Water as a green solvent for sustainable sample preparation: single drop microextraction of N-nitrosamines from losartan tablets.

Water, renowned for its sustainability and minimal toxicity, is an ideal candidate for environmentally friendly solvent-based microextraction. However, its potential as an extractant solvent in miniaturized sample preparation remains largely unexplored. This paper pioneers using water as the extraction solvent in headspace single-drop microextraction (HS-SDME) for N-nitrosamines from losartan tablets. Autonomous HS-SDME is executed by an Arduino-controlled, lab-made Cartesian robot, using water for the online preconcentration of enriched extracts through direct injection into a column-switching system. Critical experimental parameters influencing HS-SDME performance are systematically explored through univariate and multivariate experiments. While most previously reported methods for determining N-nitrosamines in pharmaceutical formulations rely on highly selective mass spectrometry detection techniques to handle the strong matrix effects typical of pharmaceutical samples, the water-based HS-SDME method efficiently eliminates the interfering effects of a large amount of the pharmaceutical active ingredient and tablet excipients, allowing straightforward analysis using high-performance liquid chromatography with ultraviolet detection (HPLC-UV-Vis). Under optimized conditions, the developed method exhibits linear responses from 100 to 2400 ng g-1, demonstrating appropriate detectability, precision, and accuracy for the proposed application. Additionally, the environmental sustainability of the method is assessed using the AGREEprep methodology, positioning it as an outstanding green alternative for determining hazardous contaminants in pharmaceutical products.

Analysis of human biological samples using porous graphitic carbon columns and liquid chromatography-mass spectrometry: a review.

Liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful analytical technique for analyzing complex biological samples. Among various chromatographic stationary phases, porous graphitic carbon (PGC) columns have attracted significant attention due to their unique properties-such as the ability to separate both polar and non-polar compounds and their stability through all pH ranges and to high temperatures-besides the compatibility with LC-MS. This review discusses the applicability of PGC for SPE and separation in LC-MS-based analyses of human biological samples, highlighting the diverse applications of PGC-LC-MS in analyzing endogenous metabolites, pharmaceuticals, and biomarkers, such as glycans, proteins, oligosaccharides, sugar phosphates, and nucleotides. Additionally, the fundamental principles underlying PGC column chemistry and its advantages, challenges, and advances in method development are explored. This comprehensive review aims to provide researchers and practitioners with a valuable resource for understanding the capabilities and limitations of PGC columns in LC-MS-based analysis of human biological samples, thereby facilitating advancements in analytical methodologies and biomedical research.

Advances and Applications of Hybrid Graphene-Based Materials as Sorbents for Solid Phase Microextraction Techniques.

The advancement of traditional sample preparation techniques has brought about miniaturization systems designed to scale down conventional methods and advocate for environmentally friendly analytical approaches. Although often referred to as green analytical strategies, the effectiveness of these methods is intricately linked to the properties of the sorbent utilized. Moreover, to fully embrace implementing these methods, it is crucial to innovate and develop new sorbent or solid phases that enhance the adaptability of miniaturized techniques across various matrices and analytes. Graphene-based materials exhibit remarkable versatility and modification potential, making them ideal sorbents for miniaturized strategies due to their high surface area and functional groups. Their notable adsorption capability and alignment with green synthesis approaches, such as bio-based graphene materials, enable the use of less sorbent and the creation of biodegradable materials, enhancing their eco-friendly aspects towards green analytical practices. Therefore, this study provides an overview of different types of hybrid graphene-based materials as well as their applications in crucial miniaturized techniques, focusing on offline methodologies such as stir bar sorptive extraction (SBSE), microextraction by packed sorbent (MEPS), pipette-tip solid-phase extraction (PT-SPE), disposable pipette extraction (DPX), dispersive micro-solid-phase extraction (d-µ-SPE), and magnetic solid-phase extraction (MSPE).

Automated microextraction by packed sorbent of endocrine disruptors in wastewater using a high-throughput robotic platform followed by liquid chromatography-tandem mass spectrometry.

An automated microextraction by packed sorbent followed by liquid chromatography-tandem mass spectrometry (MEPS-LC-MS/MS) method was developed for the determination of four endocrine disruptors-parabens, benzophenones, and synthetic phenolic antioxidants-in wastewater samples. The method utilizes a lab-made repackable MEPS device and a multi-syringe robotic platform that provides flexibility to test small quantities (2 mg) of multiple extraction phases and enables high-throughput capabilities for efficient method development. The overall performance of the MEPS procedure, including the investigation of influencing variables and the optimization of operational parameters for the robotic platform, was comprehensively studied through univariate and multivariate experiments. Under optimized conditions, the target analytes were effectively extracted from a small sample volume of 1.5 mL, with competitive detectability and analytical confidence. The limits of detection ranged from 0.15 to 0.30 ng L-1, and the intra-day and inter-day relative standard deviations were between 3 and 21%. The method's applicability was successfully demonstrated by determining methylparaben, propylparaben, butylated hydroxyanisole, and oxybenzone in wastewater samples collected from the São Carlos (SP, Brazil) river. Overall, the developed method proved to be a fast, sensitive, reliable, and environmentally friendly analytical tool for water quality monitoring.

Microextraction by packed sorbent of N-nitrosamines from Losartan tablets using a high-throughput robot platform followed by liquid chromatography-tandem mass spectrometry.

The development of a fast, cost-effective, and efficient microextraction by packed sorbent setup was achieved by combining affordable laboratory-repackable devices of microextraction with a high-throughput cartesian robot. This setup was evaluated for the development of an analytical method to determine N-nitrosamines in losartan tablets. N-nitrosamines pose a significant concern in the pharmaceutical market due to their carcinogenic risk, necessitating their control and quantification in pharmaceutical products. The parameters influencing the performance of this sample preparation for N-nitrosamines were investigated through both univariate and multivariate experiments. Microextractions were performed using just 5.0 mg of carboxylic acid-modified polystyrene divinylbenzene copolymer as the extraction phase. Under the optimized conditions, the automated setup enabled the simultaneous treatment of six samples in less than 20 min, providing reliable analytical confidence for the proposed application. The analytical performance of the automated high-throughput microextraction by the packed sorbent method was evaluated using a matrix-matching calibration. Quantification was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry with chemical ionization at atmospheric pressure. The method exhibited limits of detection as low as 50 ng/g, good linearity, and satisfactory intra-day (1.38-18.76) and inter-day (2.66-20.08) precision. Additionally, the method showed accuracy ranging from 80% to 136% for these impurities in pharmaceutical formulations.

Open tubular liquid chromatography: Recent advances and future trends.

Nano-liquid chromatography (nanoLC) is gaining significant attention as a primary analytical technique across various scientific domains. Unlike conventional high-performance LC, nanoLC utilizes columns with inner diameters (i.ds.) usually ranging from 10 to 150 µm and operates at mobile phase flow rates between 10 and 1000 nl/min, offering improved chromatographic performance and detectability. Currently, most exploration of nanoLC has focused on particle-packed columns. Although open tubular LC (OTLC) can provide superior performance, optimized OTLC columns require very narrow i.ds. (< 10 µm) and demand challenging instrumentation. At the moment, these challenges have limited the success of OTLC. Nevertheless, remarkable progress has been made in developing and utilizing OTLC systems featuring narrow columns (< 2 µm). Additionally, significant efforts have been made to explore larger columns (10-75 µm i.d), demonstrating practical applicability in many situations. Due to their perceived advantages, interest in OTLC has resurged in the last two decades. This review provides an updated outlook on the latest developments in OTLC, focusing on instrumental challenges, achievements, and advancements in column technology. Moreover, it outlines selected applications that illustrate the potential of OTLC for performing targeted and untargeted studies.

NanoLC-EI-MS: Perspectives in Biochemical Analysis.

Although LC-MS with atmospheric pressure ionization (API) sources is the primary technique used in modern bioanalytical studies, electron ionization mass spectrometry (EI-MS) can provide some substantial advantages over it. EI-MS is a matrix effect-free technique that provides reproducible and comparable mass spectra, serving as a compound fingerprint for easy identification through automated comparison with spectral libraries. Leveraging EI-MS in biochemical studies can yield critical analytical benefits for targeted and untargeted analyses. However, to fully utilize EI-MS for heavy and non-volatile molecules, a new technology that enables the coupling of liquid chromatography with EI-MS is needed. Recent advancements in nanoLC have addressed the compatibility issues between LC and EI-MS, and innovative interfacing strategies such as Direct-EI, liquid electron ionization (LEI), and Cold-EI have extended the application of EI-MS beyond the determination of volatile organic molecules. This review provides an overview of the latest developments in nanoLC-EI-MS interfacing technologies, discussing their scope and limitations. Additionally, selected examples of nanoLC-EI-MS applications in the field of biochemical analysis are presented, highlighting the potential prospects and benefits that the establishment of this technique can bring to this field.

Applicability and Limitations of a Capillary-LC Column-Switching System Using Hybrid Graphene-Based Stationary Phases.

Graphene oxide sheets fixed over silica particles (SiGO) and their modification functionalized with C18 and endcapped (SiGO-C18ec) have been reported as sorbents for extraction and analytical columns in LC. In this study, a SiGO column was selected as the extraction column and a SiGO-C18ec as the analytical column to study the applicability and limitations of a column-switching system composed exclusively of columns packed with graphene-based sorbents. Pyriproxyfen and abamectin B1a were selected as the analytes, and orange-flavored carbonated soft drinks as the matrix. The proposed system could be successfully applied to the pyriproxyfen analysis in a concentration range between 0.5 to 25 µg/mL presenting a linearity of R2 = 0.9931 and an intra-day and inter-day accuracy of 82.2-111.4% (RSD < 13.3%) and 95.5-99.8% (RSD < 12.7%), respectively. Furthermore, the matrix composition affected the area observed for the pyriproxyfen: the higher the concentration of orange juice in the soft drink, the higher the pyriproxyfen the signal observed. Additionally, the SiGO extraction column presented a life use of 120 injections for this matrix. In contrast, the proposed system could not apply to the analysis of abamectin B1a, and the SiGO-C18ec analytical column presented significant tailing compared to a similar approach with a C18 analytical column.

Packed in-tube SPME-LC-MS/MS for fast and straightforward analysis of cannabinoids and metabolites in human urine.

Cannabinoids are pharmacologically active compounds present in cannabis plants, which have become important research topics in the modern toxicological and medical research fields. Not only is cannabis the most used drug globally, but also cannabinoids have a growing use to treat a series of diseases. Therefore, new, fast, and efficient analytical methods for analyzing these substances in different matrices are demanded. This study developed a new packed-in-tube solid-phase microextraction (IT-SPME) method coupled to liquid chromatography with tandem mass spectrometry (LC-MS/MS), for the automated microextraction of seven cannabinoids from human urine. Packed IT-SPME microcolumns were prepared in (508 µm i.d. × 50 mm) stainless-steel hardware; each one required only 12 mg of sorbent phase. Different sorbents were evaluated; fractional factorial design 24-1 and a central composite design were employed for microextraction optimization. Under optimized conditions, the developed method was a fast and straightforward approach. Only 250 µl of urine sample was needed, and no hydrolysis was required. The sample pretreatment included only dilution and centrifugation steps (8 min), whereas the complete IT-SPME-LC-MS/MS method took another 12 min, with a sample throughput of 3 samples h-1 . The developed method presented adequate precision, accuracy and linearity; R2 values ranged from 0.990 to 0.997, in the range of 10-1000 ng ml-1 . The lower limits of quantification varied from 10 to 25 ng ml-1 . Finally, the method was successfully applied to analyze 20 actual urine samples, and the IT-SPME microcolumn was reused over 150 times.

Electron ionization mass spectrometry: Quo vadis?

Mass spectrometry (MS) is a fundamental technique to identify compounds by their mass-to-charge ratio. It is known that MS can only detect target compounds when they are converted to ions in the gas phase. The ionization procedure is considered one of the most critical steps, and there are distinct techniques for it. One of them is electron ionization (EI), a widely used hard-ionization technique capable of generating several ions due to the excess energy employed. The existence of distinct ionization mechanisms turns EI capable of producing a fingerprint-like spectrum for each molecule. So, it is an essential technique for obtaining structural information. EI is often combined with chromatography to obtain a practical introduction of pretreated samples despite its excellent performance. EI-MS has been applied coupled with gas chromatography (GC) since the 1960s as both are very compatible. Currently, analytes of interest are more suitable for liquid chromatography (LC) analysis, so there are researchers dedicated to developing suitable interfaces for coupling LC and EI-MS. EI excels, as a reliable technique to fill the gap between GC and LC, possibly allowing them to coexist in a single instrument. In this work, the authors will present the fundamentals of EI-MS, emphasizing the development over the years, coupling with gas and LC, and future trends.

Determination of parabens in wastewater samples via robot-assisted dynamic single-drop microextraction and liquid chromatography-tandem mass spectrometry.

Dynamic single-drop microextraction (SDME) was automatized employing an Arduino-based lab-made Cartesian robot and implemented to determine parabens in wastewater samples in combination with liquid chromatography-tandem mass spectrometry. A dedicated Arduino sketch controls the auto-performance of all the stages of the SDME process, including syringe filling, drop exposition, solvent recycling, and extract collection. Univariate and multivariate experiments investigated the main variables affecting the SDME performance, including robot-dependent and additional operational parameters. Under selected conditions, limit of detections were established at 0.3 µg/L for all the analytes, and the method provided linear responses in the range between 0.6 and 10 µg/L, with adequate reproducibility, measured as intraday relative standard deviations (RSDs) between 5.54% and 17.94%, (n = 6), and inter-days RSDs between 8.97% and 16.49% (n = 9). The robot-assisted technique eased the control of dynamic SDME, making the process more feasible, robust, and reliable so that the developed setup demonstrated to be a competitive strategy for the automated extraction of organic pollutants from water samples.

Microextraction by packed sorbent of selected pesticides in coffee samples employing ionic liquids supported on graphene nanosheets as extraction phase.

This paper describes the synthesis, characterization, and use of ionic liquids supported on silica, functionalized with graphene oxide through covalent bonding (ILz/Si@GO), as sorbents for microextraction by packed sorbent (MEPS). Seven selected pesticides (diazinon, heptachlor, aldrin, endrin, dieldrin, endosulfan, and methoxychlor), used for the prevention of pests in coffee crops, and endosulfan sulfate-an endosulfan metabolite-were selected for this study as model compounds for evaluating the sorbent performance of the synthesized materials in the MEPS device. The cycles of each of the stages were previously optimized through univariate experiments to carry out the extraction. The ILz/Si@GO phase was compared to other sorbents used in MEPS (GO, DVB-MMA, C4/SiO2, C8/SiO2, ILz/SiO2, and bare silica) and also with graphene functionalized through other methodologies, where ILz/Si@GO showed the best results. The material was characterized using a range of techniques. The selectivity of the sorbent material and its adsorption capacity were evaluated by gas chromatography coupled with tandem mass spectrometry. The precision and accuracy of the method showed a relative standard deviation lower than 10% and recoveries from 35 to 97%. Finally, the proposed method was employed for the determination of pesticide residues in coffee samples.

A cartridge-based device for automated analyses of solid matrices by online sample prep-capillary LC-MS/MS.

Sample preparation is an essential step focused on eliminating interfering compounds while pre-concentrating the analytes. However, its multiple steps are laborious, time-consuming, and a source of errors. Currently, automated approaches represent a promising alternative to overcome these drawbacks. Similarly, miniaturisation has been considered an ideal strategy for creating greener analytical workflows. The combination of these concepts is currently highly desired by analytical chemists. However, most automated and miniaturised sample preparation techniques are primarily concerned with liquid samples, while solids are frequently overlooked. We present an approach based on a cartridge packed with solids (soil samples) coupled with a capillary LC-MS, combining sample preparation and analytical steps into a unique platform. As a proof-of-concept, nine pesticides used in sugarcane crops were extracted and analysed by our proposed method. For optimisation, a fractional factorial design (25-1) was performed with the following variables: aqueous dilution of the sample (V1), extraction strength (V2), matrix washing time (V3), extraction flow (V4), and analytical flow (V5). After, the most influential ones (V1, V2, and V3) were taken into a central composite design (23) to select their best values. Under optimised conditions, the method reported linear ranges between 10 and 125 ng g-1 with R2 > 0.985. Accuracy and precision were in accordance with the values established by the International Council for Harmonisation (Q2(R1)). Therefore, the proposed approach was effective in extracting and analysing selected pesticides in soil samples. Also, we carried out initial qualitative tests for pesticides in honeybees to see if there is the possibility to apply our method in other solids.

Neonicotinoids exposure assessment in Africanized honey bees (Apis mellifera L.) by using an environmentally-friendly sample preparation technique followed by UPLC-MS/MS.

A miniaturized QuEChERS extraction method followed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed to analyze thiamethoxam and imidacloprid in 0.3 g of lyophilized Africanized honey bees (Apis mellifera L.). The work aimed to estimate honey bees' exposure to pesticides at the nanogram/gram (ng g-1) levels, using small sample and reagent quantities. Low amounts of solvents and salts were employed - 15× less than used in traditional methods. Average recoveries ranged from 64.5% to 99.7%, with repeatability below 20% for samples spiked at 3 and 167 ng g-1. LOD and LOQ were 0.7, and 3 ng g-1 for both pesticides. Applying the proposed approach, honey bee samples from different apiaries from the State of São Paulo (Brazil) were analyzed. The pesticides were detected in concentrations between 7.0 and 27.0 ng g-1. Thus, the proposed method can be used as a greener alternative to analyze the two neonicotinoids at trace levels in small quantities of bees, consequently saving chemicals and waste.

Miniaturized liquid chromatography applied to the analysis of residues and contaminants in food: A review.

The humankind is pretty dependent on food to control several biological processes into the organism. As the world population increases, the demand for foodstuffs follows the same trend claiming for a high food production situation. For this reason, a substantial amount of chemicals is used in agriculture and livestock husbandries every year, enhancing the likelihood of contaminated foodstuffs being commercialized. This outlook becomes a public health concern; thus, the governmental regulatory agencies impose laws to control the residues and contaminants in food matrices. Currently, one of the most important analytical techniques to perform it is LC. Despite its already recognized effectiveness, it is often time consuming and requires significant volumes of reagents, which are transformed into toxic waste. In this context, miniaturized LC modes emerge as a greener and more effective analytical technique. They have remarkable advantages, including higher sensitivity, lower sample amount, solvent and stationary phase requirements, and more natural coupling to MS. In this review, most of the critical characteristics of them are discussed, focusing on the benchtop instruments and their related analytical columns. Additionally, a discussion regarding the last 10 years of publications reporting miniaturized LC application for the analysis of natural and industrial food samples is categorized. The main chemical classes as applied in the crops are highlighted, including pesticides, veterinary drugs, and mycotoxins.

Multidimensional capillary liquid chromatography-tandem mass spectrometry for the determination of multiclass pesticides in "sugarcane spirits" (cachaças).

Cachaça or "sugarcane spirit" is a Brazilian beverage considered the third most consumed beverage worldwide. Sugarcane, its raw material, is one of the main crops developed in the country, placing Brazil as the largest producer of this commodity on a global scale. Considering the growth in sugarcane production, many farmers use pesticides in their crops. However, excess pesticides can be accumulated in products derived from sugarcane, creating an environmental and public health concern. In this context, the development of analytical methods capable of identifying residues of pesticides in cachaças and other sugarcane-derived products is essential to ensure the beverage's quality. This work presents a method to quantify multiclass pesticides in Brazilian sugarcane spirits (cachaças) through an automated multidimensional system. The first dimension consists of an extraction column packed with a graphene-silica phase, followed by a capillary liquid chromatography-tandem mass spectrometry system as the second dimension. The method was optimized by an experimental design, in which the influence of three variables was evaluated on the extraction process: percentage of acetonitrile, loading flow, and loading time. Afterward, twenty-two cachaças were analyzed to ascertain the applicability of the proposed method. The analyses reported five samples containing clomazone (a type of herbicide widely used in sugarcane production). The method showed good linearity under optimized conditions, with correlation coefficients greater than 0.981, and limits of detection and quantification of 5 µg L-1 and 10 µg L-1, respectively. The herein discussed results suggest that the proposed method could be a practical option for identifying pesticides in beverages. Graphical Abstract.

ß-Cyclodextrin coupled to graphene oxide supported on aminopropyl silica as a sorbent material for determination of isoflavones.

ß-Cyclodextrin, coupled to graphene oxide supported on aminopropyl silica, was synthesized and characterized. This material was combined with microextraction by packed sorbent to act as the sample preparation step. The analytical method optimization was carried out by employing experimental design and had its figures of merit determined. The resulting linearity ranged from 1.0 to 200 µg/L for daidzein, from 2.0 to 200 µg/L for genistein, from 3.0 to 200 µg/L for formononetin, and from 2.0 to 200 µg/L for biochanin A with all R2 values above 0.993 and limit of quantification ranging from 0.5 to 1.5 µg/L. The accuracy ranged from 93.3 to 123.3%, and intraday and interday precision reported by the relative standard deviations were <16%. This work aimed to synthesize and evaluate cyclodextrins coupled to graphene-based sorbents to be used as a high sorption capacity and selective sorbent for sample preparation of complex matrices using microextraction techniques. The synthesized material kept the high absorption characteristic of graphene-based materials while maintaining the cyclodextrins' selectivity to extract the target analyte. Four isoflavones were determined in soy-based juice samples from the local market, confirming the excellent performance of the proposed method.

Evaluation of Two Fully Automated Setups for Mycotoxin Analysis Based on Online Extraction-Liquid Chromatography-Tandem Mass Spectrometry.

Mycotoxins are secondary metabolites of fungi species widely known for their potentially toxic effects on human health. Considering their frequent presence in crops and their processed food, monitoring them on food-based matrices is now an important topic. Within such a context, the sample preparation step is usually mandatory before the chromatographic analysis, due to the complexity of matrices such as nuts, cereals, beverages, and others. For these reasons, we herein present the evaluation of two greener setups, based on the automation and miniaturization of the sample preparation step for mycotoxin analysis in different beverages. Firstly, we describe an analytical method based on a multidimensional assembly, coupling a lab-made microextraction column (508 µm i.d. × 100 mm) to a UPLC-MS/MS for the analysis of ochratoxin A in beverages. This configuration used a synthesized sorbent phase containing C18-functionalized graphene-silica particles, which exhibited excellent extraction performance, as well as being reusable and cheaper than commercially available extractive phases. Sequentially, a second setup, based on a multidimensional capillary LC coupled to MS/MS, was assessed for the same purpose. In this case, a graphene oxide-based capillary extraction column (254 µm i.d. × 200 mm) was used as the first dimension, while a C18 analytical capillary column performed the mycotoxin separation in beverages. Although this second one has similarities with the first, we focused mainly on the benefits related to the link between a miniaturized/automated sample preparation device with a capillary LC-MS/MS system, which made our analysis greener. Additionally, the chromatographic efficiency could even be enhanced.

Multidimensional Liquid Chromatography Employing a Graphene Oxide Capillary Column as the First Dimension: Determination of Antidepressant and Antiepileptic Drugs in Urine.

Human mental disorders can be currently classified as one of the most relevant health topics. Including in this are depression and anxiety, which can affect us at any stage of life, causing economic and social problems. The treatments involve cognitive psychotherapy, and mainly the oral intake of pharmaceutical antidepressants. Therefore, the development of analytical methods for monitoring the levels of these drugs in biological fluids is critical. Considering the current demand for sensitive and automated analytical methods, the coupling between liquid chromatography and mass spectrometry, combined with suitable sample preparation, becomes a useful way to improve the analytical results even more. Herein we present an automated multidimensional method based on high-performance liquid chromatography-tandem mass spectrometry using a lab-made, graphene-based capillary extraction column connected to a C8 analytical column to determined five pharmaceutical drugs in urine. A method enhancement was performed by considering the chromatographic separation and the variables of the loading phase, loading time, loading flow, and injection volume. Under optimized conditions, the study reports good linearity with R2 > 0.98, and limits of detection in the range of 0.5-20 µg L-1. Afterward, the method was applied to the direct analysis of ten untreated urine samples, reporting traces of citalopram in one of them. The results suggest that the proposed approach could be a promising alternative that provides direct and fully automated analysis of pharmaceutical drugs in complex biological matrices.

Determination of ring-substituted amphetamines through automated online hollow fiber liquid-phase microextraction-liquid chromatography.

The present paper describes an original method for the online preconcentration and analysis of ring-substituted amphetamines in urine samples, used on the integration of robot-assisted hollow fiber liquid-phase microextraction (HF-LPME), high-performance liquid chromatography (HPLC), and fluorescence detection (FLD). A lab-made autosampler, actuating a 100-µL syringe and equipped with a three-way solenoid microvalve, allowed the acceptor phase to flow through and be withdrawn from the lumen fiber, enabling the automated online transference of the enriched acceptor phase for chromatographic analysis, through a six-port switching valve. The developed online HF-LPME-LC/FLD method demonstrated high analytical throughput and confidence, facilitating the efficient extraction and determination of the target analytes, with minimal solvent consumption and sample manipulation, in a straightforward way. Sample cleanup, analyte uptake, and analysis were carried out in 14.5 min. Under optimal conditions, automated online HF-LPME showed excellent linearity, precision, and trueness, obtaining intraday RSDs between 2.9 and 9.2% (n = 6) and interday RSDs between 5.3 and 9.3% (n = 6). Enrichment factors (EFs) ranged between 14.2 and 15.7, extraction recoveries (ERs) ranged between 17.7 and 19.5%, and the limits of detection (S/N = 3) were 2.0, 3.0, and 3.0 µg L-1 for MDA, MDMA, and MDEA, respectively. The method proved to be an effortless, rapid, reliable, and environment-friendly approach for the determination of drug abuse in urine samples. Graphical abstract.