Explore the vast range of titles available.

Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.

This protocol describes how in-solution protein FTIR can be used to obtain information about the relative contributions of α-helices, β-sheets, β-turn, and random coil structures to a protein's secondary structure. Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. The repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I–VII). Amide I bands (1,700–1,600 cm −1 ) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the determination of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample preparation, instrument operation, FTIR spectra collection and spectra analysis in order to estimate protein secondary-structural components in aqueous (both H 2 O and deuterium oxide (D 2 O)) solution using algorithms, such as second-derivative, deconvolution and curve fitting. Small amounts of high-purity (>95%) proteins at high concentrations (>3 mg ml −1 ) are needed in this protocol; typically, the procedure can be completed in 1–2 d.

Fabric phase sorptive extraction (FPSE) is an evolutionary sample preparation approach which was introduced in 2014, meeting all green analytical chemistry (GAC) requirements by implementing a natural or synthetic permeable and flexible fabric substrate to host a chemically coated sol–gel organic–inorganic hybrid sorbent in the form of an ultra-thin coating. This construction results in a versatile, fast, and sensitive micro-extraction device. The user-friendly FPSE membrane allows direct extraction of analytes with no sample modification, thus eliminating/minimizing the sample pre-treatment steps, which are not only time consuming, but are also considered the primary source of major analyte loss. Sol–gel sorbent-coated FPSE membranes possess high chemical, solvent, and thermal stability due to the strong covalent bonding between the fabric substrate and the sol–gel sorbent coating. Subsequent to the extraction on FPSE membrane, a wide range of organic solvents can be used in a small volume to exhaustively back-extract the analytes after FPSE process, leading to a high preconcentration factor. In most cases, no solvent evaporation and sample reconstitution are necessary. In addition to the extensive simplification of the sample preparation workflow, FPSE has also innovatively combined the extraction principle of two major, yet competing sample preparation techniques: solid phase extraction (SPE) with its characteristic exhaustive extraction, and solid phase microextraction (SPME) with its characteristic equilibrium driven extraction mechanism. Furthermore, FPSE has offered the most comprehensive cache of sorbent chemistry by successfully combining almost all of the sorbents traditionally used exclusively in either SPE or in SPME. FPSE is the first sample preparation technique to exploit the substrate surface chemistry that complements the overall selectivity and the extraction efficiency of the device. As such, FPSE indeed represents a paradigm shift approach in analytical/bioanalytical sample preparation. Furthermore, an FPSE membrane can be used as an SPME fiber or as an SPE disk for sample preparation, owing to its special geometric advantage. So far, FPSE has overwhelmingly attracted the interest of the separation scientist community, and many analytical scientists have been developing new methodologies by implementing this cutting-edge technique for the extraction and determination of many analytes at their trace and ultra-trace level concentrations in environmental samples as well as in food, pharmaceutical, and biological samples. FPSE offers a total sample preparation solution by providing neutral, cation exchanger, anion exchanger, mixed mode cation exchanger, mixed mode anion exchanger, zwitterionic, and mixed mode zwitterionic sorbents to deal with any analyte regardless of its polarity, ionic state, or the sample matrix where it resides. Herein we present the theoretical background, synthesis, mechanisms of extraction and desorption, the types of sorbents, and the main applications of FPSE so far according to different sample categories, and to briefly show the progress, advantages, and the main principles of the proposed technique.

X-ray single-crystal diffraction (SCD) analysis has the intrinsic limitation that the target molecules must be obtained as single crystals. Here we report a protocol for SCD analysis that does not require the crystallization of the sample. In our method, tiny crystals of porous complexes are soaked in a solution of the target, such that the complexes can absorb the target molecules. Crystallographic analysis clearly determines the absorbed guest structures along with the host frameworks. Because the SCD analysis is carried out on only one tiny crystal of the complex, the required sample mass is of the nanogram–microgram order. We demonstrate that as little as about 80 nanograms of a sample is enough for the SCD analysis. In combination with high-performance liquid chromatography, our protocol allows the direct characterization of multiple fractions, establishing a prototypical means of liquid chromatography SCD analysis. Furthermore, we unambiguously determined the structure of a scarce marine natural product using only 5 micrograms of the compound. Absorption of target molecules into a porous matrix permits single-crystal X-ray diffraction analysis of the ‘guest’ molecules, avoiding the need to obtain them in single-crystal form and making analysis possible using as little as 80 nanograms of sample. Crystal structure without the crystals X-ray single crystal diffraction provides direct structural information of molecules at the atomic level and is recognized as a reliable structure determination method. However, as its name implies, the technique has a limitation, the sample needs to be available as a single crystal, the growth of which can be a time consuming process of trial-and-error. This paper describes a new X-ray analysis protocol that does not require crystallization of the sample itself. Instead, crystalline 'sponges' known as metal organic frameworks are used to soak up a drop of a liquid guest containing the target molecule. The sponges contain pores that recognize the target molecules and bind them in an ordered array, enabling the crystallographic structure analysis of the absorbed guest along with the host framework. The method is demonstrated with the absolute structure determination of a scarce natural product, miyakosyne A, using little more than a trace, 5 μg of sample. Corrected 8 April 2013

A method, filter-aided sample preparation (FASP) combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics, allowing deeper proteomic coverage in a shorter analysis time, using small sample amounts. We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.

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

In recent years, green solvents have emerged as promising alternatives in the field of analytical chemistry, replacing conventional organic solvents known for their toxicity, volatility, and flammability. The combination of these solvents with liquid–liquid microextraction techniques has facilitated the development of simpler, faster, more economical, and environment-friendly methodologies for the analysis of samples of varying complexity. This review discusses the fundamental physicochemical properties and advantages of using deep eutectic solvents, ionic liquids, switchable-hydrophilicity solvents, supramolecular solvents, and surfactants as extractants. Furthermore, analytical methods based on liquid–liquid microextraction techniques developed in the last 5 years for the determination of organic compounds and metals in biological and environmental samples are presented and discussed, highlighting their applications and benefits to improve analytical performance and sustainability. Graphical Abstract

Miniaturised sampling and extraction are redefining therapeutic drug monitoring (TDM) by enabling low-volume sampling, simplifying collection, and improving patient acceptability, while also promoting decentralised workflows and more sustainable laboratory practices. This review critically appraises the current landscape, with emphasis on analytical performance, matrix compatibility, and readiness for clinical implementation. It examines validation requirements, the extent of alignment and existing gaps across major regulatory guidelines, and recurrent challenges such as haematocrit bias, real-world stability and transport, incurred sample reanalysis, device variability, commutability with conventional matrices, and inter-laboratory reproducibility. To make the evidence actionable, operational recommendations are distilled into a practical ten-point checklist designed to support validation and translation of miniaturised approaches into routine laboratory practice. Looking ahead, priorities include automation and portable platforms, advanced functional materials, and integration with digital tools and biosensors, alongside the development of harmonised frameworks tailored to miniaturised methods and prospective clinical studies that demonstrate impact on dosing decisions, adherence, and clinical outcomes. Overall, this review aims to equip researchers, laboratory professionals, and regulators with the knowledge to implement miniaturised bioanalysis and advance personalised medicine through TDM.

Raman imaging of plant cell walls represents a nondestructive technique that can provide insights into chemical composition in context with structure at the micrometer level (<0.5.mu m). The major steps of the experimental procedure are described: sample preparation (embedding and microcutting), setting the mapping parameters, and finally the calculation of chemical images on the basis of the acquired Raman spectra. Every Raman image is based on thousands of spectra, each being a spatially resolved molecular 'fingerprint' of the cell wall. Multiple components are analyzed within the native cell walls, and insights into polymer composition as well as the orientation of the cellulose microfibrils can be gained. The most labor-intensive step of this process is often the sample preparation, as the imaging approach requires a flat surface of the plant tissue with intact cell walls. After finishing the map (acquisition time is similar to 10 min to 10 h, depending on the size of the region of interest and scanning parameters), many possibilities exist for the analysis of spectral data and image generation.

Functionalized magnetic nanoparticles have attracted much attention in sample preparation because of their excellent performance compared with traditional sample-preparation sorbents. In this review, we describe the application of magnetic nanoparticles functionalized with silica, octadecylsilane, carbon-based material, surfactants, and polymers as adsorbents for separation and preconcentration of analytes from a variety of matrices. Magnetic solid-phase extraction (MSPE) techniques, mainly reported in the last five years, are presented and discussed.