Fiber-in-tube SPME-CapLC-MS/MS method to determine Aß peptides in cerebrospinal fluid obtained from Alzheimer's patients.

Mass spectrometry is characterized by its high sensitivity, ability to measure very low analyte concentrations, specificity to distinguish between closely related compounds, availability to generate high-throughput methods for screening, and high multiplexing capacity. This technique has been used as a platform to analyze fluid biomarkers for Alzheimer's disease. However, more effective sample preparation procedures, preferably antibody-independent, and more automated mass spectrometry platforms with improved sensitivity, chromatographic separation, and high throughput are needed for this purpose. This short communication discusses the development of a fiber-in-tube SPME-CapLC-MS/MS method to determine Aß peptides in cerebrospinal fluid obtained from Alzheimer's disease patients. To obtain the fiber-in-tube SPME capillary, we longitudinally packed 22 nitinol fibers coated with a zwitterionic polymeric ionic liquid into the same length of the PEEK tube. In addition, this communication compares this fiber-in-tube SPME method with the conventional HPLC scale (HPLC-MS/MS) and when directly coupled to CapESI-MS/MS without chromatographic separation, and, as a case study, discusses the benefits and challenges inherent in miniaturizing the flow scale of the sample preparation technique (fiber-in-tube SPME) to the CapLC-MS/MS system. Fiber-in-tube SPME-CapLC-MS/MS provided LLOQ ranging from 0.09 to 0.10 ng mL-1, accuracy ranging from 91 to 117 % (recovery), and reproducibility of less than 18 % (RSD). Analysis of the cerebrospinal fluid samples obtained from Alzheimer's disease patients evidenced that the method is robust. At the capillary scale (10 µL min-1), this innovative method presented higher analytical sensitivity than the conventional HPLC-MS/MS scale. Although fiber-in-tube SPME directly coupled to CapESI-MS/MS offers advantages in terms of high throughput, the sample was dispersed and non-quantitatively desorbed from the capillary at low flow rate. These results highlighted that chromatographic separation is important to decrease the matrix effect and to achieve higher detectability, which is indispensable for bioanalysis.

Development of Wall-Coated Open Tubular Columns and Their Application to Nano Liquid Chromatography Coupled to Tandem Mass Spectrometry.

This work presents a study on the application of wall open tubular column (WCOT) in liquid chromatography coupled with tandem mass spectrometry. Each process step reports the column preparation method in detail, subdivided into column pretreatment, silanization, stationary phase coating, and immobilization. Then, an evaluation of the parameters that can affect the efficiency of these columns was made. Atrazine, clomazone, and metolachlor were used as probes during this step. Factors such as stationary phase composition, length, internal diameter, stationary phase mass employed, and injection volume were investigated. In addition, with the help of Knox and Poppe graphs, the columns' performance was evaluated to determine the optimal flow rate and the speed-efficiency relationship, respectively. Based on the results, the best configurations for the WCOT column application to the LC system were defined: length-8 m; inner diameter-25 µm; mass of OV-210-2.5% m/v; and, injection volume-100 nL. Finally, the optimized WCOT column developed in this work was coupled with a commercially-packed trapping column in the nano liquid chromatography system (nanoLC). In this configuration, more significant results were obtained regarding separation resolution, with Rs = 5.9 achieved for the most retained pair of analytes (clomazone and metolachlor).

Recent Trends in Graphene-Based Sorbents for LC Analysis of Food and Environmental Water Samples.

This review provides an overview of recent advancements in applying graphene-based materials as sorbents for liquid chromatography (LC) analysis. Graphene-based materials are promising for analytical chemistry, including applications as sorbents in liquid chromatography. These sorbents can be functionalized to produce unique extraction or stationary phases. Additionally, graphene-based sorbents can be supported in various materials and have consequently been applied to produce various devices for sample preparation. Graphene-based sorbents are employed in diverse applications, including food and environmental LC analysis. This review summarizes the application of graphene-based materials in food and environmental water analysis in the last five years (2019 to 2023). Offline and online sample preparation methods, such as dispersive solid phase microextraction, stir bar sorptive extraction, pipette tip solid phase extraction, in-tube solid-phase microextraction, and others, are reviewed. The review also summarizes the application of the columns produced with graphene-based materials in separating food and water components and contaminants. Graphene-based materials have been reported as stationary phases for LC columns. Graphene-based stationary phases have been reported in packed, monolithic, and open tubular columns and have been used in LC and capillary electrochromatography modes.

Modern automated microextraction procedures for bioanalytical, environmental, and food analyses.

Sample preparation frequently is considered the most critical stage of the analytical workflow. It affects the analytical throughput and costs; moreover, it is the primary source of error and possible sample contamination. To increase efficiency, productivity, and reliability, while minimizing costs and environmental impacts, miniaturization and automation of sample preparation are necessary. Nowadays, several types of liquid-phase and solid-phase microextractions are available, as well as different automatization strategies. Thus, this review summarizes recent developments in automated microextractions coupled with liquid chromatography, from 2016 to 2022. Therefore, outstanding technologies and their main outcomes, as well as miniaturization and automation of sample preparation, are critically analyzed. Focus is given to main microextraction automation strategies, such as flow techniques, robotic systems, and column-switching approaches, reviewing their applications to the determination of small organic molecules in biological, environmental, and food/beverage samples.

Online Extraction Followed by LC-MS/MS Analysis of Lipids in Natural Samples: A Proof-of-Concept Profiling Lecithin in Seeds.

Sample preparation is usually a complex and time-consuming procedure, which can directly affect the quality of the analysis. Recent efforts have been made to establish analytical methods involving minimal sample preparation, automatized and performed online with the analytical techniques. Online Extraction coupled with Liquid Chromatography-Mass Spectrometry (OLE-LC-MS) allows a fully connected extraction, separation, and analysis system. In this work, the lecithin profile was investigated in commercial sunflower, almonds, peanuts, and pistachio seeds to demonstrate that the concept of extraction, followed by the online analysis of the extract, could be applied to analyze this class of analytes in such complex solid matrices without a prior off-line solvent extraction step. The extraction phase gradient method was optimized. Two different analytical columns were explored, one being a conventional C18 (50 × 2.1 mm, 1.7 µm SPP) and the other a novel self-packed SIGO-C18ec (100 × 0.5, 5 µm FPP), which resulted in better separation. The analysis repeatability was investigated, and suggestions to improve it were pointed out. A characteristic ion with a m/z of 184, related to lysophosphatidylcholine structure, was used to identify the lecithin compounds. The temperature effect on the chromatograms was also explored. In short, it was found that the OLE-LC-MS approach is suitable for the analysis of lecithin compounds in seeds, being a promising alternative for lipidomics approaches in the near future.

Application of an in-house packed octadecylsilica-functionalized graphene oxide column for capillary liquid chromatography analysis of hormones in urine samples.

Graphene oxide-based LC stationary phases were developed and applied for separating hormones from urine using capillaryLC-MS/MS. Using two analytical approaches - direct injection and column-switching arrangement - it was possible to evaluate the chromatographic parameters and perform tests on the raw biological fluid. Two stationary phases (SPs) were produced, varying the amino silica support particle diameter (Si, 5, and 10 µm). Graphene oxide was covalently bonded to the surface of Si particles, and this material was functionalized by the insertion of octadecylsilica groups, generating the SiGO-C18. Infra-red spectroscopy assays revealed that both steps were successful - supporting GO onto Si and further C18 customization. Scanning electron microscopy showed spherical geometries with minor irregularities and narrow particle size distribution for the produced SPs. The GO-coating rate was higher on the Si particles of 10 µm. As a result, the 10 µm produced column reported better resolution, efficiency, and peak capacity. Therefore, this SiGO-C18 capillary column (100 mm × 0.32 mm i.d., 10 µm dp) was applied successfully in a column-switching method to separate hormones in urine. Linearity (R2 above 0.99), quantification limits (between 1.0 and 5 µg/L), and other figures of merit of the method were determined. It is worth mentioning that the SiGO-C18 capillaryLC column performed adequately, separating the target compounds in less than 6 min. We hope this work could significantly contribute to shedding some light on graphene-based materials as a promising class of stationary phase for miniaturized liquid chromatography.

Investigation of the applicability of silica-graphene hybrid materials as stationary phases for capillary liquid chromatography.

Graphene and graphene-derived substances are cutting-edge materials receiving increasing attention in the analytical chemistry field. Graphene oxide sheets bonded to amino silica particles functionalized with octadecyl (C18) groups and endcapped, also known as SiGO-C18ec, have been successfully employed as extraction phases and in analytical columns associated with conventional liquid chromatography (LC). In this work, SiGO-C18ec particles of 3, 5, and 10 µm nominal id were employed to pack capillary LC columns (100 mm long x 0.3 mm id), and their performance in the gradient mode was evaluated and compared. A 3 µm C18 capillary LC column (50 x 0.3 mm) was used as a reference column. Eight analytes having different polarities and topological surface areas were selected as a probe in this study: carbofuran clomazone, hexazinone, carbamazepine, citalopram, clomipramine, desipramine, and ochratoxin A. Studies about orthogonality were performed to investigate the orthogonality between the SiGO-C18ec and C18 phases. Among the SiGO-C18ec phases investigated, the column packed with 5 µm SiGO-C18ec particles presented the best peak capacity (29) in 15 min. Additionally, the performance of the columns packed with 5 µm SiGO-C18ec particles overcame the performance of the C18 columns used. Significant orthogonality was found between C18 and SiGO-C18ec packed columns; however, no significant differences were found between columns packed with SiGO-C18ec particles of different diameters.

Environmentally friendly analysis of sulphonamides in Brazilian honey through automated and miniaturised sample preparation coupled with LC-MS/MS.

Increased use of environmentally friendly practices has become a trend in science because of the current awareness regarding climate change and related issues. Similarly for analytical chemistry, considering the development of greener methods for reducing the use of reagents and samples and also toxic waste generation. To meet such goals, automation, and miniaturisation of sample preparation-a well-recognised laborious and time-consuming analytical step-are two promising strategies. This work associates the greener aspects of miniaturisation and the performance of automated sample preparation. Therefore, we proposed an analytical method using a miniaturised extraction column for pre-concentrating sulphamerazine, sulphamethazine, sulphamethoxazole, sulphadimethoxine, sulphathiazole, and sulphachlorpyridazine from honey and cleaning-up the samples. Several variables were optimised: extractive phase, loading flow, loading phase, and loading time. Under optimised conditions, the method showed adequate linearity between 5.0 and 60 ng g-1 with R > 0.99, and also good selectivity and recovery (114.6-124.1%) which are acceptable according to Brazilian legislation. Intra and inter-day precision were in the range 3.0-5.0%. Although sulphonamides were detected in one of the eight commercial honey samples, the value was below the established MRL. The method showed efficiency, while also exhibiting greener characteristics resulting from miniaturisation and automation, representing a promising environmentally friendly alternative for conventional sample preparation methods.

Modified graphene-silica as a sorbent for in-tube solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry. Determination of xanthines in coffee beverages.

Given the increasing need for analyzing natural or contaminating compounds in complex food matrices in a simple and automated way, coupling miniaturized sample preparation techniques with chromatographic systems have become a growing field of research. In this regard, given the low extraction efficiency of conventional sorbent phases, the development of materials with enhanced extraction capabilities is of particular interest. Here we present several synthesized graphene-based materials supported on aminopropyl silica as sorbents for the extraction of xanthines. The synthesized materials were characterized by infrared spectroscopy and scanning electron microscopy. Aminopropyl silica coated with graphene oxide and functionalized with octadecylsilane/end-capped (SiGOC18ecap) showed the best performance for xanthines extraction. Hence, this material was employed as an in-tube solid phase microextraction (in-tube SPME) device coupled online with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and applied for the analysis of xanthines in roasted coffee samples. Extraction parameters and detection conditions were optimized. The method showed low limits of quantification (0.3-1.0 µg L-1), precision as relative standard deviation (RSD) values lower than 10%, recoveries between 73 and 109%, and pre-concentration factors from 5.6 to 7.2. Caffeine was determined in all ground roasted and instant coffee samples, in a wide range (0.9 to 36.8 mg g-1), and small amounts of theobromine and theophylline were also detected in some samples. This work demonstrated that functionalized graphene-based materials represent a promising new sorbent class for in-tube SPME, showing improved extraction capacity. The method was efficient, simple, and fast for the analysis of xanthines, demonstrating an excellent potential to be applied in other matrices.

Silica modified with polymeric amphiphilic nanoparticles as first dimension for multidimensional separation techniques.

This paper addresses the evaluation of a new amphiphilic nanoparticle supported on silica and its application as sorbent in on-line solid phase extraction. The investigated sorbent material is a copolymer composed by [2- (Acryloyloxy) ethyl] trimethylammonium chloride (block A) and butyl acrylate (block B) prepared by reversible addition-fragmentation chain transfer. After polymerization the nanoparticles were adsorbed into silica surface by electrostatic attraction providing to the sorbent both, apolar and polar characteristics. After the fundamental studies to understand the main features of the synthesized nanoparticles supported on silica, this sorbent material was packed into an extraction column. This column was connected on line with liquid chromatography-tandem mass spectrometry and utilized for automated online determination of several analytes as Ochratoxin A, azoxystrobin, cyproconazole and difenoconazole, in wine and water samples.

Packed in-tube solid phase microextraction with graphene oxide supported on aminopropyl silica: Determination of target triazines in water samples.

On-line in-tube solid phase microextraction (in-tube SPME) coupled to high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) was successfully applied to the determination of selected triazines in water samples. The method based on the employment of a packed column containing graphene oxide (GO) supported on aminopropyl silica (Si) showed that the extraction phase has a high potential for triazines extraction aiming to its physical-chemical properties including ultrahigh specific surface area, good mechanical and thermal stability and high fracture strength. Injection volume and loading time were both investigated and optimized. The method validation using Si-GO to extract and concentrate the analytes showed satisfactory results, good sensitivity, good linearity (0.2-4.0 µg L-1) and low detection limits (1.1-2.9 ng L-1). The high extraction efficiency was determined with enrichment factors ranging from 1.2-2.9 for the lowest level, 1.3-4.9 intermediate level and 1.2-3.0 highest level (n = 3). Although the analytes were not detected in the real samples evaluated, the method has demonstrated to be efficient through its application in the analysis of spiked triazines in ground and mineral water samples.

Determination of Ochratoxin A in wine by packed in-tube solid phase microextraction followed by high performance liquid chromatography coupled to tandem mass spectrometry.

Ochratoxin A (OTA), a widely studied mycotoxin, can be found in a variety of food matrices. As its concentration in food is generally low (in the order of µg kg-1), sample preparation techniques are necessary for the analyte purification and pre-concentration in order to achieve the required low detection limits. The separation and detection methods used for OTA analysis should also offer proper sensitivity in order to allow the adequate quantification of the analyte. This manuscript addresses the development of a methodology aiming the analysis of OTA in wine samples by packed in-tube SPME in flow through extraction mode coupled to HPLC-MS/MS. The in-tube SPME set up utilized a PEEK tube packed with C18 particles as the extraction column. The method was optimized by a central composite design 22+3 extra central points, having as factors the percentage of ACN and time in the sample load step. The functionalities of the method were attested and its analytical conditions, enhanced by using 22% of ACN and 6min in the sample load step. Validation of the method was also accomplished prior to analyses of both dry red wine and dry white wine samples. The method demonstrated proper sensitivity, with detection and quantification limits equal to 0.02 and 0.05µgL-1, respectively. Linearity and precision exhibited a 0.996 correlation coefficient and RSD under 6%, respectively. The method proved to be accurate at medium and higher concentration levels with a maximum recovery of 73% at higher concentration levels. OTA was not detected in either dry red and dry white wine samples evaluated in this work. If present, it would be at concentrations lower than the detection and quantification limits established for the proposed method, and considered not a potential danger to human health according to our present knowledge.

Evolution in miniaturized column liquid chromatography instrumentation and applications: An overview.

The purpose of this article is to underline the miniaturized LC instrumental system and describe the evolution of commercially available systems by discussing their advantages and drawbacks. Nowadays, there are already many miniaturized LC systems available with a great variety of pump design, interface and detectors as well as efficient columns technologies and reduced connections devices. The solvent delivery systems are able to drive the mobile phase without flow splitters and promote gradient elution using either dual piston reciprocating or syringe-type pumps. The mass spectrometry as detection system is the most widely used detection system; among many alternative ionization sources direct-EI LC-MS is a promising alternative to APCI. In addition, capillary columns are now available showing many possibilities of stationary phases, inner diameters and hardware materials. This review provides a discussion about miniaturized LC demonstrating fundamentals and instrumentals' aspects of the commercially available miniaturized LC instrumental system mainly nano and micro LC formats. This review also covers the recent developments and trends in instrumentation, capillary and nano columns, and several applications of this very important and promising field.

Unified chromatography: Fundamentals, instrumentation and applications.

The concept of unified chromatography has been in existence for 50 years after the work of Giddings proposing that all modes of chromatography (gas chromatography, liquid chromatography, supercritical fluid chromatography and so on) may be treated together under a single unified theory. His idea was partially fulfilled 23 years later by Ishii, Takeuchi and colleagues, who demonstrated for the first time the possibility to analyze a complex sample containing substances with a wide range of boiling points and polarities in the same instrument and column, just by varying the mobile phase pressure and temperature to change from one chromatographic mode to another. This approach has been demonstrated through application to the separation of complex mixtures in several areas including crude oil, edible oils and polymers. Still, unified chromatography has not yet been fully developed. In the present work, we will review the fundamentals, instrumentation and several applications of the technique. Also discussed are the drawbacks that still hinder development, as well as the recent developments and trends in instrumentation and columns that suggest the most feasible ways forward to the full development of unified chromatography.

Leaf-cutter ant fungus gardens are biphasic mixed microbial bioreactors that convert plant biomass to polyols with biotechnological applications.

Leaf-cutter ants use plant matter to culture the obligate mutualistic basidiomycete Leucoagaricus gongylophorus. This fungus mediates ant nutrition on plant resources. Furthermore, other microbes living in the fungus garden might also contribute to plant digestion. The fungus garden comprises a young sector with recently incorporated leaf fragments and an old sector with partially digested plant matter. Here, we show that the young and old sectors of the grass-cutter Atta bisphaerica fungus garden operate as a biphasic solid-state mixed fermenting system. An initial plant digestion phase occurred in the young sector in the fungus garden periphery, with prevailing hemicellulose and starch degradation into arabinose, mannose, xylose, and glucose. These products support fast microbial growth but were mostly converted into four polyols. Three polyols, mannitol, arabitol, and inositol, were secreted by L. gongylophorus, and a fourth polyol, sorbitol, was likely secreted by another, unidentified, microbe. A second plant digestion phase occurred in the old sector, located in the fungus garden core, comprising stocks of microbial biomass growing slowly on monosaccharides and polyols. This biphasic operation was efficient in mediating symbiotic nutrition on plant matter: the microbes, accounting for 4% of the fungus garden biomass, converted plant matter biomass into monosaccharides and polyols, which were completely consumed by the resident ants and microbes. However, when consumption was inhibited through laboratory manipulation, most of the plant polysaccharides were degraded, products rapidly accumulated, and yields could be preferentially switched between polyols and monosaccharides. This feature might be useful in biotechnology.

Determination of steroids, caffeine and methylparaben in water using solid phase microextraction-comprehensive two dimensional gas chromatography-time of flight mass spectrometry.

Analysis of several emerging contaminants (steroids, caffeine and methylparaben) in water using automated solid-phase microextraction with comprehensive two dimensional gas chromatography coupled to time of flight mass spectrometry (SPME-GCxGC-ToF/MS) is presented. Experimental design was used to determine the best SPME extraction conditions and the steroids were not derivatized prior to injection. SPME-GCxGC-ToF/MS provided linear ranges from 0.6 to 1200µgL(-1) and limits of detection and quantitation from 0.02 to 100µgL(-1). A series of river water samples obtained locally were subjected to analysis. SPME-GCxGC-ToF/MS is readily automated, straightforward and competitive with other methods for low level analysis of emerging contaminants.

Development of a new stir bar sorptive extraction coating and its application for the determination of six pesticides in sugarcane juice.

In this article, a novel polydimethylsiloxane/activated carbon (PDMS-ACB) material is proposed as a new polymeric phase for stir bar sorptive extraction (SBSE). The PDMS-ACB stir bar, assembled using a simple Teflon(®)/glass capillary mold, demonstrated remarkable stability and resistance to organic solvents for more than 150 extractions. The SBSE bar has a diameter of 2.36 mm and a length of 2.2 cm and is prepared to contain 92 µL of polymer coating. This new PDMS-ACB bar was evaluated for its ability to determine the quantity of pesticides in sugarcane juice samples by performing liquid desorption (LD) in 200 µL of ethyl acetate and analyzing the solvent through gas chromatography coupled with mass spectrometry (GC-MS). A fractional factorial design was used to evaluate the main parameters involved in the extraction procedure. Then, a central composite design with a star configuration was used to optimize the significant extraction parameters. The method used demonstrated a limit of quantification (LOQ) of 0.5-40 µg/L, depending on the analyte detected; the amount of recovery varied from 0.18 to 49.50%, and the intraday precision ranged from 0.072 to 8.40%. The method was used in the analysis of real sugarcane juice samples commercially available in local markets.

Biocompatible in-tube solid phase microextraction coupled with liquid chromatography-fluorescence detection for determination of interferon α in plasma samples.

The present work demonstrates the successful application of automated biocompatible in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC) for determination of interferon alpha(2a) (IFN α(2a)) in plasma samples for therapeutic drug monitoring. A restricted access material (RAM, protein-coated silica) was employed for preparation of a lab-made biocompatible in-tube SPME capillary that enables the direct injection of biological fluids as well as the simultaneous exclusion of macromolecules by chemical diffusion barrier and drug pre-concentration. The in-tube SPME variables, such as sample volume, draw/eject volume, number of draw-eject cycles, and desorption mode were optimized, to improve the sensitivity of the proposed method. The IFN α(2a) analyses in plasma sample were carried out within 25min (sample preparation and LC analyses). The response of the proposed method was linear over a dynamic range, from 0.06 to 3.0MIUmL(-1), with correlation coefficient equal to 0.998. The interday precision of the method presented coefficient of variation lower than 8%. The proposed automated method has adequate analytical sensitivity and selectivity for determination of IFN α(2a) in plasma samples for therapeutic drug monitoring.

Refrigerated sorptive extraction: determination of BTEX in water samples.

Benzene, toluene, ethyl benzene, o-xylene, m-xylene, and p-xylene (BTEX) enrichment from aqueous samples is performed using refrigerated sorptive extraction (RSE). RSE implies the sorption of volatiles compounds into polydimethylsiloxane (approximately 0.39 mg) coated on an open stainless steel tube internally refrigerated by cold water. A metal tube is inserted into a 30-mL headspace vial through a hole at both sides of the vial. The sample inside the vial is equilibrated with stirring for 60 min at 40 degrees C. After the extraction, the analytes are desorbed with 280 microL of methanol under ultrasonic bath. BTEXs are analyzed by high resolution gas chromatography-flame ionization detection. Optimization of temperature and extraction time were carried out, as well validation in terms of linearity, precision, recuperation, limits of detection and quantification. Linearity is the range 0.999-0.998, linear range between 50 and 1000 microg/L, precision is 6.1% to 14.7% in the 0.1 microg/mL level and 3.5% to 5.6% in the 1 microg/mL level. Recovery ranged from 30% to 50%, detection limits from 1.7 to 19.6 microg/L, and quantification limits from 6.7 to 64.6 microg/L. The optimized method was applied to the analysis of water samples collected near a gas station, which was suspected of contamination by gas oil leaking from storage tanks.