Analytical and Bioanalytical Chemistry (v.410, #27)
Be unafraid … to try something new or challenging by Adam T. Woolley (6973-6974).
Yiyang Dong (Ed.): Direct analysis in real time mass spectrometry: principles and practices of DART-MS by Jürgen H. Gross (6975-6976).
An assessment of retention behavior for gold nanorods in asymmetrical flow field-flow fractionation by Hind El Hadri; Julien Gigault; Jiaojie Tan; Vincent A. Hackley (6977-6984).
Applications of asymmetrical flow field-flow fractionation (AF4) continue to expand rapidly in the fields of nanotechnology and biotechnology. In particular, AF4 has proven valuable for the separation and analysis of particles, biomolecular species (e.g., proteins, bacteria) and polymers (natural and synthetic), ranging in size from a few nanometers to several micrometers. The separation of non-spheroidal structures (e.g., rods, tubes, etc.) with primary dimensions in the nanometer regime, is a particularly challenging application deserving of greater study and consideration. The goal of the present study was to advance current understanding of the mechanism of separation of rod-like nano-objects in the AF4 channel. To achieve this, we have systematically investigated a series of commercially available cetyltrimethylammonium bromide stabilized gold nanorods (AuNRs), with aspect ratios from 1.7 to 10. Results show clearly that the retention time is principally dependent on the translational diffusion coefficient of the AuNRs. Equations used to calculate translational and rotational diffusion coefficients (cylinder and prolate ellipsoid models) yield similarly good fits to experimental data. Well characterized gold nanorods (length and diameter by transmission electron microscopy) can be used as calibrants for AF4 measurements allowing one to determine the aspect ratio of nanorod samples based on their retention times. Graphical abstractᅟ
Keywords: Aspect ratio; Diffusion coefficient; Field flow fractionation; Gold nanorod; Retention; Separation
A critical analysis of L-asparaginase activity quantification methods—colorimetric methods versus high-performance liquid chromatography by Agnes Magri; Matheus F. Soler; André M. Lopes; Eduardo M. Cilli; Patrick S. Barber; Adalberto Pessoa Jr; Jorge F. B. Pereira (6985-6990).
L-asparaginase or ASNase (L-asparagine aminohydrolase, E.C.18.104.22.168) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma through the depletion of L-asparagine (L-Asn) resulting in cytotoxicity to leukemic cells. ASNase is also important in the food industry, preventing acrylamide formation in processed foods. Several quantification techniques have been developed and used for the measurement of the ASNase activity, but standard pharmaceutical quality control methods were hardly reported, and in general, no official quality control guidelines were defined. To overcome this lack of information and to demonstrate the advantages and limitations, this work properly compares the traditional colorimetric methods (Nessler; L-aspartic acid β-hydroxamate (AHA); and indooxine) and the high-performance liquid chromatography (HPLC) method. A comparison of the methods using pure ASNase shows that the colorimetric methods both overestimate (Nessler) and underestimate (AHA and indooxine) the ASNase activity when compared to the values obtained with HPLC, considered the most precise method as this method monitors both substrate consumption and product formation, allowing for overall mass-balance. Correlation and critical analysis of each method relative to the HPLC method were carried out, resulting in a demonstration that it is crucial to select a proper method for the quantification of ASNase activity, allowing bioequivalence studies and individualized monitoring of different ASNase preparations. Graphical abstractᅟ
Keywords: L-asparaginase; Enzymatic activity; L-aspartic acid; Nessler; L-aspartic acid β-hydroxamate; Indooxine
Quantification of BSA-loaded chitosan/oligonucleotide nanoparticles using reverse-phase high-performance liquid chromatography by Chun Y. Wong; Jorge Martinez; Hani Al-Salami; Crispin R. Dass (6991-7006).
obtained B. Pharm (Honours) from Curtin University in 2016. He is currently a pharmaceutical science PhD candidate, developing novel oral peptide nanoparticulate and microparticulate formulations, in both Curtin Health Innovation Research Institute and School of Pharmacy and Biomedical Science. is a pharmaceutical technology officer and he is responsible for the management and operation of the Pharmaceutical Technology Facility, Curtin University. His field of expertise includes tableting, particle sizing, microencapsulation, HPLC, spray drying, aseptic compounding, drug release and stability studies. is an Otago School of Pharmacy graduate and an Australian and a New Zealand registered pharmacist, who owned a Dunedin-based pharmacy, before working at Otago School of Pharmacy (New Zealand) and McGill Faculty of Medicine (Canada). Hani is currently teaching and researching in the area of bio-nanotechnology and drug delivery, and is the founder of Biotechnology and Drug Development Research Laboratory at Curtin University. is a Charles Sturt University Medical Biotechnology graduate, who has worked in academia, private industry and hospitals for the past 20 years. He currently teaches Pharmacy and Biomedical Science students at Curtin University. Therapeutic proteins are administered subcutaneously because of their instability in the gastrointestinal tract. Current research suggests that polymeric-based nanoparticles, microparticles and liposomes are ideal nanocarriers to encapsulate proteins for disease management. In order to develop a successful drug delivery system, it is crucial to determine drug release profile and stability. However, the non-active excipients in polymeric formulations can influence the quantification of proteins in analytical techniques. This study investigated the effect of nine common polymers on quantification of bovine serum albumin (BSA) using RP-HPLC method. The technique offers advantages such as short analytical time, high accuracy and selectivity. In the meantime, the technique can be employed to separate proteins including BSA, insulin and pigment epithelium-derived factor (PEDF). Furthermore, the RP-HPLC method was applied to quantify the drug release pattern of a novel BSA-loaded nanoparticulate formulation in simulated gastric and intestinal fluids. The nanoparticles were formulated by natural polymer (chitosan) and oligonucleotide (Dz13Scr) using complex coacervation. The prepared particles were found to have small size (337.87 nm), low polydispersity index (0.338) and be positively charged (10.23 mV). The in vitro drug release patterns were characterised using the validated RP-HPLC method over 12 h. Graphical abstractᅟ
Keywords: BSA; Insulin; PEDF; Polymers; Protein; RP-HPLC
An ESIPT-based fluorescent probe for the determination of hypochlorous acid (HClO): mechanism study and its application in cell imaging by Yonghuan He; Yan Xu; Yuting Shang; Shuwen Zheng; Weihua Chen; Yi Pang (7007-7017).
is a postdoctoral fellow in Prof. Chen’s group. He graduated from the Institute of Chemistry, Chinese Academy of Science, and joined Prof. Chen’s group in 2015. His research focuses on the design and synthesis of novel fluorescence probe for fast detection in food quality and safety. is a former graduate student in Prof. Chen’s group from 2015 to 2018. Her graduate research was the development of a novel fluorescent probe for an illegally added antiviral drug in animal feeds. She is currently working in Meiduoke Food in Tangshan city, Hebei province. is a former graduate student in Prof. Chen’s group from 2013 to 2018. Her graduate thesis was about the synthesis of a fluorescent probe for detecting and imaging of zinc in plants. She is currently pursuing her PhD at Jiangnan University, working in the field of foodborne pathogen detection. is a second-year graduate student in Prof. Chen’s group. Her research is focused on the utilization of natural pigments to fabricate intelligent colorimetric sensors for the onsite monitoring of freshness of seafood. is a “Young Talents Program” Professor in the Institute of Food Science and Technology CAAS which he joined after obtaining a PhD in chemistry from The University of Akron in 2012. His current research is focusing on the development of fluorescent probes and sensors for food safety detection and food quality analysis. is the Lester E. and Kathleen A. Coleman Professor in the Department of Chemistry at The University of Akron. His current research interests include luminescent polymers, fluorescent molecular probes for metal cations, and biologically important anions. A highly selective and sensitive probe for the detection of hypochlorous acid (HClO) in real samples was designed and synthesized by using the specific reaction between HClO and phenyl azo group. Upon reaction with HClO, the nonfluorescent probe generated a highly fluorescent 2-(2-hydroxy-4-chlorophenyl)benzimidazole (HBI-Cl) fluorophore, which underwent the excited state intramolecular proton transfer process to give strong fluorescence turn-on. The sensing mechanism, conversion of the nonfluorescent azo moiety into the fluorescent derivative of HBI upon reaction with HClO, was verified by independent synthesis of HBI-Cl (ϕfl ≈ 0.75). The theoretical computing results were in agreement with the experimental results that the azo moiety was the reactive site to realize fluorescence detection for HClO. Additionally, the probe was successfully utilized to determine HClO in tap water, exogenous HClO in HeLa cells, and endogenous HClO in MCF-7 cells with a low detection limit and cytotoxicity. Graphical abstractᅟ
Keywords: Fluorescence; HClO; ESIPT; HBI; Azo
A sample-in-digital-answer-out system for rapid detection and quantitation of infectious pathogens in bodily fluids by Haowen Yang; Zhu Chen; Xiaobao Cao; Zhiyang Li; Stavros Stavrakis; Jaebum Choo; Andrew J. deMello; Philip D. Howes; Nongyue He (7019-7030).
joined the deMello group as a research assistant at ETH Zurich in 2015. His research interests lie primarily in the field of microfluidics and nanotheranostics. is currently a lecturer at Hunan University of Technology. His research interest is molecular diagnostic systems. is currently a PhD student in the deMello group ETH Zurich, with a focus on microfluidics. is the Head of the scientific research group in the Department of Clinical Laboratory at Nanjing Drum Tower Hospital. His research focuses on nanotechnology-based biomacromolecules for diagnosis and therapy, biochips and biosensors, and functional nanomaterials. is currently a Senior Scientist in the deMello group in the Department of Chemistry and Applied Biosciences at ETH Zurich. His current research is focused upon developing new microfluidic/optofluidic platforms for single molecule enzymology, single-cell screening, high-throughput imaging flow cytometry, and fast enzyme kinetics using fluorescence lifetime detection. has been in the faculty of the Bionano Engineering Department at Hanyang University since 1995. His current research programs are centered on the development of ultra-sensitive optical detection systems for rapid and accurate in vitro diagnostics. is Professor of Biochemical Engineering in the Department of Chemistry and Applied Biosciences at ETH Zurich. His research interests cover a broad range of activities in the general area of microfluidics and nanoscale science, including the development of microfluidic devices for high-throughput biological and chemical analysis, ultra-sensitive optical detection techniques, nanofluidic reaction systems for chemical synthesis, novel methods for nanoparticle synthesis, the exploitation of semiconducting materials in diagnostic applications, the development of intelligent microfluidics, and the processing of living organisms. is a Research Associate in the deMello group at ETH Zurich. His research focuses on the synthesis of biomolecular-nanoparticle conjugates and their application in biological sensing and imaging. is a Professor in the School of Biological Science and Medical Engineering at Southeast University. His research interests are focused on biochips and biosensors, functional nanomaterials, controlled drug release, and tissue engineering. A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care. Graphical abstractᅟ
Keywords: Sample-in-answer-out; Microfluidics; Quantitation; Digital RPA; Molecular diagnostics; Tuberculosis
Aniline-based catalysts as promising tools to improve analysis of carbonyl compounds through derivatization techniques: preliminary results using dansylacetamidooxyamine derivatization and LC-fluorescence by Stéphan Houdier; Justine Lévêque; Tiphaine Sabatier; Véronique Jacob; Jean-Luc Jaffrezo (7031-7042).
Derivatization techniques based on α-effect amines and H+ catalysis are commonly used for the measurement of carbonyl compounds (CCs), whether in environmental, food, or biological samples. Here, we investigated the potential of aniline-based catalysts to improve derivatization rates of selected carbonyls by using dansylacetamidooxyamine (DNSAOA) as a reagent. Kinetic experiments were performed in aqueous solutions by varying catalyst and CC concentrations and delivered insights into the reaction mechanism. Using anilinium acetate (AnAc), rate constants varied linearly with the catalyst concentration with rate enhancements toward H+-catalyzed reactions as high as ca. 90 and 200 for acetone and benzaldehyde, respectively. Owing to contamination problems when using AnAc, anilinium chloride (AnCl) was chosen for the optimized analysis of real samples at low concentration. Rate enhancements for derivatization reaction of 4.4 (methylglyoxal), 6.0 (glyoxal), 12 (acetone), 20 (formaldehyde), and 47 (hydroxyacetaldehyde) were obtained using 0.1 M AnCl. The optimized method was successfully applied to the determination of the above compounds in natural snow and meltwater samples. Limits of detection (LODs) and limits of quantification (LOQs) were in the 2–14 and 7–41 nM range, respectively, i.e., low enough to allow for the analysis of most natural samples. Satisfactory relative recoveries (92.8 ± 3.8–118.3 ± 4.4%) and intra-day precision (2.7–11.3%) were achieved. Finally, we think that this approach could be applied not only to every α-effect nitrogen reagent—with the most evident profit of lowering derivatization times and particularly those required for low-reactive ketones—but also to the derivatization of CCs onto coated solid sorbents.
Keywords: Carbonyl compounds; Derivatization; HPLC; Catalysts; Fluorescence/luminescence
Probing some organic ukiyo-e Japanese pigments and mixtures using non-invasive and mobile infrared spectroscopies by Carole Biron; Gwénaëlle Le Bourdon; Josefina Pérez-Arantegui; Laurent Servant; Rémy Chapoulie; Floréal Daniel (7043-7054).
Non-invasive identification of organic colourants in paintings still remains a challenging issue, especially in the case of extremely thin layers of paint on printed paper such as Japanese ukiyo-e prints. Because prints are fragile artworks, various non-invasive analytical methods need to be employed. The present work focuses on results obtained by combining fibre optic reflectance spectroscopy in the near-infrared range (FORS NIR) with mid-infrared (MIR) spectroscopy. The first step consists of identifying spectroscopic marker bands typical of some organic pigments (indigo, gamboge, cochineal, turmeric, safflower, dragon’s blood). Some reference printouts involving paper substrate, binder and pigments (seldom used or as mixtures) were then investigated in order to establish a straightforward way to extract the marker bands of the pigments. Some data post-treatments were applied to the spectra, such as spectral subtraction, in order to abstract the signal from overlapping bands originating from both substrate and binder, and second derivative calculation to emphasise the pigment marker bands’ frequency positions. These data treatments turned out to be relevant to extract information on the organic pigments of interest, even within complex mixtures.
Keywords: Infrared spectroscopy; Organic pigments; Japanese woodblock prints; Non-invasive method; Data post-treatment; FORS NIR
13C quantification in heterogeneous multiphase natural samples by CMP-NMR using stepped decoupling by Paris Ning; Ronald Soong; Wolfgang Bermel; Daniel Lane; Myrna J. Simpson; André J. Simpson (7055-7065).
Many natural and environmental samples contain combinations of liquids, gels, and solids, yet quantification in the intact state and across multiple phases is highly challenging. Comprehensive multiphase nuclear magnetic resonance (CMP-NMR) combines all the capabilities of high-resolution magic angle spinning (HR-MAS), with the addition of full solids power handling, permitting all phases (i.e., mixtures of liquids, gels, and solids) to be studied and differentiated in intact samples without pre-treatment or extraction. Here, quantification in CMP-NMR is considered. As 1H NMR is considerably broadened in the solid-state, quantification is easier to achieve through 13C which can be observed easily in all the phases. Accurate 13C quantification requires effective 1H decoupling for all the phases, but each phase requires different decoupling conditions. To satisfy these conditions, a pulse sequence termed stepped decoupling is introduced. This sequence can be used to study all components under ideal decoupling conditions resulting in high-resolution spectra without truncation artifacts and provides accurate integrals of components in all phases. The approach is demonstrated on standards and then applied to natural samples including broccoli, soil, and Arabidopsis. The approach permits accurate quantification of chemical categories (for example total carbohydrates) as well as individual species (for example glucose). Further, as the samples are studied intact, volatile species such as methanol and ethylene which are normally hard to detect in plants can be easily quantified in Arabidopsis. Graphical abstractᅟ
Keywords: CMP-NMR; Multiphase NMR; Decoupling; Multiphase quantification; Natural intact samples
Measuring the bioactivity of anti-IL-6/anti-IL-6R therapeutic antibodies: presentation of a robust reporter gene assay by Chuanfei Yu; Junxia Cao; Lan Wang; Yalan Yang; Yongbo Ni; Junzhi Wang (7067-7075).
IL-6 has an important role in the pathogenesis of autoimmunity and chronic inflammation. Several mAbs that target IL-6 or the IL-6 receptor (IL-6R) have been established and approved for the treatment of various diseases such as multicentric Castleman's disease and rheumatoid arthritis. Quality control of therapeutic antibodies requires accurate determination of bioactivity. However, current cell-based anti-proliferation assays are tedious, time consuming, and result in high variation. We therefore developed a reporter gene assay (RGA) based on an IL-6-dependent DS-1 cell line that stably expressed the reporter luciferase controlled by the serum-induced element (SIE) response element, which was a key element located downstream of the IL-6 signaling pathway. The RGA method demonstrated good performance characteristics after careful optimization, including high specificity, stability, accuracy, precision, and robustness. It also had superior precision and sensitivity. The assay is simple compared with the traditional anti-proliferation assay. This novel RGA based on the IL-6-IL-6R-STAT3 pathway can be useful, in conjunction with the anti-proliferation bioassay, to determine the bioactivity of anti-IL-6/anti-IL-6R therapeutic mAbs. Graphical abstractThe mechanism sketch of the reporter gene assay for the bioactivity determination of anti-IL-6/anti-IL-6Rα mAbs
Keywords: IL-6; IL-6R; Tocilizumab; Bioassay; Reporter gene assay
Matrix solid-phase dispersion coupled with gas chromatography–tandem mass spectrometry for simultaneous determination of 13 organophosphate esters in vegetables by Qing Luo; Shiyu Wang; Yue Shan; Li-na Sun; Hui Wang (7077-7084).
In this study, matrix solid-phase dispersion coupled with gas chromatography–tandem mass spectrometry (GC–MS/MS) was developed for the analysis of 23 organophosphate esters (OPEs) in vegetables. Under the optimal conditions, 0.5 g vegetables was dispersed with use of 2 g Florisil, 2 g anhydrous sodium sulfate, and 0.1 g graphitized carbon black, and it was transferred to an empty solid-phase extraction cartridge. The analytes were eluted with 15 mL n-hexane/acetone (1:1, v/v) and analyzed by GC–MS/MS. The method detection limits and quantitation limits ranged from 0.05 to 0.33 ng/g and from 0.16 to 1.10 ng/g, respectively. The recoveries ranged from 65.1% to 109.1%, and the relative standard deviations were less than 15%. The analysis of eight kinds of vegetables shows that the vegetables had been contaminated by OPEs; the concentrations of the sum of the OPEs ranged from 5.89 to 26.8 ng/g. The proposed method is applicable to analyze OPEs in vegetables. Graphical abstractᅟ
Keywords: Matrix solid-phase dispersion; Gas chromatography–tandem mass spectrometry; Organophosphate esters; Vegetables
Extreme cosmetics and borderline products: an analytical-based survey of European regulation compliance by Marta Lores; Maria Celeiro; Laura Rubio; Maria Llompart; Carmen Garcia-Jares (7085-7102).
The cosmetic industry currently focuses on products with magnified or exaggerated effects or extremely long-lasting characteristics. There are also a number of related commercial products for which the regulatory framework is far from clear; they are called ‘borderline’, and the European authorities only recommend which regulations they need to comply with. In any case, all these products must be safe under reasonable conditions of use in accordance with the applicable laws in force in the European Union (EU) framework. In this context, adequate analytical methodology is needed to evaluate the degree of compliance. Ultrasound Assisted Extraction (UAE) procedures for the analysis of 70 cosmetic ingredients have therefore been developed in this work. Moreover, for cosmetics with plastic applicators, a Supported-UAE (Sup-UAE) method was also opportunely optimized to check if a partial transfer of plasticizers to the cosmetics—and thereby to the consumers—could happen. In a survey of 50 commercial products (30 ‘extreme’ and 20 ‘borderline’), the methods afforded mean recoveries of about 100% and RSD values lower than 5% for UAE and 10% for Sup-UAE, and with detection limits far below the legal requirements, for all the target compounds, thereby demonstrating their analytical suitability. Results are discussed in detail for phthalates, fragrances (musks and allergens) and some frequent preservatives. Additionally, a labelling study was performed to check if the consumer is correctly and fully informed. Graphical abstractᅟ
Keywords: Phthalates; Fragrances; Preservatives; Ultrasound assisted extraction; Gas chromatography–mass spectrometry
A fluorescent material for the detection of chlortetracycline based on molecularly imprinted silica–graphitic carbon nitride composite by Shengnan Xu; Jie Ding; Ligang Chen (7103-7112).
A new fluorescent probe based on graphitic carbon nitride (g-C3N4) combined with molecularly imprinted silica was successfully fabricated and used to selectively recognize chlortetracycline (CTC). The g-C3N4 used in this study has the characteristics of low toxicity and high chemical stability. This synthetic composite was characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, UV spectroscopy, X-ray diffraction, and fluorescence spectroscopy. The material was used to detect CTC by the fluorescence quenching technique. The fluorescence quenching was due to g-C3N4 and the benzene ring of CTC through π–π electron donor–acceptor interaction and electrostatic force. Hydrogen bonds formed between CTC and 3-aminopropyltriethoxysilane during the polymerization process. Eventually, a considerable amount of selective recognition holes were formed in the composite material and could specifically recognize the template molecule CTC. In addition, the probe strategy was successfully applied to milk analysis, and the recoveries ranged from 90.1% to 95.7%, with relative standard deviations of 1.8–2.8%; the detection limit for CTC was 8 ng mL-1. The results indicate that this method combined the sensitivity of fluorescence detection with the excellent selectivity of a molecularly imprinted polymer. The new material can be widely used in the detection of dairy products. Graphical abstractSchematic of synthesis of the MIP-capped g-C3N4 by sol-gel reaction
Keywords: Graphitic carbon nitride; Molecularly imprinted; π–π electron donor–acceptor interaction; Chlortetracycline
Imaging of growth factors on a human tooth root canal by surface-enhanced Raman spectroscopy by Václav Ranc; Radovan Žižka; Zuzana Chaloupková; Juraj Ševčík; Radek Zbořil (7113-7120).
Endodontic treatment of immature permanent teeth with necrotic pulp poses several clinical challenges and is one of the most demanding interventions in endodontics. Recently, with new discoveries in the field of tissue engineering, novel treatment protocols have been established. The most promising treatment modality is revascularization, whose integral part is the exposure of collagen matrix and embedded growth factors. However, optimization of the treatment protocol requires a development of analytical procedures able to analyze growth factors directly on the sample surface. In this work, method based on surface-enhanced Raman spectroscopy (SERS) was developed to investigate the influence of the time of the medical treatment using EDTA on exposure and accessibility of the growth factors, namely TGF-ß1, BMP-2, and bFGF on the dentine surface. The nanotags, which consist of magnetic Fe3O4@Ag nanocomposite covalently functionalized by tagged antibodies (anti-TGF-ß1-Cy3, anti-BMP-2-Cy5, and anti-bFGF-Cy7), were employed as a SERS substrate. Each antibody was coupled with a unique label allowing us to perform a parallel analysis of all three growth factors within one analytical run. Developed methodology presents an interesting alternative to a fluorescence microscopy and in contrary allows evaluating a chemical composition and thus minimizing possible false-positive results. Graphical abstract
Keywords: SERS; Nanocomposites; Growth factors; Imaging
Lipidomic differentiation of Graves’ ophthalmopathy in plasma and urine from Graves’ disease patients by Seul Kee Byeon; Se Hee Park; Jong Cheol Lee; Sena Hwang; Cheol Ryong Ku; Dong Yeob Shin; Jin Sook Yoon; Eun Jig Lee; Myeong Hee Moon (7121-7133).
Approximately 50% of patients with Graves’ disease (GD) develop retracted eyelids with bulging eyes, known as Graves’ ophthalmopathy (GO). However, no simple diagnostic blood marker for distinguishing GO from GD has been developed yet. The objective of this study was to conduct comprehensive profiling of lipids using plasma and urine samples from patients with GD and GO undergoing antithyroid therapy using nanoflow ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry. Plasma (n = 86) and urine (n = 75) samples were collected from 23 patients with GD without GO, 31 patients with GO, and 32 healthy controls. Among 389 plasma and 273 urinary lipids that were structurally identified, 281 plasma and 191 urinary lipids were quantified in selected reaction monitoring mode. High-abundance lipids were significantly altered, indicating that the development of GD is evidently related to altered lipid metabolism in both plasma and urine. Several urinary lysophosphatidylcholine species were found to be increased (3- to 10-fold) in both GD and GO. While the overall lipid profiles between GD and GO were similar, significant changes (area under receiver operating curve > 0.8) in GO vs. GD were observed in a few lipid profiles: 58:7-TG and (16:1,18:0)-DG from plasma, 16:1-PC and 50:1-TG from urine, and d18:1-S1P from both plasma and urine samples. An altered metabolism of lipids associated with the additional development of ophthalmopathy was confirmed with the discovery of several candidate markers. These can be suggested as candidate markers for differentiating the state of GO and GD patients based on plasma or urinary lipidomic analysis. Graphical abstract
Keywords: Graves’ disease; Ophthalmopathy; Lipidomic analysis; Plasma; Urine; nUPLC-ESI-MS/MS
Characterization and mapping of secondary metabolites of Streptomyces sp. from caatinga by desorption electrospray ionization mass spectrometry (DESI–MS) by Júlia Pereira Rodrigues; Shamina Saiyara Prova; Luiz Alberto Beraldo Moraes; Demian Rocha Ifa (7135-7144).
The discovery of new secondary metabolites is a challenge to biotechnologists due to the emergence of superbugs and drug resistance. Knowledge about biodiversity and the discovery of new microorganisms have become major objectives; thus, new habitats like extreme ecosystems have become places of interest to research. In this context, caatinga is an unexplored biome. The ecosystem caatinga is a rich habitat for thermophilic microbes. Its high temperature and dry climate cause selective microbes to flourish and become established. Actinobacteria (Caat 1-54 genus Streptomyces sp.) isolated from the soil of caatinga was investigated to characterize and map its secondary metabolites by desorption electrospray ionization mass spectrometry imaging (DESI–MSI). With this technique, the production of bioactive metabolites was detected and associated with the different morphological differentiation stages within a typical Streptomyces sp. life cycle. High-resolution mass spectrometry, tandem mass spectrometry, UV–Vis profiling and NMR analysis were also performed to characterize the metabolite ions detected by DESI–MS. A novel compound, which is presumed to be an analogue of the antifungal agent lienomycin, along with the antimicrobial compound lysolipin I were identified in this study to be produced by the bacterium. The potency of these bioactive compounds was further studied by disc diffusion assays and their minimum inhibitory concentrations (MIC) against Bacillus and Penicillium were determined. These bioactive metabolites could be useful to the pharmaceutical industry as candidate compounds, especially given growing concern about increasing resistance to available drugs with the emergence of superbugs. Consequently, the unexplored habitat caatinga affords new possibilities for novel bioactive compound discovery. Graphical Abstractᅟ
Keywords: Ambient ionization; Desorption electrospray ionization (DESI); Secondary metabolites; NMR; UV–Vis; Antibiotics
Ultrathin ZIF-67 nanosheets as a colorimetric biosensing platform for peroxidase-like catalysis by Shujuan Wang; Dongpo Xu; Lan Ma; Jingxuan Qiu; Xiang Wang; Qingli Dong; Qi Zhang; Jing Pan; Qing Liu (7145-7152).
In this work, we report a zeolitic imidazolate framework (ZIF-67) which could catalyze 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a yellow chromogenic reaction. ZIF-67 showed high peroxidase-like activity compared with copper-based metal−organic framework nanoparticles (Cu-MOF), zinc-based metal−organic framework nanoparticles (ZIF-8), and horseradish peroxidase (HPR). We discovered for the first time that the cobalt-based metal−organic framework nanoparticles possess intrinsic peroxidase-like activity without H2O2, which can be employed to quantitatively monitor the H2O2. Graphical abstractᅟ
Keywords: Chromogenic reaction, in the absence of H2O2 ; Peroxidase-like activity; Zeolitic imidazolate framework
High-throughput liquid chromatography differential mobility spectrometry mass spectrometry for bioanalysis: determination of reduced and oxidized form of glutathione in human blood by Sophie Bravo-Veyrat; Gérard Hopfgartner (7153-7161).
Currently, the measure of the oxidative stress, from oxidized and reduced glutathione (GSSG and GSH respectively), for large cohorts of samples, is generally limited to spectrometric methods. In this study, a high-throughput assay for GSH after derivatization with N-ethylmaleimide and GSSG in blood sample was developed with an analysis time of 1.5 min. The method combines protein precipitation and a short LC (10-mm length) column where compounds were trapped in front-flush mode and eluted in back-flush mode. This setup is combined with modifier-assisted differential ion mobility spectrometry (DMS, SelexIon) and detection is performed in the selected reaction monitoring mode using positive electrospray ionization. In DMS, various modifiers were investigated including N2, methanol, toluene, ethanol, acetonitrile, and isopropanol to improve assay selectivity. Using EtOH as modifier, the limit of quantification (LOQ) was found to be 0.4 μM for GSSG and 3.2 μM for GS-N-ethylmaleimide (NEM) using a blood volume of 60 μL. The method is linear over a wide dynamic concentration range of 0.4 to 400 μM for GSSG and from 3.2 to 3200 μM for GS-NEM. The inter-assay precision of QC samples were ≤ 6.7%, with accuracy values between 98.3 and 103%. The method was further cross-validated with a LC Hypercarb-DMS-MS/MS method by the analysis of human blood samples. The bias between both assays ranged from − 0.3 to 0.2%. Graphical abstractᅟ
Keywords: Differential mobility spectrometry; Short LC; Quantification; Blood; Glutathione
Targeted LC-MS/MS for the evaluation of proteomics biomarkers in the blood of neonates with necrotizing enterocolitis and late-onset sepsis by Anastasia Chrysovalantou Chatziioannou; Justina Clarinda Wolters; Kosmas Sarafidis; Agathi Thomaidou; Charalampos Agakidis; Natalia Govorukhina; Jan Albert Kuivenhoven; Rainer Bischoff; Georgios Theodoridis (7163-7175).
Late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) are severe life-threatening conditions for neonates. Accurate, early diagnosis and timely initiation of treatment are crucial. Non-specific overlapping clinical signs along with the non-sensitive/specific diagnostic tools set obstacles to speedy, trustful diagnosis including differential diagnosis. The objective of this study was to evaluate the potential of targeted LC-MS/MS proteomics in identifying diagnostic biomarkers of NEC or LOS. We conducted a prospective case-control study evaluating serum proteomics profiles of 25 NEC, 18 LOS, and an equal number of matched control neonates, over three sampling points. Eighty-three concatemers and synthetic peptides belonging to 47 protein markers of the two diseases were selected after thorough literature search. A novel selected reaction monitoring (SRM), LC-MS/MS method was developed for their analysis and evaluation as potential biomarkers. Multivariate and univariate statistical analyses highlighted significant proteins in differentiating LOS and NEC neonates and diseased from controls. Moreover, panels of proteins were tested for their ability to distinguish LOS from NEC and controls. We suggest two panels of three proteins each, exhibiting very high diagnostic value for LOS and excellent diagnostic performance at the critical LOS-NEC differentiation, reaching an AUC ROC value close to 1 (0.999). These panels constitute a valuable starting point for further validation with broader cohorts of neonates, aiming to improve the clinical practice. Graphical abstractᅟ
Keywords: Mass spectrometry; Proteome; Blood; Biomarker; Systems biology; Differential diagnosis
Capillary-based chemiluminescence immunoassay for C-reactive protein with portable imaging device by Haiying Shen; Rizwanullah Khan; Xiaoqian Wang; Zulan Li; Feng Qu (7177-7183).
A capillary-based chemiluminescence immunoassay system using a charge-coupled device (CCD) camera as detector was established in this paper. The fused quartz capillary was easily activated in one step for immobilizing capture antibody, and the chemiluminescence immunoassay was carried out in the capillary in double-antibody sandwich format. Chemiluminescence signals were recorded by the portable imaging device which was installed with the CCD camera and the results were analyzed through gray intensity. The total cost time, which included not only the time for test but also the time for the preparation of experimental materials, was only 2 h. The immunoassay was performed without any complicated or expensive instruments. The consumption of the sample was only 0.8 μL in one test, which was significantly less than other methods. In this work, C-reactive protein (CRP), as a target, was quantitatively detected from 0.3 to 160.0 μg mL−1 with high specificity and low sample volume. The reproducibility and accuracy were tested in clinic human serum samples and shown good results. Thus, this rapid, easy preparation and using, portable immunoassay system indicated its usefulness as a novel technology platform. Graphical abstractᅟ
Keywords: Immunoassay; Capillary; C-reactive protein; Portable imaging device
A hierarchically porous composite monolith polypyrrole/octadecyl silica/graphene oxide/chitosan cryogel sorbent for the extraction and pre-concentration of carbamate pesticides in fruit juices by Pattamaporn Klongklaew; Thamolwan Naksena; Proespichaya Kanatharana; Opas Bunkoed (7185-7193).
A hierarchically porous structured composite monolith sorbent of polypyrrole-coated graphene oxide and octadecyl silica incorporated in chitosan cryogel (PPY/GOx/C18/chitosan) was synthesized and used as solid-phase extraction sorbent for the determination of carbamate pesticides. Various factors affecting the characteristics of the adsorbents (chemistry of the sorbent, polymerization time, concentrations of graphene oxide and octadecyl silica) and the extraction efficiency using the prepared sorbents, such as sample loading, desorption conditions, sample volume, sample flow rate, sample pH, and ionic strength, were investigated and optimized. Under the optimal conditions of sorbent preparation and extraction, the developed composite monolith sorbent provided wide linear responses from 1.0 to 500 μg L−1 for carbofuran and diethofencarb, from 0.5 to 500 μg L−1 for carbaryl, and from 2.0 to 500 μg L−1 for isoprocarb. The limits of detection using HPLC-UV at 203, 220, and 208 nm were in the range of 0.5–2.0 μg L−1. When the composite monolith sorbent was applied for the pre-concentration and determination of carbamate in fruit juices, good recoveries (84.1–99.5%) were achieved. The developed sorbents were porous and exhibited low back pressure enabling their use at high flow rates during sample loading. Extraction and clean-up were highly efficient, and the good physical and chemical stability of the sorbent enables reuse up to 13 times. Graphical abstractᅟ
Keywords: Carbamate; Polypyrrole; Octadecyl silica; Graphene oxide; Chitosan cryogel; Monolith
Rapid and quantitative analysis of impurities in silicon powders by glow discharge mass spectrometry by Jianying Zhang; Tao Zhou; Yichuan Tang; Yanjie Cui; Dan Song (7195-7201).
High-purity silicon power was doped with standard solutions containing 15 elements, and a high-purity indium tablet was prepared by the melting of indium pellets. An In–Si tablet, which is mechanically stable and thus suitable as a calibration sample, was prepared by our pressing the doped silicon power on the high-purity indium tablet. The matrix effect was studied by our investigating the variations of measured mass fractions (standard relative sensitivity factor, StdRSF, calibration) of doped impurities in the In–Si tablet, which provides a series of matrixes with different mass ratios of In to Si. For all the elements, the relative standard deviations of the measured mass fractions of impurities were less than 30%. The RSFs of the glow discharge mass spectrometer (Element GD) were obtained, and the results showed that three RSFs derived from the In–Si tablet with low, intermediate and high mass ratios of In to Si, respectively, agreed with the mean RSF within an uncertainty interval of 30%. The measurement of Fe and Al matrix certified reference materials further demonstrated that the RSFs generated from a matrix can be used for the calibration of another matrix, and the uncertainty was within 30%. Finally, another doped silicon powder was measured with the glow discharge mass spectrometer, which was calibrated by the mean RSFs from the In–Si tablet, and the analytical results obtained by glow discharge mass spectrometry are in good agreement with the analytical results obtained by high-resolution inductively coupled plasma mass spectrometry. Graphical abstractᅟ
Keywords: Glow discharge mass spectrometry; Quantitative analysis; Impurities; Matrix effect; Relative sensitivity factors; Silicon powders
Sensitive determination of aldehyde metabolites in exhaled breath condensate using capillary electrophoresis with laser-induced fluorescence detection by Tingting Wang; Dan Luo; Zheyan Chen; Yining Qu; Xiuhua Ma; Jiannong Ye; Qingcui Chu; Dongping Huang (7203-7210).
A novel capillary electrophoresis with laser-induced fluorescence detection method has been developed for the analysis of aldehyde metabolism biomarkers for oxidative stress in exhaled breath condensate (EBC), and fluorescein 5-thiosemicarbazide was used as a derivatization reagent. In a simple capillary zone electrophoresis mode, ten low molecular weight aldehydes (LMWAs) could be well separated within 30 min. The reaction efficiency was doubled by increasing sample solution pH and magnetic stirring, and the LODs of this method reached 0.16–3.4 nM (S/N = 3). Acceptable recoveries (82.1–115%) were obtained for EBC samples, and the RSD data were within 7.9%. This developed method has been applied for the analyses of EBC samples and evaluation of the correlation between smoking and the contents of aldehyde metabolites in EBC. Due to no need of buffer additives and sample preconcentration, this proposed method may provide an appealing alternative for the trace analyses of LMWAs in noninvasive biofluids. Graphical abstractᅟ
Keywords: Aldehyde metabolites; Oxidative stress markers; Exhaled breath condensate; Noninvasive biofluids; Capillary electrophoresis with laser-induced fluorescence detection
Critical assessment of different methods for quantitative measurement of metallodrug-protein associations by Luis Galvez; Sarah Theiner; Márkó Grabarics; Christian R. Kowol; Bernhard K. Keppler; Stephan Hann; Gunda Koellensperger (7211-7220).
Quantitative screening for potential drug–protein binding is an essential step in developing novel metal-based anticancer drugs. ICP–MS approaches are at the core of this task; however, many applications lack in the capability of large-scale high-throughput screenings and proper validation. In this work, we critically discuss the analytical figures of merit and the potential method-based quantitative differences applying four different ICP–MS strategies to ex vivo drug–serum incubations. Two candidate drugs, more specifically, two Pt(IV) complexes with known differences of binding affinity towards serum proteins were selected. The study integrated centrifugal ultrafiltration followed by flow injection analysis, turbulent flow chromatography (TFC), and size exclusion chromatography (SEC), all combined with inductively coupled plasma-mass spectrometry (ICP–MS). As a novelty, for the first time, UHPLC SEC-ICP–MS was implemented to enable rapid protein separation to be performed within a few minutes at > 90% column recovery for protein adducts and small molecules. Graphical abstractQuantitative screening for potential drug–protein binding is an essential step in developingnovel metal-based anticancer drugs
Keywords: Metal-based anticancer drugs; High-throughput; ICP–MS; Elemental speciation analysis; Metal–protein interaction
Multi-contrast diffraction enhanced computed laminography at Beijing Synchrotron Radiation Facility by Jian Fu; Xianhong Shi; Qingxi Yuan; Wanxia Huang; Wei Guo; Peng Peng (7221-7228).
Synchrotron radiation X-ray computed tomography (CT) enables nondestructive visualization of 3D morphological and chemical changes inside a sample and has become a powerful analysis tool to monitor reactive parts and their chemical states. However, synchrotron radiation CT imaging of specimens with lateral extensions much larger than the acceptance window of detectors is rather problematic due to strong absorption of X-rays in the lateral directions. On the other hand, X-ray computed laminography (CL) permits 3D imaging of flat samples while X-ray diffraction enhanced imaging (DEI) can provide high-quality results with different imaging contrasts such as absorption, phase and dark-field for samples with weak absorptions. Combining CL and DEI together, we have developed a multi-contrast DEI-CL system at the 4W1A beamline of the Beijing Synchrotron Radiation Facility for this kind of sample. Here we reported its design, implementation, and preliminary experimental results of carbon fiber reinforced polymer laminates with three kinds of imaging contrasts. The results have demonstrated the validity of this DEI-CL system. It will be helpful to push the applications of the state-of-the-art synchrotron radiation methods and instruments towards cutting-edge research. Graphical abstractᅟ
Keywords: Synchrotron radiation X-ray; Diffraction enhanced imaging; Computed laminography; Phase contrast imaging; Dark-field imaging
Chitosan-magnetite nanocomposite as a sensing platform to bendiocarb determination by Raissa C. de Oliveira; Camila P. Sousa; Tiago M. Freire; Rafael M. Freire; Juliano C. Denardin; Pierre B. A. Fechine; Helena Becker; Simone Morais; Pedro de Lima-Neto; Adriana N. Correia (7229-7238).
A novel platform for carbamate-based pesticide quantification using a chitosan/magnetic iron oxide (Chit-Fe3O4) nanocomposite as a glassy carbon electrode (GCE) modifier is shown for an analytical methodology for determination of bendiocarb (BND). The BND oxidation signal using GCE/Chit-Fe3O4 compared with bare GCE was catalyzed, showing a 37.5% of current increase with the peak potential towards less positive values, showing method’s increased sensitivity and selectivity. Using square-wave voltammetry (SWV), calibration curves for BND determination were obtained (n = 3), and calculated detection and quantification limits values were 2.09 × 10−6 mol L−1 (466.99 ppb) and 6.97 × 10−6 mol L−1 (1555.91 ppb), respectively. The proposed electroanalytical methodology was successfully applied for BND quantification in natural raw waters without any sample pretreatment, proving that the GCE/Chit-Fe3O4 modified electrode showed great potential for BND determination in complex samples. ᅟGraphical abstract
Keywords: Bendiocarb; Magnetic nanoparticles; Nanocomposite; Chitosan; Electrochemical sensor
Rapid monitoring of plant growth regulators in bean sprouts via automated on-line polymeric monolith solid-phase extraction coupled with liquid chromatography tandem mass spectrometry by Qixun Nian; Lianfeng Ai; Dongmei Li; Xuelei Chen; Lei Zhang; Manman Wang; Xuesheng Wang (7239-7247).
An automated on-line solid-phase extraction (SPE) following liquid chromatography tandem mass spectrometry was established for the fast determination of plant growth regulator residues in soybean sprout and mung bean sprout. The crude extracted specimens were directly purified on a poly (2-(dimethylamino) ethyl methacrylate-co-ethylene dimethacrylate) monolithic column which was well-defined as the on-line SPE adsorbent. Under the optimized conditions, the developed method gave the linear range of 0.3–50 ng/mL for gibberellin and 2,4-dichlorophenoxyacetic acid, 0.2–50 ng/mL for 4-chlorophenoxyacetic acid, and 0.5–50 ng/mL for 1-naphthaleneacetic acid (r ≥ 0.998). The detection limits (S/N = 3) ranged from 1.0 to 2.5 μg/kg and the recoveries for spiked soybean sprout samples were in the range of 75.0–93.3%. Besides, the total time for one analysis was 16 min. The reusability of the monolith was up to 600 extractions. The proposed process facilitated fully automated SPE and accurate determination in one step with rapidity, simplicity, and reliability. Graphical abstractᅟ
Keywords: Polymeric monolithic column; On-line solid-phase extraction; Liquid chromatography tandem mass spectrometry; Plant growth regulators; Bean sprouts
Minimally invasive technique for measuring transdermal glucose with a fluorescent biosensor by Sheniqua Brown; Paige N. Zambrana; Xudong Ge; Dayanand Bagdure; Audra L. Stinchcomb; Govind Rao; Leah Tolosa (7249-7260).
There is a need for blood glucose monitoring techniques that eliminate the painful and invasive nature of current methods, while maintaining the reliability and accuracy of established medical technology. This research aims to ultimately address these shortcomings in critically ill pediatric patients. Presented in this work is an alternative, minimally invasive technique that uses microneedles (MN) for the collection of transdermal glucose (TG). Due to their comparable skin properties, diffusion studies were performed on full thickness Yucatan miniature pig skin mounted to an in-line diffusion flow cell and on different skin sites of human subjects. Collected TG samples were measured with a L255C mutant of the E. coli glucose-binding protein (GBP) with an attached fluorescent probe. The binding constant (K d = 0.67 μM) revealed the micromolar sensitivity and high selectivity of the his-tagged GBP biosensor for glucose, making it suitable for TG measurements. In both the animal and human models, skin permeability and TG diffusion across the skin increased with MN application. For intact and MN-treated human skin, a significant positive linear correlation (r > 0.95, p < 0.01) existed between TG and BG. The micromolar sensitivity of GBP minimized the volume required for interstitial fluid glucose analysis allowing MN application time (30 s) to be shortened compared to other studies. This time reduction can help in eliminating skin irritation issues and improving practical use of the technique by caregivers in the hospital. In addition, the his-tagged optical biosensor used in this work can be immobilized and used with a portable sensing fluorometer device at the point of care (POC) making this minimally invasive technology more ideal for use in the pediatric intensive care unit. Graphical abstractᅟ
Keywords: Optical biosensor; In-line flow through diffusion cell; Transdermal glucose monitoring; Microneedles