Electromembrane extraction in microfluidic formats.

Electromembrane extraction is a microextraction technique where charged analytes are extracted across a supported liquid membrane and selectively isolated from the sample based on an electrical field. Since the introduction in 2006, there has been continuously increasing interest in electromembrane extraction, and currently close to 50 new articles are published per year. Electromembrane extraction can be performed in different technical configurations, based on standard laboratory glass vials or 96-well plate systems, and applications are typically related to pharmaceutical, environmental, and food and beverages analysis. In addition to this, conceptual research has developed electromembrane extraction into different milli- and microfluidic formats. These are much more early-stage activities, but applications among others related to organ-on-chip systems and smartphone detection indicate unique perspectives. To stimulate more research in this direction, the current article reviews the scientific literature on electromembrane extraction in milli- and microfluidic formats. About 20 original research articles have been published on this subject so far, and these are discussed critically in the following. Based on this and the authors own experiences with the topic, we discuss perspectives, challenges, and future research.

Continuous electromembrane extraction coupled with mass spectrometry - Perspectives and challenges.

This tutorial discusses continuous electromembrane extraction (c-EME) coupled directly to mass spectrometry (MS), and the applicability of such systems for on-line and real-time monitoring of in-vitro drug metabolism. Parent drug substances and corresponding drug metabolites are extracted from the metabolic reaction mixture, through a supported liquid membrane (SLM), and into an acceptor solution on the other side. Extraction is accomplished using an external electrical field sustained over the SLM. The acceptor solution is continuously pumped into the mass spectrometer, and the decline of parent drug as well as the development of metabolites is followed directly with the mass spectrometer. The purpose of the extraction is to avoid proteins and salts from the reaction mixture from entering the mass spectrometer. This tutorial first discusses the principles and theory of operation. Second, technical development is highlighted with special focus on major challenges associated with c-EME-MS systems. Third, operational parameters and performance are discussed, and finally future perspectives and challenges are considered.

Direct coupling of electromembrane extraction to mass spectrometry - Advancing the probe functionality toward measurements of zwitterionic drug metabolites.

A triple-flow electromembrane extraction (EME) probe was developed and coupled directly to electrospray-ionization mass spectrometry (ESI-MS). Metabolic reaction mixtures (pH 7.4) containing drug substances and related metabolites were continuously drawn (20 µL/min) into the EME probe in one flow channel, and mixed inside the probe with 7.5 µL min-1 of 1 M formic acid as make-up flow from a second flow channel. Following this acidification, the drug substances and their related metabolites were continuously extracted by EME at 400 V, across a supported liquid membrane (SLM) comprising 2-nitrophenyl octyl ether (and for some experiments containing 30% triphenyl phosphate (TPP)), and into 20 µL min-1 of formic acid as acceptor phase, which was introduced through a third flow channel. The acceptor phase was pumped directly to the MS system, and the ion intensity of extracted analytes was followed continuously as function of time. The triple-flow EME probe was used for co-extraction of positively charged parent drugs and their zwitterionic drug metabolites (hydroxyzine and its carboxylic acid metabolite cetirizine; and vortioxetine and its carboxylic acid metabolite Lu AA34443). While the zwitterionic metabolites could not be extracted at pH 7.4, it was shown that by acidifying the sample solution the zwitterionic metabolites could be extracted effectively. Various extraction parameters like make-up flow, extraction voltage and SLM composition were optimized for simultaneous extraction of parent drugs and metabolites. It was found that TPP added to the SLM improved extraction efficiencies of certain drug metabolites. Finally the optimized and characterized triple-flow EME probe was used for online studying the in-vitro metabolism of hydroxyzine and vortioxetine by rat liver microsomes. Due to the automated pre-extraction acidification of the rat liver microsomal solutions, it was possible to continuously monitor formation of the zwitterionic drug metabolites. As the triple-flow EME probe allowed modification of the pH of the sample without changing the pH in the bulk sample, the system can potentially be used for direct analysis of various kinds of chemical reactions that have to be run at pH conditions unfavorable for direct analyte extractions.

Fully Automated Electro Membrane Extraction Autosampler for LC-MS Systems Allowing Soft Extractions for High-Throughput Applications.

The current work describes the implementation of electro membrane extraction (EME) into an autosampler for high-throughput analysis of samples by EME-LC-MS. The extraction probe was built into a luer lock adapter connected to a HTC PAL autosampler syringe. As the autosampler drew sample solution, analytes were extracted into the lumen of the extraction probe and transferred to a LC-MS system for further analysis. Various parameters affecting extraction efficacy were investigated including syringe fill strokes, syringe pull up volume, pull up delay and volume in the sample vial. The system was optimized for soft extraction of analytes and high sample throughput. Further, it was demonstrated that by flushing the EME-syringe with acidic wash buffer and reverting the applied electric potential, carry-over between samples can be reduced to below 1%. Performance of the system was characterized (RSD, <10%; R(2), 0.994) and finally, the EME-autosampler was used to analyze in vitro conversion of methadone into its main metabolite by rat liver microsomes and for demonstrating the potential of known CYP3A4 inhibitors to prevent metabolism of methadone. By making use of the high extraction speed of EME, a complete analytical workflow of purification, separation, and analysis of sample could be achieved within only 5.5 min. With the developed system large sequences of samples could be analyzed in a completely automated manner. This high degree of automation makes the developed EME-autosampler a powerful tool for a wide range of applications where high-throughput extractions are required before sample analysis.

Direct coupling of a flow-flow electromembrane extraction probe to LC-MS.

A fully integrated and automated electromembrane extraction LC-MS (EME-LC-MS) system has been developed and characterized. Hyphenation of a flow-flow EME probe to LC-MS was accomplished by using an in-built 10-port switching valve of the LC-MS system. The 10-port switching valve decoupled the high pressure of the UHPLC-system from the low pressure required for operation of the EME-probe by automated switching between a sample extraction/analysis and a sample load position. In the sample load position the extracted analytes were loaded into a HPLC sample loop. By switching the valve to the sample extraction/analysis position the setup allowed simultaneous analysis of previously loaded analytes while extracting a new sample. Performance of the system was characterized with respect to precision and linearity (RSD < 2.5%, R(2): 0.998) and the setup was applied for studying the in-vitro metabolism of methadone by rat liver microsomes. As the metabolic reaction proceeded, methadone and its metabolites were extracted and analyzed in parallel by LC-MS using either isocratic or gradient elution. Compared to a conventional in-vitro metabolism analysis based on protein precipitation followed by LC-MS analysis the fully automated EME-LC-MS system offers a significant time saving and in addition demonstrates increased sensitivity as the analytes were automatically enriched during the extraction process. The experiment revealed 6 to 16 times higher S/N ratios of the EME-LC-MS method compared to protein precipitation followed by LC-MS and thus concomitantly lower LOD and LOQ. The setup integrates a complete analytical workflow of rapid extraction, enrichment, separation and detection of analytes in a fully automated manner. These attributes make the developed system a powerful alternative approach for a wide range of analytical applications.

Real Time Extraction Kinetics of Electro Membrane Extraction Verified by Comparing Drug Metabolism Profiles Obtained from a Flow-Flow Electro Membrane Extraction-Mass Spectrometry System with LC-MS.

A simple to construct and operate, "dip-in" electromembrane extraction (EME) probe directly coupled to electrospray ionization-mass spectrometry (ESI-MS) for rapid extraction and real time analysis of various analytes was developed. The setup demonstrated that EME-MS can be used as a viable alternative to conventional protein precipitation followed by liquid chromatography-mass spectrometry (LC-MS) for studying drug metabolism. Comparison of EME-MS with LC-MS for drug metabolism analysis demonstrated for the first time that real time extraction of analytes by EME is possible. Metabolism kinetics were investigated for three different drugs: amitriptyline, promethazine, and methadone. By comparing the EME-MS extraction profiles of the drug substances and formed drug metabolites with the metabolism profiles obtained by conventional protein precipitation followed by LC-MS good correlation was obtained with only very limited time delay in the extraction. The results indicate that, by tuning the electromembrane properties, for example, by optimizing the extraction voltage, extremely fast extraction kinetics can be obtained. A metabolic profile could be generated while the drug was metabolized offering a significant time saving as compared to conventional LC-MS where laborious protein precipitation or other sample pretreatments are required before analysis. This makes the developed EME-MS setup a highly promising sample preparation method for various kinds of applications where fast and real-time analysis of analytes is of interest.

Application of temperature-correlated mobility theory for optimizing the MEKC separation of the main lignans from Schisandra Chinensis Fructus and its prescription Yuye Decoction.

The present work shows the application of the temperature-correlated mobility theory for the optimization of the separation and peak alignment of the main lignans from water extracts of traditional Chinese medicine Schisandra Chinensis Fructus as well as its prescription Yuye Decoction (Jade Fluid Decoction; YYD). This is the first application of this theory for MEKC separations, and the data presentation allows a much easier peak tracking and thereby identification of the analytes. Most interestingly, the data obtained and presented in the mobility scale at 298 K, show that Schisantherin A, which is easily mistaken as one of the analytes using traditional time scale, was actually not detected in Schisandra Chinensis Fructus and Yuye Decoction (Jade Fluid Decoction) water extracts. This proves the value of the temperature-correlated mobility scale for method optimization of complex samples. Thus, in the temperature-correlated mobility scale, the optimization of the system conditions for the MEKC separations can easily be achieved by correcting for viscosity changes. Also, the influence of the operating temperature can be monitored in a more distinct way.

Design and implementation of an automated liquid-phase microextraction-chip system coupled on-line with high performance liquid chromatography.

An automated liquid-phase microextraction (LPME) device in a chip format has been developed and coupled directly to high performance liquid chromatography (HPLC). A 10-port 2-position switching valve was used to hyphenate the LPME-chip with the HPLC autosampler, and to collect the extracted analytes, which then were delivered to the HPLC column. The LPME-chip-HPLC system was completely automated and controlled by the software of the HPLC instrument. The performance of this system was demonstrated with five alkaloids i.e. morphine, codeine, thebaine, papaverine, and noscapine as model analytes. The composition of the supported liquid membrane (SLM) and carrier was optimized in order to achieve reasonable extraction performance of all the five alkaloids. With 1-octanol as SLM solvent and with 25 mM sodium octanoate as anionic carrier, extraction recoveries for the different opium alkaloids ranged between 17% and 45%. The extraction provided high selectivity, and no interfering peaks in the chromatograms were observed when applied to human urine samples spiked with alkaloids. The detection limits using UV-detection were in the range of 1-21 ng/mL for the five opium alkaloids presented in water samples. The repeatability was within 5.0-10.8% (RSD). The membrane liquid in the LPME-chip was regenerated automatically between every third injection. With this procedure the liquid membrane in the LPME-chip was stable in 3-7 days depending on the complexity of sample solutions with continuous operation. With this LPME-chip-HPLC system, series of samples were automatically injected, extracted, separated, and detected without any operator interaction.

Development and characterization of a small electromembrane extraction probe coupled with mass spectrometry for real-time and online monitoring of in vitro drug metabolism.

A small and very simple electromembrane extraction probe (EME-probe) was developed and coupled directly to electrospray ionization mass spectrometry (ESI-MS), and this system was used to monitor in real time in vitro metabolism by rat liver microsomes of drug substances from a small reaction (incubation) chamber (37 °C). The drug-related substances were continuously extracted from the 1.0 mL metabolic reaction mixture and into the EME-probe by an electrical potential of 2.5 V. The extraction probe consisted of a 1-mm long and 350-µm ID thin supported liquid membrane (SLM) of 2-nitrophenyl octyl ether. The drugs and formed metabolites where extracted through the SLM and directly into a 3 µL min(-1) flow of 60 mM HCOOH inside the probe serving as the acceptor solution. The acceptor solution was directed into the ESI-MS-system, and the MS continuously monitored the drug-related substances extracted by the EME-probe. The extraction efficiency of the EME-probe was dependant on the applied electrical potential and the length of the SLM, and these parameters as well as the volume of the reaction chamber were set to the values mentioned above to avoid serious depletion from the reaction chamber (soft extraction). Soft extraction was mandatory in order not to affect the reaction kinetics by sample composition changes induced by the EME-probe. The EME-probe/MS-system was used to establish kinetic profiles for the in vitro metabolism of promethazine, amitriptyline and imipramine as model substances.

Nano-electromembrane extraction.

The present work has for the first time described nano-electromembrane extraction (nano-EME). In nano-EME, five basic drugs substances were extracted as model analytes from 200 µL acidified sample solution, through a supported liquid membrane (SLM) of 2-nitrophenyl octyl ether (NPOE), and into approximately 8 nL phosphate buffer (pH 2.7) as acceptor phase. The driving force for the extraction was an electrical potential sustained over the SLM. The acceptor phase was located inside a fused silica capillary, and this capillary was also used for the final analysis of the acceptor phase by capillary electrophoresis (CE). In that way the sample preparation performed by nano-EME was coupled directly with a CE separation. Separation performance of 42,000-193,000 theoretical plates could easily be obtained by this direct sample preparation and injection technique that both provided enrichment as well as extraction selectivity. Compared with conventional EME, the acceptor phase volume in nano-EME was down-scaled by a factor of more than 1000. This resulted in a very high enrichment capacity. With loperamide as an example, an enrichment factor exceeding 500 was obtained in only 5 min of extraction. This corresponded to 100-times enrichment per minute of nano-EME. Nano-EME was found to be a very soft extraction technique, and about 99.2-99.9% of the analytes remained in the sample volume of 200 µL. The SLM could be reused for more than 200 nano-EME extractions, and memory effects in the membrane were avoided by effective electro-assisted cleaning, where the electrical potential was actively used to clean the membrane.

On-chip electromembrane extraction for monitoring drug metabolism in real time by electrospray ionization mass spectrometry.

A temperature controlled (37 °C) metabolic reaction chamber with a volume of 1 mL was coupled directly to electrospray ionization mass spectrometry (ESI-MS) by the use of a 50 µm deep counter flow micro-chip electromembrane extraction (EME) system. The EME/ESI-MS system was used to study the in vitro metabolism of amitriptyline in real time. There was no need to stop the metabolisms by protein precipitation as in conventional metabolic studies, since the EME selectively extracted the drug and metabolites from the reaction solution comprised of rat liver microsomes in buffer. Compositional changes in the reaction chamber were continuously detected 9 seconds later in the MS. Most of this time delay was due to transport of the purified extract towards the ESI source. The EME step effectively removed the enzymatic material, buffer and salts from the reaction mixture, and prevented these species from being introduced into the ESI-MS system. The on-chip EME/ESI-MS system provided repeatability for the amitriptyline signal intensity within 3.1% relative standard deviation (RSD) (n = 6), gave a linear response for amitriptyline in the tested concentration range of 0.25 to 15 µM, and was found not to be prone to ion-suppression from major metabolites introduced simultaneously into the EME/ESI-MS system. The setup allowed the study of fast reactions kinetics. The half-life, t(1/2), for the metabolism of 10 µM amitriptyline was 1.4 minutes with a 12.6% RSD (n = 6).

Liquid-phase microextraction in a microfluidic-chip--high enrichment and sample clean-up from small sample volumes based on three-phase extraction.

In this work, a microfluidic-chip based system for liquid-phase microextraction (LPME-chip) was developed. Sample solutions were pumped into the LPME-chip with a micro-syringe pump at a flow rate of 3-4 µL min(-1). Inside the LPME chip, the sample was in direct contact with a supported liquid membrane (SLM) composed of 0.2 µL dodecyl acetate immobilized in the pores of a flat membrane of polypropylene (25 µm thickness). On the other side of the SLM, the acceptor phase was present. The acceptor phase was either pumped at 1 µL min(-1) during extraction or kept stagnant (stop-flow). Amitriptyline, methadone, haloperidol, loperamide, and pethidine were selected as model analytes, and they were extracted from alkaline sample solution, through the SLM, and into 10 mM HCl or 100mM HCOOH functioning as acceptor phase. Subsequently, the acceptor phase was either analyzed off-line by capillary electrophoresis for exact quantification, or on-line by UV detection or electrospray ionization mass spectrometry for time profiling of concentrations. The LPME-chip was found to be highly effective, and extraction efficiencies were in the range of 52-91%. When the flow of acceptor phase was turned off during extraction (stop-flow), analyte enrichment increased linearly with the extraction time. After 10 min as an example, amitriptyline was enriched by a factor of 42 from only 30 µL sample solution, and after 120 min amitriptyline was enriched by a factor of 500 from 320 µL sample solution. This suggested that the LPME-chip has great potentials for very efficient analyte enrichments from limited sample volumes in the future.

Selective electromembrane extraction at low voltages based on analyte polarity and charge.

Electromembrane extraction (EME) at low voltage (0-15 V) of 29 different basic model drug substances was investigated. The drug substances with logP<2.3 were not extracted at voltages less than 15 V. Extraction of drug substances with logP≥2.3 and with two basic groups were also effectively suppressed by the SLM at voltages less than 15 V. Drug substances with logP≥2.3 and with one basic group were all extracted at low voltages and with a strong compound selectivity which appeared to have some influence from the polar surface area of the compound. For this group of substances, recoveries varied between 0 and 23% at 5 V, whereas, recoveries varied between 5.5 and 51% at 15 V. Based on mass transfer differences related to charge, polarity, and polar surface, highly selective extractions of drug substances were demonstrated from human plasma, urine, and breast milk. An initial evaluation at low voltage (5 V) was compared with similar extractions at a more normal voltage level (50 V), and this supported that reliable data can be obtained under these low-voltage (mild) conditions by EME.

Electromembrane extraction from biological fluids.

Electro-assisted extraction of ionic drugs from biological fluids through a supported liquid membrane (SLM) and into an aqueous acceptor solution was recently introduced as a new sample preparation technique termed electromembrane extraction (EME). The applied electrical potential across the SLM has typically been in the range of 1-300 V. Successful extractions have been demonstrated even with common batteries (9 V) instead of a power supply. The chemical composition of the SLM has been crucial for the selectivity and for the recoveries of the extraction. Compared to other liquid-phase microextraction techniques (LPME), extraction times have been reduced by a factor of 6-17, and successful extractions have been obtained at extraction times of 1-5 min, and even down to a few seconds with online microfluidic EME devices. The technique has provided very efficient sample clean-up and has been found well suited for the extraction of sample sizes in the low µL range. Extractions have been performed with both rod-shaped hydrophobic porous fibers and with flat hydrophobic porous sheets as SLM support. The technique has been successfully downscaled into the micro-chip format. The nature of the SLM has been tuned for extraction of drugs with different polarity allowing extractions to be tailored for specific applications depending on the analyte of interest. The technique has been found to be compatible with a wide range of biological fluids and extraction of drugs directly from untreated human plasma and whole blood has been demonstrated. EME selectively extracts the compounds from the complex biological sample matrix as well as allowing concentration of the drugs. With home-built equipment fully acceptable validation results have been obtained.

On-chip electro membrane extraction with online ultraviolet and mass spectrometric detection.

Electro membrane extraction was demonstrated in a microfluidic device. The device was composed of a 25 µm thick porous polypropylene membrane bonded between two poly(methyl methacrylate) (PMMA) substrates, each having 50 µm deep channel structures facing the membrane. The supported liquid membrane (SLM) consisted of 2-nitrophenyl octyl ether (NPOE) immobilized in the pores of the membrane. The driving force for the extraction was a 15 V direct current (DC) electrical potential applied across the SLM. Samples containing the basic drugs pethidine, nortriptyline, methadone, haloperidol, loperamide, and amitriptyline were used to characterize the system. Extraction recoveries were typically in the range of 65-86% for the different analytes when the device was operated with a sample flow of 2.0 µL/min and an acceptor flow of 1.0 µL/min. With the sample flow at 9.0 µL/min and the acceptor flow at 0.0 µL/min, enrichment factors exceeding 75 were obtained during 12 min of operation from a total sample volume of only 108 µL. The on-chip electro membrane system was coupled online to electrospray ionization mass spectrometry and used to monitor online and real-time metabolism of amitriptyline by rat liver microsomes.

Drop-to-drop microextraction across a supported liquid membrane by an electrical field under stagnant conditions.

Electromembrane extraction (EME) of basic drugs from 10 microL sample volumes was performed through an organic solvent (2-nitrophenyl octyl ether) immobilized as a supported liquid membrane (SLM) in the pores of a flat polypropylene membrane (25 microm thickness), and into 10 microL 10 mM HCl as the acceptor solution. The driving force for the extractions was 3-20 V d.c. potential sustained over the SLM. The influence of the membrane thickness, extraction time, and voltage was investigated, and a theory for the extraction kinetics is proposed. Pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted from pure water samples with recoveries ranging between 33% and 47% after only 5 min of operation under totally stagnant conditions. The extraction system was compatible with human urine and plasma samples and provided very efficient sample pretreatment, as acidic, neutral, and polar substances with no distribution into the organic SLM were not extracted across the membrane. Evaluation was performed for human urine, providing linearity in the range 1-20 microg/mL, and repeatability (RSD) in average within 12%.