Recent trends on the implementation of reticular materials in column-centered separations.

Advances in the development of column-based analytical separations are strongly linked to the development of novel materials. Stationary phases for chromatographic separation are usually based on silica and polymer materials. Nevertheless, recent advances have been made using porous crystalline reticular materials, such as metal-organic frameworks and covalent organic frameworks. However, the direct packing of these materials is often limited due to their small crystal size and nonspherical shape. In this review, recent strategies to incorporate porous crystalline materials as stationary phases for liquid-phase separations are covered. Moreover, we discuss the potential future directions in their development and integration into suitable supports for analytical applications. Finally, we discuss the main challenges to be solved to take full advantage of these materials as stationary phases for analytical separations.

Zeolitic imidazolate frameworks in analytical sample preparation.

Zeolitic imidazolate frameworks are a class of metal-organic frameworks that are topologically isomorphic with zeolites. Zeolitic imidazolate frameworks are composed of tetrahedrally coordinated metal ions connected by imidazolate linkers and have a high porosity and chemical stability. Here, we summarize the progress made in the application of zeolitic imidazolate frameworks in sample preparation for analytical purposes. This review is focused on analytical methods based on liquid chromatography, gas chromatography, or capillary electrophoresis, where the use of zeolitic imidazolate frameworks has contributed to increasing the sensitivity and selectivity of the method. While bulk zeolitic imidazolate frameworks have been directly used in analytical sample preparation protocols, a variety of strategies for their magnetization or their incorporation into sorbent particles, monoliths, fibers, stir bars, or thin films, have been developed. These modifications have facilitated the handling and application of zeolitic imidazolate frameworks for a number of analytical sample treatments including magnetic solid-phase extraction, solid-phase microextraction, stir bar sorptive extraction, or thin film microextraction, among other techniques.

Lab-In-Syringe with Bead Injection Coupled Online to High-Performance Liquid Chromatography as Versatile Tool for Determination of Nonsteroidal Anti-Inflammatory Drugs in Surface Waters.

We report on the hyphenation of the modern flow techniques Lab-In-Syringe and Lab-On-Valve for automated sample preparation coupled online with high-performance liquid chromatography. Adopting the bead injection concept on the Lab-On-Valve platform, the on-demand, renewable, solid-phase extraction of five nonsteroidal anti-inflammatory drugs, namely ketoprofen, naproxen, flurbiprofen, diclofenac, and ibuprofen, was carried out as a proof-of-concept. In-syringe mixing of the sample with buffer and standards allowed straightforward pre-load sample modification for the preconcentration of large sample volumes. Packing of ca. 4.4 mg microSPE columns from Oasis HLB® sorbent slurry was performed for each sample analysis using a simple microcolumn adapted to the Lab-On-Valve manifold to achieve low backpressure during loading. Eluted analytes were injected into online coupled HPLC with subsequent separation on a Symmetry C18 column in isocratic mode. The optimized method was highly reproducible, with RSD values of 3.2% to 7.6% on 20 µg L-1 level. Linearity was confirmed up to 200 µg L-1 and LOD values were between 0.06 and 1.98 µg L-1. Recovery factors between 91 and 109% were obtained in the analysis of spiked surface water samples.

3D-Printed Magnetic Stirring Cages for Semidispersive Extraction of Bisphenols from Water Using Polymer Micro- and Nanofibers.

A magnetic stirring device allowing semidispersive solid phase extraction of eight bisphenols (A, AF, AP, C, BP, G, M, and Z) from river waters using polymer nano- and microfibers followed by HPLC with spectrophotometric detection has been developed and applied. About 50 mg of fibers was placed in a round, cage-like housing consisting of two identical 3D printed pieces that were locked together by a magnetic stirring bar. Magnetic stirring action of the cage devices enabled highly efficient interaction of the fibers housed inside with the aqueous samples and analyte transfer without risking fiber compaction and/or damaging. Polypropylene was found to be the best-suited filament material for the cage 3D printing, and polycaprolactone fibers appeared the most efficient sorbent out of eight tested polymers. Experimental design revealed that analytes extraction from 100 mL aqueous samples was completed within 50 min and stripping in methanol required less than 35 min. Cage housing enabled simple and robust handling of the fibrous sorbent that could be used repeatedly up to at least 5 times. Procedural repeatability was less than 5% RSD, and limits of detection and quantitation were 0.1-2.1 and 0.4-7.0 µg L-1, respectively. Analyte recoveries at 50 µg L-1 level ranged from 87.1% to 106.5% in the analysis of two spiked river and two lake waters.

Prototyping of a Microfluidic Modulator Chip and Its Application in Heart-Cut Strong-Cation-Exchange-Reversed-Phase Liquid Chromatography Coupled to Nanoelectrospray Mass Spectrometry for Targeted Proteomics.

A novel multilayer modulator chip offering a robust miniaturized interface for multidimensional liquid chromatography has been developed. The thermoplastic microfluidic device comprises five tailor-made functional layers, and the chip is compatible with commercially available switching-valve technology. The modulator chip allows for robust ultrahigh-pressure operation up to 65 MPa. Peak-dispersion characteristics of system peaks were assessed directly at the valve outlet by monitoring fluorescein injection profiles with laser-induced fluorescence detection. Integration of a microporous monolithic mixing entity in the microchannels significantly narrows the resulting peak profile. Proof-of-concept of the applicability of the microfluidic modulator chip is demonstrated in a heart-cut multidimensional strong-cation-exchange-reversed-phase liquid chromatography proteomics analysis workflow coupled to nanoelectrospray mass spectrometry for the target analysis of Glu-1-Fibrinopeptide B spiked in a protein digest mixture of bovine serum albumin.

The Automation Technique Lab-In-Syringe: A Practical Guide.

About eight years ago, a new automation approach and flow technique called "Lab-In-Syringe" was proposed. It was derived from previous flow techniques, all based on handling reagent and sample solutions in a flow manifold. To date Lab-In-Syringe has evidently gained the interest of researchers in many countries, with new modifications, operation modes, and technical improvements still popping up. It has proven to be a versatile tool for the automation of sample preparation, particularly, liquid-phase microextraction approaches. This article aims to assist newcomers to this technique in system planning and setup by overviewing the different options for configurations, limitations, and feasible operations. This includes syringe orientation, in-syringe stirring modes, in-syringe detection, additional inlets, and addable features. The authors give also a chronological overview of technical milestones and a critical explanation on the potentials and shortcomings of this technique, calculations of characteristics, and tips and tricks on method development. Moreover, a comprehensive overview of the different operation modes of Lab-In-Syringe automated sample pretreatment is given focusing on the technical aspects and challenges of the related operations. We further deal with possibilities on how to fabricate required or useful system components, in particular by 3D printing technology, with over 20 different elements exemplarily shown. Finally, a short discussion on shortcomings and required improvements is given.

Where are modern flow techniques heading to?

This article aims to provide an overview on the transition from earlier laboratory automation using analytical flow approaches toward today's applications of flow methodologies, recent developments, and future trends. The article is directed to flow practitioners while serving as a valuable reference to newcomers in the field in providing insight into flow techniques and conceptual differences in operation across the distinct flow generations. In the focus are the recently developed and complementary techniques Lab-On-Valve and Lab-In-Syringe. In the following, a brief comparison of the different application niches and contributions of flow techniques to past and modern analytical chemistry is given, including (i) the development of sample pretreatment approaches, (ii) the potential applicability for in-situ/on-site monitoring of environmental compartments or technical processes, (iii) the ability of miniaturization of laboratory chemistry, (iv) the unique advantages for implementation of kinetic assays, and finally (v) the beneficial online coupling with scanning or separation analytical techniques. We also give a critical comparison to alternative approaches for automation based on autosamplers and robotic systems. Finally, an outlook on future applications and developments including 3D prototyping and specific needs for further improvements is given. Graphical abstract ᅟ.

Fully Automatic In-Syringe Magnetic Stirring-Assisted Dispersive Liquid-Liquid Microextraction Hyphenated to High-Temperature Torch Integrated Sample Introduction System-Inductively Coupled Plasma Spectrometer with Direct Injection of the Organic Phase.

A proof of concept study involving the online coupling of automatic dispersive liquid-liquid microextraction (DLLME) to inductively coupled plasma optical emission spectrometry (ICP OES) with direct introduction and analysis of the organic extract is herein reported for the first time. The flow-based analyzer features a lab-in-syringe (LIS) setup with an integrated stirring system, a Meinhard nebulizer in combination with a heated single-pass spray chamber, and a rotary injection valve, used as an online interface between the microextraction system and the detection instrument. Air-segmented flow was used for delivery of a fraction of the nonwater miscible extraction phase, 12 µL of xylene, to the nebulizer. All sample preparative steps including magnetic stirring assisted DLLME were carried out inside the syringe void volume as a size-adaptable yet sealed mixing and extraction chamber. Determination of trace level concentrations of cadmium, copper, lead, and silver as model analytes has been demonstrated by microextraction as diethyldithiophosphate (DDTP) complexes. The automatic LIS-DLLME method features quantitative metal extraction, even in troublesome sample matrixes, such as seawater, salt, and fruit juices, with relative recoveries within the range of 94-103%, 93-100%, and 92-99%, respectively. Furthermore, no statistically significant differences at the 0.05 significance level were found between concentration values experimentally obtained and the certified values of two serum standard reference materials.

In-syringe magnetic-stirring-assisted liquid-liquid microextraction for the spectrophotometric determination of Cr(VI) in waters.

A fully automated method for the determination of chromate is described. It is based on the selective reaction of Cr(VI) with diphenylcarbazide in acidic media to form a colored complex of Cr(III) with the oxidation product diphenylcarbazone. The reaction was performed within the syringe of an automatic burette containing a magnetic stirrer for homogenization of the sample and the required reagents. In-syringe stirring was made possible using a specially designed driving device placed around the syringe barrel to achieve a rotating magnetic field in the syringe, forcing the stirrer to spin. In a second step, the reaction mixture in the syringe was neutralized to allow in-syringe magnetic-stirring-assisted dispersive liquid-liquid microextraction of the complex into 125 µL of n-hexanol. After phase separation by droplet flotation over 30 s, the organic phase was propelled into a coupled spectrophotometric detection cell. The entire multistep procedure including in-system standard preparation was done within 270 s. The method was used for the analysis of natural waters, achieving average analyte recovery of 103%, a limit of detection of 0.26 µg L(-1), and a repeatability of less than 4% relative standard deviation.

Lab-in-syringe automated protein precipitation and salting-out homogenous liquid-liquid extraction coupled online to UHPLC-MS/MS for the determination of beta-blockers in serum.

A sample preparation method involving tandem implementation of protein precipitation and salting-out homogenous liquid-liquid extraction was developed for the determination of beta-blockers in serum. The entire procedure was automated using a computer-controlled syringe pump following the Lab-In-Syringe approach. It is based on the denaturation of serum proteins with acetonitrile followed by salt-induced phase separation upon which the proteins accumulate as a compact layer at the interphase of the solutions. The extract is then separated and diluted in-syringe before being submitted to online coupled UHPLC-MS/MS. A 1 mL glass syringe containing a small stir bar for solution mixing at up to 3000 rpm, was used to deal with sample volumes as small as 100 µL. A sample throughput of 7 h-1 was achieved by performing the chromatographic run and sample preparation procedure in parallel. Linear working ranges were obtained for all analytes between 5 and 100 ng mL-1, with LOD values ranging from 0.4 to 1.5 ng mL-1. Accuracy values in the range of 88.2-106% and high precision of <11% RSD suggest applicability for routine analysis that can be further improved using deuterated standards.

Solvent-assisted dispersive micro-solid phase extraction of bisphenols using iron(III) thenoyltrifluoroacetonate complex (Fe(TTA)3) as a new nanostructured sorbent: a proof of concept.

In this work, we describe for the first time the use of iron(III) thenoyltrifluoroacetonate complex (Fe(TTA)3) as a novel sorbent for solvent-assisted dispersive micro-solid phase extraction (SA-dµSPE) of bisphenols from water samples. The extraction procedure is based on the formation of nanoparticles in situ following the rapid injection of a methanolic solution of Fe(TTA)3 into the stirred aqueous sample. Herein, the synthesis of Fe(TTA)3 and study of the essential parameters of the preparative procedure are described. The optimized procedure allowed for efficient enrichment of bisphenols from various water samples, chosen as model contaminants and matrix, within 2.5 min. The sorbent was collected by centrifugation, dissolved in methanol, and injected to perform HPLC with spectrophotometric detection. The limits of detection and quantification obtained ranged from 1.0-3.1 and 3.1-7.5 µg L-1, respectively. Intraday and interday precisions of <7% relative standard deviation (RSD) and <8% RSD with analyte recoveries ranging between 70-117% (103.8% on average) were obtained for the analysis of river water, wastewater treatment plant effluent, and bottled water.

Fully-automated fluorimetric determination of aluminum in seawater by in-syringe dispersive liquid-liquid microextraction using lumogallion.

A sensitive and selective automated in-syringe dispersive liquid-liquid microextraction (DLLME) method is presented. It was successfully applied to the determination of aluminum in coastal seawater samples. The complete analytical procedure including sampling, buffering, reaction of the analyte with fluorescence reagent lumogallion (LMG), extraction, phase separation, and quantification was completely automized and carried out within 4 min. DLLME was done using n-hexanol as an extracting solvent and ethanol as a dispersing solvent in a 1:8 v/v percent mixture. The Al-LMG complex was extracted by an organic solvent and separated from the aqueous phase within the syringe of an automated syringe pump. Two devices were specially developed for this work. These were (a) the fluorescence detector and accompanying flow cell for the organic phase enriched with the reaction product and (b) a heating device integrated into the holding coil to accelerate the slow reaction kinetics. The limits of detection (3σ) and quantification (10σ) were 8.0 ± 0.5 nmol L(-1) and 26.7 ± 1.6 nmol L(-1), respectively. The relative standard deviation for eight replicate determinations of 200 nmol L(-1) Al(3+) was <1.5%. The calibration graph using the preconcentration system was linear up to 1000 nmol L(-1) with a correlation coefficient of 0.999. Ambient concentrations of samples were quantifiable with found concentrations ranging from 43 to 142 nmol L(-1). Standard additions gave analyte recoveries from 97% to 113% proving the general applicability and adequateness of the analyzer system to real sample analysis.

Lab in a syringe: fully automated dispersive liquid-liquid microextraction with integrated spectrophotometric detection.

A new approach for the integration of various analytical steps inside a syringe (Lab in a Syringe) is presented. Fully automated dispersive liquid-liquid microextraction with integrated spectrophotometric detection is carried out in-syringe using a very simple instrumental setup. The lighter-than-water organic droplets released in the extraction step accumulate at the head of the syringe, where two optical fibers are placed on both sides of the syringe, facing each other and enabling the in situ quantification of the extracted compounds. By this, monitoring of the progressively accumulating droplet in the head of the syringe was further possible. In this first report, the developed instrumental setup has been applied to the determination of the dye rhodamine B in water samples and soft drinks. The main parameters influencing the extraction such as the selection of the extractant and disperser solvents, extractant/disperser and organic/water phase ratios, pH of the aqueous phase, extraction flow rates, and extraction time were investigated. Under the selected conditions, rhodamine B was quantified in a working range of 0.023-2 mg L(-1) with a limit of detection of 0.007 mg L(-1). Good repeatability values of up to 3.2% (RSD) were obtained for ten consecutive extractions. The enrichment factor for a 1 mg L(-1) rhodamine B standard was 23, and up to 51 extractions were accomplished in 1 h.

Renewable sorbent dispersive solid phase extraction automated by Lab-In-Syringe using magnetite-functionalized hydrophilic-lipophilic balanced sorbent coupled online to HPLC for determination of surface water contaminants.

An automated methodology for magnetic dispersive solid phase microextraction integrating bead injection approach for renewable sorbent introduction is presented for the first time and was successfully applied to the enrichment of water contaminants. For this purpose, a simple procedure was developed for the functionalization of commercial SupelTM-Select HLB (Hydrophilic modified styrene polymer) sorbent beads that allowed embedding magnetite nanoparticles (Fe3O4). The sorbent was then used in a dispersive solid phase extraction procedure that was carried out entirely inside the void of an automatic syringe pump following the flow-batch concept of Lab-In-Syringe including automated renewal of the sorbent for each analysis. Mixing processes, sorbent dispersion, and sorbent recovery were enabled by using a strong magnetic stirring bar, fabricated from a 3D printed polypropylene casing and neodymium magnets, inside the syringe. The final extract was submitted to online coupled liquid chromatography with spectrometric detection. System and methodology were applied to determine mebendazole, bisphenol A, benzyl 4-hydroxybenzoate, diclofenac, and triclosan selected as models from different groups of environmental contaminants of current concern. Experimental parameters including extraction and elution times, composition and volume of eluent, and bead recollection were optimized. Required system elements were produced by 3D printing. Enlarging the sample volume by repeated extraction to enhance the sensitivity of the method was studied. Using double extraction from 3.5 mL, limits of detection ranged from 1.2 µg L-1 to 6.5 µg L-1 with an RSD (n = 6) value less than 7% for all the analytes at 25 µg L-1 level. The method was linear in the range of 5-200 µg L-1 and was successfully implemented for the analysis of surface waters with analyte recoveries ranging from 78.4% to 105.6%.

Automated centrifugation-less milk deproteinization and homogenous liquid-liquid extraction of sulfonamides for online liquid chromatography.

A novel approach to the determination of sulfonamides in milk based on the Lab-In-Syringe technique is presented. The method involves automated salting-out liquid-liquid extraction of the analytes, allowing simultaneous sample deproteination without requiring centrifugation or manual sample handling. The procedural parameters, including salt type, solvent-to-sample ratio, and salt solution volume, were studied. The extracts obtained were diluted in situ and transferred to online coupled liquid chromatography for analyte separation carried out in parallel to the subsequent extraction. In this way, a sample throughput of approximately 6 h-1 was achieved. The detection limits ranged from 25 to 32 µg L-1 using a 500 µL milk sample and spectrophotometric detection. The applicability of the developed method to sample analysis was proven by recovery values ranging from 73.2% to 94.1% (86.3% on average) for milk samples of different fat content spiked at 3 µg mL-1 level. Straightforward automation of one of the most laborious preparation steps in food analysis, i.e., deproteination, was demonstrated.

Lab-In-Syringe automation of deep eutectic solvent-based direct immersion single drop microextraction coupled online to high-performance liquid chromatography for the determination of fluoroquinolones.

Lab-In-Syringe direct immersion single drop microextraction is proposed as an automated sample pretreatment methodology and coupled online to HPLC with fluorescence detection for the determination of fluoroquinolones in environmental waters. For the first time, a drop of a natural deep eutectic solvent (NADES), synthesized from hexanoic acid and thymol, has been used as an extractant in automated single-drop microextraction. The extraction procedure was carried out within the 5 mL void of an automatic syringe pump. A 9-position head valve served the aspiration of all required solutions, air, waste disposal, and hyphenation with the HPLC instrument. Sample mixing during extraction was done by a magnetic stirring bar placed inside the syringe. Only 60 µL of NADES were required omitting toxic classical solvents and improving the greenness of the proposed methodology. By direct injection, linear working ranges between 0.1 and 5 µg L-1 were achieved for all fluoroquinolones. The limit of quantification values and enrichment factors ranged from 20 ng L-1 to 30 ng L-1 and 35 to 45, respectively. Accuracies obtained from the analysis of spiked surface water and wastewater treatment plant effluent analysis at two concentration levels (0.5 and 4 µg L-1) ranged from 84.6% to 119.7%, with RSD values typically <3%.

Sweeping-micellar electrokinetic chromatography with tandem mass spectrometry as an alternative methodology to determine neonicotinoid and boscalid residues in pollen and honeybee samples.

In this work, it is proposed for the first time an electrophoretic approach based on micellar electrokinetic chromatography coupled with tandem mass spectrometry (MEKC-MS/MS) for the simultaneous determination of nine neonicotinoids (NNIs) together with the fungicide boscalid in pollen and honeybee samples. The separation was performed using ammonium perfluorooctanoate (50 mM, pH 9) as both volatile surfactant and electrophoretic buffer compatible with MS detection. A stacking strategy for accomplishing the on-line pre-concentration of the target compounds, known as sweeping, was carried out in order to improve separation efficiency and sensitivity. Furthermore, a scaled-down QuEChERS was developed as sample treatment, involving a lower organic solvent consumption and using Z-Sep+ as dispersive sorbent in the clean-up step. Regarding the detection mode, a triple quadrupole mass spectrometer was operating in positive ion electrospray mode (ESI+) under multiple reaction monitoring (MRM). The main parameters affecting MS/MS detection as well as the composition of the sheath-liquid (ethanol/ultrapure water/formic acid, 50:49.5:0.5 v/v/v) and other electrospray variables were optimized in order to achieve satisfactory sensitivity and repeatability. Procedural calibration curves were established in pollen and honeybee samples with LOQs below 11.6 µg kg-1 and 12.5 µg kg-1, respectively. Precision, expressed as RSD, lower than 15.2% and recoveries higher than 70% were obtained in both samples. Two positive samples of pollen were found, containing imidacloprid and thiamethoxam. Imidacloprid was also found in a sample of honeybees. The obtained results highlight the applicability of the proposed method, being an environmentally friendly, efficient, sensitive and useful alternative for the determination of NNIs and boscalid in pollen and honeybee samples.

Real-time monitoring of Metridia luciferase release from cells upon interaction with model toxic substances by a fully automatic flow setup - A proof of concept.

This manuscript reports on a fully automatic sequential injection system incorporating a 3D printed module for real-time monitoring of the release of Metridia luciferase from a modified liver epithelial cell line. To this end, a simple and effective approach for the automation of flash-type chemiluminescence assays was developed. The 3D printed module comprised an apical and a basal compartment that enabled monitoring membrane processes on both sides of the cell monolayer aimed at elucidating the direction of luciferase release. A natural release was observed after transfection with the luciferase plasmid by online measurement of the elicited light from the reaction of the synthesized luciferase with the coelenterazine substrate. Model substances for acute toxicity from the group of cholic acids - chenodeoxycholic and deoxycholic acids - were applied at the 1.0 and 0.5 mmol L-1 levels. The tested cholic acids caused changes in cell membrane permeability that was accompanied by an increased luciferase release. The obtained kinetic profiles were evaluated based on the delay between the addition of the toxic substance and the increase of the chemiluminescence signal. All experiments were carried out in a fully automatic system in ca. 5 min per sample in 30 min intervals and no manual interventions were needed for a sampling period of at least 6 h.

Nanofibrous Online Solid-Phase Extraction Coupled with Liquid Chromatography for the Determination of Neonicotinoid Pesticides in River Waters.

Polymeric nano- and microfibers were tested as potential sorbents for the extraction of five neonicotinoids from natural waters. Nanofibrous mats were prepared from polycaprolactone, polyvinylidene fluoride, polystyrene, polyamide 6, polyacrylonitrile, and polyimide, as well as microfibers of polyethylene, a polycaprolactone nano- and microfiber conjugate, and polycaprolactone microfibers combined with polyvinylidene fluoride nanofibers. Polyimide nanofibers were selected as the most suitable sorbent for these analytes and the matrix. A Lab-In-Syringe system enabled automated preconcentration via online SPE of large sample volumes at low pressure with analyte separation by HPLC. Several mat layers were housed in a solvent filter holder integrated into the injection loop of an HPLC system. After loading 2 mL sample on the sorbent, the mobile phase eluted the retained analytes onto the chromatographic column. Extraction efficiencies of 68.8-83.4% were achieved. Large preconcentration factors ranging from 70 to 82 allowed reaching LOD and LOQ values of 0.4 to 1.7 and 1.2 to 5.5 µg·L-1, respectively. Analyte recoveries from spiked river waters ranged from 53.8% to 113.3% at the 5 µg·L-1 level and from 62.8% to 119.8% at the 20 µg·L-1 level. The developed methodology proved suitable for the determination of thiamethoxam, clothianidin, imidacloprid, and thiacloprid, whereas matrix peak overlapping inhibited quantification of acetamiprid.

Lab-In-Syringe for automated double-stage sample preparation by coupling salting out liquid-liquid extraction with online solid-phase extraction and liquid chromatographic separation for sulfonamide antibiotics from urine.

A double-stage Lab-In-Syringe automated extraction procedure coupled online to HPLC for the determination of four sulfonamides in urine has been developed. Our method is based on homogeneous liquid-liquid extraction at pH 3 using water-miscible acetonitrile with induction of phase separation by the addition of a saturated solution of kosmotropic salts MgSO4 and NaCl. The procedure allowed extraction of the moderately polar model analytes and the use of a solvent that is compatible with the used separation technique. The automated sample preparation system based on the stirring-assisted Lab-In-Syringe approach was coupled on-line with HPLC-UV for the subsequent separation of the sulfonamide antibiotics. To improve both preconcentration factor and extract cleanup, the analytes were trapped at pH 10 in an anion-exchange resin cartridge integrated into the HPLC injection loop thus achieving a double-stage sample clean-up. Analytes were eluted using an acidic HPLC mobile phase in gradient elution mode. Running the analytes separation and the two-step preparation of the following sample in parallel reduced the total time of analysis to mere 13.5 min. Limits of detection ranged from 5.0 to 7.5 µg/L with linear working ranges of 50-5000 µg/L (r2 > 0.9997) and RSD ≤ 5% (n = 6) at a concentration level of 50 µg/L. Average recovery values were 102.7 ± 7.4% after spiking of urine with sulfonamides at concentrations of 2.5 and 5 mg/L followed by 5 times dilution. To the best of our knowledge, the use of Lab-In-Syringe for the automation of coupled homogeneous liquid-liquid extraction and SPE for preparation of the complex matrices suitable for separation techniques is here presented for the first time.