). Similarly, the presence of basic analytes with different initial concentrations (0, 0.5 and 2 µg/mL) showed no effect on the recoveries of acidic analytes with an initial concentration of 2 µg/mL by LPME (Table 6). Thus, we believe that the simultaneous EME/LPME has high potential to be used for real samples, where both basic and acidic drugs may be present in the same sample but with different concentrations.

Simultaneous EME/LPME from human plasma. Simultaneous EME/LPME was initially carried out for 15 min from human plasma diluted six times with 100 mM HCl. The recoveries for CIT, SER, KET and IBU were 22, 38, 52 and 43%, respectively. Following this, simultaneous EME/LPME was carried out for 15 min from 3, 6, 12, 24, and 36 times diluted human plasma (with 100 mM HCl), respectively. The plot of recoveries versus the dilution factor is shown in Figure 4, which demonstrates that the recoveries increased with increasing dilution factor as expected. In addition, the recoveries for all the analytes could also be improved further by using longer extraction time (45 min) from both 6-fold and 36-fold diluted human plasma (Table 7). When simultaneous EME/LPME was performed for 45 min from 36-fold diluted human plasma, the recoveries for CIT, SER, KET and IBU were 63, 95, 94 and 82%, respectively. However, with the extraction time of 45 min, coextraction of a small fraction of acidic drugs into the EME acceptor solution was observed as mentioned previously (Figure S1).

Evaluation. Finally, this simultaneous EME/LPME approach was evaluated for the determination of CIT, SER, KET and IBU from diluted human plasma. The acceptor solutions after EME and LPME were analyzed by LC–MS in positive and negative ion mode, respectively. Fluoxetine-d5 and ibuprofen-d3 were used as the internal standards for the basic and acidic


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analytes, respectively. To cover the therapeutic ranges, the investigated linearity range for basic drugs and acidic drugs were in the range of 10-600 ng/mL and 1-60 µg/mL, respectively.38 The obtained data are summarized in Table 7. The R2 values were no less than 0.997 within the tested linearity range. The repeatability was tested at 10, 60, and 300 ng/mL for basic drugs, and at 1, 6, and 30 µg/mL for acidic drugs (n = 6). All RSD-values were below 15%. The limits of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) shown in Table 7 indicated that simultaneous EME/LPME can be used to quantify the basic and acidic drugs concentration in real samples. Accordingly, the evaluation from human plasma meets the requirements of FDA for validation of bioanalytical methods.39 CONCLUSIONS In the current work, the combination of electromembrane extraction (EME) and liquid-phase microextraction (LPME) has been investigated for the first time to achieve simultaneous and clear group separation of basic and acidic drugs in a single step from sample at low pH, which was optimum for EME of basic drugs and for LPME of acidic drugs theoretically and practically. With this simultaneous EME/LPME system, basic drugs were extracted exhaustively by EME; however, slightly lower recoveries for acidic drugs were obtained due to a small fraction of acidic drugs that were trapped in SLMs of both EME and LPME. After the optimization, clear group separation was obtained with high recovery after 15 min extraction. The evaluation of this simultaneous EME/LPME from human plasma using LC-MS suggested that the proposed method has high potential for real biological samples. In conclusion, except that the low cost device was used only for a single extraction to avoid carry over, combined EME and LPME enabled rapid and efficient group separation of basic and acidic analytes from biological fluids


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using only 6 µL of organic solvent based on two different microextraction principles under optimum operation conditions for both. ASSOCIATED CONTENT Supporting information Supporting information includes the LC-MS method, the selection of the background electrolyte in the sample and in the acceptor solutions, the effect of extraction time on group separation. This information is available free of charge via the Internet at http://pubs.acs.org/. AUTHOR INFORMATION Corresponding Authors * S. Pedersen-Bjergaard. E-mail: [email protected] * X. Shen. E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work has been performed as part of the 'Robust affinity materials for applications in proteomics and diagnostics' (PEPMIP) project, supported by the Seventh Research Framework Program of the European Commission. Grant agreement number: 264699. REFERENCES (1) Martı´n-Esteban, A.; Sellergren, B. Comprehensive Sampling and Sample Preparation; Pawliszyn, J., Ed.; Academic Press: Oxford, 2012; pp 331-344. (2) Rutkowska, M.; Dubalska, K.; Konieczka, P.; Namieśnik, J. Molecules 2014, 19, 75817609. (3) Tankiewicz, M.; Fenik, J.; Biziuk, M. Talanta 2011, 86, 8-22.


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(4) WELLS, D. E. Pure &Appl. Chem. IUPAC: Great Britain, 1988; pp 1437-1448. (5) Lakshmana Prabu, S.; Suriyaprakash, T. N. K. Applied Biological Engineering - Principles and Practice; Naik, G. R., Ed.; InTech: Croatia, 2012; pp 479-506. (6) Hunter, G. J. clin. Path. 1957, 10, 161-164. (7) Gracia-Lor, E.; Sancho, J.V.; Hernández, F. J. Chromatogr. A 2010, 1217, 622-632. (8) Kudo, K.; Usumoto, Y.; Usui, K.; Hayashida, M.; Kurisaki, E.; Saka, K.; Tsuji, A.; Ikeda, N. Forensic Toxicol. 2014, 32, 97-104. (9) Arthur, C. L.; Killam, L. M.; Motlagh, S.; Lim, M.; Potter, D. W.; Pawliszyn, J. Environ. Sci. Technol. 1992, 26, 979-983. (10) Pawliszyn, J. J. Chromatogr. Sci. 2000, 38, 270-278. (11) Boyac, E.; Gorynski, K.; Rodriguez-Lafuente, A.; Bojko, B.; Pawliszyn, J. Anal. Chim. Acta. 2014, 809, 69-81. (12) Togunde, O. P.; Lord, H.; Oakes, H. D.; Servos, M. R.; Pawliszyn, J. J. Sep. Sci. 2013, 36, 219-223. (13) Liu, H. H.; Daasgupta, P. K. Anal. Chem. 1996, 68, 1817-1821. (14) Jeannot, M. A.; Cantwell, F. F. Anal. Chem. 1996, 68, 2236-2240. (15) Liu, H. H.; Daasgupta, P. K. TrAC-Trend. Anal. Chem. 1996, 15, 468-475. (16) Pedersen-Bjergaard, S.; Rasmussen, K. E. Anal. Chem. 1999, 71, 2650-2256. (17) Namera, A.; Saito, T. Bioanalysis 2013, 5, 915-932. (18) Bostrom, M. L.; Huang, C.; Engstrom, H.; Larsson, E.; Berglund, O.; J¨onsson, J. Å. Anal. Methods 2014, 6, 6031-6037. (19) Pedersen-Bjergaard, S.; Rasmussen, K. E. J. Chromatogr. A 2006,1109, 183-190. (20) Lee, J.; Lee, H. K.; Rasmussen, K. E.; Pedersen-Bjergaard, S. Anal. Chim. Acta 2008, 624, 253-268. (21) Ramos-Payán, M.; Villar-Navarro, M.; Fernández-Torres, R.; Callejón-Mochón, M.; BelloLópez, M. A. Anal. Bioanal. Chem. 2013, 405, 2575-2584. (22) Middelthon-Bruer, T. M.; Gjelstad, A.; Rasmussen, K. E.; Pedersen-Bjergaard, S. J. Sep. Sci. 2008, 31, 753-759. (23) Gjelstad, A.; Rasmussen, K. E.; Pedersen-Bjergaard, S. Anal. Bioanal. Chem. 2009, 393, 921-928.


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(24) Balchen, M.; Rasmussen, K. E.; Gjelstad, A.; Pedersen-Bjergaard, S. J. Chromatogr. A 2007, 1152, 220-225. (25) Huang, C.; Eibak, L. E. E.; Gjelstad, A.; Shen, X.; Trones, R.; Jensen, H.; PedersenBjergaard, S. J. Chromatogr. A 2014, 1326, 7-12. (26) Gjelstad, A.; Pedersen-Bjergaard, S. Anal. Methods 2013, 5, 4549-4557. (27) Basheer, C.; Lee, J.; Pedersen-Bjergaard, S.; Rasmussen, K. E.; Lee, H. K. J. Chromatogr. A 2010, 1217, 6661-6667. (28) Seidi, S.; Yamini, Y.; Rezazadeh, M.; Esrafili, A. J. Chromatogr. A 2012, 1243, 6-13. (29) Tabani, H.; Fakhari, A. R.; Shahsavani, A. Electrophoresis 2013, 34, 269-276. (30) Sun, J. N.; Shi, Y. P.; Chen, J. RSC Adv, 2015, 5, 37682-37690. (31) Dindore, V. Y.; Brilman, D. W. F.; Geuzebroek, F. H.; Versteeg, G. F. Sep. Purif. Technol. 2004, 40, 133-145. (32) Eibak, L. E. E.; Gjelstad, A.; Rasmussen, K. E.; Pedersen-Bjergaard, S. J. Pharm. Biomed. Anal. 2012, 57, 33-38. (33) Larsson, E.; Al-Hamimi, S.; Jönsson, J. Å. Sci. Total Environ. 2014, 485–486, 300-308. (34) Payan, M. R.; Lopez, M. A.; Torres, R. F.; Navarro, M. V.; Mochon, M. C.; Talanta 2011, 85, 394-399. (35) Gjelstad, A.; Rasmussen, K. E.; Pedersen-Bjergaard, S. J. Chromatogr. A 2007, 1174, 104111. (36) Fakhari, A. R.; Sahragard, A.; Ahmar, H.; Tabani, H. J. Electroanal. Chem. 2015, 747, 1219. (37) Kubáˇn, P.; Boˇcek, P. J. Chromatogr. A 2015, 1398, 11-19. (38) Schulz, M.; Schmoldt, A. Pharmazie. 2003, 447-474. (39) http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf.


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Table 1. Related parameters for all model substances.a Analytes Protein binding (%) pKa Log P Citalopram (CIT)

80

9.78

3.76

Sertraline (SER)

98

9.85

5.15

Ketoprofen (KET)

99

3.88

3.61

Ibuprofen (IBU)

90-99

4.85

3.84

a http://www.drugbank.ca/drugs.

Table 2. Analytes (%) in different phases after EME for 15 min with an SLM of NPOE and a voltage of 300 V from 6-fold diluted standard solution with 100 mM HCl. Analytes Acceptor (RSD)/% Donor (RSD)/% SLM/% CIT

103 (3)

nd

0

SER

93 (4)

nd

7

KET

nd

9 (6)

91

IBU

nd

2 (49)

98

nd: not detected.

Table 3. Analytes (%) in different phases after LPME for 15 min with an SLM of DHE from 6fold diluted standard solution with 100 mM HCl. Analytes Acceptor (RSD)/% Donor (RSD)/% SLM/% CIT

nd

96 (4)

4

SER

nd

101 (7)

0

KET

97 (7)

nd

3

IBU

103 (2)

nd

0

nd: not detected.


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Table 4. Analytes (%) in different phases after simultaneous EME/LPME from 6-fold diluted standard solution with 100 mM HCl for 15 min with an SLM of NPOE and DHE, respectively. EME acceptor LPME acceptor Donor SLMs (EME Analytes (RSD)/% (RSD)/% /% +LPME)/% CIT

97 (6)

nd

nd

3

SER

97 (1)

nd

nd

3

KET

nd

86 (3)

nd

14

IBU

nd

76 (10)

nd

24

nd: not detected.

Table 5. Effect of the coexisting acidic drugs’ concentration on the extraction recovery (Rec). 0 2 10 Conc. (µg/mL) Analytes

Rec (RSD)/% Rec (RSD)/% Rec (RSD) /%

CIT

96 (6)

95(2)

106 (10)

SER

102 (7)

93 (3)

100 (3)

KET

nd

92 (7)

82 (6)

IBU

nd

72 (5)

70 (3)

nd: not detected.

Table 6. Effect of the coexisting basic drugs’ concentration on the extraction recovery (Rec). 0 0.5 2 Conc. (µg/mL) Analytes

Rec (RSD)/% Rec (RSD)/% Rec (RSD) /%

CIT

nd

95 (2)

94 (6)

SER

nd

93 (3)

96 (2)

KET

87 (7)

92 (7)

86 (12)

IBU

70 (6)

72 (5)

72 (8)

nd: not detected.


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Table 7. Recoveries of the analytes from 6-fold and 36-fold diluted human plasma using different extraction time (15 min and 45 min). Simultaneous EME/LPME was performed with NPOE and DHE as the SLM for EME and LPME, respectively. The voltage for EME was 300 V. 6-fold diluted human plasma 36-fold diluted human plasma Analytes

15 min

45 min

15 min

45 min

Rec (RSD)/% Rec (RSD)/% Rec (RSD) /% Rec (RSD) /% CIT

22 (15)

55 (4)

38 (13)

63 (14)

SER

38 (14)

65 (4)

65 (9)

95 (5)

KET

52 (7)

83 (7)

69 (6)

94 (5)

IBU

43 (8)

70 (10)

65 (6)

82 (5)

Table 8. Evaluation results with simultaneous EME/LPME-LC/MS from spiked acidified human plasma. Analyte Bases CIT SER

R2

Therapeutic levela

Linearity range

RSD (n=6) LOD LOQ (S/N=3) (S/N=10) Lowb Mediumb Highb

ng/mL 0.997 0.999

20-200 50-250

10-600

1.2 1.9

4.0 6.3

14 14

% 10 7

10 8

Acids µg/mL % KET 0.999 1-6 0.2 0.6 10 7 6 1-60 IBU 0.997 15-30 0.3 0.9 11 9 9 a Data were adapted from Ref. [38]. b Low, medium and high were 10, 60 and 300 ng/mL for bases, but 1, 6 and 30 µg/mL for acids.


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Scheme 1. Schematic representation of simultaneous EME/LPME.


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Figure 1. Group separation using simultaneous EME/LPME. Chromatograms of the acceptor solution after EME and LPME, and of the standard solution (STD, 10 µg/mL for each analyte). EME was performed with an SLM of NPOE and a voltage of 300 V for 15 min from 6-fold diluted standard solution with 100 mM HCl to 100 mM HCl. LPME was performed with an SLM of DHE for 15 min from spiked 100 mM HCl to 100 mM NaOH.

Figure 2. Selection of the organic solvent for LPME.


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Figure 3. Effects of the LPME acceptor solution volume (a) and the extraction time (b) on the extraction recoveries (Rec) of all analytes obtained from simultaneous EME/LPME (n = 3, RSD < 10%).

Figure 4. Effects of the dilution factor of the human plasma on the recoveries (n = 3, RSD < 15%) of all analytes. Simultaneous EME/LPME were performed for 15 min with NPOE and DHE as the SLM, respectively. The voltage for EME was 300.


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For TOC only


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Combination of Electromembrane Extraction and Liquid-Phase

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