Artificial Liquid Membrane Research Articles

Immobilised artificial membrane liquid chromatography vs liposome electrokinetic capillary chromatography: Suitability in drug/bio membrane partitioning studies and effectiveness in the assessment of the passage of drugs through the respiratory mucosa

This study pioneers a comparison of the application of biomimetic techniques, immobilised artificial membrane liquid chromatography (IAM LC) and liposome electrokinetic capillary chromatography (LEKC), for the prediction of pulmonary drug permeability. The pulmonary absorption profiles of 26 structurally unrelated drug-like molecules were evaluated using their IAM hydrophobicity index (CHI IAM) measured in IAM LC, and the logarithm of distribution constants (log KLEKC) derived from the LEKC experiments. Lipophilicity (phospholipids) parameters obtained from IAM LC and most LEKC analyses were linearly related to the n-octanol/water partitioning coefficients of the neutral forms (i.e., log Po/w values) to a moderate extent. However, the relationship with distribution coefficients at the experimental pH (7.4) (i.e., log D7.4) were weaker overall for IAM LC data and sigmoidal for some liposome compositions (phosphatidyl choline (PC): phosphatidyl inositol (PI) 85:15 mol% and 90:10 mol%) and concentrations (4 mM) in LEKC. This suggests that phospholipid partitioning supports both hydrophobic and electrostatic interactions occurring between ionised drugs and charged phospholipid moieties. The latter interactions are original when compared to those taking place in the more established n-octanol/water partitioning systems.A stronger correlation (R2 > 0.65) was identified between the LEKC retention parameters, and the experimental apparent lung permeability (i.e., log Papp values) as opposed to the values obtained by IAM LC. Therefore, LEKC offers unprecedented advantages over IAM LC in simulating cell membrane partitioning processes in the pulmonary delivery of drugs. Although LEKC has the advantage of more effectively simulating the electrostatic and hydrophobic forces in drug/pulmonary membrane interactions in vitro, the technique is unsuitable for analysing highly hydrophilic neutral or anionic compounds at the experimental pH. Conversely, IAM LC is useful for analysing compounds spanning a wider range of lipophilicity. Its simpler and more robust implementation, and propensity for high-throughput automation make it a favourable choice for researchers in drug development and pharmacological studies.

Solvent-free parallel artificial liquid membrane extraction for drugs of abuse in plasma samples using LC-MS/MS

BackgroundParallel artificial liquid membrane extraction (PALME) is a 96-well plate setup variant of liquid-phase microextraction. Basic or acidic analytes are extracted in neutral form from the sample, through a supported liquid membrane (SLM), and into aqueous acceptor. PALME is already considered a green extraction technique, but in the current conceptual work, we sought to make it even greener by replacing the use of organic solvents with essential oils (EO). PALME was combined with LC-MS/MS for analysis of plasma samples and multiple drugs of abuse with toxicological relevance (amphetamines, phenethylamines, synthetic cathinones, designer benzodiazepines, ayahuasca alkaloids, lysergic acid diethylamide, and ketamine). ResultsFourteen EO were compared to organic solvents frequently used in PALME. The EO termed smart & sassy yielded the best analyte recovery for all drugs studied and was thus selected as SLM. Then, factorial screening and Box-Behnken were employed to optimize the technique. The extraction time, concentration of base, sample volume, and percentage of trioctylamine significantly impacted analyte recovery. The optimum values were defined as 120 min, 10 mmol/L of NaOH, 150 μL, and 0%, respectively. Once optimized, validation parameters were 1–100 ng mL-1 as linear range, accuracy ±16.4%, precision >83%, 1 ng mL-1 as limit of quantitation, 0.1–0.75 ng mL-1 as limit of detection, matrix effect <20%, and recovery 20–106%. Additionally, EO purchased from different production batches were tested and achieved acceptable reproducibility. Data were in compliance with requirements set by internationally accepted validation guidelines and the applicability of the technique was proven using authentic samples. SignificanceIn this study, the use of an EO provided a solvent-free sample preparation technique suited to extract different classes of drugs of abuse from plasma samples, dismissing the use of hazardous organic solvents. The method also provided excellent sample clean-up, thus being a simple and efficient tool for toxicological applications that is in agreement with the principles of sustainable chemistry.

Parallel artificial liquid membrane extraction coupled with UPLC-ESI-MS/MS method for high-throughput quantitation of repaglinide in diabetic patients

A high-throughput therapeutic monitoring method was developed for repaglinide (RPG) in diabetic patients, combining parallel artificial liquid membrane extraction (PALME) with ultraperformance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). PALME was performed using a 96-well donor plate comprising a donor solution containing a plasma sample, 50 mM phosphate buffer (pH = 8.0), and cetirizine (CTZ) as internal standard. A polypropylene (PP) porous membrane served as a selective support for the liquid membrane (SLM), preventing nonspecific binding produced by other membranes. The extraction was accomplished across SLM made of PP membrane with dodecyl acetate and 1 % trioctylamine (w/w), and the acceptor solution comprised DMSO and 200 mM formic acid (50:50, v/v). The simple workflow for PALME provided analyte enrichment, highly efficient sample cleanup, high throughput analysis, and excellent reproducibility. Method validation met FDA criteria, with a linear plasma calibration range (0.1–100 ng mL−1, r = 0.9995) and a lower limit of quantitation (LLOQ) of 0.1 ng mL−1. Recovery results at 98.9 % affirmed method reliability. The ability to analyze 198 samples per hour, coupled with a reduced amount of solvents, underscores the method's high throughput and eco-friendly profile. The PALME-UPLC-ESI-MS/MS method was successfully applied to therapeutic drug monitoring of RPG in diabetic patients following 2 mg RPG tablet administration, establishing its effectiveness.

Liquid-phase microextraction of aromatic amines: hollow fiber–liquid-phase microextraction and parallel artificial liquid membrane extraction comparison

Aromatic amines (AA) are carcinogenic compounds that can enter the human body through many sources, one of the most important being tobacco smoke. They are excreted with the urine, from which they can be extracted and measured. To that end, hollow fiber-liquid-phase microextraction (HF-LPME) and parallel artificial liquid membrane extraction (PALME) were optimized for the analysis of representative aromatic amines, as alternatives to liquid–liquid extraction (LLE). Relevant extraction parameters, namely organic solvent, extraction time, agitation speed, and acceptor solution pH, were studied, and the two optimized techniques—HF-LPME: dihexyl ether, 45 min, 250 rpm, and pH 1; PALME: undecane, 20 min, 250 rpm and pH 1—were compared. Comparison of the optimized methods showed that significantly higher recoveries could be obtained with PALME than with HF-LPME. Therefore, PALME was further validated. The results were successful for nine different AA, with regression coefficients (R2) of at least 0.991, limits of detection (LOD) of 45–75 ng/L, and repeatability and peak area relative standard deviations (RSD) below 20%. Furthermore, two urine samples from smokers were measured as proof of concept, and 2-methylaniline was successfully quantified in one of them. These results show that PALME is a great green alternative to LLE. Not only does it use much smaller volumes of toxic organic solvents, and sample—enabling the study of samples with limited available volumes—but it is also less time consuming and labor intensive, and it can be automated.Graphical

Determination of nerve agent biomarkers in human urine by a natural hydrophobic deep eutectic solvent-parallel artificial liquid membrane extraction technique

Alkyl methyl phosphonic acids (AMPAs) are the major metabolites of organophosphorus nerve agents. A method based on the use of natural hydrophobic deep eutectic solvents as supported liquid membrane in parallel artificial liquid microextraction (PALME) combined with LC-MS/MS analysis was developed and applied to their extraction from urine samples.PALME is a miniaturized liquid-phase extraction method performed in a multiwell plate format where the aqueous sample and the aqueous acceptor phase are separated by a flat membrane impregnated with an organic solvent. In this study, we investigated the possibility of replacing the harmful conventional organic solvent by an emerging green solvent, a coumarin/thymol-based deep eutectic solvent, in ordered to raise the greenness of the sample preparation method.Linear response was obtained in an interval of 0.5, 5 or 10–100 ng/ml depending on the AMPAs with a determination coefficients (R2s) ranging from 0.9751 to 0.9989 for their determination in not treated urine samples. Enrichment factors (EFs) up to 12.65 were obtained, and repeatability was within 8.90–16.28% RSD (n = 12). The limit of quantifications (LOQs: S/N ≥ 10) of the whole analytical procedure were in the range from 0.04 to 5.35 ng/ml. In addition to its good sensitivity, the presented method permitted the treatment of 192 samples in 120 min (equivalent to 37.5 s/sample), which places it as one of the most powerful preparation technique for biomonitoring of civilian or military people exposed to nerve agents in case of public health emergency. Indeed, the developed procedure combined sensitivity, high-throughput, greenness, simplicity and practicality for the determination of five acidic polar AMPAs in urine samples.

Parallel artificial liquid membrane extraction of organophosphorus nerve agent degradation products from environmental samples

An emerging miniaturized high-throughput microextraction technique named Parallel artificial liquid membrane extraction (PALME) was, for the first time, investigated for the extraction of polar alkyl methylphosphonic acids (AMPAs) that are the degradation products of organophosphorus nerve agents. The effect of the key-parameters of the extraction method (nature of the membrane, of the extraction solvent, of the pH values of both donor and acceptor phases, agitation speed, extraction time, temperature and ionic strength) on the extraction recoveries was studied in spiked pure water samples. This led to extraction recoveries in the range of 25–102% for the 5 targeted analytes from water with enrichment factors in the range of 4.50–42.75.The developed PALME-LC-MS/MS method was first evaluated with spiked pure water. LOQs (S/N ≥ 10) were in the range of 0.009–1.141 ng mL−1, linearity above 0.9973 for all the AMPAs and with RSD values below 11%. This method was then applied on simulated waste water, river water and aqueous soil extracts. The achieved LOQs were in the range of 0.011–1.210, 0.013–1.196 and 0.016–6.810 ng mL−1, respectively. A detailed comparison of the performances of this PALME method with those of a previously developed hollow fiber liquid-phase microextraction methods already applied to AMPAs was done thus allowing to demonstrate the easy transfer of methods from HF-LPME to PALME. Moreover, the high-throughput potential of PALME was revealed since 192 samples were processed in parallel during 120 min (37.5 s/sample).

Quantitative analysis of fentanyl, several analogues and metabolites in urine by parallel artificial liquid membrane extraction and liquid chromatography tandem mass spectrometry analysis

The rapid introduction of new psychoactive substances (NPS) has definitively changed the drug market. Among the several NPS that were identified in the last decades, fentanyl and its analogues deserve special attention. These are synthetic opioids with high potency and are associated with increasing number of deaths; for this reason, forensic toxicologists are paying close attention to these analytes and sensitive analytical methods for their detection in biological samples of drug users are needed.The aim of this study was the development of a LC–MS/MS method for the determination of fentanyl, 23 analogues and metabolites in urine by exploiting parallel artificial liquid membrane extraction (PALME). This technique was shown to be particularly suitable for fentanyl extraction and allowed to obtain a high enrichment factor by using a few microliters of organic solvent (1-octanol) immobilized into a polyvinylidene fluoride (PVDF) membrane. The extraction was carried out on a 96 well plate providing high laboratory throughput.The applied strategy allowed to measure concentrations ranging from 0.1 ng mL−1 for fentanyl and most analogues to 5 ng mL−1 for metabolites, by using an entry level mass spectrometer. Because of the different concentration levels generally found in real samples, linearity was studied in different ranges i.e. LOQ to 50 ng mL−1 for parent drugs and LOQ to 200 ng mL−1 for metabolites. All the validation parameters were found within the imposed limits, and notably matrix effect was not significant for all the analytes, showing the selectivity achieved by PALME extraction.

Green and sustainable drug analysis – Combining microsampling and microextraction of drugs of abuse

A novel method combining microsampling of whole blood containing drugs of abuse with 96-well liquid solution microextraction (Parallel Artificial Liquid Membrane Extraction, PALME) and analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was evaluated. Different donor solutions, supported liquid membranes and acceptor solutions were tested, and the most promising set-up was validated. The method included common classes of drugs of abuse, such as opioids, amphetamines, cocaine, benzodiazepines and z-hypnotics. Extraction recoveries above 70% were found for 13 of the compounds, while the last four compounds had recoveries of 10–58%. THC was not extracted with the current method. A linear calibration model was found for all drugs but morphine. Limits of quantitation were between 1 and 5 ng/mL and inter-day precision and accuracy was within 20% for all compounds except for morphine and zopiclone that had a CV of 25% at LOQ. All matrix effects were within 78–123%. Samples were stable for 14 days except for zopiclone and zolpidem. With low-cost, high sample throughput, semi-automated miniaturized sample preparation in combination with dried blood microsamples, and an Eco-Scale score of 78, the proposed method fulfills green chemistry principles.

Liquid-phase microextraction in 96-well plates - calibration and accurate quantification of pharmaceuticals in human plasma samples

The use of miniaturized systems with the possibility for automation has become increasingly popular in the field of bioanalysis. As a new approach to liquid phase microextraction in the 96-well format, parallel artificial liquid membrane extraction (PALME) was introduced in 2013. In the present work, the reliability of the quantitative data obtained with PALME was thoroughly evaluated. Amitriptyline, nortriptyline, quetiapine, venlafaxine, o-desmethylvenlafaxine, and fluoxetine were selected as model analytes. The analytes were extracted under non-exhaustive conditions from human plasma samples and the extracts were analyzed directly by LC-MS/MS. Accuracy was within ±15% and precision was <15% when the QC samples were prepared in both pooled plasma and in plasma from multiple sources. Accuracy and precision was superior when stable isotopically labelled (SIL) internal standards were used, as compared to structural analogue internal standards in the plasma samples from multiple sources. SIL internal standards are therefore recommended as the first choice. Assessment of accuracy and precision was also carried out with four different operators performing the extraction procedure, providing accuracy within ±15% and precision <15%. The extraction recoveries were in the range from 48 to 85 %, and non-exhaustive extraction of the analytes did not affect the accuracy and precision of the method. With the method described, up to 96 samples can be extracted with a total extraction time of 60 min and with a total consumption of organic solvent less than 0.4 mL for the whole wellplate. PALME is therefore a new approach to high-throughput sample preparation, providing accurate quantification, along with simple workflow, low consumption of organic solvent, and extensive sample clean-up.

Rapid determination of designer benzodiazepines, benzodiazepines, and Z-hypnotics in whole blood using parallel artificial liquid membrane extraction and UHPLC-MS/MS.

Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R2> 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0ngmL-1 and 3.2 to 160ngmL-1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10days at 10 and - 20°C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis. Graphical abstract.

Cytotoxicity of seven bisphenol analogues compared to bisphenol A and relationships with membrane affinity data

Bisphenol A (BPA) is a chemical used in numerous industrial applications. Due to its well ascertained toxicity as endocrine disruptor, industries have started to replace it with other bisphenols whose alleged greater safety is scarcely supported by literature studies. In this study, the toxicity of seven BPA analogues was evaluated using both in silico and in vitro techniques, as compared to BPA toxicity. Furthermore, their affinity indexes for phospholipids (i.e. phospholipophilicity) were determined by immobilized artificial membrane liquid chromatography (IAM-LC) and possible relationships with in vitro toxic activity were also investigated. The results on four different cell cultures yielded similar ranking of toxicity for the bisphenols considered, with IC50 values confirming their poor acute toxicity. As compared to BPA, bisphenol AF, bisphenol B, bisphenol M, and bisphenol A diglycidyl ether resulted more toxic, while bisphenol S, bisphenol F and bisphenol E were found as the less toxic congeners. These results are partly consistent with the scale of phospholipid affinity showing that toxicity increases at increasing membrane affinity. Therefore, phospholipophilicity determination can be assumed as a useful preliminary tool to select less toxic congeners to surrogate BPA in industrial applications.

Parallel artificial liquid membrane extraction of psychoactive analytes: a novel approach in therapeutic drug monitoring.

Liquid-liquid extraction is widely used in therapeutic drug monitoring of antipsychotics, but difficulties in automation of the technique can result in long operational time. In this paper, parallel artificial liquid membrane extraction was used for extraction of serotonin-and serotonin-norepinephrine reuptake inhibitors from human plasma, and an approach to automate the technique was investigated. Eight model analytes were extracted from 125μl human plasma with recoveries in the range 72-111% (relative standard deviation [RSD] ≤12.8%). A semiautomated pipettor was successfully utilized in the procedure, reducing the manual handling time. Real patient samples were analyzed with satisfying accuracy. A semiautomated extraction of serotonin-and serotonin-norepinephrine reuptake inhibitors by parallel artificial liquid membrane extraction extraction was successfully performed.

Screening therapeutics according to their uptake across the blood-brain barrier: A high throughput method based on immobilized artificial membrane liquid chromatography-diode-array-detection coupled to electrospray-time-of-flight mass spectrometry

The Blood-Brain Barrier (BBB) plays an essential role in protecting the brain tissues against possible injurious substances. In the present work, 79 neutral, basic, acidic and amphoteric structurally unrelated analytes were considered and their chromatographic retention coefficients on immobilized artificial membrane (IAM) stationary phase were determined employing a mass spectrometry (MS)-compatible buffer based on ammonium acetate. Their BBB passage predictive strength was evaluated and the statistical models based on IAM indexes and in silico physico-chemical descriptors showed solid statistics (r2 (n − 1) = 0.78). The predictive strength of the indexes achieved by the MS-compatible method was comparable to that achieved by employing the more “biomimetic” Dulbecco’s phosphate buffered saline, even if some differences in the elution order were observed. The method was transferred to the MS, employing a diode-array-detection coupled to an electrospray ionization source and a time-of-flight analyzer. This setup allowed the simultaneous analysis of up to eight analytes, yielding a remarkable acceleration of the analysis time.

Dried blood spots and parallel artificial liquid membrane extraction–A simple combination of microsampling and microextraction

In this paper, parallel artificial liquid membrane extraction (PALME) was used for the first time to clean-up dried blood spots (DBS) prior to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Fundamental studies exploring amongst others desorption from the DBS in alkaline or acidic aqueous conditions, total extraction time and absolute recoveries were executed. Desorption and PALME were performed using a set of two 96-well plates, one of them housing the sample and the other comprising the supported liquid membrane (SLM) and the acceptor solution. In one procedure, amitriptyline and quetiapine (basic model analytes) were desorbed from the DBS using 250 μL of 10 mM sodium hydroxide solution (aqueous), and subsequently extracted through the SLM consisting of 4 μL of 1% trioctylamine in dodecyl acetate, and further into an acceptor solution consisting of 50 μL of 20 mM formic acid. In a second procedure, ketoprofen, fenoprofen, flurbiprofen, and ibuprofen (acidic model analytes) were desorbed from the DBS into 20 mM formic acid, extracted through an SLM with dihexyl ether, and further into an acceptor solution of 25 mM ammonia. Within 60 min of PALME, both basic and acidic model analytes were effectively desorbed from the DBS and extracted into the acceptor solution, which was injected directly into the analytical instrument. Recoveries between 63 and 85% for the six model analytes were obtained. PALME provided excellent clean-up from the DBS samples, and acceptor solutions were free from phospholipids. Linearity was obtained with r2 > 0.99 for five of the six analytes. Accuracy, precision and UHPLC-MS/MS matrix effects were in accordance with the European Medicines Agency (EMA) guideline. Based on these experiments, PALME shows great potential for future processing of DBS in a short and simple way, and with the presented setup, up to 96 DBS can be processed within a total extraction time of 60 min.

Determination of in Vitro and in Silico Indexes for the Modeling of Blood-Brain Barrier Partitioning of Drugs via Micellar and Immobilized Artificial Membrane Liquid Chromatography.

In the present work, 79 structurally unrelated analytes were taken into account and their chromatographic retention coefficients, measured by immobilized artificial membrane liquid chromatography (IAM-LC) and by micellar liquid chromatography (MLC) employing sodium dodecyl sulfate (SDS) as surfactant, were determined. Such indexes, along with topological and physicochemical parameters calculated in silico, were subsequently used for the development of blood-brain barrier passage-predictive statistical models using partial least-squares (PLS) regression. Highly significant relationships were observed either using IAM (r2 (n - 1) = 0.78) or MLC (r2 (n - 1) = 0.83) derived indexes along with in silico descriptors. This hybrid approach proved fast and effective in the development of highly predictive BBB passage oriented models, and therefore, it can be of interest for pharmaceutical industries as a high-throughput BBB penetration oriented screening method. Finally, it shed new light into the molecular mechanism involved in the BBB uptake of therapeutics.

Electrochemical Analysis on the Biomimetic Coupling Reaction Between Ion Transport and Electron Transport through an Artificial Liquid Membrane

Introduction In the energy-transducing membrane as mitochondria, some ion transports are coupled with electron transports. In the biological membrane, the ion transport region and the electron transport region are located separately on the membrane. Under this condition, in order to maintain the electroneutrality inside and outside the membrane, same amounts of ions and electrons should be transported through a membrane under the established membrane potential. The coupling of the ion transport and the electron transport in different regions of the membrane would be different from that in same region of the membrane such as a liquid membrane where ions are distributed with electrons across the aqueous/membrane interface to keep electroneutrality in an aqueous phase and membrane phase [1]. In the present study, an artificial membrane system with separated charge transport sites is constructed, and novel coupling mechanism between ion transport and electron transport is demonstrated. Experimental Two liquid membrane systems, W1/O1/W2 and W1’/O2/W2, were connected through a common phase of W2. The former is the ion transport system and the latter is the electron transport system. The organic solvent used in O1 and O2 was nitrobenzene. W1 and O1 contained the transported ion (K+ or tetraethyl ammonium ion, TEA+) as hydrophilic and hydrophobic salts, respectively. W1’ and W2 contained [Fe(CN)6]4- and [Fe(CN)6]3-, respectively. O2 contained 7,7,8,8-tetracyanoquinodimethane, TCNQ, and TCNQ·-. When W1 and W1’ were connected through Ag/AgCl electrodes, K+ or TEA+, was transported from W1 to W2, and an electron was transported from W1’ to W2. That is, the electric current accompanied with the coupling between the ion transport and the electron transport flowed through an outer circuit from W1’ to W1. The electric current was measured by using a galvanometer, and simultaneously the membrane potential between W1 and W2 and that between W1’ and W2 were recorded using electrometers. Furthermore, the voltammogram for ion transport and that for electron transport were recorded independently in W1/O1/W2 system and W1’/O2/W2 system. Results and Discussion The membrane potential for the coupling reaction was predicted from the viewpoint of the conservation of electroneutrality in all phases of W1, W1’, O1, O2 and W2, which was determined as a potential where positive current for ion transport is equal to negative current for electron transport in comparison of voltammograms recorded in W1/O1/W2 and W1’/O2/W2 systems. When two liquid membrane systems were connected, the membrane potentials of W1-W2 and W1’-W2 approached each other after connecting W1 and W1’, and the electric current decreased gradually. The finally attained membrane potential agreed with the potential expected from the voltammogram for ion transport and that for electron transport. Further, when the concentration of [Fe(CN)6]3- in W2 changed from 1 x 10-4 mol dm-3 to 5 x 10-4 mol dm-3, the membrane potential attained in the coupling reaction shifted positively and the current decreased, which agreed with the prediction obtained from voltammograms. These results indicate that the coupling reaction in the separated ion transfer site and electron transfer site proceeds under the identical membrane potential in both charge transport region to keep electroneutrality in all phases. In addition, the possibility of the violation of electroneutrality is discussed in the transient period immediately after connecting W1 and W1’. Especially, the difference in capacitance component between two charge transport systems influenced the disturbance of balance in Faradaic component. [1] H. Ohde, K. Maeda, O. Shirai, Y. Yoshida and S. Kihara, J. Electroanal. Chem., 438, 139-145 (1997).

Parallel artificial liquid membrane extraction of new psychoactive substances in plasma and whole blood.

Parallel artificial liquid membrane extraction (PALME) was combined with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the potential for screening of new psychoactive substances (NPS) was investigated for the first time. PALME was performed in 96-well format comprising a donor plate, a supported liquid membrane (SLM), and an acceptor plate. Uncharged NPS were extracted from plasma or whole blood, across an organic SLM, and into an aqueous acceptor solution, facilitated by a pH gradient. MDAI (5,6-methylenedioxy-2-aminoindane), methylone, PFA (para-fluoroamphetamine), mCPP (meta-chlorophenylpiperazine), pentedrone, methoxetamine, MDPV (methylenedioxypyrovalerone), ethylphenidate, 2C-E (2,5-dimethoxy-4-ethylphenethylamine), bromo-dragonfly, and AH-7921 (3,4-dichloro-N-{[1-(dimethylamino)cyclohexyl]methyl}benzamide) were selected as representative NPS. Optimization of operational parameters was necessary as the NPS were novel to PALME, and because PALME was performed from whole blood for the very first time. In the PALME method developed for plasma, NPS were extracted from a 250μL alkalized donor solution consisting of 125μL plasma sample, 115μL 40mM NaOH, and 10μL internal standard. In the PALME method from whole blood, the 250μL alkalized donor solution consisted of 100μL whole blood, 50μL deionized water, 75μL 80mM NaOH, and 25μL internal standard. In both methods, extraction was accomplished across an SLM of 5μL dodecyl acetate with 1% trioctylamine (w/w), and further into an acidic acceptor solution of 50μL 20mM formic acid. The extraction was promoted by agitation at 900rpm and was carried out for 120min. Method validation was performed and the following parameters were considered: linearity, limits of quantification (LOQ), intra- and inter-day precision, accuracy, extraction recoveries, carry-over, and matrix effects. The validation results were in accordance with FDA guidelines.

One-step extraction of polar drugs from plasma by parallel artificial liquid membrane extraction.

The new microextraction technique named parallel artificial liquid membrane extraction (PALME) was introduced as an alternative approach to liquid-liquid extraction of charged analytes from aqueous samples. The concept is based on extraction of analytes across a supported liquid membrane sustained in the pores of a thin polymeric membrane, a well-known extraction principle also used in hollow fiber liquid-phase microextraction (HF-LPME). However, the new PALME technique offers a more user-friendly setup in which the supported liquid membrane is incorporated in a 96 well plate system. Thus, high-throughput is achievable, in addition to the green chemistry offered by using PALME. The consumption of organic solvent is minimized to 3-5μL per sample. With a sample volume of 250μL and acceptor solution volume of 50μL, a maximal enrichment factor of five is achievable. Based on these parameters, a new method for extraction of polar basic drugs was developed in the present work. The basic drugs hydralazine, ephedrine, metaraminol, salbutamol, and cimetidine were used as model analytes, and were extracted from alkalized human plasma into an aqueous solution via the supported liquid membrane. The extraction was promoted by a carrier dissolved in the membrane, creating a temporary ion-pair complex between the hydrophilic drug and the carrier. As the model analytes were extracted directly into an aqueous solution, there was no need for evaporation of the extract before injection into LC-MS. Hence, the sample preparation is performed in one step. With optimized conditions, the extraction recoveries were in the range 50-89% from human plasma after 45min extraction. The data from the method evaluation were satisfactory and in line with current guidelines, and revealed an extraction method with substantial potential for high throughput bioanalysis of polar basic drugs.

Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma

Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC–MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines.

Photocurrent generation based on a light-driven proton pump in an artificial liquid membrane

Biological light-driven proton pumps use light to move protons across a cell membrane, creating a proton gradient. Although photochromic compounds such as spiropyrans can reversibly convert between two structures with differing pKa values, spiropyrans have not been used to generate either a light-driven proton pump or an electrical current. Here, we report an artificial light-harvesting system based on a supported liquid membrane doped with a spiropyran. Irradiating the membrane with ultraviolet light induces a ring-opening reaction, converting spiropyran to merocyanine, whereas irradiation with visible light induces the reverse reaction. When the membrane is irradiated with ultraviolet and visible light on opposite sides, H(+) is taken up by merocyanine, carried through the polymeric membrane and released on the other side. We show that this system produces a light-induced proton flux, an electrical current with an efficiency of ∼0.12%, an open-circuit voltage of ∼210mV and a membrane gradient of ∼3.6ΔpH units. Alternating the sides illuminated with ultraviolet and visible light generates an alternating current.