Analytical and Bioanalytical Chemistry (v.408, #29)

Estrogen receptor preparation effects on the receptor–DNA interaction by surface plasmon resonance by Sandrine Bayle; Joel Chopineau; Benoit Roig; Denis Habauzit (8257-8262).
is an Associate Professor at the Ecole des Mines d’Alès. She is a member of Odor and VOC research teams of the Laboratory of Industrial Environment Engineering (LGEI, Ecole des Mines d’Alès, Alès). Her research focuses on the detection and quantification of environmental pollutant in several matrices mainly in air or in water and on the evaluation of their effect on living. is a Professor of Biochemistry at the University of Nimes. He performs his research in the group “Advanced Materials for Catalysis and Health” of the Institute for Molecular Chemistry and Material Sciences in Montpellier (UMR CNRS 5253). His research interests concern the design and development of membrane models and platforms dedicated to protein and nanoparticle–membrane interactions. is a Professor at the University of Nimes. He is the head of the CHROME team (EA7352) involved in the characterization and management of chronic and emerging risks. He works since 15 years in the field of the analysis of environmental contaminants (chemical and biological) and the impact on the populations (aquatic and terrestrial including humans). His research concerns, in particular, measures of exposure and hazard to emerging contaminants as well as the development of field methods. completed his PhD in 2008 in the University of Montpellier 1. After a first postdoc in UMR CNRS 6026, he joined in 2011 the Research Institute for Environmental and Occupational Health (IRSET, Inserm U1085) in the University of Rennes 1. His current research focuses on the possible interaction between living and chemical and physical disruptors such as endocrine disruptors (especially in the interaction with estrogen receptor) or electromagnetic fields (millimeter waves or extremely low frequency) by several methodologies such as biosensor, cell culture, and genomic approaches. Up to now, several studies have investigated estrogen receptor (ER)–estrogen response element (ERE) interaction using biosensors such as surface plasmon resonance. These strategies have aimed to understand the molecular mechanism of such interaction as well as the effect of the ligand on this interaction. These approaches start to be used to determine the mechanisms of protein/DNA interaction, in particular in the context of drug discovery or environmental applications. However, some physical and biochemical parameters (incubation time, temperature, protease inhibitor cocktail, and bovine serum albumin (BSA)) are not completely described in the literature and could deeply modify the obtained results. This paper aims to focus not only on the preliminary steps of sample preparation such as protein thawing and incubation conditions (time and temperature) but also on the evaluation of protease inhibitor cocktail and BSA effect on the measurement of ER–ERE interactions.
Keywords: Surface plasmon resonance; Estrogen; Estrogen receptor; Dimerization; Temperature

is a master student of Biomedical Engineering at School of Biological Science and Medical Engineering, Southeast University in China. Her research topic is DNA sequencing. is a professor at School of Biological Science and Medical Engineering, Southeast University in China. His research interests include DNA sequencing, DNA microarray, and biosensor applications. Molecular haplotyping is becoming increasingly important for studying the disease association of a specific allele because of its ability of providing more information than any single nucleotide polymorphism (SNP). Computational analysis and experimental techniques are usually performed for haplotypic determination. However, established methods are not suitable for analyzing haplotypes of massive natural DNA samples. Here we present a simple molecular approach to analyze haplotypes of conventional polymerase chain reaction (PCR) products quantitatively in a single sequencing run. In this approach, specific types and proportions of haplotypes in both individual and pooled samples could be determined by solving equations constructed from nonsynchronous pyrosequencing with di-base addition. Two SNPs (rs11176013 and rs11564148) in the gene for leucine-rich repeat kinase 2 (LRRK2) related to Parkinson’s disease were selected as experimental sites. A series of DNA samples, including these two heterozygous loci, were investigated. This approach could accurately identify multiple DNA samples indicating that the approach is likely to be applied for haplotyping of unrestricted conventional PCR products from natural samples, and be especially applicable for analyzing short sequences in clinical diagnosis. Graphical Abstract One DNA sample consisting of 4 different DNA templates with different proportion are sequenced by nonsynchronous pyrosequencing with di-base addition. The number of incorporated nucleotides produced by a single sequencing reaction equals to the total of incorporated nucleotides. Four independent equations are constructed from the pyrograms of nonsynchronous pyrosequencing data. Molecular haplotypes of two adjacent SNPs can be quantitatively identified by solving these equations.
Keywords: Haplotype; Quantitative analysis; Nonsynchronous pyrosequencing; Di-base; Equations

High performance liquid chromatography coupled with triple-quadrupole mass spectrometry was applied in the determination of in vitro metabolism products of selected antibiotic drugs (cefotaxime, ciprofloxacin, fluconazole, gentamicin, clindamycin, linezolid, and metronidazole). The analytes were separated on a reversed phase C18 column, with acetonitrile and 0.1 % aqueous formic acid as the mobile phase. Tandem mass spectrometry with positive electrospray ionization was used to facilitate the structural characterization of the potential metabolites. Metabolism studies on human liver microsomes were performed via cytochromes P450 (phase I) and via NADPH/UDP-glucuronosyltransferase (phase II) mediated reactions. LC-MS/MS experiments allowed potential metabolite peaks, including sum formulae suggestions, to be identified; high resolution MS/MS experiments led to the identification of various oxidative and reductive modifications of target compounds in phase I biotransformation, and conjugation products with glucuronic acid in phase II reactions. A total of 11 potential metabolites and their proposed structures were characterized during the incubation of human liver microsomes by comparing their retention times and spectral patterns with those of the parent drug. Dehydrogenation and reactions of side chains such as hydroxylation and hydrolysis of ester bonds constituted the major metabolic pathways. Finally, LC-MS/MS spectrometry was revealed to be a suitable analytical tool to procure a feasible analytical base for the envisioned in vivo experiments. Graphical Abstract Workflow overview of in vitro drug metabolism studies.
Keywords: Antibiotic drugs; Human liver microsomes; In-vitro; Metabolism; Mass spectrometry

In this study, stable cadmium(II)-based metal–organic nanotubes (Cd-MONTs) were prepared and used as a coating material for solid-phase microextraction (SPME) of polychlorinated biphenyls (PCBs) from environmental water samples. The as-prepared Cd-MONT SPME coating material was characterized by thermal gravimetric analysis, scanning electron microscopy, and X-ray diffraction. The synthesized Cd-MONTs exhibited high thermal stability (385 °C) and excellent extraction performance toward PCBs. The important conditions were optimized systematically by the response surface method. Under the optimal conditions, the new fiber achieved high enrichment factors (938–3417), low limits of detection (1.80–8.73 pg L−1), and wide linearity (10–5000 pg L−1). The method developed was used in ultratrace-level analysis of PCBs in seawater samples, with satisfactory results for each sample.
Keywords: Metal–organic nanotubes; Gas chromatography–tandem mass spectrometry; Solid-phase microextraction; Polychlorinated biphenyls; Water samples

Development and validation of the modular Feed-code method for qualitative and quantitative determination of feed botanical composition by Luca Braglia; Silvia Gianì; Diego Breviario; Floriana Gavazzi; Francesco Mastromauro; Laura Morello (8299-8316).
The analysis of feed composition in terms of ingredients is addressed by Regulation (EC) 767/2009 and is important for detecting economic fraud and for monitoring feed safety. Within the framework of the EU project Feed-code, we developed and internally validated a modular assay, relying on intron polymorphism, for the complete qualitative analysis of the botanical composition of feed and the quantitative determination of six target plant species. Main performance parameters of each module, such as applicability, repeatability, specificity, and limit of detection, were evaluated. The whole assay was applied to a set of feed-like samples and results were in agreement with the expected composition. Application to a large set of compound feed and individual raw materials revealed the occurrence of botanical impurities. When compared with microscopic analysis, the proposed method gave more reliable results. We conclude that the Feed-code prototype, readily upgradable to include more plant species, is worthy of consideration for a full validation through a collaborative trial. Graphical Abstract The modular Feed-code method for the authentication of feed botanical composition
Keywords: Feed; Traceability; Intron; TBP; Feed-code; qPCR

A non-enzymatic urine glucose sensor with 2-D photonic crystal hydrogel by Zequn Yan; Min Xue; Qian He; Wei Lu; Zihui Meng; Dan Yan; Lili Qiu; Lijun Zhou; Yingjie Yu (8317-8323).
A novel polymerized crystalline colloidal array (PCCA) sensing material for the detection of urine glucose was developed by embedding a two-dimensional (2-D) polystyrene crystalline colloidal array (CCA) in 3-acrylamidophenylboronic acid (3-APBA)-functionalized hydrogel. After adjusting the cross-linker concentration, this material showed significant sensitivity for glucose under lab conditions, the particle spacing of the PCCA changed from 917 to 824 nm (93 nm) within 3 min as the glucose concentration increased from 0 to 10 mM, and the structural color of the PCCA changed from red through orange, to green, and finally, to cyan. In further experiments, this material was used to semi-quantitatively detect glucose in 20 human urine (HU) samples. Compared with the traditional dry-chemistry method, which was applied widely in clinical diagnosis, the PCCA method was more accurate and cost-effective. Moreover, this method can efficiently avoid the errors induced by most of the urine-interfering elements like vitamin C and ketone body. With a homemade portable optical detector, this low-cost intelligent sensing material can provide a more convenient and efficient strategy for the urine glucose detection in clinical diagnosis and point-of-care monitoring.
Keywords: Photonic crystal; Visual detection; Urine glucose; Point-of-care monitoring

Quantification of antibody coupled to magnetic particles by targeted mass spectrometry by Nicole A. Schneck; Karen W. Phinney; Sang Bok Lee; Mark S. Lowenthal (8325-8332).
Quantifying the amount of antibody on magnetic particles is a fundamental, but often overlooked step in the development of magnetic separation-based immunoaffinity enrichment procedures. In this work, a targeted mass spectrometry (MS)-based method was developed to directly measure the amount of antibody covalently bound to magnetic particles. Isotope-dilution liquid chromatography-tandem MS (ID-LC-MS/MS) has been extensively employed as a gold-standard method for protein quantification. Here, we demonstrate the utility of this methodology for evaluating different antibody coupling processes to magnetic particles of different dimensions. Synthesized magnetic nanoparticles and pre-functionalized microparticles activated with glutaraldehyde or epoxy surface groups were used as solid supports for antibody conjugation. The key steps in this quantitative approach involved an antibody-magnetic particle coupling process, a wash step to remove unreacted antibody, followed by an enzymatic digestion step (in situ with the magnetic particles) to release tryptic antibody peptides. Our results demonstrate that nanoparticles more efficiently bind antibody when compared to microparticles, which was expected due to the larger surface area per unit mass of the nanoparticles compared to the same mass of microparticles. This quantitative method is shown to be capable of accurately and directly measuring antibody bound to magnetic particles and is independent of the conjugation method or type of magnetic particle. Graphical Abstract Schematic illustration of the isotope-dilution mass spectrometry-based workflow to directly measure antibody bound to magnetic particles (MP)
Keywords: Bioanalytical methods; Immunoaffinity enrichment; Mass spectrometry; Nanoparticles/Nanotechnology

Diffusive gradients in thin films measurement of sulfur stable isotope variations in labile soil sulfate by Ondrej Hanousek; Jakob Santner; Sean Mason; Torsten W. Berger; Walter W. Wenzel; Thomas Prohaska (8333-8341).
A diffusive gradient in thin films (DGT) technique, based on a strongly basic anion exchange resin (Amberlite IRA-400), was successfully tested for 34S/32S analysis in labile soil sulfate. Separation of matrix elements (Na, K, and Ca) that potentially cause non-spectral interferences in 34S/32S analysis by MC ICP-MS (multi-collector inductively coupled plasma–mass spectrometry) during sampling of sulfate was demonstrated. No isotopic fractionation caused by diffusion or elution of sulfate was observed below a resin gel disc loading of ≤79 μg S. Above this threshold, fractionation towards 34S was observed. The method was applied to 11 different topsoils and one mineral soil profile (0–100 cm depth) and compared with soil sulfate extraction by water. The S amount and isotopic ratio in DGT-S and water-extractable sulfate correlated significantly (r 2 = 0.89 and r 2 = 0.74 for the 11 topsoils, respectively). The systematically lower 34S/32S isotope ratios of the DGT-S were ascribed to mineralization of organic S.
Keywords: Diffusive gradients in thin films; MC ICP-MS; Sulfur; Sulfate; Isotope ratio

Targeted proteomics for the indirect detection of dexamethasone treatment in bovines by Roberto Stella; Federica Barrucci; Roberto Angeletti; Peter James; Clara Montesissa; Giancarlo Biancotto (8343-8353).
The illegal use of pharmacologically active compounds for growth promotion in food-producing species poses risks for consumer health and animal welfare. Surveillance relies on the quantification of drug residues in animal fluids or tissues, but the efficacy can be negatively affected due to undetectable residual concentrations in biological matrices. Consequently, techniques focusing on the indirect biological effects of exogenous compound administration have been proposed as more sensitive detection methods. The purpose of the present study is to develop a tandem mass spectrometry analytical method based on low-energy collision-induced dissociation (CID-MS/MS) using multiple reaction monitoring (MRM) for the quantification of 12 potential protein markers of skeletal muscle to detect anabolic treatments with dexamethasone. Protein markers identified in a previous study applying a 2D-DIGE proteomics approach have been quantified using the signature peptide method. A group of proteins were confirmed as reliable markers. Quantitative results enabled a predictive model to be defined based on logistic regression for the detection of treated animals. The developed model was finally cross-validated in an independent animal set. Graphical abstract Analytical workflow used for the quantification of indirect protein markers of dexamethasone treatment
Keywords: Bovine; Illegal treatments; Dexamethasone; Multiple reaction monitoring; Absolute quantification; Protein markers

Eco-friendly LC–MS/MS method for analysis of multi-class micropollutants in tap, fountain, and well water from northern Portugal by Marta O. Barbosa; Ana R. Ribeiro; Manuel F. R. Pereira; Adrián M. T. Silva (8355-8367).
Organic micropollutants present in drinking water (DW) may cause adverse effects for public health, and so reliable analytical methods are required to detect these pollutants at trace levels in DW. This work describes the first green analytical methodology for multi-class determination of 21 pollutants in DW: seven pesticides, an industrial compound, 12 pharmaceuticals, and a metabolite (some included in Directive 2013/39/EU or Decision 2015/495/EU). A solid-phase extraction procedure followed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (offline SPE–UHPLC–MS/MS) method was optimized using eco-friendly solvents, achieving detection limits below 0.20 ng L−1. The validated analytical method was successfully applied to DW samples from different sources (tap, fountain, and well waters) from different locations in the north of Portugal, as well as before and after bench-scale UV and ozonation experiments in spiked tap water samples. Thirteen compounds were detected, many of them not regulated yet, in the following order of frequency: diclofenac > norfluoxetine > atrazine > simazine > warfarin > metoprolol > alachlor > chlorfenvinphos > trimethoprim > clarithromycin ≈ carbamazepine ≈ PFOS > citalopram. Hazard quotients were also estimated for the quantified substances and suggested no adverse effects to humans. Graphical Abstract Occurrence and removal of multi-class micropollutants in drinking water, analyzed by an eco-friendly LC–MS/MS method
Keywords: Drinking water; Priority substances; Contaminants of emerging concern; Solid-phase extraction; Ultra-high-performance liquid chromatography–tandem mass spectrometry

Magnetic bead (MB)-based chemiluminescence (CL) ELISA can be a sample-thrifty, time-saving tool for evaluation of cigarette smoke-induced DNA single-strand breaks (SSBs) with high specificity. This article describes a novel approach using immobilized oligonucleotide on MBs to determine cigarette smoke-induced DNA SSBs and screen some protective natural compounds. Typically, fluorescein-labeled DNA (FAM-DNA) was immobilized on the MBs and then oxidized by the smoke in the absence or presence of natural compounds, and a part of FAM-DNA was fragmented due to cigarette smoke-induced DNA SSB and then detached from MBs whereas other non-broken FAM-DNA still remained on MBs. Then, any broken FAM-DNA fragments, complex tobacco smoke matrix, and other stuff related with natural compounds were conveniently washed away by a magnetic force, and thus possible interfering substances were completely removed. Finally, those remaining non-broken FAM-DNA on MBs were reacted with HRP-labeled anti-fluorescein antibody and then detected by CL ELISA. CL signal was converted to molar concentrations of the FAM-DNA by interpolation from a pre-determined standard linear calibration curve. The level of DNA SSBs induced by cigarette smoke was thus calculated using the method. A library of 30 natural products was subsequently screened, and two among them were found to protect DNA from oxidative damage and thus may be promising compounds for the development of new drugs. The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of DNA SSBs. Graphical abstract ᅟ
Keywords: Chemiluminescence; DNA; Magnetic beads; Natural products; Screening

Analysis of environmental microplastics by vibrational microspectroscopy: FTIR, Raman or both? by Andrea Käppler; Dieter Fischer; Sonja Oberbeckmann; Gerald Schernewski; Matthias Labrenz; Klaus-Jochen Eichhorn; Brigitte Voit (8377-8391).
The contamination of aquatic ecosystems with microplastics has recently been reported through many studies, and negative impacts on the aquatic biota have been described. For the chemical identification of microplastics, mainly Fourier transform infrared (FTIR) and Raman spectroscopy are used. But up to now, a critical comparison and validation of both spectroscopic methods with respect to microplastics analysis is missing. To close this knowledge gap, we investigated environmental samples by both Raman and FTIR spectroscopy. Firstly, particles and fibres >500 μm extracted from beach sediment samples were analysed by Raman and FTIR microspectroscopic single measurements. Our results illustrate that both methods are in principle suitable to identify microplastics from the environment. However, in some cases, especially for coloured particles, a combination of both spectroscopic methods is necessary for a complete and reliable characterisation of the chemical composition. Secondly, a marine sample containing particles <400 μm was investigated by Raman imaging and FTIR transmission imaging. The results were compared regarding number, size and type of detectable microplastics as well as spectra quality, measurement time and handling. We show that FTIR imaging leads to significant underestimation (about 35 %) of microplastics compared to Raman imaging, especially in the size range <20 μm. However, the measurement time of Raman imaging is considerably higher compared to FTIR imaging. In summary, we propose a further size division within the smaller microplastics fraction into 500–50 μm (rapid and reliable analysis by FTIR imaging) and into 50–1 μm (detailed and more time-consuming analysis by Raman imaging). Graphical Abstract Marine microplastic sample (fraction <400 μm) on a silicon filter (middle) with the corresponding Raman and IR images
Keywords: Microplastics; Raman spectroscopy; FTIR spectroscopy; Environmental

Ciprofloxacin: pH-dependent SERS signal and its detection in spiked river water using LoC-SERS by Izabella J. Hidi; Jan Heidler; Karina Weber; Dana Cialla-May; Jürgen Popp (8393-8401).
Monitoring the successful removal of antibiotics in waste and surface waters is of high interest to overcome the occurrence of antibacterial resistance in the ecosystem. Among the newly developed analytical methods, the lab-on-a-chip surface-enhanced Raman spectroscopic (LoC-SERS) technique has gained the interest of the scientific community in the last few years. Ciprofloxacin, a second-generation fluoroquinolone, is widely used and administered to patients in dosages up to 1000 mg. In addition, more than 50 % of the antibiotic is excreted in urine as the parental drug. Thus, ciprofloxacin in environmental samples may exceed the minimum inhibitory concentration (MIC) values. The present study aims to assess the potential of the LoC-SERS technique to detect the target analyte in spiked river water samples at MIC concentrations. As sample clean-up procedure, a simple filtration is proposed, while as SERS, active substrates silver nanoparticles prepared at room temperature are employed. Ciprofloxacin was successfully quantified in the 0.7–10 μM concentration range with data that were measured on two different days. Furthermore, because of the low solubility of the antibiotic at the neutral pH range, insights into the effect of pH on the SERS signal of the target molecule are also presented. Graphical Abstract Ciprofloxacin detected at MIC values by LOC-SERS
Keywords: Ciprofloxacin; SERS; Microfluidics; Surface water; LoC

Structural modifications of cellulose samples after dissolution into various solvent systems by Jérémy Rebière; Maëlie Heuls; Patrice Castignolles; Marianne Gaborieau; Antoine Rouilly; Frédéric Violleau; Vanessa Durrieu (8403-8414).
This work deals with the modifications resulting from the dissolution of four commercial cellulosic samples, with different crystallinity rates and degrees of polymerization (DPs), in four solvent systems, known and used to dissolve cellulose. The dissolution conditions were optimized for the 16 various systems and followed by turbidity measurements. After regeneration, the samples were analyzed by thermal gravimetric analysis (TGA), scanning electron microscopy (SEM), and X-ray diffractometry (XRD) to study their modification. Viscosimetry measurements were used to evaluate the potential decrease of the DP after dissolution. The observed structural modifications established that, for low DP, all the solvent systems were efficient in dissolving the cellulose without altering the DP, except BMIM [Cl], which provoked a decrease of up to 40 % and a decrease of around 20 % of the degradation temperature (onset temperature, T o). For high molecular weight (MW) celluloses, DMSO/TBAF was the only system to allow a complete dissolution without any molar mass loss and degradation temperature modification.
Keywords: Cellulose; Dissolution; Underivatizing solvent; Structural effects; Crystallinity index; Degree of polymerization

The adhesion, spreading, and proliferation of human umbilical vein endothelial cell line (HUVEC-C) cells, on a gold electrode were monitored using quartz crystal microbalance (QCM) measurements. The viscodensity effect caused by the normal action of the cells led to a decrease of the resonant frequency and increase of the motional resistance. The oxidative injury of HUVEC-C cells appeared immediately with the addition of H2O2, exhibiting the decline of cellular spreading area and cell coverage on the electrode surface and resulting in inverted QCM responses. The injured extent of the cells was found to be related to the content of H2O2. It is found that 0.05 mM quercetin added beforehand in the growth medium could remove completely the oxidative action of 1.0 mM H2O2. Quercetin with increased dosage still exerted a partial protective effect on HUVEC-C cells against oxidative injury induced by 2.5 mM H2O2. The microscope observations, electrochemical measurements, and MTT analysis validate the QCM assay results, indicating that quercetin is a valuable flavonoid anti-oxidant in the precaution and treatment for the oxidative injury of vascular endothelium. Graphical Abstract Upper part: Microscope images (×400) of 7.5×104 HUVEC-C cells adhered to the substrate at 48 h in the presence of H2O2. Middle part: Real-time Δf 0 and ΔR 1 responses to the addition of 7.5×104 HUVEC-C cells onto QCM gold electrode in the presence of H2O2 added at 24 h after the introduction of the cells. Lower part: Microscope images (×400) of 7.5×104 HUVEC-C cells adhered to the substrate at 48 h in the presence of quercetin added at 18 h and H2O2 added at 24 h after the introduction of the cells
Keywords: Vascular endothelial cells; H2O2 ; Oxidative injury; Quercetin; Protective effect; Quartz crystal microbalance

Evaluation of a minimally invasive glucose biosensor for continuous tissue monitoring by Sanjiv Sharma; Zhenyi Huang; Michelle Rogers; Martyn Boutelle; Anthony E. G. Cass (8427-8435).
We describe here a minimally invasive glucose biosensor based on a microneedle array electrode fabricated from an epoxy-based negative photoresist (SU8 50) and designed for continuous measurement in the dermal compartment with minimal pain. These minimally invasive, continuous monitoring sensor devices (MICoMS) were produced by casting the structures in SU8 50, crosslinking and then metallising them with platinum or silver to obtain the working and reference electrodes, respectively. The metallised microneedle array electrodes were subsequently functionalised by entrapping glucose oxidase in electropolymerised polyphenol (PP) film. Sensor performance in vitro showed that glucose concentrations down to 0.5 mM could be measured with a response times (T90) of 15 s. The effect of sterilisation by Co60 irradiation was evaluated. In preparation for further clinical studies, these sensors were tested in vivo in a healthy volunteer for a period of 3–6 h. The sensor currents were compared against point measurements obtained with a commercial capillary blood glucometer. The epoxy MICoMS devices showed currents values that could be correlated with these. Graphical Abstract Microneedle arrays for continuous glucose monitoring in dermal interstitial fluid
Keywords: Microneedles; Electrochemical sensor; Continuous glucose monitoring (CGM)

A novel protocol for enzymatic digestion based on covalent binding by protein immobilization by Qian Qi; Guoquan Yan; Chunhui Deng; Xiangmin Zhang (8437-8445).
The process of protein digestion is a critical step for successful protein identification in proteomic analysis. Many efforts have been dedicated to enhancing the digestion efficiency for sufficient digestion. Among these approaches, protein complete denaturation with denaturants is a common process for better digestion. However, the removal of denaturants was tedious or would cause protein loss and other problems. In this work, a feasible digestion approach, immobilized protein digestion (IPD), based on covalent binding has been developed. Proteins can be completely denatured and immobilized on the surface of functional materials by covalent binding to form a monolayer. Subsequently, varieties of denaturants or contaminants would be removed thoroughly by washing. To achieve fast immobilization and high digestion efficiency, different functional materials and denaturants were selected. Compared with traditional in-solution digestion, the method achieved a prominent increase in identified peptides numbers and sequence coverage of proteins. Data analysis also showed that covalent binding could evidently decrease enzymatic missed cleavage for various protein sequences. Furthermore, possible peptide losses due to covalent binding were also investigated. Also, it has been proved to be efficient for complex biological sample digestion. Graphical abstract Workflow of the IPD method, including protein denaturation, immobilization, digestion, and identification
Keywords: Protein digestion; Denaturation; Functional microspheres; Covalent binding

Surface-enhanced Raman probe for rapid nanoextraction and detection of erythropoietin in urine by Yesim Somay Selbes; Mehmet Gokhan Caglayan; Merve Eryilmaz; Ismail Hakki Boyaci; Necdet Saglam; Arif Ahmet Basaran; Ugur Tamer (8447-8456).
We present a surface-enhanced Raman probe (SERS) platform for the determination of a prohibited substance, recombinant erythropoietin (rEPO), in urine matrix, using nanoparticles as substrate. Rod-shaped gold nanoparticles (GNR) were modified with a Raman label and an antibody as SERS probe. We developed two SERS-based immunoassays for detection and quantification of rEPO in urine. In the first assay, rEPO was determined by a sandwich assay with gold surfaces and GNR. In the second assay, rEPO was extracted by using core shell-structured magnetic iron oxide gold nanoparticles, and again sandwich assay was performed by using GNR. We also demonstrated the ability of the proposed method to discriminate rEPO and urinary erythropoietin (uEPO). A good linear correlation was obtained between logarithms of rEPO concentrations in urine and Raman intensities within the range of 10−1–103 pg mL−1 rEPO concentrations. Detection limits which are smaller than 0.1 pg mL−1 levels were achieved owing to the high extractive performance of the nanoextraction techniques. Graphical Abstract Schematic represantation of surface-enhanced Raman probe for rapid nanoextraction and detection of erythropoietin
Keywords: Erythropoietin; Nanoextraction; Western blot; SERS

In this study, a novel boronic acid-fumed silica nanoparticles incorporated hybrid monolithic stationary phase for glycoprotein separation by nano-liquid chromatography was developed. The stationary phase was prepared in 100 μm capillary by an in situ copolymerization of methacryloyl-fumed silica nanoparticle (MFSNP), 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl), and ethylene dimethacrylate (EDMA) in a binary porogenic solvent composed of cyclohexanol and dodecanol. The preparation of the monolith was optimized by investigating the ratio of functional monomer to crosslinker and the effect of MFSNP content. The resulting monolithic column was functionalized with 3-aminophenylboronic acid (3-APBA). The column morphology, pore size, and specific surface area of the fabricated monolith were characterized by scanning electron microscopy, X-ray photo electron spectroscopy, and BET equation by means of the nitrogen adsorption–desorption isotherm, respectively. Good permeability stability and column efficiency were observed on the monolith with nano-flow. The results also indicated that the MFSNP content was very important for final preparation of the monolithic stationary phase. The monolith with MFSNP could achieve better separation than one without MFSNP. The chromatographic performance of the monolith with respect to hydrophobic/affinity interactions was evaluated by the separation of alkylbenzene derivatives, proteins, and glycoprotein, respectively. The column efficiencies for alkylbenzenes on the hybrid monolithic column reached to 15,600–25,000 plates/m at the velocity of 1.2 mm/s in nano-liquid chromatography.
Keywords: Boronic acid; Nano-LC; Proteomics; Monolith; Silica nanoparticles; Affinity

Guilty by dissociation—development of gas chromatography–mass spectrometry (GC-MS) and other rapid screening methods for the analysis of 13 diphenidine-derived new psychoactive substances (NPSs) by Pierre M. Geyer; Matthew C. Hulme; Joseph P. B. Irving; Paul D. Thompson; Ryan N. Ashton; Robert J. Lee; Lucy Johnson; Jack Marron; Craig E. Banks; Oliver B. Sutcliffe (8467-8481).
The prevalence of new psychoactive substances (NPSs) in forensic casework has increased prominently in recent years. This has given rise to significant legal and analytical challenges in the identification of these substances. The requirement for validated, robust and rapid testing methodologies for these compounds is obvious. This study details the analysis of 13 synthesised diphenidine derivatives encountered in casework using presumptive testing, thin layer chromatography and gas chromatography–mass spectrometry (GC-MS). Specifically, the validated GC-MS method provides, for the first time, both a general screening method and quantification of the active components for seized solid samples, both in their pure form and in the presence of common adulterants. Graphical Abstract Chemical synthesis and forensic analysis of 13 diphenidine-derived new psychoactive substance(s)
Keywords: New psychoactive substances; Characterisation (NMR, FT-IR); Diphenidine; Methoxphenidine; GC–MS; Triage

Evaluation of dilution and normalization strategies to correct for urinary output in HPLC-HRTOFMS metabolomics by Franziska C. Vogl; Sebastian Mehrl; Leonhard Heizinger; Inga Schlecht; Helena U. Zacharias; Lisa Ellmann; Nadine Nürnberger; Wolfram Gronwald; Michael F. Leitzmann; Jerome Rossert; Kai-Uwe Eckardt; Katja Dettmer; Peter J. Oefner (8483-8493).
Reliable identification of features distinguishing biological groups of interest in urinary metabolite fingerprints requires the control of total metabolite abundance, which may vary significantly as the kidneys adjust the excretion of water and solutes to meet the homeostatic needs of the body. Failure to account for such variation may lead to misclassification and accumulation of missing data in case of less concentrated urine specimens. Here, different pre- and post-acquisition methods of normalization were compared systematically for their ability to recover features from liquid chromatography-mass spectrometry metabolite fingerprints of urine that allow distinction between patients with chronic kidney disease and healthy controls. Methods of normalization that were employed prior to analysis included dilution of urine specimens to either a fixed creatinine concentration or osmolality value. Post-acquisition normalization methods applied to chromatograms of 1:4 diluted urine specimens comprised normalization to creatinine, osmolality, and sum of all integrals. Dilution of urine specimens to a fixed creatinine concentration resulted not only in the least number of missing values, but it was also the only method allowing the unambiguous classification of urine specimens from healthy and diseased individuals. The robustness of classification could be confirmed for two independent patient cohorts of chronic kidney disease patients and yielded a shared set of 49 discriminant metabolite features. Graphical Abstract Dilution to a uniform creatinine concentration across urine specimens yields more comparable urinary metabolite fingerprints
Keywords: Metabolic fingerprinting; LC-MS; Urine; Creatinine; Osmolality; Normalization

Use of a versatile, easy, and rapid atmospheric monitor (VERAM) passive samplers for pesticide determination in continental waters by Thalita Dallapícula Ramos; Ricardo J. Cassella; Miguel de la Guardia; Agustín Pastor; Francesc A. Esteve-Turrillas (8495-8503).
The versatile, easy, and rapid atmospheric monitor (VERAM), a passive sampler device widely used for air monitoring, was evaluated as passive sampler for the determination of 23 pesticides in water. Gas chromatography with mass spectrometry detection was employed for determination of pesticides after microwave-assisted-extraction and specific clean-up of deployed samplers. The proposed methodology reached method detection levels from 2 to 10 ng pesticide per sampler. Sampling rate (Rs) was determined for every pesticide from an uptake isotherm study performed at three different concentration levels (50, 125, and 250 ng L–1). The obtained RS values ranged from 0.06 to 0.76 L d–1. The obtained limits of detection for a 24-h passive sampling were from 4 to 50 ng L–1. The effect of water parameters, such as temperature, pH, and ionic strength, were evaluated for their effect on pesticides retention using VERAMs. Pesticide RS values were independent of the water composition and increased on increasing temperature. Finally, the VERAM uptake was compared with that obtained using classic semipermeable membrane devices (SPMDs). This study is the first precedent for the use of VERAMs as passive samplers for the adsorption and concentration of pesticides in water and it confirms the satisfactory analytical figures of merit of VERAM as passive sampler of water. Graphical Abstract Scheme of water sampling of pesticides using versatile, easy, and rapid atmospheric monitor (VERAM) passive samplers
Keywords: VERAM; SPMD; Pesticide; Water sampling; Passive sampler; Sampling rate

Pitfalls and capabilities of various hydrogen donors in evaluation of peroxidase-like activity of gold nanoparticles by Marcin Drozd; Mariusz Pietrzak; Paweł G. Parzuchowski; Elżbieta Malinowska (8505-8513).
Catalytic nanomaterials, widely used as substitutes of peroxidase, exhibit unique properties, which are unattainable for native enzymes. However, their activity is usually examined by means of substrates developed and methods standardized for horseradish peroxidase (HRP). The aim of the presented work was to determine the scope of usefulness of chromogenic substrates for gold nanoparticle (AuNP) activity studies under conditions which significantly extend beyond the activity range of a native HRP. The applicability of chromogens such as 3,3′5,5′-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) beyond the typical range of pH, and for the samples of high concentration of hydrogen peroxide was examined. The conducted research confirmed the usefulness of ABTS and TMB in acidic media (pH 2.5–3.5). At the same time, potential interferences from chloride anion, unobservable for HRP-based assays, were indicated. Moreover, a number of potentially useful hints concerning relations of concentration of substrates and catalyst for aromatic amine oxidation (TMB and OPD) were proposed. By increasing the concentration of chromogens and thanks to assuring the relatively low conversion of the reaction, the stability of TMB and OPD oxidation product was improved even in acidic media. The comparative studies of H2O2 affinity to the surface of AuNPs in the presence of various hydrogen donors underlined the superiority of phenolic compounds over aromatic amines and ABTS in the case of the samples of relatively low H2O2 concentration. This work highlights some improvements in the methods of HRP-like activity characterization of NPs. It provides a critical analysis of the major challenges, which may emerge in a case of bioanalytical assays employing the catalytic nanoparticles as labels.
Keywords: Catalytic activity; Gold nanoparticles; Peroxidase mimetics; Nanozyme

In the presented work, metal-organic framework (MOF) material MIL-101(Cr) (MIL, Matérial Institute Lavoisier) was used as a sorbent for vortex assisted dispersive micro-solid-phase extraction (VA-D-μ-SPE) of trace amount of metronidazole (MNZ), ronidazole (RNZ), secnidazole (SNZ), dimetridazole (DMZ), tinidazole (TNZ), and ornidazole (ONZ) in different environmental water samples. Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was used to quantify the target analytes. The extraction conditions, including type of sorbents, amount of MIL-101(Cr), solution pH, extraction method, extraction time, effect of salt, and elution conditions were investigated. Upon the optimal conditions, the developed method showed an excellent extraction performance with the average recovery ranging from 75.2 to 98.8 %. Good sensitivity levels were achieved with the detection limits of 0.03∼0.06 μg/L and the quantitation limits of 0.09∼0.20 μg/L. The linear ranges were varied from 0.1 to 20 for SNZ and ONZ and from 0.2 to 40 μg/L for MNZ, RNZ, DMZ, and TNZ (r 2 > 0.992), and repeatability of the method was satisfactory with the relative standard deviations (RSD) <8 %. Ultimately, the applicability of the method was successfully confirmed by the extraction and determination of 5-nitroimidazoles (5-NDZs) in 12 real water samples, showing the positive findings of MNZ and TNZ ranging from 0.3 to 1.0 μg/L. Furthermore, molecular docking was applied to explain the molecular interactions and free binding energies between MIL-101(Cr) and 5-NDZs, providing a deep insight into the adsorption mechanism. The proposed method exhibited the advantages of simplicity, rapidly, less solvent consumption, ease of operation, higher sensitivity, and lower matrix effect. Graphical abstract Schematic diagram of the extraction process and molecular docking investigation
Keywords: MIL-101(Cr); Vortex assisted dispersive micro-solid-phase extraction; 5-Nitroimidazoles; Molecular docking; Adsorption mechanism; Environmental waters analysis

Quantitative characterization of capsaicin-induced TRPV1 ion channel activation in HEK293 cells by impedance spectroscopy by Maxi Weyer; Heinz-Georg Jahnke; Dana Krinke; Franziska D. Zitzmann; Kerstin Hill; Michael Schaefer; Andrea A. Robitzki (8529-8538).
The analysis of receptor activity, especially in its native cellular environment, has always been of great interest to evaluate its intrinsic but also downstream biological activity. An important group of cellular receptors are ion channels. Since they are involved in a broad range of crucial cell functions, they represent important therapeutic targets. Thus, novel analytical techniques for the quantitative monitoring and screening of biological receptor activity are of great interest. In this context, we developed an impedance spectroscopy-based label-free and non-invasive monitoring system that enabled us to analyze the activation of the transient receptor potential channel Vanilloid 1 (TRPV1) in detail. TRPV1 channel activation by capsaicin resulted in a reproducible impedance decrease. Moreover, concentration response curves with an EC50 value of 0.9 μM could be determined. Control experiments with non TRPV1 channel expressing HEK cells as well as experiments with the TRPV1 channel blocker ruthenium red validated the specificity of the observed impedance decrease. More strikingly, through correlative studies with a cytoskeleton restructuring inhibitor mixture and equivalent circuit analysis of the acquired impedance spectra, we could quantitatively discriminate between the direct TRPV1 channel activation and downstream-induced biological effects. In summary, we developed a quantitative impedimetric monitoring system for the analysis of TRPV1 channel activity as well as downstream-induced biological activity in living cells. It has the capabilities to identify novel ion channel activators as well as inhibitors for the TRPV1 channel but could also easily be applied to other ion channel-based receptors.
Keywords: TRPV1 channel activation monitoring; Time-resolved quantification of ion channel activity; Impedance spectroscopy; Interdigital electrode arrays; Equivalent circuit modeling

Rapid determination of β-lactam antimicrobial resistance in bacteria by a liquid chromatography-mass spectrometry-based method by JeongWoo Kang; Md Akil Hossain; Hae-chul Park; Yangho Jang; Seonhwa Kim; Jae Young Song; Kwang-jick Lee; Tae-Wan Kim (8539-8549).
Conventional antimicrobial susceptibility tests (ASTs) are very time consuming and insufficiently precise to promptly select a proper antimicrobial treatment. This difficulty disrupts the management of infections and exacerbates the development of antimicrobial resistance. Generally, antimicrobial resistance involves the chemical modification of an antimicrobial compound to an inactive form by an enzyme released by bacteria. This modification causes a structural change and is followed by a characteristic mass shift of the antimicrobials. Using this mechanism, we developed a new liquid chromatography-mass spectrometry method to rapidly determine the degree of resistance of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium), Escherichia coli, and Staphylococcus aureus to amoxicillin, ampicillin, and penicillin G, respectively. This method was successfully applied to 20 bacterial isolates from Korean slaughterhouses and farms. There were 18-Da mass shifts in resistant strains compared with susceptible strains of Salmonella Typhimurium, E. coli, and S. aureus, and the intensities of the hydrolyzed penicillin mass spectra were much higher in resistant strains than those in susceptible strains, which together indicate the reliability of this method. A comparison of the mass spectrometry-derived results with that from conventional ASTs revealed an identical classification of the tested bacteria according to sensitivity and resistance. Notably, this assay method requires only 2 h for determining the susceptibility status of a strain. This newly developed method is able to determine the extent of antimicrobial resistance qualitatively and quantitatively within a very short time and could be used to replace conventional AST methods. Graphical abstract Rapid determination of β-lactam antimicrobial resistance in bacteria by LC-MS/MS
Keywords: Rapid analysis; β-lactam antimicrobial resistance; Antimicrobial susceptibility; LC-MS/MS determination

We report a simple, facile, and reliable colorimetric system for detection of chromium(III) ions (Cr3+) using citrate- and thiourea-modified gold nanoparticles (AuNPs). The colorimetric sensing strategy is based on the synergistic coordination interaction of citrate and thiourea toward Cr3+ on the surface of AuNPs, leading to the aggregation of AuNPs which produces a color change from red to purple. Under the optimal conditions, this colorimetric sensing system shows an excellent selectivity and sensitivity for Cr3+, and the limit of detection (LOD) is estimated to be 0.05 μM at a signal-to-noise ratio of 3, which is far below the current standard stipulated by U.S. Environmental Protection Agency (1.9 μM). Moreover, this LOD is one and a half orders of magnitude lower than those of previously reported modified AuNPs-based colorimetric methods. Visual color change can be observed when 50 μM of Cr3+ was introduced to the sensing system. Furthermore, this colorimetric sensing system can be employed for detection of Cr3+ in diluted natural water samples.
Keywords: Thiourea; Citrate; Colorimetry; Gold nanoparticles; Cr3+

Highly selective solid-phase extraction sorbents for chloramphenicol determination in food and urine by ion mobility spectrometry by Sergio Armenta; Miguel de la Guardia; Antonio Abad-Fuentes; Antonio Abad-Somovilla; Francesc A. Esteve-Turrillas (8559-8567).
Different highly selective sorbents have been evaluated for the treatment of food and biological samples to determine chloramphenicol residues by ion mobility spectrometry (IMS). Combination of a selective solid-phase extraction (SPE) and dispersive liquid-liquid microextraction allowed a highly sensitive determination of chloramphenicol in water, milk, honey, and urine samples. The performance of selective SPE supports such as immunoaffinity chromatography (IAC) and molecular imprinted polymers (MIP) have been compared in terms of selectivity, sensitivity, trueness, precision, and reusability. Quantitative recoveries were obtained for chloramphenicol residues, ranging from 91 to 123 % for water, from 99 to 120 % for skimmed milk, and from 95 to 124 % for urine using IAC-IMS and MIP-IMS methods. Quantitative recoveries (from 88 to 104 %) were also achieved for honey samples using IAC-IMS, but low recoveries were obtained using MIP-IMS. The limit of quantification was set at 0.1 μg L−1 which is lower than the minimum required performance limit established by the EU. The proposed methodology is a simple and cost affordable alternative to chromatography methods for the highly sensitive and selective analysis of chloramphenicol residues in food and urine. Graphical Abstract Scheme for chloramphenicol determination by selective solid-phase extraction and ion mobility spectrometry
Keywords: Chloramphenicol; Ion mobility spectrometry; Immunoaffinity chromatography; Molecular imprinted polymer; Solid-phase extraction