The widespread occurrence of veterinary drug residues in animal-derived foods demands analytical methods that are not only sensitive and reliable, but also efficient and environmentally sustainable. To meet this need, this study developed a novel high-throughput platform based on 96-well electromembrane extraction (EME) using a designed deep eutectic solvent (DES) composed of 6-methylcoumarin and thymol (1:1, w/w) as a green and efficient supported liquid membrane. When coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), the proposed DES-EME method enabled the simultaneous determination of 22 veterinary drugs in three kinds of complex food samples: honey, milk, and eggs. Under optimized conditions (10 mM trifluoroacetic acid, 50 V, 15 min), the method demonstrated excellent analytical performance, featuring wide linear ranges of 0.01–100 ng/mL with r ≥ 0.993, and limits of quantification (LOQs) expressed as original sample concentrations of 1.0 ng/g for honey and 25 ng/g for milk and eggs. Recoveries ranged from 40 to 99

The selective enrichment of trace hydrophilic peptides from complex biological matrices remains a pivotal challenge in biomarker discovery. To address this, we developed a high-throughput and environmentally friendly electromembrane extraction-liquid chromatograph/mass spectrometry (EME-LC/MS) method for the analysis of 21 hydrophilic endogenous peptides in urine. Two different liquid membranes were optimized: P1, a conventional organic liquid membrane consisting of 2-nitrophenyl octyl ether (NPOE) and carvacrol (2:1, v/v) with 2% di(2-ethylhexyl) phosphate (DEHP); and P2, a deep eutectic solvent (DES)-based membrane composed of camphor and decanoic acid (1:1, w/w) with 0.5% DEHP. Mechanistic studies revealed that P1 extraction relies on cation-t[, t[-t[ stacking, and ion-pair interactions, enriching 13 peptides within 20 min at 30 V. In contrast, P2 operates via hydrogen bonding and ionic interactions, achieving more rapid extraction of 14 peptides within only 10 min at 30 V, along with a broader operational window and enhanced green solvent potential. Importantly, we established a log P-charge dual-factor model, demonstrating that peptide electromigration was co-determined by lipophilicity (log P) and net charge under operational buffer, with its predictive reliability confirmed by external validation. Implemented in a 96-well format, the EME method was high-throughput, solvent-minimized, and environmentally friendly with AGREEprep score of 0.72. Beyond providing a robust analytical tool, this work established a fundamental mechanistic framework for the design of electromembrane extraction systems, advancing both biomarker analysis and the understanding of transmembrane transfer for polar biomolecules.

Neonatal meconium is a widely accepted biological matrix for assessing prenatal exposure to drugs of abuse. Its complex and heterogeneous nature, along with typically low target analyte concentrations, possess significant analytical challenges. This study reports the first application of electromembrane extraction for processing meconium, providing a protocol for isolating amphetamines and synthetic cathinones from only 50 mg of sample. Extraction parameters, including the supported liquid membrane composition, donor solution composition and pH, voltage, and shaking intensity, were systematically optimized. Electromembrane extraction followed by UHPLC-MS/MS assay was validated, achieving low limits of quantification (2 ng·g−1), along with consistent recoveries (55–65%) and high reproducibility (RSD < 5%) for all analytes. The method was successfully applied to a real neonatal meconium sample, in which amphetamine and methamphetamine were found. Several analytical performance parameters were compared with those of the previously published LC-MS based assays. The environmental impact was assessed using the Analytical Greenness Metric for Sample Preparation, showing that electromembrane extraction substantially reduces organic solvent and plastic consumption, operator exposure and preparation time, compared with conventional methods such as solid-phase extraction and salting-out assisted liquid–liquid extraction. Overall, electromembrane extraction provides a robust, high-throughput, and sustainable strategy for neonatal toxicology screening, with potential for broader adoption in clinical and forensic laboratories.

Electromembrane extraction (EME) is a microextraction technique where charged analytes are transferred across a liquid membrane, under the influence of an electrical field, and enables selective and efficient sample cleanup. In this paper, we present for the first time a microfluidic device for electromembrane extraction where the liquid membrane was held in place by rows of micro-pillars. This design allows rapid membrane replacement, low-voltage extractions, and extended reuse, addressing key limitations of conventional EME formats. The microchip was fabricated in a stoichiometric thiol-ene polymer, offering a balance between hydrophobic and hydrophilic properties and supporting controlled flow of both aqueous and organic phases. Lidocaine was selected as the test substance for the experiments. Extraction parameters were optimized, and with a sample flow of 0.5 µL min−1, acceptor flow of 2.0 µL min−1, 0.1 µL 2-nitrophenyl octyl ether (NPOE) as the liquid membrane, and a 10-min extraction time, recoveries up to 50

Commercialization of sample preparation techniques is crucial for ensuring standard operating procedures and routine analysis capability. However, current devices for liquid-phase microextraction (LPME) are usually homemade and hardly commercialized due to the storage difficulty of manually prepared supported liquid membranes (SLMs). To address this challenge, a novel strategy for automated formation of SLMs by molecular self-assembly was proposed in this study. Specifically, the SLM solvent was directly added into the aqueous acceptor solution and self-assembled into the pores of the polymeric membrane with the assistance of a surfactant, automatically forming the SLM (in situ SLM). The in situ SLM successfully formed during the initial phase of extraction and caused no delay of the mass transfer, which could rationally solve the storage difficulty for manually prepared SLMs. Based on this strategy, a mass-producible three-dimensional printed extraction device incorporated with the in situ SLM was developed for drug analysis in urine samples. Under optimal conditions, exhaustive extraction (recovery >85%) was attained using the electrical-assisted LPME (i.e., electromembrane extraction, EME). Finally, the electromembrane extraction-liquid chromatography-tandem mass spectrometry (EME-LC-MS/MS) method based on the in situ SLM was validated using spiked urine samples, showing satisfactory limits of detection (0.87-2.97 ng mL-1), accuracy (87-105%), precision (RSD ≤ 9%), and matrix effects (92-110%). In summary, this approach could further simplify the extraction process, ensure the standard operating procedures, and enhance operational robustness, which is essential for facilitating the commercialization and routine application of SLM-based microextraction techniques.

For the first time, gel electromembrane extraction was demonstrated in a 96-well system. Gel membranes of 3% w/v agarose and with a thickness of 3.5 mm were immobilized in hydrophilic polyvinylidene fluoride (PVDF) filters in a 96-well filter plate. The filters provided mechanical support for the gel membranes, and were important for the stability and robustness of the system. A selection of 90 basic pharmaceuticals in the polarity range -4.2 < log P < 8.1 was used as model analytes (compounds). The compounds were extracted from 200 μL sample (water or human plasma) adjusted to pH 4.0 with dilute formic acid, through the gel membrane, and into 200 μL of 100 mM formic acid as acceptor. The extraction potential was 25 V, and the extraction time was 20 min for careful operation to limit Joule heating and electroendosmosis. The majority of the compounds in the polarity range -4.0 < log P < 3.0 were extracted with high recovery (40-100%). Compounds with log P > 3.0 were discriminated due to interactions with the gel membrane. Proteins and phospholipids were not extracted, and the system therefore provided efficient cleanup from human plasma samples. The 96-well agarose-gel electromembrane extraction (EME) system showed great potential. Selectivity was controlled by interactions with the aqueous gel membrane. This is fundamentally very different from traditional EME with oil membranes, where selectivity is controlled by electro-assisted partition in and out of the oil membrane. 96-Well electromembrane extraction with gel membranes of agarose is favorable in terms of greenness and efficiency for polar analytes, as compared with systems based on oil membranes.

Electromembrane extraction (EME) is a membrane-based miniaturized microextraction technique used to extract ionized analytes from complex mixtures. EME extracts can be analyzed using all major analytical instrumental techniques. The major advantages of EME include short extraction time, low consumption of organic solvents and chemicals, high extraction capability, high selectivity, and efficient sample cleanup. Numerous modifications to EME, such as the use of microfluidic devices, green solvents, biobased renewable membranes, and hyphenation with other separation techniques, have increased the selectivity and sensitivity of EME. Furthermore, nanomaterials have been used to improve the efficiency, selectivity, and stability of EME systems. Various nanomaterials have been proposed for the modification of EME-based separation systems. The larger surface area, high porosity, and various interactions with the target analytes are the most important properties of nanomaterials and nanocomposites in improving the figures of merit of EME. Nanomaterials have mainly been used to modify the chemical composition of the liquid membrane in EME, but modifications of the polymeric support membrane and the electrodes have also been reported. Therefore, this review highlights the transformative role of nanomaterials in EME, focusing on their application in enhancing extraction efficiency, selectivity, and stability. Key advancements include modifying supported liquid membranes (SLMs), membrane decoration, and optimizing electrode performance. The review also critically examines challenges, such as pore blockage and electrolysis-induced instability, offering insights into future directions for nanomaterial-enhanced EME. Despite of the numerous benefits of nanomaterials, their environmental toxicity cannot be overlooked and should be carefully examined for each new case. A bio-based and biopolymer-based nanomaterials in future EME studies can significantly address these issues while remaining aligned with green chemistry principles. Artificial intelligence-based models should be applied to predict effective nanomaterials in EME, thus significantly reduce chemical costs and consumption while also increasing the greenness level of developed EME approaches. Finally, long-term stability of new developed solutions should be an obligatory part of each new research in this field.

Volumetric absorptive microsampling (VAMS) enables accurate collection of low blood volumes, independent of hematocrit. Electromembrane extraction (EME) is a sustainable sample clean-up technique; however, its wider applicability to extract analytes directly from VAMS tips remains unexplored. This study aimed to evaluate applicability of the first commercially available conductive vial EME device (with 2-nitrophenyl octyl ether as liquid membrane) for isolating 41 basic pharmaceuticals (log P 2-6) from 10 μL of blood on VAMS tips. The following extraction parameters were optimized: donor solution composition and volume, conductive vials size, applied voltage, extraction time and agitation speed. It was found that: 1/large conductive vials (600 μL) and 300 μL of donor solution provide higher process efficiency and reproducibility compared to smaller vials (200 μL) or larger donor solution volumes; 2/methanol in donor solution improve reproducibility and 3/sonication of VAMS tips in donor solution within a conductive vial prior to extraction enhances process efficiency. The EME protocol, followed by UHPLC-MS/MS analysis, was evaluated for process efficiency, linearity (1-1000 ng/mL), precision, and accuracy. Eleven analytes met most of the predefined acceptance criteria: process efficiencies 34.9-65.8 %, linearity (R2) 0.9933-0.9995, accuracy 85.9-111.1 % and precision 1.4-13.3 % RSD. The extraction was not impacted by hematocrit variation. EME demonstrated superior reproducibility and reduced matrix effects when compared to conventional VAMS tips treatment. This study confirms the reliability of a commercial conductive vial EME device for isolating basic pharmaceuticals from whole blood on VAMS tips, highlighting its potential for routine bioanalytical applications.

BACKGROUND:Three-phase liquid membrane extraction (LME) of acids involves mass transfer from an acidified sample, through an organic liquid membrane into an alkaline aqueous acceptor. However, this approach presents challenges for acids with pKa > 9-10, as their efficient extraction often requires extreme pH conditions in the acceptor, which can compromise chemical stability and compatibility with chromatographic analysis. Alternatively, a polar organic solvent can be used as acceptor, but this may challenge the stability of the liquid membrane and the integrity of the extraction system. RESULTS:In this work, commercial 96-well plates were used for the extraction of nine weakly acidic model analytes (phenols and bisphenols). With an alkaline acceptor, the presence of a boundary layer between the liquid membrane and the acceptor hindered the extraction of the analytes, requiring the use of pH 13.0. To overcome this, an alternative system was developed with acceptor based on dimethyl sulfoxide diluted with pure water. In both systems, different liquid membranes were evaluated, and extraction kinetics were studied. Both systems were applied to human plasma and provided exhaustive extraction of the analytes. Under the final conditions, the DMSO-water acceptor system was evaluated obtaining satisfactory analytical parameters in terms of linearity (r2 > 0.990) and precision (RSD ≤15 %). SIGNIFICANCE:This new approach enhances the applicability of the three-phase aqueous-organic-organic system, making it milder towards the liquid membrane. It enables the extraction of the analytes as neutral species, broadening the scope of extractable compounds in the three-phase system. Additionally, it offers an alternative to the well-established ionization mode.

Effective monitoring of veterinary drug residues in food is essential for legislation compliance and food safety, yet remains challenging due to low concentrations and complex matrices. This study introduced a miniaturized 96-well electromembrane extraction (EME) technique for pre-concentration and isolation 80 prohibited/ restricted veterinary drugs from honey samples. Three liquid membranes were developed and characterized: V1 ("V" for veterinary), a mixture of 2-undecanone and 0.5 % 2-nitro-p-cymene, was optimal for extraction of quinolones, tetracyclines, and (1-agonists. V2, a mixture of 2-nitrophenyl octyl ether and a deep eutectic solution (formed upon mixing 6-methylcoumarin with thymol in a 1:1 w/w ratio) in a 1:1 v/v ratio, with the addition of 0.5 % di(2-ethylhexyl) phosphate (DEHP), was suitable for macrolides and sulfonamides extraction. V3, a deep eutectic solvent formed upon 1-decanoic acid and camphor in a 1:1 w/w ratio with 1 % DEHP, was suitable for aminoglycosides and (1-lactams. Operational parameters were fine-tuned to achieve optimal extraction efficiencies, with recoveries ranging from 20.1-117.5 %, 20.3-91.9 %, and 20.1-118.8 % for V1, V2, and V3, respectively. High precision was observed for compounds with recoveries over 40 % (intra-day: 0.4-27.7 %; inter-day: 2.6-33.0 %), and matrix effects were generally within 80-120 %. This work established a theoretical framework for EME in food analysis, highlighting its potential as a powerful sample pretreatment method for the detection of veterinary drug residues. The results demonstrated the feasibility and advantages of using EME for ensuring food safety and regulatory compliance.

This work evaluates the greenness of 174 standard methods with a sample preparation step and their 332 sub-method variations from CEN, ISO, and Pharmacopoeias, used in laboratories for environmental/organic, food, trace element, or pharmaceutical analyses. The widely adopted AGREEprep metric was applied to assess the greenness of the standard methods. The discussion begins with the overall scores of each method, followed by a detailed analysis of individual criteria, that highlights the strengths and weaknesses of the evaluated sample preparation methods in terms of greenness.The results revealed a generally poor greenness performance, as 67 % of the methods scored below 0.2 on the AGREEprep scale, where 1 represents the highest possible score. Specifically, the percentage of methods scoring below 0.2 was 86 % for methods related to the environmental analysis of organic compounds, 62 % for methods used in food analysis, 62 % for those applied to inorganic and trace metals analysis, and 45 % for methods used in pharmaceutical analysis. The findings obtained in this work reveal that many official methods still rely on resource-intensive, outdated techniques, scoring poorly on key greenness criteria. This discrepancy highlights the urgent need to update standard methods by including contemporary and mature sample preparation methods, as the traditional methodologies currently used often conflict with global sustainability efforts and increase regulatory and societal pressures. As such, this contribution serves not only as a critique of the current state of official standard methods but also as a call to action for their reform. This work was conducted within the framework of the IUPAC project ''Greenness of official standard sample preparation methods'' (2021-015-2-500).

Hair analysis can provide chronological insights into past drug use for months to years after drug administration. In comparison to analyses from other biological matrices, such as blood and urine, sample pretreatment is often tedious and not environmental friendly. In this study, we present a more environmental friendly approach to hair analysis using micropulverized hair and electromembrane extraction for the efficient extraction of 15 drugs of abuse, prescription drugs, and metabolites from hair. The optimized extraction method, involving micropulverization, demonstrated comparable yields to the standard approach of cutting and overnight incubation. A 15-min extraction method using a commercial electromembrane extraction prototype was developed and validated according to forensic guidelines, using only 10 mu l of organic solvent per sample. The final method, employing HPLC-MS-MS with a biphenyl column, exhibited good linearity, precision, and sensitivity. An AgreePrep assessment comparing the environmental impact of our method with the standard routine method, involving overnight incubation and conventional liquid-liquid extraction, was conducted. This is the first time micropulverized hair has been subjected to electromembrane extraction.

For the first time, we present targeted protein detection by tryptic digestion of human chorionic gonadotropin (hCG) followed by electromembrane extraction (EME). Operational parameters were optimized, and urine and serum samples spiked with hCG underwent tryptic digestion followed by EME of the (3T5 signature peptide. The liquid membrane comprised nitrophenyl octyl ether (NPOE), carvacrol, and di(2-ethyl hexyl) phosphate (DEHP) at ratios of 49:49:2 (w/w/w). Extractions were performed in a conductive vial format for 45 min at 5 V. Even from highly complex digested samples of serum and urine, the signature peptide (3T5 was extracted by EME and detected by LC-MS/MS. While attempts to extract intact hCG protein were unsuccessful, the extraction of the signature peptide was efficient. The extraction recovery from undigested and digested urine was 71 % (RSD = 17 %) and 116 % (RSD = 17 %), respectively. For serum, the extraction recoveries were 11 % (RSD = 23 %) for undigested samples and 110 % (RSD = 14 %) for digested samples. This study demonstrates both the potential and challenges of EME for protein analysis. Experiments regarding EME of intact proteins provided new insights into protein phase distribution. This fundamental case study underscores the potential of EME as a sample preparation technique for the targeted determination of protein biomarkers and drugs.

Sample preparation plays a pivotal role in chemical analysis, serving to isolate target analytes from diverse matrices and enhance measurement selectivity and sensitivity. This review examines microextraction's eco-friendliness versus tradition, focusing on LPME and EME systems for acidic compounds. Different solid supports (polymeric and gel membranes) implemented in different configurations are evaluated. These innovative techniques reduce the consumption of chemicals and offer enhanced environmental safety. To determine the greenness of these techniques, we employ three widely recognized metrics: Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), and Analytical GREEnness (AGREE). Our comparative analysis provides insights into the strengths and weaknesses of these metrics and offers a holistic perspective on the greenness of microextraction techniques. This review contributes to the ongoing efforts in Green Analytical Chemistry by facilitating the selection of environmentally benign sample preparation methods, thus promoting sustainable laboratory practices, and minimizing adverse environmental impacts.

Electromembrane extraction (EME) is a miniaturized technique in which charged molecules are extracted from biological samples, through a porous membrane, into an acceptor solution. This process occurs in the presence of an electric field that enables increased analyte recovery in short extraction times. Studies are being carried out to establish recommended extraction conditions for different groups of compounds based on their physicochemical properties. In this work, pre-established generic liquid membranes for the extraction of basic compounds were tested using synthetic cathinones within a wide log P range as basic model analytes. First, recommended organic solvents for the chosen analytes were evaluated in terms of recovery. The best candidates were included in an optimization study with other relevant EME conditions (voltage, extraction time, and agitation rate). All analyses were performed using a UPLC-MS/MS with the multiple reaction monitoring mode. 2-Undecanone was chosen as the liquid membrane providing the best extraction efficiency in combination with 40 V, 35 min, and 725 rpm. Validation showed r(2) > 0.99 in the 1-500 ng/mL range, accuracy of +/- 12%, imprecision of <= 19%, 1 ng/mL as limit of quantitation, 0.1-0.5 ng/mL as limit of detection, matrix effect 97-118%, and recovery 63-110%. Among the generic liquid membranes studied in this work, 2-Undecanone was the best choice for the target basic analytes. High recoveries and low matrix effects were achieved, thus demonstrating the applicability of recommended generic liquid membranes in the 96-well plate setup and with whole blood samples. Our findings contribute to further establishing EME as an actual sample preparation technique for routine analyses that could be further explored in a commercial format.

Electromembrane extraction (EME) is a miniaturized sample preparation technique used to preconcentrate various analytes from complex matrices. Several modifications to EME and hyphenation with other preconcentration techniques have been introduced to improve its efficiency, sensitivity, accuracy, automation, sample clean-up capacity, and environmental friendliness. This work summarizes up-to-date approaches that have been introduced for the modification of EME and EME-hyphenated techniques (EME combined with other microextraction techniques), as well as discusses the need and significance of modification in EME and EME-hyphenation and the purpose of the presented approaches. In addition, the experimental work of modified-EME and EME-hyphenated techniques are explained in detail for the solid and liquid food analysis. The review will assist them with various modified and hyphenated-EME sample preparation approaches and the applications of green solvents, nanomaterials, Lab-on-a-chip technology (LOC), and biodegradable materials to their respective fields of study. It also forecasts the vision for further enhancing the automation, environment friendliness and hyphenation of EME.

BACKGROUND:Microsampling of biological fluids followed by innovative sample pre-treatment reflects trends in bioanalytical chemistry. Volumetric absorptive microsampling (VAMS) enables exact whole blood volume collection and reduces the impact of hematocrit on the assay. In animal studies, it complies with the 3R principles (refine, reduce, replace). It allows for a gentle bleeding technique and a reduction in the number of laboratory animals by enabling ethically acceptable repeated blood collection from a single animal. Treating VAMS tips with electromembrane extraction (EME) in 96-well format offers a smart combination of non-invasive, low-volume blood collection with effective, environmentally friendly sample clean-up. RESULTS:This study introduces the first application of EME in 96-well format for direct isolation of analytes from 10 μL of whole blood collected onto a VAMS device. Doxorubicin, a clinically used anticancer drug also utilized in cancer/cardio-oncology research involving rodents, where microsampling offers important advantages, and its metabolite doxorubicinol, were selected as relevant analytes. The optimized EME yielded reproducible recoveries for both analytes regardless of hematocrit levels, different anticoagulants, or free multivalent ions in the sample. Compared to conventional VAMS tips treatment, EME reduced matrix effects, increased throughput, and an environmental friendliness of the extraction. The EME followed by the UHPLC-MS/MS assay was validated for both analytes in whole blood absorbed onto VAMS tips. The same protocol was implemented to treat plasma to determine the blood-to-plasma ratio of the analytes in the same experiments. The practical utility was demonstrated by analyzing real samples collected from the doxorubicin-treated nude mice. SIGNIFICANCE:The study offers a novel assay combining whole blood microsampling and sample clean-up in microextraction scale for preclinical pharmacokinetic studies with doxorubicin in rodents and for pharmacokinetic/pharmacodynamic modeling. This advancement in bioanalytical chemistry promotes scalable environmentally friendly procedures compatible with the 3R ethical principles in animal studies. Moreover, the concept of direct VAMS tips treatment with EME may also be easily translatable to clinical settings.

The suggested work brings a novel knowledge of an electric-induced mass transfer occurring during the gel electro-membrane extraction (G-EME) followed by HPLC UV detection. The natural deep eutectic solvent(s) (DESs), such as choline chloride mixed with itaconic acid (mole ratio 1:1) and choline chloride mixed with methacrylic acid (mole ratio 1:1), were used as the green additives in the agarose gel membrane. The impact of DESs was tested for the extraction of codeine, dasatinib, imatinib, morphine, and nilotinib from human plasma and urine samples. The DESs were incorporated into silica nanoparticles (SiNPs) through porous polymerization to form a SiNPs@P(DES), subsequently dispersed in the gel structure. As a result, the agarose membrane was stabilized, extraction efficiency increased, and the EEO flow diminished. In addition, testing of nonporous polymerization versus porous polymerization showed that extraction efficiencies are higher using the latter approach due to the higher surface area of the porous SiNPs@P(DES). The optimal extraction conditions were found to be 3.0 % w/v agarose gel (pH 3.5) containing 0.02 % w/v SiNPs@P(DES), applied voltage at 60 V, extraction time at 10 min, pH of the donor phase at 6.0, and pH of the acceptor phase at 4.0. The obtained extraction recoveries were in the range of 88.1 - 92.9 %. The limits of detection (LODs) and quantification (LOQs) were 1.6 - 14.5 ng mL -1 and 5.3 - 47.8 ng mL -1 , respectively. The intra- and inter-day repeatability (n = 4) were within 3.3 % and 6.4 % RSD, respectively.

Background: Parallel artificial liquid membrane extraction (PALME) is a 96-well plate setup variant of liquidphase 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). Results: Fourteen 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 mu L, 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. Significance: In 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 , efficient tool for toxicological appli- cations that is in agreement with the principles of sustainable chemistry.