Charge site manipulation to enhance top-down fragmentation efficiency.

In recent years, top-down mass spectrometry has become a widely used approach to study proteoforms; however, improving sequence coverage remains an important goal. Here, two different proteins, α-synuclein and bovine carbonic anhydrase, were subjected to top-down collision-induced dissociation (CID) after electrospray ionisation. Two high-boiling solvents, DMSO and propylene carbonate, were added to the protein solution in low concentration (2%) and the effects on the top-down fragmentation patterns of the proteins were systematically investigated. Each sample was measured in triplicate, which revealed highly reproducible differences in the top-down CID fragmentation patterns in the presence of a solution additive, even if the same precursor charge state was isolated in the quadrupole of the instrument. Further investigation supports the solution condition-dependent selective formation of different protonation site isomers as the underlying cause of these differences. Higher sequence coverage was often observed in the presence of additives, and the benefits of this approach became even more evident when datasets from different solution conditions were combined, as increases up to 35% in cleavage coverage were obtained. Overall, this approach therefore represents a promising opportunity to increase top-down fragmentation efficiency.

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.

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.

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.

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.

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.

The Current Role of Graphene-Based Nanomaterials in the Sample Preparation Arena.

Since its discovery in 2004 by Novoselov et al., graphene has attracted increasing attention in the scientific community due to its excellent physical and chemical properties, such as thermal/mechanical resistance, electronic stability, high Young's modulus, and fast mobility of charged atoms. In addition, other remarkable characteristics support its use in analytical chemistry, especially as sorbent. For these reasons, graphene-based materials (GBMs) have been used as a promising material in sample preparation. Graphene and graphene oxide, owing to their excellent physical and chemical properties as a large surface area, good mechanical strength, thermal stability, and delocalized π-electrons, are ideal sorbents, especially for molecules containing aromatic rings. They have been used in several sample preparation techniques such as solid-phase extraction (SPE), stir bar sorptive extraction (SBSE), magnetic solid-phase extraction (MSPE), as well as in miniaturized modes as solid-phase microextraction (SPME) in their different configurations. However, the reduced size and weight of graphene sheets can limit their use since they commonly aggregate to each other, causing clogging in high-pressure extractive devices. One way to overcome it and other drawbacks consists of covalently attaching the graphene sheets to support materials (e.g., silica, polymers, and magnetically modified supports). Also, graphene-based materials can be further chemically modified to favor some interactions with specific analytes, resulting in more efficient hybrid sorbents with higher selectivity for specific chemical classes. As a result of this wide variety of graphene-based sorbents, several studies have shown the current potential of applying GBMs in different fields such as food, biological, pharmaceutical, and environmental applications. Within such a context, this review will focus on the last five years of achievements in graphene-based materials for sample preparation techniques highlighting their synthesis, chemical structure, and potential application for the extraction of target analytes in different complex matrices.

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 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.

Evaluation of the tubing material and physical dimensions on the performance of extraction columns for on-line sample preparation-LC-MS/MS.

Nowadays, high analytical throughputs are required considering an increasing demand for faster, simple and improved methods to analyze contaminants in a considerable number of samples. Generally, these compounds are present in complex matrices in contact with a high number of interferents becoming their determination difficult at low concentration. In this context, on-line extraction techniques arose to improve the extraction as well as separation power, while minimizing errors related to human sample manipulation. This paper describes a study regarding the development and optimization of columns used as an extraction device in multidimensional liquid chromatography. The main goals were the evaluation of the material used as column body as well as the investigation of the tube dimensions (internal diameter and length) in the extraction performance. Firstly, several tube materials were tested (steel, fused silica, PEEK, among others) being steel whose reported the best performance and was consequently chose for further studies. The investigation about the effects of the columns physical dimensions revealed a linear relationship between performance and the amount of sorbent utilized as extractive phase. However, when different columns with same amount of sorbent were tested results suggests that both i.d. and lengths play an important role in extraction efficiency. The longest columns with lower internal diameter showed the best results favoring the radial as well as axial analytes diffusion into the extraction column. After evaluation of these column variables, applications were carried out employing several different analytes belonging to various chemical classes and practical utilization, in order to reinforce the versatility as well as the robustness of this proposed study.

Current status and future trends on automated multidimensional separation techniques employing sorbent-based extraction columns.

Determination of target analytes present in complex matrices requires a suitable sample preparation approach to efficiently remove the analytes of interest from a medium containing several interferers while at the same time preconcentrating them aiming to improve the output signal detection. Online multidimensional solid-phase separation techniques have been widely used for the analysis of different contaminants in complex matrices such as food, environmental, and biological samples, among others. These online techniques usually consist of two steps performed in two different columns (extraction and analytical column), the first being employed to extract the analytes of interest from the original medium and the latter to separate them from the interferers. The extraction column in multidimensional techniques presents a relevant role since their variations as building material (usually a tube), sorbent material, modes of application, and so on can significantly influence the extraction success. The main features of such columns are subject of constant research aiming improvements directly related to the performance of the separation techniques that utilize multidimensional analysis. The present review highlights the main features of extraction columns online coupled to chromatographic techniques, inclusive for in-tube solid-phase microextraction, online solid phase and turbulent flow, aiming the determination of analytes present at very low concentrations in complex matrices. It will critically describe and discuss some of the most common instrumental set up as well as comments on recent applications of these multidimensional techniques. Besides that, the authors have described some properties and enhancements of the extraction columns that are used as first dimension on these systems, such as type of column material (poly (ether ether ketone), fused silica, stainless steel, and other materials) and the way that the extractive phase is accommodated inside the tubing (filled and open tubular). Practical applications of this approach in fields such as environment, food, and bioanalysis are also presented and discussed.

Online fully automated SPE-HPLC-MS/MS determination of ceftiofur in bovine milk samples employing a silica-anchored ionic liquid as sorbent.

Solid-phase extraction coupled online with high performance liquid chromatography and tandem mass spectrometry was successfully applied to determine low concentrations of ceftiofur antibiotic in bovine milk samples. A silica-anchored ionic liquid was applied as sorbent material to be used as extraction phase in the proposed online system. The material was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. In order to improve the system reproducibility, the following experimental parameters were optimized: organic solvent percentage, time and sample loading flow rate. Subsequently, the method was validated presenting satisfactory results as adequate selectivity, good linearity and correlation coefficient higher than 0.98. The limit of detection and quantification were 0.1 and 0.7 µg/L, respectively. The precision of the methodology was evaluated as repeatability and intermediate precision, with relative standard deviation values lower than 15%. The accuracy of the method ranged from 72.8 to 137% and the minimum and maximum recovery values were 73.4 and 111.3%, respectively. After the validation, seven milk samples were analyzed and although ceftiofur was not detected in any of them the method was demonstrated to be efficient when applied to the analysis of milk samples fortified with the pollutant of interest.

The role of graphene-based sorbents in modern sample preparation techniques.

The application of graphene-based sorbents in sample preparation techniques has increased significantly since 2011. These materials have good physicochemical properties to be used as sorbent and have shown excellent results in different sample preparation techniques. Graphene and its precursor graphene oxide have been considered to be good candidates to improve the extraction and concentration of different classes of target compounds (e.g., parabens, polycyclic aromatic hydrocarbon, pyrethroids, triazines, and so on) present in complex matrices. Its applications have been employed during the analysis of different matrices (e.g., environmental, biological and food). In this review, we highlight the most important characteristics of graphene-based material, their properties, synthesis routes, and the most important applications in both off-line and on-line sample preparation techniques. The discussion of the off-line approaches includes methods derived from conventional solid-phase extraction focusing on the miniaturized magnetic and dispersive modes. The modes of microextraction techniques called stir bar sorptive extraction, solid phase microextraction, and microextraction by packed sorbent are discussed. The on-line approaches focus on the use of graphene-based material mainly in on-line solid phase extraction, its variation called in-tube solid-phase microextraction, and on-line microdialysis systems.

Recent approaches for on-line analysis of residues and contaminants in food matrices: A review.

This review highlights recent developments for on-line determination of residues and contaminants in complex matrices such as food samples. This involves the on-line coupling of a sample preparation technique (as the first "dimension") with a chromatographic system (second "dimension"), usually followed by mass spectrometry. Although frequently treated as quite distinct techniques, the role of all devices utilized as the first dimension in this approach aims to decrease the sample complexity while eliminating as much as possible the matrix contaminants to facilitate the qualitative and quantitative determination of the compounds of interest. This review will focus on the following techniques as the first dimension: (i) on-line solid-phase extraction; (ii) in-tube solid-phase microextraction; (iii) matrix solid-phase dispersion; and (iv) turbulent flow chromatography. The second dimension is usually performed using a chromatographic column to isolate the analyte(s) of interest for further mass spectrometry determination. A description of the basis of this on-line approach and its distinct set up possibilities is presented, which is followed by a critical review of the literature covering this subject in the last ten years (focusing on the last five years) with emphasis on the analysis of residue and contaminants in food samples.