Showing papers in "Analytical and Bioanalytical Chemistry in 2017"

Glycans and glycoproteins as specific biomarkers for cancer

Abstract: Protein glycosylation and other post-translational modifications are involved in potentially all aspects of human growth and development. Defective glycosylation has adverse effects on human physiological conditions and accompanies many chronic and infectious diseases. Altered glycosylation can occur at the onset and/or during tumor progression. Identifying these changes at early disease stages may aid in making decisions regarding treatments, as early intervention can greatly enhance survival. This review highlights some of the efforts being made to identify N- and O-glycosylation profile shifts in cancer using mass spectrometry. The analysis of single or panels of potential glycoprotein cancer markers are covered. Other emerging technologies such as global glycan release and site-specific glycosylation analysis and quantitation are also discussed.

Zeta potential: a case study of cationic, anionic, and neutral liposomes.

Abstract: Zeta potential is often used to approximate a nanoparticle’s surface charge, i.e., cationic, anionic, or neutral character, and has become a standard characterization technique to evaluate nanoparticle surfaces. While useful, zeta potential values provide only very general conclusions about surface charge character. Without a thorough understanding of the measurement parameters and limitations of the technique, these values can become meaningless. This case study attempts to explore the sensitivity of zeta potential measurement using specifically formulated cationic, anionic, and neutral liposomes. This study examines zeta potential dependence on pH and ionic strength, resolving power, and highlights the sensitivity of zeta potential to charged liposomes. Liposomes were prepared with cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and varying amounts of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS). A strong linear relationship was noted between zeta potential values and the mole percentage of charged lipids within a liposome (e.g., cationic DOTAP or anionic DOPS). This finding could be used to formulate similar liposomes to a specific zeta potential, potentially of importance for systems sensitive to highly charged species. In addition, cationic and anionic liposomes were titrated with up to two mole percent of the neutral lipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (lipid-PEG; LP). Very small amounts of the lipid-PEG (<0.2 mol%) were found to impart stability to the DOTAP- and DOPS-containing liposomes without significantly affecting other physicochemical properties of the formulation, providing a simple approach to making stable liposomes with cationic and anionic surface charge.

Raman spectroscopy as a process analytical technology for pharmaceutical manufacturing and bioprocessing

Abstract: Adoption of Quality by Design (QbD) principles, regulatory support of QbD, process analytical technology (PAT), and continuous manufacturing are major factors effecting new approaches to pharmaceutical manufacturing and bioprocessing. In this review, we highlight new technology developments, data analysis models, and applications of Raman spectroscopy, which have expanded the scope of Raman spectroscopy as a process analytical technology. Emerging technologies such as transmission and enhanced reflection Raman, and new approaches to using available technologies, expand the scope of Raman spectroscopy in pharmaceutical manufacturing, and now Raman spectroscopy is successfully integrated into real-time release testing, continuous manufacturing, and statistical process control. Since the last major review of Raman as a pharmaceutical PAT in 2010, many new Raman applications in bioprocessing have emerged. Exciting reports of in situ Raman spectroscopy in bioprocesses complement a growing scientific field of biological and biomedical Raman spectroscopy. Raman spectroscopy has made a positive impact as a process analytical and control tool for pharmaceutical manufacturing and bioprocessing, with demonstrated scientific and financial benefits throughout a product’s lifecycle.

Resolution-optimized headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) for non-targeted olive oil profiling.

Abstract: A prototype gas chromatography-ion mobility spectrometry (GC-IMS) system, hyphenating temperature-ramped headspace GC to a modified drift time IMS cell, was evaluated and compared to a conventional, isothermal capillary column (CC)-IMS system on the example of the geographical differentiation of extra virgin olive oils (EVOO) from Spain and Italy. It allows orthogonal, 2D separation of complex samples and individual detection of compounds in robust and compact benchtop systems. The information from the high-resolution 3D fingerprints of volatile organic compound (VOC) fractions of EVOO samples were extracted by specifically developed chemometric MATLAB® routines to differentiate between the different olive oil provenances. A combination of unsupervised principal component analysis (PCA) with two supervised procedures, linear discriminant analysis (LDA) and k-nearest neighbors (kNN), was applied to the experimental data. The results showed very good discrimination between oils of different geographical origins, featuring 98 and 92% overall correct classification rate for PCA-LDA and kNN classifier, respectively. Furthermore, the results showed that the higher resolved 3D fingerprints obtained from the GC-IMS system provide superior resolving power for non-targeted profiling of VOC fractions from highly complex samples such as olive oil. Graphical abstract Principle of the determination of geographic origins of olive oils by chemometric analysis of three-dimensional HS-GC-IMS fingerprints.

Moving from millifluidic to truly microfluidic sub-100-μm cross-section 3D printed devices

Abstract: Three-dimensional (3D) printing has generated considerable excitement in recent years regarding the extensive possibilities of this enabling technology. One area in which 3D printing has potential, not only for positive impact but also for substantial improvement, is microfluidics. To date many researchers have used 3D printers to make fluidic channels directed at point-of-care or lab-on-a-chip applications. Here, we look critically at the cross-sectional sizes of these 3D printed fluidic structures, classifying them as millifluidic (larger than 1 mm), sub-millifluidic (0.5–1.0 mm), large microfluidic (100–500 μm), or truly microfluidic (smaller than 100 μm). Additionally, we provide our prognosis for making 10–100-μm cross-section microfluidic features with custom-formulated resins and stereolithographic printers. Such 3D printed microfluidic devices for bioanalysis will accelerate research through designs that can be easily created and modified, allowing improved assays to be developed.

Glycomic and glycoproteomic analysis of glycoproteins—a tutorial

Abstract: The structural analysis of glycoproteins is a challenging endeavor and is under steadily increasing demand, but only a very limited number of labs have the expertise required to accomplish this task. This tutorial is aimed at researchers from the fields of molecular biology and biochemistry that have discovered that glycoproteins are important in their biological research and are looking for the tools to elucidate their structure. It provides brief descriptions of the major and most common analytical techniques used in glycomics and glycoproteomics analysis, including explanations of the rationales for individual steps and references to published literature containing the experimental details necessary to carry out the analyses. Glycomics includes the comprehensive study of the structure and function of the glycans expressed in a given cell or organism along with identification of all the genes that encode glycoproteins and glycosyltransferases. Glycoproteomics which is subset of both glycomics and proteomics is the identification and characterization of proteins bearing carbohydrates as posttranslational modification. This tutorial is designed to ease entry into the glycomics and glycoproteomics field for those without prior carbohydrate analysis experience.

Current applications of high-resolution mass spectrometry for the analysis of new psychoactive substances: a critical review

Abstract: The proliferation of new psychoactive substances (NPS) in recent years has resulted in the development of numerous analytical methods for the detection and identification of known and unknown NPS derivatives. High-resolution mass spectrometry (HRMS) has been identified as the method of choice for broad screening of NPS in a wide range of analytical contexts because of its ability to measure accurate masses using data-independent acquisition (DIA) techniques. Additionally, it has shown promise for non-targeted screening strategies that have been developed in order to detect and identify novel analogues without the need for certified reference materials (CRMs) or comprehensive mass spectral libraries. This paper reviews the applications of HRMS for the analysis of NPS in forensic drug chemistry and analytical toxicology. It provides an overview of the sample preparation procedures in addition to data acquisition, instrumental analysis, and data processing techniques. Furthermore, it gives an overview of the current state of non-targeted screening strategies with discussion on future directions and perspectives of this technique.

Reversed-phase separation methods for glycan analysis

Abstract: Reversed-phase chromatography is a method that is often used for glycan separation. For this, glycans are often derivatized with a hydrophobic tag to achieve retention on hydrophobic stationary phases. The separation and elution order of glycans in reversed-phase chromatography is highly dependent on the hydrophobicity of the tag and the contribution of the glycan itself to the retention. The contribution of the different monosaccharides to the retention strongly depends on the position and linkage, and isomer separation may be achieved. The influence of sialic acids and fucoses on the retention of glycans is still incompletely understood and deserves further study. Analysis of complex samples may come with incomplete separation of glycan species, thereby complicating reversed-phase chromatography with fluorescence or UV detection, whereas coupling with mass spectrometry detection allows the resolution of complex mixtures. Depending on the column properties, eluents, and run time, separation of isomeric and isobaric structures can be accomplished with reversed-phase chromatography. Alternatively, porous graphitized carbon chromatography and hydrophilic interaction liquid chromatography are also able to separate isomeric and isobaric structures, generally without the necessity of glycan labeling. Hydrophilic interaction liquid chromatography, porous graphitized carbon chromatography, and reversed-phase chromatography all serve different research purposes and thus can be used for different research questions. A great advantage of reversed-phase chromatography is its broad distribution as it is used in virtually every bioanalytical research laboratory, making it an attracting platform for glycan analysis. Graphical Abstract Glycan isomer separation by reversed phase liquid chromatography.

Spectroscopic sensors for in-line bioprocess monitoring in research and pharmaceutical industrial application

Abstract: The use of spectroscopic sensors for bioprocess monitoring is a powerful tool within the process analytical technology (PAT) initiative of the US Food and Drug Administration. Spectroscopic sensors enable the simultaneous real-time bioprocess monitoring of various critical process parameters including biological, chemical, and physical variables during the entire biotechnological production process. This potential can be realized through the combination of spectroscopic measurements (UV/Vis spectroscopy, IR spectroscopy, fluorescence spectroscopy, and Raman spectroscopy) with multivariate data analysis to obtain relevant process information out of an enormous amount of data. This review summarizes the newest results from science and industry after the establishment of the PAT initiative and gives a critical overview of the most common in-line spectroscopic techniques. Examples are provided of the wide range of possible applications in upstream processing and downstream processing of spectroscopic sensors for real-time monitoring to optimize productivity and ensure product quality in the pharmaceutical industry.

Development of an optimal filter substrate for the identification of small microplastic particles in food by micro-Raman spectroscopy.

Abstract: When analysing microplastics in food, due to toxicological reasons it is important to achieve clear identification of particles down to a size of at least 1 μm. One reliable, optical analytical technique allowing this is micro-Raman spectroscopy. After isolation of particles via filtration, analysis is typically performed directly on the filter surface. In order to obtain high qualitative Raman spectra, the material of the membrane filters should not show any interference in terms of background and Raman signals during spectrum acquisition. To facilitate the usage of automatic particle detection, membrane filters should also show specific optical properties. In this work, beside eight different, commercially available membrane filters, three newly designed metal-coated polycarbonate membrane filters were tested to fulfil these requirements. We found that aluminium-coated polycarbonate membrane filters had ideal characteristics as a substrate for micro-Raman spectroscopy. Its spectrum shows no or minimal interference with particle spectra, depending on the laser wavelength. Furthermore, automatic particle detection can be applied when analysing the filter surface under dark-field illumination. With this new membrane filter, analytics free of interference of microplastics down to a size of 1 μm becomes possible. Thus, an important size class of these contaminants can now be visualized and spectrally identified.

Divide and conquer: cleavable cross-linkers to study protein conformation and protein-protein interactions.

Abstract: Chemical cross-linking combined with mass spectrometry (MS) and computational modeling has evolved as an alternative method to address fundamental questions in structural biology. The constraints revealed by the cross-links yield valuable distance information and allow one to deduce three-dimensional structural information on very large and transient protein complexes. During the past few years, technical advances in the cross-linking/MS approach have been enormous, mainly owing to the fantastic advances in MS technology, and it is easily overlooked that significant progress has been made in the design of novel cross-linking reagents. In this review, the advent of cleavable cross-linking reagents will be highlighted. In particular, gas-phase (MS-) cleavable cross-linkers offer unique properties for an automated, data-dependent assignment of cross-linked products based on the generation of characteristic fragment ion signatures in MS/MS and MS3 spectra. Therefore, MS-cleavable cross-linkers are envisioned to hold the key for proteome-wide applications of the chemical cross-linking/MS approach, not only to delineate the conformation of single proteins but also to decipher protein interaction networks. Graphical Abstract Outline of the analytical strategy using cleavable cross-linkers in combination with MS to conduct protein conformational and protein interaction studies.

Rapid analysis of bile acids in different biological matrices using LC-ESI-MS/MS for the investigation of bile acid transformation by mammalian gut bacteria

Abstract: Bile acids are important signaling molecules that regulate cholesterol, glucose, and energy homoeostasis and have thus been implicated in the development of metabolic disorders. Their bioavailability is strongly modulated by the gut microbiota, which contributes to generation of complex individual-specific bile acid profiles. Hence, it is important to have accurate methods at hand for precise measurement of these important metabolites. Here, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and quantitation of primary and secondary bile acids as well as their taurine and glycine conjugates was developed and validated. Applicability of the method was demonstrated for mammalian tissues, biofluids, and cell culture media. The analytical approach mainly consists of a simple and rapid liquid-liquid extraction procedure in presence of deuterium-labeled internal standards. Baseline separation of all isobaric bile acid species was achieved and a linear correlation over a broad concentration range was observed. The method showed acceptable accuracy and precision on intra-day (1.42–11.07 %) and inter-day (2.11–12.71 %) analyses and achieved good recovery rates for representative analytes (83.7–107.1 %). As a proof of concept, the analytical method was applied to mouse tissues and biofluids, but especially to samples from in vitro fermentations with gut bacteria of the family Coriobacteriaceae. The developed method revealed that the species Eggerthella lenta and Collinsella aerofaciens possess bile salt hydrolase activity, and for the first time that the species Enterorhabdus mucosicola is able to deconjugate and dehydrogenate primary bile acids in vitro.

Parameterizing amylose chain-length distributions for biosynthesis-structure-property relations.

Abstract: Amylose, one of the components of starch, is a glucose polymer consisting largely of long, linear chains with a few long-chain branch points. The chain-length (molecular weight) distribution (CLD) of the component chains of amylose can provide information on amylose biosynthesis-structure-property relations, as has been done previously by fitting amylopectin CLDs to a model with physically meaningful parameters. Due to the presence of long chains, the CLD of amylose can currently best be obtained by size-exclusion chromatography, a technique that suffers from band-broadening effects which alter the observed distribution. The features of the multiple regions present in amylose chain-length distributions are also difficult to resolve, an issue that combines with band broadening to compound the difficulty of analysis and subsequent parameterization of the structural characteristics of amylose. A new method is presented to fit these distributions with biologically meaningful parameters in a way that accounts for band broadening. This is achieved by assuming that band broadening takes the form of a simple Gaussian over a relatively small region and that chain stoppage is a random process independent of the length of the substrate chain over the same region; these assumptions are relatively weak and expected to be frequently applicable. The method provides inbuilt consistency tests for its applicability to a given data set and, in cases where it is applicable, allows for the first nonempirical parameterization of amylose biosynthesis-structure-property relations from CLDs by using parameters directly linked to the activities of the enzymes responsible for chain growth and chain stoppage.

Identifying known unknowns using the US EPA’s CompTox Chemistry Dashboard

Abstract: Chemical features observed using high-resolution mass spectrometry can be tentatively identified using online chemical reference databases by searching molecular formulae and monoisotopic masses and then rank-ordering of the hits using appropriate relevance criteria. The most likely candidate “known unknowns,” which are those chemicals unknown to an investigator but contained within a reference database or literature source, rise to the top of a chemical list when rank-ordered by the number of associated data sources. The U.S. EPA’s CompTox Chemistry Dashboard is a curated and freely available resource for chemistry and computational toxicology research, containing more than 720,000 chemicals of relevance to environmental health science. In this research, the performance of the Dashboard for identifying known unknowns was evaluated against that of the online ChemSpider database, one of the primary resources used by mass spectrometrists, using multiple previously studied datasets reported in the peer-reviewed literature totaling 162 chemicals. These chemicals were examined using both applications via molecular formula and monoisotopic mass searches followed by rank-ordering of candidate compounds by associated references or data sources. A greater percentage of chemicals ranked in the top position when using the Dashboard, indicating an advantage of this application over ChemSpider for identifying known unknowns using data source ranking. Additional approaches are being developed for inclusion into a non-targeted analysis workflow as part of the CompTox Chemistry Dashboard. This work shows the potential for use of the Dashboard in exposure assessment and risk decision-making through significant improvements in non-targeted chemical identification.

Extracellular matrix proteomics in schizophrenia and Alzheimer’s disease

Abstract: Brain extracellular matrix (ECM) is a highly organized system that consists of collagens, noncollagenous proteins, glycoproteins, hyaluronan, and proteoglycans. Recognized physiological roles of ECM include developmental regulation, tissue homeostasis, cell migration, cell proliferation, cell differentiation, neuronal plasticity, and neurite outgrowth. Aberrant ECM structure is associated with brain neurodegenerative conditions. This review focuses on two neurodegenerative conditions, schizophrenia and Alzheimer’s disease, and summarizes recent findings of altered ECM components, including proteoglycans, glycosaminoglycans, proteins, and glycoproteins, and proteins and genes related to other brain components. The scope includes immunohistochemical, genomics, transcriptomics, proteomics, and glycomics studies, and a critical assessment of current state of proteomic studies for neurodegenerative disorders. The intent is to summarize the ECM molecular alterations associated with neurodegenerative pathophysiology.

Asymmetrical flow field flow fractionation methods to characterize submicron particles: application to carbon-based aggregates and nanoplastics

Abstract: In the last 10 years, asymmetrical flow field flow fractionation (AF4) has been one of the most promising approaches to characterize colloidal particles. Nevertheless, despite its potentialities, it is still considered a complex technique to set up, and the theory is difficult to apply for the characterization of complex samples containing submicron particles and nanoparticles. In the present work, we developed and propose a simple analytical strategy to rapidly determine the presence of several submicron populations in an unknown sample with one programmed AF4 method. To illustrate this method, we analyzed polystyrene particles and fullerene aggregates of size covering the whole colloidal size distribution. A global and fast AF4 method (method O) allowed us to screen the presence of particles with size ranging from 1 to 800 nm. By examination of the fractionating power F d, as proposed in the literature, convenient fractionation resolution was obtained for size ranging from 10 to 400 nm. The global F d values, as well as the steric inversion diameter, for the whole colloidal size distribution correspond to the predicted values obtained by model studies. On the basis of this method and without the channel components or mobile phase composition being changed, four isocratic subfraction methods were performed to achieve further high-resolution separation as a function of different size classes: 10-100 nm, 100-200 nm, 200-450 nm, and 450-800 nm in diameter. Finally, all the methods developed were applied in characterization of nanoplastics, which has received great attention in recent years. Graphical Absract Characterization of the nanoplastics by asymmetrical flow field flow fractionation within the colloidal size range.

Field flow fractionation techniques to explore the “nano-world”

Abstract: Field flow fractionation (FFF) techniques are used to successfully characterize several nanomaterials by sizing nano-entities and producing information about the aggregation/agglomeration state of nanoparticles. By coupling FFF techniques to specific detectors, researchers can determine particle-size distributions (PSDs), expressed as mass-based or number-based PSDs. This review considers FFF applications in the food, biomedical, and environmental sectors, mostly drawn from the past 4 y. It thus underlines the prominent role of asymmetrical flow FFF within the FFF family. By concisely comparing FFF techniques with other techniques suitable for sizing nano-objects, the advantages and the disadvantages of these instruments become clear. A consideration of select recent publications illustrates the state of the art of some lesser-known FFF techniques and innovative instrumental set-ups.

Identification of terpenes and essential oils by means of static headspace gas chromatography-ion mobility spectrometry.

Abstract: Static headspace gas chromatography-ion mobility spectrometry (SHS GC-IMS) is a relatively new analytical technique that has considerable potential for analysis of volatile organic compounds (VOCs). In this study, SHS GC-IMS was used for the identification of the major terpene components of various essential oils (EOs). Based on the data obtained from 25 terpene standards and 50 EOs, a database for fingerprint identification of characteristic terpenes and EOs was generated utilizing SHS GC-IMS for authenticity testing of fragrances in foods, cosmetics, and personal care products. This database contains specific normalized IMS drift times and GC retention indices for 50 terpene components of EOs. Initially, the SHS GC-IMS parameters, e.g., drift gas and carrier gas flow rates, drift tube, and column temperatures, were evaluated to determine suitable operating conditions for terpene separation and identification. Gas chromatography-mass spectrometry (GC-MS) was used as a reference method for the identification of terpenes in EOs. The fingerprint pattern based on the normalized IMS drift times and retention indices of 50 terpenes is presented for 50 EOs. The applicability of the method was proven on examples of ten commercially available food, cosmetic, and personal care product samples. The results confirm the suitability of SHS GC-IMS as a powerful analytical technique for direct identification of terpene components in solid and liquid samples without any pretreatment. Graphical abstract Fingerprint pattern identification of terpenes and essential oils using static headspace gas chromatography-ion mobility spectrometry.

Enhancing glycan isomer separations with metal ions and positive and negative polarity ion mobility spectrometry-mass spectrometry analyses

Abstract: Glycomics has become an increasingly important field of research since glycans play critical roles in biology processes ranging from molecular recognition and signaling to cellular communication. Glycans often conjugate with other biomolecules, such as proteins and lipids, and alter their properties and functions, so glycan characterization is essential for understanding the effects they have on cellular systems. However, the analysis of glycans is extremely difficult due to their complexity and structural diversity (i.e., the number and identity of monomer units, and configuration of their glycosidic linkages and connectivities). In this work, we coupled ion mobility spectrometry with mass spectrometry (IMS-MS) to characterize glycan standards and biologically important isomers of synthetic αGal-containing O-glycans including glycotopes of the protozoan parasite Trypanosoma cruzi, which is the causative agent of Chagas disease. IMS-MS results showed significant differences for the glycan structural isomers when analyzed in positive and negative polarity and complexed with different metal cations. These results suggest that specific metal ions or ion polarities could be used to target and baseline separate glycan isomers of interest with IMS-MS.

Chemometrics in analytical chemistry-part I: history, experimental design and data analysis tools.

Abstract: Chemometrics has achieved major recognition and progress in the analytical chemistry field. In the first part of this tutorial, major achievements and contributions of chemometrics to some of the more important stages of the analytical process, like experimental design, sampling, and data analysis (including data pretreatment and fusion), are summarised. The tutorial is intended to give a general updated overview of the chemometrics field to further contribute to its dissemination and promotion in analytical chemistry.

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization.

Abstract: The biosynthesis of glycans is a template-free process; hence compositionally identical glycans may contain highly heterogeneous structures Meanwhile, the functions of glycans in biological processes are significantly influenced by the glycan structure Structural elucidation of glycans is an essential component of glycobiology Although NMR is considered the most powerful approach for structural glycan studies, it suffers from low sensitivity and requires highly purified glycans Although mass spectrometry (MS)-based methods have been applied in numerous glycan structure studies, there are challenges in preserving glycan structure during ionization Permethylation is an efficient derivatization method that improves glycan structural stability In this report, permethylated glycans are isomerically separated; thus facilitating structural analysis of a mixture of glycans by LC-MS/MS Separation by porous graphitic carbon liquid chromatography at high temperatures in conjunction with tandem mass spectrometry (PGC-LC-MS/MS) was utilized for unequivocal characterization of glycan isomers Glycan fucosylation sites were confidently determined by eliminating fucose rearrangement and assignment of diagnostic ions, achieved by permethylation and PGC-LC at high temperatures, respectively Assigning monosaccharide residues to specific glycan antennae was also achieved Galactose linkages were also distinguished from each other by CID/HCD tandem MS This was attainable because of the different bond energies associated with monosaccharide linkages Graphical Abstract LC-MS and tandem MS of terminal galactose isomers

Quantification of three chlorinated dialkyl phosphates, diphenyl phosphate, 2,3,4,5-tetrabromobenzoic acid, and four other organophosphates in human urine by solid phase extraction-high performance liquid chromatography-tandem mass spectrometry

Abstract: Polybrominated diphenyl ethers (PBDEs), produced as flame retardants worldwide, have been phased-out in many countries, and chlorinated and non-chlorinated organophosphates and non-PBDE brominated formulations (e.g., Firemaster 550 (FM550)) have entered the consumers’ market. Recent studies show that components of organophosphate esters and FM550 are frequently detected in many products common to human environments. Therefore, urinary metabolites of these compounds can be used as human exposure biomarkers. We developed a method to quantify nine compounds in 0.4 mL urine: diphenyl phosphate (DPhP), bis(1,3-dichloro-2-propyl) phosphate (BDCPP), bis-(1-chloro-2-propyl) phosphate, bis-2-chloroethyl phosphate, di-p-cresylphosphate, di-o-cresylphosphate (DoCP), di-n-butyl phosphate, dibenzyl phosphate (DBzP), and 2,3,4,5-tetrabromobenzoic acid. The method relies on an enzymatic hydrolysis of urinary conjugates of the target analytes, automated off-line solid phase extraction, reversed phase high performance liquid chromatography separation, and isotope dilution-electrospray ionization tandem mass spectrometry detection. The method is high-throughput (96 samples/day) with detection limits ranging from 0.05 to 0.16 ng mL−1. Spiked recoveries were 90–113 %, and interday imprecision was 2–8 %. We assessed the suitability of the method by analyzing urine samples collected from a convenience sample of adults (n = 76) and from a group of firefighters (n = 146). DPhP (median, 0.89; range, 0.26–5.6 ng mL−1) and BDCPP (median, 0.69; range, 0.31–6.8 ng mL−1) were detected in all of the non-occupationally exposed adult samples and all of the firefighter samples (DPhP [median, 2.9; range, 0.24–28 ng mL−1], BDCPP [median, 3.4; range, 0.30–44 ng mL−1]); DBzP and DoCP were not detected in any samples.

Perspectives and challenges of photon-upconversion nanoparticles - Part I: routes to brighter particles and quantitative spectroscopic studies

Abstract: Lanthanide-doped photon-upconversion nanoparticles (UCNPs) have been the focus of many research activities in materials and life sciences in the last 15 years because of their potential to convert light between different spectral regions and their unique photophysical properties. To fully exploit the application potential of these fascinating nanomaterials, a number of challenges have to be overcome, such as the low brightness, particularly of small UCNPs, and the reliable quantification of the excitation-power-density-dependent upconversion luminescence. In this series of critical reviews, recent developments in the design, synthesis, optical-spectroscopic characterization, and application of UCNPs are presented with special focus on bioanalysis and the life sciences. Here we guide the reader from the synthesis of UCNPs to different concepts to enhance their luminescence, including the required optical-spectroscopic assessment to quantify material performance; surface modification strategies and bioanalytical applications as well as selected examples of the use of UCNPs as reporters in different assay formats are addressed in part II. Future trends and challenges in the field of upconversion are discussed with special emphasis on UCNP synthesis and material characterization, particularly quantitative luminescence studies. Graphical Abstract Both synthesis and spectroscopy as well bioanalytical applications of UCNPs are driven and supported by COST Action CM1403 "The European Upconversion Network".

Quantification of urinary mono-hydroxylated metabolites of polycyclic aromatic hydrocarbons by on-line solid phase extraction-high performance liquid chromatography-tandem mass spectrometry.

Abstract: Human exposure to polycyclic aromatic hydrocarbons (PAHs) can be assessed through monitoring of urinary mono-hydroxylated PAHs (OH-PAHs) Gas chromatography (GC) has been widely used to separate OH-PAHs before quantification by mass spectrometry in biomonitoring studies However, because GC requires derivatization, it can be time consuming We developed an on-line solid phase extraction coupled to isotope dilution-high performance liquid chromatography-tandem mass spectrometry (on-line-SPE-HPLC-MS/MS) method for the quantification in urine of 1-OH-naphthalene, 2-OH-naphthalene, 2-OH-fluorene, 3-OH-fluorene, 1-OH-phenanthrene, the sum of 2-OH and 3-OH-phenanthrene, 4-OH-phenanthrene, and 1-OH-pyrene The method, which employed a 96-well plate platform and on-line SPE, showed good sensitivity (ie, limits of detection ranged from 0007 to 009 ng/mL) and used only 100 μL of urine Accuracy, calculated from the recovery percentage at three spiking levels, varied from 94 to 113 %, depending on the analyte The inter- and intra-day precision, calculated from 20 repeated measurements of two quality control materials, varied from 52 to 167 % Adequate method performance was also confirmed by acceptable recovery (83–102 %) of two NIST standard reference materials (3672 and 3673) This high-throughput on-line-SPE-HPLC-MS/MS method can be applied in large-scale epidemiological studies

Rapid urinary tract infection diagnostics by surface-enhanced Raman spectroscopy (SERS): identification and antibiotic susceptibilities

Abstract: SERS spectra of 12 bacterial strains of urinary tract infection (UTI) clinical isolates grown and enriched from urine are reported. A partial least squares-discriminant analysis (PLS-DA) classification treatment of these SERS spectra results in strain level identification with >95% sensitivity and >99% specificity. The classification model successfully identified the SERS spectra of a urine-cultured strain not used to build this statistical model. Enrichment was accomplished by a filtration and centrifugation protocol. The predetermined drug susceptibility profiles of these clinical isolates thus allowed the SERS methodology to provide appropriate UTI antibiotic information in less than 1 h. Most of this time was used for sample preparation procedures (enrichment and washing) for this proof of principle study. SERS spectra of the enriched bacterial samples are dominated by nucleotide degradation metabolites: adenine, hypoxanthine, xanthine, guanine, uric acid, AMP, and guanosine. Strain-specific specificity is due to the different relative amounts of these purines contributing to the corresponding SERS spectra of these clinical isolates. All measurements were made at the minimal bacterial concentration in urine for UTI diagnosis (105 cfu/mL).

Electrochemical aptamer-based biosensor developed to monitor PSA and VEGF released by prostate cancer cells

Abstract: Early prostate cancer (PCa) diagnostic is crucial to enhance patient survival rates; besides, non-invasive platforms have been developed worldwide in order to precisely detect PCa biomarkers. Therefore, the aim of the present study is to develop a new aptamer-based biosensor through the self-assembling of thiolated aptamers for PSA and VEGF on the top of gold electrodes. This biosensor was tested in three prostate cell lines (RWPE-1, LNCaP and PC3). The results evidenced a stable and sensitive sensor presenting wide linear detection ranges (0.08–100 ng/mL for PSA and 0.15 ng–100 ng/mL for VEGF). Therefore, the aptasensor was able to detect the patterns of PSA and VEGF released in vitro by PCa cells, which gave new insights about the prostate cancer protein dynamics. Thus, it could be used as a non-invasive PCa clinical diagnosis instrument in the near future.

Cobalt oxyhydroxide nanoflakes with intrinsic peroxidase catalytic activity and their application to serum glucose detection.

Abstract: Cobalt oxyhydroxide (CoOOH) nanoflakes, an emerging type of two-dimensional nanomaterial, show great potential for use in molecular detection. Previous assays utilizing such materials have largely been based on their outstanding fluorescence quenching ability and oxidizing power. Herein, we report the intrinsic peroxidase-like activity of cobalt oxyhydroxide (CoOOH) nanoflakes, and we show how this activity can be employed for glucose detection. We found that, in the presence of hydrogen peroxide (H2O2), the nanoflakes accelerated the conversion of peroxidase substrates such as 3,3′,5,5′-tetramethylbenzidine (TMB) into colored products. By combining the CoOOH nanoflakes with the biological enzyme glucose oxidase (GOx), we developed a colorimetric method for the detection of glucose within the concentration range 5.3–500 μM. The proposed method was applied to detect elevated blood glucose levels in diabetic patients, and the intense color change induced by elevated glucose levels was found to be readily apparent to the naked eye, proving the utility of our assay for point-of-care testing.

Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals

Abstract: Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.

Perspectives and challenges of photon-upconversion nanoparticles - Part II: bioanalytical applications

Abstract: In Part II of this review series on lanthanide-doped photon-upconversion nanoparticles (UCNPs), we present and critically discuss the performance and suitability of UCNPs as background-free luminescent reporters in bioimaging and bioanalytical applications. The preparation of a biocompatible nanoparticle surface is an integral step for all life - science-related applications. UCNPs have found their way into a large number of diagnostic platforms, homogeneous and heterogeneous assay formats, and sensor applications. Many bioanalytical detection schemes involve Forster resonance energy transfer (FRET), which is still debated for UCNPs and needs to be much improved. The need for dedicated and standardized instruments as well as recent studies on the dissolution and potential toxicity of UCNPs are addressed. Finally we outline future trends and challenges in the field of upconversion.

Two-dimensional capillary zone electrophoresis-mass spectrometry for the characterization of intact monoclonal antibody charge variants, including deamidation products.

Abstract: Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE–CZE–MS setup with an implemented mechanical four-port valve interface was developed that used a generic e-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE–CZE–MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes.