Molecularly imprinted polymers in online extraction liquid chromatography methods: Current advances and recent applications.

BACKGROUND: One of the primary objectives in green analytical practices is the seamless integration of extraction and separation steps, resulting in the augmentation of both analytical throughput and method performance. Consequently, the exploration of prospective sorbent materials has drawn significant attention in the scientific community, particularly concerning the potential for online procedures. Employing the optimal sorbent material within an automated analytical approach holds the promise of elevating the precision of the analytical evaluation. Molecularly imprinted polymers (MIPs) excel in specific analyte interaction within complex matrices. However, MIPs' full potential was not widely exploring especially for online analytical methodologies. RESULTS: Here is presented a comprehensive overview of the current applications of MIPs as sorbent materials within integrated and automated separation methodologies applied to diverse matrices including biological, food, and environmental samples. Notably, their primary advantage, as evidenced in the literature, lies in their exceptional selectivity for the target analyte discussed according to the adopted synthesis protocol. Furthermore, the literature discussed here illustrates the versatility of MIPs in terms of modification with one or more phases which are so-called hybrid materials, such as molecularly imprinted monoliths (MIM), the molecularly imprinted ionic liquid polymer (IL-MIP), and restricted access to molecularly imprinted polymer (RAMIP). The reported advantages enhance their applicability in integrated and automated separation procedures, especially to the column switching methods, across a broader spectrum of applications. SIGNIFICANCE: This revision aims to demonstrate the MIP's potential as a sorbent phase in integrated and automated methods, this comprehensive overview of MIP polymers in integrated and automated separation methodologies can be used as a valuable guide, inspiring new research on developing novel horizons for MIP applications to have their potential emphasized in analytical science and enhanced to the great analytical methods achievement.

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.

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.

An open-source smart fraction collector for isocratic preparative liquid chromatography.

Preparative liquid chromatography is a technique for separating complex samples or isolating pure compounds from complex extracts. It involves eluting samples through a packed column and selectively collecting or isolating the separated bands in a sequence of fractions. Depending on the column length and the sample complexity, a large number of fractions may be obtained, making fraction collection a laborious and time-consuming process. Manual fraction collection is also tedious, error-prone, less reproducible, and susceptible to contamination. Several commercial and lab-made solutions are available for automated fraction collection, but most systems do not synchronize with the instrument detector and collect fractions at fixed volumes or time intervals. We have assembled a low-cost Arduino-based smart fraction collector that can record the signal from the UV-vis detector of the chromatography instrument and enable the automated selective collection of the targeted bands. The system consists of a robot equipped with position sensors and a 3-way solenoid valve that switches the column effluent between the waste or collection positions. By proper programming, an Arduino board records the detector response and actuates the solenoid valve, the position sensors, and the stepper motors to collect the target chromatographic bands.

The overshadowed role of electron ionization-mass spectrometry in analytical biotechnology.

Target and untargeted analysis of several compounds are crucial methods in important areas such as omics sciences. Gas chromatography coupled to mass spectrometry (GC-MS) is widely used for volatile and thermally stable compounds. In this case, the electron ionization technique (EI) is preferable as it produces highly fragmented and reproducible spectra comparable to spectral libraries. However, only a fraction of target compounds is analyzable by GC without chemical derivatization. Therefore, liquid chromatography (LC) coupled with MS is the most used technique. Contrary to EI, electrospray ionization does not produce reproducible spectra. That is why researchers have been working on interfaces between LC and EI-MS to bridge the gap between those techniques. This short review will discuss advancements, applications, and perspectives on biotechnological analysis.

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.

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.

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.

Determination of selected herbicides in sugarcane-derived foods by graphene-oxide based disposable pipette extraction followed by liquid chromatography-tandem mass spectrometry.

Sugarcane is widely cultivated in Brazil. Although there are Maximum Residue Limits of pesticides determined for this plant, there is no legislation covering alimentary products from sugarcane. In this study, Disposable Pipette Tip Extraction (DPX) technique was evaluated as a sample preparation technique for simultaneous determination of eleven herbicides followed by LC-MS/MS analysis in three sugarcane-derived food matrices: juice, candy, and syrup. First, graphene oxide anchored to silica functionalized with octadecyl silane and endcapped was synthesized, which was evaluated as a sorbent in DPX. Then, after evaluating the parameters involved in DPX extraction, the method was validated following the ICH guide. As a result, the method showed acceptable linearity (r ≥ 0.99), limits of quantification (1.0 - 5.0 ng mL-1 for juice and 5.0 - 25.0 ng g - 1 for candy and syrup, varying according to the pesticide), precision, and accuracy within the limits of the literature, and recoveries ranging from 48 - 69% (juice), 34 - 89% (candy), and 28 - 76% (syrup). Finally, the developed method was successfully applied in actual samples of the three studied matrices.

Porous layer open tubular nano liquid chromatography directly coupled to electron ionization mass spectrometry.

A 25 µm i.d x 1.2 m length PS-DVB porous layer open tubular column (PLOT) was prepared and assessed in the configuration of a nano liquid chromatography coupled to an electron ionization mass spectrometry system (OT-nanoLC-EI-Ms), via the direct insertion of the column outlet into the ionization source. The developed system's operational parameters were comprehensively studied, and the setup performance was investigated employing both unidimensional and column switching configurations. As a result, the OT-nanoLC-EI-MS system demonstrated competitive applicability in separating non-amenable ESI compounds, such as polyaromatic hydrocarbons (PAHs) and non-amenable GC compounds such as thermolabile pesticides. Furthermore, with excellent chromatographic performance, the PLOT columns can work under more compatible EI-detection conditions - such as the elution with 100% organic solvent. For example, PAHs retention factors ranged between 1.5 and 2.2 for 100% MeCN mobile phase, and more than 33,000 plates per meter for naphthalene at 50 nL/min flow rate. In analyzing thermolabile pesticides, the column switching PLOT-nanoLC-EI-MS system provided LODs of 25 µg/L, demonstrating suitable intra e interday reproducibility (% RSD < 13%, n = 3), and possibilities the direct injection of raw samples with suitable robustness.

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.

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.

Current advances and applications of online sample preparation techniques for miniaturized liquid chromatography systems.

Miniaturized liquid chromatography (LC) has been recognized as one of the most important analytical methods in several research fields. Reduced analytical work-scale provides superior chromatographic resolution and decreases sample and organic solvent consumption. However, frequent clogging of tubing connections and use of small sample volumes are significant limitations when high throughput and sensitive analyses are required. Effective sample preparation could help to overcome these limitations. Online coupling of sample preparation techniques (such as column switching and in-tube solid-phase microextraction) with these miniaturized systems may result in more sensitive and reproducible analyses, improving analytical efficiency. This review describes the most common online miniaturized LC configurations, and the main applications of current online sample preparation techniques coupled to miniaturized LC systems in the bioanalytical, omics, and environmental areas. Relevant features, and challenges of these systems, and innovative sorbents, including restricted access materials, monoliths, and immunosorbents is also discussed.

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.

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.

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.

An overview of open tubular liquid chromatography with a focus on the coupling with mass spectrometry for the analysis of small molecules.

Open tubular liquid chromatography (OT-LC) can provide superior chromatographic performance and more favorable mass spectrometry (MS) detection conditions. These features could provide enhanced sensitivity when coupled with electrospray ionization sources (ESI-) and lead to unprecedented detection capabilities if interfaced with a highly structural informative electron ionization (EI) source. In the past, the exploitation of OT columns in liquid chromatography evolved slowly. However, the recent instrumental developments in capillary/nanoLC-MS created new opportunities in developing and applying OT-LC-MS. Currently, the analytical advantages of OT-LC-MS are mainly exploited in the fields of proteomics and biosciences analysis. Nevertheless, under the right conditions, OT-LC-MS can also offer superior chromatographic performance and enhanced sensitivity in analyzing small molecules. This review will provide an overview of the latest developments in OT-LC-MS, focusing on the wide variety of employed separation mechanisms, innovative stationary phases, emerging column fabrication technologies, and new OT formats. In the same way, the OT-LC's opportunities and shortcomings coupled to both ESI and EI will be discussed, highlighting the complementary character of those two ionization modes to expand the LC's detection boundaries in the performance of targeted and untargeted studies.

Automated needle-sleeve based online hyphenation of solid-phase microextraction and liquid chromatography.

A novel approach for the online coupling of solid-phase microextraction (SPME) and liquid chromatography (LC) is introduced. An innovative Si@GO@ßCD coated needle-sleeve extractant device was developed and then employed in the automated online SPME-LC-UV determination of estrogen-like isoflavones from human urine samples. The extractant SPME device is easily attachable at the endpoint of an analytical syringe needle and operated by a lab-made autosampler. Fully automated online SPME-LC is accomplished by proper autosampler programming to perform the following steps: i) the analytes extraction by direct immersion of the extractant device into the stirred sample, ii) a rinsing step iii) the analytes desorption/enrichment, iv) the online transference of the extract to the LC injection valve. Besides allowing the online SPME hyphenation, this extraction modality efficiently addressed the drawbacks associated with the clogging and dispersion of graphene-based microextraction techniques performed in packed-bed and dispersive formats. The main extraction parameters and the performance of the automated online SPME-LC method developed were carefully studied. The results show a good sensitivity, reliability, and straightforward analytical strategy for the determination of organic compounds in complex samples. The detection limit of the method was 20 µg L1 for DAI and 10 µg L-1 for GEN, FOR and BIO. The intra-day RSD was below 10% and inter-day RSD was below 13%. The total analysis time was less than 17 min per sample.

Magnetic solid-phase extraction of gingerols in ginger containing products.

In this study, a graphene oxide/magnetite (GO-Fe3O4) nanocomposite was synthesized and used as a sorbent in the magnetic solid-phase extraction (MSPE) of gingerols from fresh ginger rhizomes, ginger extracts, commercial tea samples, ginger candies, thermogenic supplements, and tonic water. An MSPE method was developed, and the main influencing parameters in the sample preparation process were investigated. After GO-Fe3O4 based MSPE, 6-gingerol, 8-gingerol, and 10-gingerol were determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The whole GO-Fe3O4-MSPE-LC-MS/MS method proved high selectivity and consistent analytical confidence. The limits of detection (LOD) ranged between 2 and 3 µg L-1. Intra-day and inter-day RSDs fluctuated between 1.7 - 13.4% and 0.4-10.9%, respectively. Weighted calibration revealed good linearity within the studied range (5-200 µg L-1) and guaranteed appropriate accuracy (relative residues < 25%). MSPE with GO-Fe3O4 demonstrated to be a practical, fast, efficient, high-throughput, and environmental-friendly sample preparation technique for determining of gingerols in commercial products, and its hyphenation with LC-MS/MS analysis yield a valuable analytical tool for the confident quality control of commercial ginger-containing products.