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Analytical and Bioanalytical Chemistry (v.405, #5)

Characterization of thin films and membranes by Pankaj Vadgama; Daniel Mandler (pp. 1433-1434).

is currently Professor of Clinical Biochemistry and Director of the Interdisciplinary Research Centre (IRC) in Biomedical Materials at Queen Mary University of London. His research interests are polymeric membranes for sample separation, electrochemical metabolite sensors for continuous monitoring and microfluidics for bioreactors. He is currently Head of Service for Clinical Biochemistry at Barts and the London NHS Trust. is a Professor of Chemistry and currently serves also as the Vice Dean for Research of the Faculty of Science at the Hebrew University of Jerusalem. His research interests span from analytical to physical electrochemistry sol-gel technology, thin films and polymers, and forensic science. He is the head of the Analytical Chemistry program at the Hebrew University, has supervised more than 40 MSc and PhD students, and has published over 150 papers.

Recent advances in the development of graphene-based surface plasmon resonance (SPR) interfaces by Sabine Szunerits; Nazek Maalouli; Edy Wijaya; Jean-Pierre Vilcot; Rabah Boukherroub (pp. 1435-1443).

Surface plasmon resonance (SPR) is a powerful technique for measurement of biomolecular interactions in real-time in a label-free environment. One of the most common techniques for plasmon excitation is the Kretschmann configuration, and numerous studies of ligand–analyte interactions have been performed on surfaces functionalized with a variety of biomolecules, for example DNA, RNA, glycans, proteins, and peptides. A significant limitation of SPR is that the substrate must be a thin metal film. Post-coating of the metal thin film with a thin dielectric top layer has been reported to enhance the performance of the SPR sensor, but is highly dependent on the thickness of the upper layer and its dielectric constant. Graphene is a single-atom thin planar sheet of sp2 carbon atoms perfectly arranged in a honeycomb lattice. Graphene and graphene oxide are good supports for biomolecules because of their large surface area and rich π conjugation structure, making them suitable dielectric top layers for SPR sensing. In this paper, we review some of the key issues in the development of graphene-based SPR chips. The actual challenges of using these interfaces for studying biomolecular interactions will be discussed and the first examples of the use of graphene-on-metal SPR interfaces for biological sensing will be presented.

Keywords: Biosensors; Thin films; Bioanalytical methods; Biological samples; Electrochemical sensors; Mass sensitive sensors; Interface; Surface analysis

Atomic force microscopy of model lipid membranes by Sandrine Morandat; Slim Azouzi; Estelle Beauvais; Amira Mastouri; Karim El Kirat (pp. 1445-1461).

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles. Figure The experimental atomic force microscopy (AFM) setup used to examine supported lipid bilayers (SLBs) under physiological conditions.

Keywords: Atomic force microscopy; Force spectroscopy; Supported lipid bilayers; Biomimetic membranes; Nanoscale organization; Nanomechanics

Mechanical characterization of polymeric thin films by atomic force microscopy based techniques by Daniele Passeri; Marco Rossi; Emanuela Tamburri; Maria Letizia Terranova (pp. 1463-1478).

Polymeric thin films have been awakening continuous and growing interest for application in nanotechnology. For such applications, the assessment of their (nano)mechanical properties is a key issue, since they may dramatically vary between the bulk and the thin film state, even for the same polymer. Therefore, techniques are required for the in situ characterization of mechanical properties of thin films that must be nondestructive or only minimally destructive. Also, they must also be able to probe nanometer-thick ultrathin films and layers and capable of imaging the mechanical properties of the sample with nanometer lateral resolution, since, for instance, at these scales blends or copolymers are not uniform, their phases being separated. Atomic force microscopy (AFM) has been proposed as a tool for the development of a number of techniques that match such requirements. In this review, we describe the state of the art of the main AFM-based methods for qualitative and quantitative single-point measurements and imaging of mechanical properties of polymeric thin films, illustrating their specific merits and limitations.

Keywords: Polymeric thin film; Atomic force microscopy; Mechanical measurement; Mechanical imaging

Structural and electronic characterization of self-assembled molecular nanoarchitectures by X-ray photoelectron spectroscopy by Antonino Gulino (pp. 1479-1495).

Molecular monolayers and similar nanoarchitectures are indicative of the promising future of nanotechnology. Therefore, many scientists recently devoted their efforts to the synthesis, characterization, and properties of mono- and multilayer-based systems. In this context, X-ray photoelectron spectroscopy is an important technique for the in-depth chemical and structural characterization of nanoscopic systems. In fact, it is a surface technique suitable for probing thicknesses of the same order of the photoelectron inelastic mean free paths (a few tens of ångströms) and allows one to immediately obtain qualitative and quantitative data, film thickness, surface coverage, molecule footprint, oxidation states, and presence of functional groups. Nevertheless, other techniques are important in obtaining a complete spectroscopic characterization of the investigated systems. Therefore, in the present review we report on X-ray photoelectron spectroscopy of self-assembled molecular mono- and multilayer materials including some examples on which other characterization techniques produced important results. Figure X-ray photoelectron spectroscopy revealed to be an important technique for an in-depth chemical and structural characterization of self-assembled molecular mono- and multi-layer materials

Keywords: XPS; Self-assembly; Monolayer; Nanoarchitecture; Sensing

Electrochemical approaches for the fabrication and/or characterization of pure and hybrid templated mesoporous oxide thin films: a review by Mathieu Etienne; Yann Guillemin; David Grosso; Alain Walcarius (pp. 1497-1512).

Electrochemistry can be used for fabrication and characterization of mesoporous oxide films. First, this review provides insight into the methods used to prepare templated mesoporous thin films on an electrode surface, i.e., evaporation-induced self-assembly (EISA) and electrochemically assisted self-assembly (EASA). Electrochemical characterization of mass transport processes in pure and organically functionalized mesoporous oxide films is then discussed. The electrochemical response can be basically restricted by the electron/mass transfer reaction at the electrode–film interface and diffusion through mesopore channels. The contributions of cyclic voltammetry, hydrodynamic voltammetry, electrochemical impedance spectroscopy, and scanning electrochemical microscopy to the characterization of films with distinct mesostructures are finally described, with special emphasis on identification of conditions that can affect the electrochemical response recorded with such modified electrodes. Figure Permeability through mesoporous thin films

Keywords: Ordered mesoporous thin films; Templated silica; Permeability; Voltammetry; SECM; Electrochemical impedance spectroscopy

Studies of the interface of conducting polymers with inorganic surfaces by Fabio Terzi; Luca Pasquali; Renato Seeber (pp. 1513-1535).

Many of the properties of multi-material systems and relevant devices depend on the interfaces between the different components. This review focuses on characterization of the interfaces between intrinsically conducting polymers and inorganic materials consisting of metals and metal oxides. These materials are chosen because of their importance in several analytical applications. Although use of conducting polymers and metals or metal oxides in analytical systems, specifically in sensing, is well established, the number of novel materials used for analytical purposes is continuously increasing. This further increases the possible number of effective combinations of different materials within multicomponent systems. As a consequence, innovative characterization techniques have become as important as more conventional techniques. On the other hand, sophisticated characterisation techniques are increasingly widespread and, consequently, also readily accessible. This critical review is not an exhaustive discussion of all possible analytical techniques suitable for characterization of interfaces. It is, instead, limited to an overview of the most effective, relatively widespread techniques, emphasising their most significant recent advances. Critical analysis of the individual techniques is complemented by a few selected examples.

Keywords: Interface/surface analysis; Polymers; Spectroscopy/instrumentation; X-ray spectroscopy (XPS | XRF | EDX); Imaging (NMR microscopy, electron microscopy)

Application of PM-IRRAS to study thin films on industrial and environmental samples by Annia H. Kycia; Kallum Koczkur; J. Jay Leitch; Jacek Lipkowski; Vlad Zamlynny; Michael W. P. Petryk (pp. 1537-1546).

Polarization modulation–infrared reflection absorption spectroscopy (PM-IRRAS) was employed to analyze two unique samples: (1) an industrially prepared alkoxysilane-pretreated aluminum alloy (AA6111) in the absence and presence of a ∼600-nm-thick lubricant coating and (2) a chemical warfare agent simulant, triethyl phosphate (TEP), on glass. For the pretreated aluminum samples, PM-IRRAS spectra were analyzed for three distinct regions; the SiO stretching vibration around 1120 cm⁻¹, the NH₂ bending mode at ∼1600 cm⁻¹ and the CH stretching region around 2900 cm⁻¹. Our results showed that increasing the curing temperature (from 55 to 100 °C) improved the overall extent of cross-linking within the siloxane network. In addition, the spectra of lubricant (top coating) and the underlying siloxane layer for the aluminum samples with lubricant were collected for the same sample. Our results show that the nature of the siloxane film remains intact and unaltered after deposition of the lubricant top-coat. For detection of TEP on glass, the band at 1268 cm⁻¹, corresponding to the P═O vibration, was monitored. A droplet of TEP solution in dichloromethane was deposited on glass. After solvent evaporation had occurred, the intensity of the P═O vibration band was used to construct calibration curves to determine the experimental limit of detection, which was found to be ∼200 μg for TEP on glass. Figure Schematic of a akoxysilane network and PM-IRRAS spectrum of the alkoxysilane film at aluminum surface

Keywords: Polarization modulation–infrared reflection absorption spectroscopy (PM-IRRAS); Aluminum oxide; Siloxane films; Lubricant; Triethyl phosphate; Glass

Characterization of nanochannel delivery membrane systems for the sustained release of resveratrol and atorvastatin: new perspectives on promoting heart health by Juliana Sih; Shyam S. Bansal; Stefano Filipini; Silvia Ferrati; Kunal Raghuwansi; Erika Zabre; Eugenia Nicolov; Daniel Fine; Mauro Ferrari; Ganesh Palapattu; Alessandro Grattoni (pp. 1547-1557).

Novel drug delivery systems capable of continuous sustained release of therapeutics have been studied extensively for use in the prevention and management of chronic diseases. The use of these systems holds promise as a means to achieve higher patient compliance while improving therapeutic index and reducing systemic toxicity. In this work, an implantable nanochannel drug delivery system (nDS) is characterized and evaluated for the long-term sustained release of atorvastatin (ATS) and trans-resveratrol (t-RES), compounds with a proven role in managing atherogenic dyslipidemia and promoting cardioprotection. The primary mediators of drug release in the nDS are nanofluidic membranes with hundreds of thousands of nanochannels (up to 100,000/mm²) that attain zero-order release kinetics by exploiting nanoconfinement and molecule-to-surface interactions that dominate diffusive transport at the nanoscale. These membranes were characterized using gas flow analysis, acetone diffusion, and scanning and transmission electron microscopy (SEM, TEM). The surface properties of the dielectric materials lining the nanochannels, SiO₂ and low-stress silicon nitride, were further investigated using surface charge analysis. Continuous, sustained in vitro release for both ATS and t-RES was established for durations exceeding 1 month. Finally, the influence of the membranes on cell viability was assessed using human microvascular endothelial cells. Morphology changes and adhesion to the surface were analyzed using SEM, while an MTT proliferation assay was used to determine the cell viability. The nanochannel delivery approach, here demonstrated in vitro, not only possesses all requirements for large-scale high-yield industrial fabrication, but also presents the key components for a rapid clinical translation as an implantable delivery system for the sustained administration of cardioprotectants.

Keywords: Implants; Sustained release; Nanochannel membranes; Drug delivery; Cardioprotection

Chemosensors and biosensors based on polyelectrolyte microcapsules containing fluorescent dyes and enzymes by Lyubov I. Kazakova; Lyudmila I. Shabarchina; Salzitsa Anastasova; Anton M. Pavlov; Pankaj Vadgama; Andre G. Skirtach; Gleb B. Sukhorukov (pp. 1559-1568).

The concept of enzyme-assisted substrate sensing based on use of fluorescent markers to detect the products of enzymatic reaction has been investigated by fabrication of micron-scale polyelectrolyte capsules containing enzymes and dyes in one entity. Microcapsules approximately 5 μm in size entrap glucose oxidase or lactate oxidase, with peroxidase, together with the corresponding markers Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride (Ru(dpp)) complex and dihydrorhodamine 123 (DHR123), which are sensitive to oxygen and hydrogen peroxide, respectively. These capsules are produced by co-precipitation of calcium carbonate particles with the enzyme followed by layer-by-layer assembly of polyelectrolytes over the surface of the particles and incorporation of the dye in the capsule interior or in the multilayer shell. After dissolution of the calcium carbonate the enzymes and dyes remain in the multilayer capsules. In this study we produced enzyme-containing microcapsules sensitive to glucose and lactate. Calibration curves based on fluorescence intensity of Ru(dpp) and DHR123 were linearly dependent on substrate concentration, enabling reliable sensing in the millimolar range. The main advantages of using these capsules with optical recording is the possibility of building single capsule-based sensors. The response from individual capsules was observed by confocal microscopy as increasing fluorescence intensity of the capsule on addition of lactate at millimolar concentrations. Because internalization of the micron-sized multi-component capsules was feasible, they could be further optimized for in-situ intracellular sensing and metabolite monitoring on the basis of fluorescence reporting.

Keywords: Encapsulation; Microsensor; Fluorescence; Lactate; Oxygen; Glucose

Sports drug testing using immuno-MS: clinical study comprising administration of human chorionic gonadotropin to males by Hanne Lund; Ann Helene Snilsberg; Elisabeth Paus; Trine Grønhaug Halvorsen; Peter Hemmersbach; Léon Reubsaet (pp. 1569-1576).

The applicability of a mass spectrometry (MS)-based method for determination of various forms of human chorionic gonadotropin (hCG) in doping analysis was demonstrated. A clinical study involving the hCG-containing pharmaceuticals Pregnyl and Ovitrelle was carried out, comprising a single injection of one pharmaceutical per participant to a total of 24 healthy male voluntaries. Hereafter, serum and urine samples were collected over a period of 14 days. The analysis of the samples using immuno-MS demonstrated elimination profiles of intact hCG for both pharmaceuticals, with last day of detection following administration at day 7 in serum, and at day 10 in urine, at limit of detections as defined by the World Anti-Doping Agency. Furthermore, the method allowed detection and differentiation of the various forms of hCG known to be present in serum and urine as a function of metabolism. For both pharmaceuticals, only the intact hCG was detected in serum, whereas in urine the injection of Pregnyl as hCG source (containing urinary hCG, i.e., most hCG variants) was shown to generate a more complex hCG variant pattern compared to Ovitrelle (contains only intact hCG). By detecting hCG using this MS-based approach in doping analysis, strong analytical evidence is provided minimizing the risk of false-positive and false-negative results.

Keywords: Doping analysis; LC-MS/MS; Human chorionic gonadotropin; Signature peptide; Tryptic digestion; Clinical study

Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy by Mingjie Tang; Gerald D. McEwen; Yangzhe Wu; Charles D. Miller; Anhong Zhou (pp. 1577-1591).

The molecular composition of mycobacteria and Gram-negative bacteria cell walls is structurally different. In this work, Raman microspectroscopy was applied to discriminate mycobacteria and Gram-negative bacteria by assessing specific characteristic spectral features. Analysis of Raman spectra indicated that mycobacteria and Gram-negative bacteria exhibit different spectral patterns under our experimental conditions due to their different biochemical components. Fourier transform infrared (FTIR) spectroscopy, as a supplementary vibrational spectroscopy, was also applied to analyze the biochemical composition of the representative bacterial strains. As for co-cultured bacterial mixtures, the distribution of individual cell types was obtained by quantitative analysis of Raman and FTIR spectral images and the spectral contribution from each cell type was distinguished by direct classical least squares analysis. Coupled atomic force microscopy (AFM) and Raman microspectroscopy realized simultaneous measurements of topography and spectral images for the same sampled surface. This work demonstrated the feasibility of utilizing a combined Raman microspectroscopy, FTIR, and AFM techniques to effectively characterize spectroscopic fingerprints from bacterial Gram types and mixtures. Figure AFM deflection images, Raman spectra, SEM images, and FTIR of Mycobacterium sp. KMS

Keywords: Mycobacteria; Gram-negative bacteria; Raman microspectroscopy; Fourier transform infrared (FTIR) spectroscopy; Atomic force microscopy (AFM)

The effects of season and soil type on microbial degradation of gasoline residues from incendiary devices by Dee A. Turner; John V. Goodpaster (pp. 1593-1599).

The primary task of a fire debris chemist is to determine if there is an ignitable liquid present in a fire debris sample and, if so, to classify it according to its boiling point and carbon number range. However, in organic-rich substrates such as soil, the ignitable liquid residue is subject to microbial degradation due to the ease with which bacteria can metabolize the various hydrocarbons present. This is a rapid process which is problematic in many forensic laboratories as fire debris is often stored for extended periods of time due to case backlog. Although microbial degradation has been studied in laboratory samples, it has not been well-studied in “real-world” samples, which have not only been exposed to microbial degradation but have also suffered the effects of weathering due to the intense heat of the fire. In this work, the effects of microbial degradation of gasoline from an incendiary device have been evaluated over time. In addition to visually monitoring chromatographic changes, this work also utilizes multivariate statistical techniques to simplify the complex data set and elucidate trends that might not otherwise be observed. Results indicate a clear difference between glass samples, which suffered the loss of low boiling compounds, and soil, which suffered the loss of the normal alkanes and lesser substituted aromatics. Also, devices deployed on lawn soil and in the winter season appear to show the most extensive degradation of gasoline. Finally, while the ratio of the C₃-alkylbenzenes is significantly altered in soil samples recovered from large devices, the overall chromatographic profile of gasoline recovered from smaller incendiary devices is significantly lower. Figure An arson investigator from the Indianapolis Fire Department deploys a Molotov Cocktail onto soil

Keywords: Fire debris; Ignitable liquids; Molotov cocktail; Microbial degradation; Chemometrics

Quality assurance in immunoassay performance—comparison of different enzyme immunoassays for the determination of caffeine in consumer products by Julia Grandke; Lidia Oberleitner; Ute Resch-Genger; Leif-Alexander Garbe; Rudolf J. Schneider (pp. 1601-1611).

Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3′,5,5′-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format. Figure Overview on the different enzyme immunoassay formats compared

Keywords: Enzyme immunoassay; Caffeine; Enzyme substrate; Competitive assay; Assay format; Direct; Indirect

Microfluidic channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A by Betty C. Galarreta; Mohammadali Tabatabaei; Valérie Guieu; Eric Peyrin; François Lagugné-Labarthet (pp. 1613-1621).

A selective aptameric sequence is adsorbed on a two-dimensional nanostructured metallic platform optimized for surface-enhanced Raman spectroscopy (SERS) measurements. Using nanofabrication methods, a metallic nanostructure was prepared by electron-beam lithography onto a glass coverslip surface and embedded within a microfluidic channel made of polydimethylsiloxane, allowing one to monitor in situ SERS fingerprint spectra from the adsorbed molecules on the metallic nanostructures. The gold structure was designed so that its localized surface plasmon resonance matches the excitation wavelength used for the Raman measurement. This optofluidic device is then used to detect the presence of a toxin, namely ochratoxin-A (OTA), in a confined environment, using very small amounts of chemicals, and short data acquisition times, by taking advantage of the optical properties of a SERS platform to magnify the Raman signals of the aptameric monolayer system and avoiding chemical labeling of the aptamer or the OTA target. Fig Aptamer detection of OTA within a SERS/microfluidic channel

Keywords: Aptamers; Ochratoxin-A; Microfluidics; SERS platforms; Plasmonics

Development of a microwave-assisted-extraction-based method for the determination of aflatoxins B₁, G₁, B₂, and G₂ in grains and grain products by Si Chen; Hong Zhang (pp. 1623-1630).

This article describes the use of microwave-assisted extraction (MAE) as a pretreatment technique for the determination of aflatoxins B₁, G₁, B₂, and G₂ in grains and grain products. The optimal operation parameters, including extraction solvent, temperature, and time, were identified to be acetonitrile as the extraction solvent at 80 °C with 15 min of MAE. The extracts were cleaned up using solid-phase extraction followed by derivatization with trifluoroacetic acid and were determined by liquid chromatography–fluorescence detection. A Sep-Pak cartridge was chosen over Oasis HLB and Bond Elut cartridges. By the use of aflatoxin M₁ as an internal standard, relative recoveries of the aflatoxins ranged from 90.7 to 105.7 % for corn and from 88.1 to 103.4 % for wheat, with relative standard deviations between 2.5 and 8.7 %. A total of 36 samples from local markets were analyzed, and aflatoxin B₁ was found to be the predominant toxin, with concentrations ranging from 0.42 to 3.41 μg/kg. Figure Methodology for aflatoxins B1, G1, B2 and G2 determination in grains and grain products.

Keywords: Aflatoxins; Microwave-assisted extraction; Corn; Wheat; Precolumn derivatization; Liquid chromatography–fluorescence detection

Development and validation of a confirmative LC-MS/MS method for the determination of ß-exotoxin thuringiensin in plant protection products and selected greenhouse crops by Theo C. de Rijk; Ruud C. J. van Dam; Paul Zomer; Ed A. M. Boers; Pieter de Waard; Hans G. J. Mol (pp. 1631-1639).

Bacterial products based on Bacillus thuringiensis are registered in many countries as plant protection products (PPPs) and are widely used as insecticides and nematocides. However, certain B. thuringiensis strains produce harmful toxins and are therefore not allowed to be used as PPPs. The serotype B. thuringiensis thuringiensis produces the beta-exotoxin thuringiensin (ßeT) which is considered to be toxic for almost all forms of life including humans (WHO 1999). The use of a non-registered PPP based on B. thuringiensis thuringiensis called bitoxybacillin was established through the determination of ßeT. First, an analytical reference standard of ßeT was characterized by nuclear magnetic resonance, liquid chromatography–high-resolution mass spectrometry and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Then, a confirmatory quantitative method for the determination of ßeT in PPPs and selected greenhouse crops based on LC-MS/MS was developed and validated. A limit of quantitation of 0.028 mg/kg was established, and average recoveries ranged from 85.6 % to 104.8 % with repeatability (RSDr) of 1.5–7.7 % and within-lab reproducibility (RSD_WLR) of 17 %. The method was used for analysis of >100 samples. ßeT was found in leaves of ornamentals, but no evidence was found for use in edible crops. Figure HMBC NMR spectrum of beta exotoxin thuringiensin

Keywords: Beta-exotoxin; Thuringiensin; Bacillus thuringiensis thuringiensis ; LC-MS/MS

MALDI-TOF mass spectrometric determination of intact phospholipids as markers of illegal bovine milk adulteration of high-quality milk by Cosima D. Calvano; Cristina De Ceglie; Antonella Aresta; Laura A. Facchini; Carlo G. Zambonin (pp. 1641-1649).

In the dairy industry one of the most common frauds is mixing high-value milk (sheep’s and goats’) with milk of lower value (cows’). This illegal practice has commercial, ethical, and serious sanitary consequences because consumers can be exposed to hidden allergens contained in the undeclared cows’ milk. Here, we investigated the possibility of using matrix-assisted laser-desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as a rapid, sensitive, and accurate technique for detection of milk adulteration by analysis of phospholipid profiles. Lipid extracts of pure raw milk, commercial milk, and binary mixtures of cows’ and goats’ milk and cows’ and sheep’s milk (the concentrations of each milk varied from 0 % to 50 %) were analyzed with α-cyano-4-chlorocinnamic acid as matrix. The abundance ratio of the ions at m/z 703 and m/z 706 was found to be species-correlated and was used as marker of cows’ milk in sheep’s and goats’ milk. Furthermore, the procedure could potentially be applied to cheese samples, because peaks at m/z 703 and 706 were also found in several commercial cheese samples. This approach proved to be an efficient, rapid, and inexpensive method of detecting milk fraud. Figure MALDI-TOF MS analysis of intact phospholipid in milk mixtures

Keywords: Milk adulteration; Goat; Cow; Sheep; MALDI; Lipid

Identification of in vitro metabolites of the novel anti-tumor thiosemicarbazone, DpC, using ultra-high performance liquid chromatography–quadrupole-time-of-flight mass spectrometry by Ján Stariat; Petra Kovaříková; Radim Kučera; Jiří Klimeš; Danuta S. Kalinowski; Des R. Richardson; Raimo A. Ketola (pp. 1651-1661).

Di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) is a promising analogue of the dipyridyl thiosemicarbazone class currently under development as a potential anti-cancer drug. In fact, this class of agents shows markedly greater anti-tumor activity and selectivity than the clinically investigated thiosemicarbazone, Triapine®. However, further development of DpC requires detailed data concerning its metabolism. Therefore, we focused on the identification of principal phase I and II metabolites of DpC in vitro. DpC was incubated with human liver microsomes/S9 fractions and the samples were analyzed using ultra-performance liquid chromatography (UPLC^TM) with electrospray ionization quadrupole-time-of-flight (Q-TOF) mass spectrometry. An Acquity UPLC BEH C₁₈ column was implemented with 2 mM ammonium acetate and acetonitrile in gradient mode as the mobile phase. The chemical structures of metabolites were proposed based on the accurate mass measurement of the protonated molecules as well as their main product ions. Ten phase I and two phase II metabolites were detected and structurally described. The metabolism of DpC occurred via oxidation of the thiocarbonyl group, hydroxylation and N-demethylation, as well as the combination of these reactions. Conjugates of DpC and the metabolite, M10, with glucuronic acid were also observed as phase II metabolites. Neither sulfate nor glutathione conjugates were detected. This study provides the first information about the chemical structure of the principal metabolites of DpC, which supports the development of this promising anti-cancer drug and provides vital data for further pharmacokinetic and in vivo metabolism studies. Figure Proposed metabolic pathways of DpC

Keywords: Di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone; DpC; Metabolism; Anti-tumor agent; Mass spectrometry

Effect of mobile phase pH on the retention of nucleotides on different stationary phases for high-performance liquid chromatography by S. Studzińska; B. Buszewski (pp. 1663-1672).

The main aim of the present investigation was to study the retention and separation of eight nucleotides with the use of seven stationary phases in RP HPLC mode. Two octadecyl columns were used; however, aminopropyl, alkylamide, cholesterol, alkyl-phosphate, and phenyl were also studied. Special attention was paid to the mobile phase buffer pH, since it appears that this factor is very influential in the case of chromatographic separation of nucleotides. The retention of nucleotides was greater for mobile phase pH 4.0 in comparison with pH 7.0 (except for octadecyl and phenyl packing). This is a consequence of protonization of polar groups present on the stationary phase surface. It was proved that aminopropyl, alkyl phosphate, alkylamide packing materials are not suitable for the resolution of nucleotides. On the other hand, cholesterol and phenyl stationary phases are alternatives for conventional octadecyl phases. Application of cholesterol packing allows separation of small, polar nucleotides, which is impossible to achieve in the case of octadecyl phase. Moreover, a phenyl support allows separation of nucleotides in a shorter time in comparison with octadecyl packing.

Keywords: High-performance liquid chromatography; Nucleotides; Stationary phases; Separation; Retention mechanism

Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method by E. den Boer; R. J. W. Meesters; B. D. van Zelst; T. M. Luider; J. M. W. Hazes; S. G. Heil; R. de Jonge (pp. 1673-1681).

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5–100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1–1,000 nM for all MTXPGn (R ² > 0.99). The coefficient of variation ranged from 1–4% for intraday precision and 6–15% for interday precision. Samples were stable for at least 1 month at −80 °C. Recovery ranged from 98–100%, and the relative matrix-effect varied from 95–99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS. Figure The seperation of methotrexate polyglutamates using UPLC.

Keywords: Methotrexate; Methotrexate polyglutamates; Erythrocytes; Rheumatoid arthritis

Tuning of Ti-doped mesoporous silica for highly efficient enrichment of phosphopeptides in human placenta mitochondria by Fuqiang Wang; Zhonghua Shi; Fan Hu; Zhengrong Xia; Ling Wang (pp. 1683-1693).

Extraction of phosphopeptides from rather complex biological samples has been a tough issue for deep and comprehensive investigation into phosphoproteomes. In this paper, we present a series of Ti-doped mesoporous silica (Ti-MPS) materials with tunable composition and controllable morphology for highly efficient enrichment of phosphopeptides. By altering the molar ratio of silicon to titanium (Si/Ti) in the precursor, the external morphology, Ti content, internal long-rang order, and surface area of Ti-MPS were all modulated accordingly with certain regularity. Tryptic digests of standard phosphoprotein α- and β-casein were employed to assess the phosphopeptide enrichment capability of Ti-MPS series. At the Si/Ti molar ratio of 8:1, the optimum enrichment performance with admirable sensitivity and capacity was achieved. The detection limit for β-casein could reach 10 fmol, and 15 phosphopeptides from the digest of α-casein were resolved in the spectrum after enrichment, both superior to the behavior of commercial TiO₂ materials. More significantly, for the digest of human placenta mitochondria, 396 phosphopeptides and 298 phosphoproteins were definitely detected and identified after enrichment with optimized Ti-MPS material, demonstrating its remarkable applicability for untouched phosphoproteomes. In addition, this research also opened up a universal pathway to construct a composition-tunable functional material in pursuit of the maximum performance in applications. Figure From human placenta mitochondria to MS

Keywords: Ti-doped mesoporous silica; Tunable composition; Controllable morphology; Phosphopeptide enrichment; Human placenta mitochondria; Mass spectrometry

Conventional liquid chromatography/triple quadrupole mass spectrometry based metabolite identification and semi-quantitative estimation approach in the investigation of in vitro dabigatran etexilate metabolism by Zhe-Yi Hu; Robert B. Parker; Vanessa L. Herring; S. Casey Laizure (pp. 1695-1704).

Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted by esterases to dabigatran (DAB), a direct inhibitor of thrombin. To elucidate the esterase-mediated metabolic pathway of DABE, a high-performance liquid chromatography/mass spectrometry based metabolite identification and semi-quantitative estimation approach was developed. To overcome the poor full-scan sensitivity of conventional triple quadrupole mass spectrometry, precursor–product ion pairs were predicted to search for the potential in vitro metabolites. The detected metabolites were confirmed by the product ion scan. A dilution method was introduced to evaluate the matrix effects on tentatively identified metabolites without chemical standards. Quantitative information on detected metabolites was obtained using “metabolite standards” generated from incubation samples that contain a high concentration of metabolite in combination with a correction factor for mass spectrometry response. Two in vitro metabolites of DABE (M1 and M2) were identified, and quantified by the semi-quantitative estimation approach. It is noteworthy that CES1 converts DABE to M1 while CES2 mediates the conversion of DABE to M2. M1 and M2 were further metabolized to DAB by CES2 and CES1, respectively. The approach presented here provides a solution to a bioanalytical need for fast identification and semi-quantitative estimation of CES metabolites in preclinical samples. Figure The scheme of the semi-quantitative estimation approach

Keywords: Dabigatran etexilate; Liquid chromatography; Mass spectrometer; Carboxylesterase; Metabolism

Investigation of the metabolism of monepantel in ovine hepatocytes by UHPLC/MS/MS by Lucie Stuchlíková; Robert Jirásko; Ivan Vokřál; Jiří Lamka; Marcel Špulák; Michal Holčapek; Barbora Szotáková; Hana Bártíková; Milan Pour; Lenka Skálová (pp. 1705-1712).

Monepantel (MOP) belongs to a new class of anthelmintic drugs known as aminoacetonitrile derivatives. It was approved for use in veterinary practice in Czech Republic in 2011. So far, biotransformation and transport of MOP in target animals have been studied insufficiently, although the study of metabolic pathways of anthelmintics is very important for the efficacy of safety of therapy and evaluation of the risk of drug–drug interactions. The aim of this study was to identify MOP metabolites and to suggest the metabolic pathways of MOP in sheep. For this purpose, primary culture of ovine hepatocytes was used as a model in vitro system. After incubation, medium samples and homogenates of hepatocytes were extracted separately using solid-phase extraction. Analysis was performed using a hybrid quadrupole-time-of-flight analyzer with respect to high mass accuracy measurements in full scan and tandem mass spectra for the confirmation of an elemental composition. The obtained results revealed S-oxidation to sulfoxide and sulfone and arene hydroxylation as MOP phase I biotransformations. From phase II metabolites, MOP glucuronides, sulfates, and acetylcysteine conjugates were found. Based on the obtained results, a scheme of the metabolic pathway of MOP in sheep has been proposed.

Keywords: Monepantel; Aminoacetonitrile derivatives; Ultrahigh-performance liquid chromatography/mass spectrometry; Biotransformation; Drug metabolism

Double stable isotope ultra performance liquid chromatographic-tandem mass spectrometric quantification of tissue content and activity of phenylethanolamine N-methyltransferase, the crucial enzyme responsible for synthesis of epinephrine by Nan Qin; Mirko Peitzsch; Mario Menschikowski; Gabriele Siegert; Karel Pacak; Graeme Eisenhofer (pp. 1713-1719).

Here, we describe a novel method utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for synthesis of the stress hormone, epinephrine. The method is based on measurement of deuterium-labeled epinephrine produced from the reaction of norepinephrine with deuterium-labeled S-adenosyl-l-methionine as the methyl donor. In addition to enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of deuterium-labeled epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n = 10) and 10.9 to 13.6 % (n = 10). We established utility of the method by showing induction of the enzyme by dexamethasone in mouse pheochromocytoma cells and strong relationships to PNMT gene expression and tissue epinephrine levels in human pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final enzyme responsible for synthesis of epinephrine, the primary fight-or-flight stress hormone. Figure Assay principle of double stale isotope UPLC-MS/MS quantification of tissue content and activity of PNMT

Keywords: UPLC-MS/MS; PNMT; Pheochromocytoma

Scrambling of autoinducing precursor peptides investigated by infrared multiphoton dissociation with electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry by Giuliana Bianco; Cristiana Labella; Antonietta Pepe; Tommaso R. I. Cataldi (pp. 1721-1732).

Two synthetic precursor peptides, H₂N-CVGIW and H₂N-LVMCCVGIW, involved in the quorum sensing of Lactobacillus plantarum WCFS1, were characterized by mass spectrometry (MS) with electrospray ionization and 7-T Fourier transform ion cyclotron resonance (ESI-FTICR) instrument. Cell-free bacterial supernatant solutions were analyzed by reversed-phase liquid chromatography with ESI-FTICR MS to verify the occurrence of both pentapeptide and nonapeptide in the bacterial broth. The structural characterization of both protonated peptides was performed by infrared multiphoton dissociation using a continuous CO₂ laser source at a wavelength of 10.6 μm. As their fragmentation behavior cannot be directly derived from the primary peptide structure, all anomalous fragments were interpreted as neutral loss of amino acids from the interior of both peptides, i.e., loss of V, G, VG and M, MC, V, CC, from H₂N-CVGIW and H₂N-LVMCCVGIW, respectively. Mechanisms of this scrambling are proposed. FTICR MS provides accurate masses of all fragment ions with very low absolute mass errors (<1.6 ppm), which facilitated the reliable assignment of their elemental compositions. The resolving power was more than sufficient to resolve closely isobaric product ions with routine subparts per million mass accuracies. Only the occurrence of pentapeptide was found in the cell-free culture of L. plantarum, grown in Waymouth’s medium broth, with a low content of 5.2 ± 2.6 μM by external calibration. Most of it was present as oxidized H₂N-CVGIW, that is, the soluble disulfide pentapeptide with a level tenfold higher (i.e., 50 ± 4 μM, n = 3). Figure IRMPD of the precursor protonated peptide, [H2N-CVGIW +H]+ at m/z 577.3 and suggested pathway showing the formation of peptide macrocycle and its selective ring opening.

Keywords: Autoinducing peptides; Infrared multiphoton dissociation (IRMPD); Tandem MS; Peptide fragmentation; Scrambling mechanism; FTICR MS (7-T)

Portable Raman versus portable mid-FTIR reflectance instruments to monitor synthetic treatments used for the conservation of monument surfaces by Claudia Conti; Jana Striova; Irene Aliatis; Chiara Colombo; Marinella Greco; Elena Possenti; Marco Realini; Luigi Brambilla; Giuseppe Zerbi (pp. 1733-1741).

This study aims to evaluate the relevance of portable Raman and portable mid-Fourier transform infrared (FTIR) reflectance instruments in monitoring the synthetic treatments applied on plaster substrates, a crucial issue in a conservation work. Some polymeric consolidants and protectives have a relatively short life owing to their degradation, and after some years the surface should be retreated. It follows that any information about the presence and composition of the products applied, their chemical transformations and their distribution on the surfaces is essential. For these purposes, conservation scientists should seek and test new in situ methods, and this is of utmost importance especially in the case of buildings, considering their large dimensions and consequent extensive mapping. The effectiveness of portable Raman and portable mid-FTIR reflectance instruments has been compared by analysing a set of laboratory specimens prepared and treated with variable amounts of products belonging to three classes of polymers; the spectroscopic investigation highlighted, for the first time, the limits and the advantages of portable Raman and portable mid-FTIR reflectance instruments in the detection of small amounts of products commonly employed for the conservation of plasters.

Keywords: Portable Raman; Portable Fourier transform infrared; Polymer; Monitoring consolidants and protectives; Reflectance; Non invasive analyses

Simultaneous determination of six hydrophilic ethers at trace levels using coconut charcoal adsorbent and gas chromatography/mass spectrometry by D. K. Stepien; W. Püttmann (pp. 1743-1751).

The main objective of the following study was to determine the efficiency of a method that uses coconut charcoal as a solid-phase extraction (SPE) adsorbent in order to simultaneously detect six hydrophilic ether species in water in the low microgram-per-liter range. The applied method was validated for quantification of ethyl tert-butyl ether, 1,4-dioxane, ethylene glycol dimethyl ether (monoglyme), diethylene glycol dimethyl ether (diglyme), triethylene glycol dimethyl ether (triglyme) and tetraethylene glycol dimethyl ether (tetraglyme). SPE followed by gas chromatography/mass spectrometry of the extracts using the selected ion monitoring mode allowed for establishing low detection limits in the range of 0.007–0.018 μg/L in ultrapure water and 0.004–0.020 μg/L in environmental samples. Examination of the method accuracy and precision resulted in a recovery greater than 86.8 % for each compound with a relative standard deviation of less than 6.6 %. A stability study established a 5-day holding time for the unpreserved water samples and extracts. Finally, 27 samples obtained from surface water bodies in Germany were analyzed for the six hydrophilic ethers. Each analyte was detected in at least eight samples at concentrations reaching 2.0 μg/L. The results of this study emphasize the advantage of the method to simultaneously determine six hydrophilic ether compounds. The outcome of the surface water analyses augments a concern about their frequent and significant presence in surface water bodies in Germany.

Keywords: 1,4-dioxane; ETBE; Glyme; Coconut charcoal; GC/MS

Development of a new porous gold SPME fiber for selective and efficient extraction of dodecanethiol followed by GC–MS analysis by Aly M. Hafez; Bernd W. Wenclawiak (pp. 1753-1758).

The efficiency of a porous gold fiber used for solid-phase microextraction (PG-SPME) was investigated for the extraction of dodecanethiol out of ethanolic solution. A commercially available standard SPME (polydimethylsiloxane, carboxen, divinylbenzene—PDMS-Carb-DVB) was used as a reference to compare the extraction efficiency and selectivity of dodecanethiol with the PG-SPME fiber. The porous gold SPME fiber allowed the analysis of self-assembled monolayers on gold by gas chromatography–mass spectrometry (GC–MS). The PG-SPME fiber showed a five times higher peak area for dodecanethiol in GC–MS compared to the standard PDMS-Carb-DVB fiber.

Keywords: SPME; SAM; GC–MS; PG-SPME; Thiol-analysis

Sensitive and simplified analysis of natural and synthetic steroids in water and solids using on-line solid-phase extraction and microwave-assisted solvent extraction coupled to liquid chromatography tandem mass spectrometry atmospheric pressure photoionization by Daniel D. Snow; Teyona Damon-Powell; Sathaporn Onanong; David A. Cassada (pp. 1759-1771).

Analytical methods for the determination of several natural and synthetic steroids in water and solid samples were developed using liquid chromatography tandem mass spectrometry. Atmospheric pressure photoionization (APPI) in positive mode was used as the source which appeared to reduce background and interferences as compared to electrospray ionization. Toluene was identified as the most appropriate dopant and its concentration optimized to enhance ionization efficiency. Method detection limits were in the low-nanogram-per-liter range for water samples using on-line solid-phase extraction and near 0.2 ng/g using microwave-assisted solvent extraction for solid samples. This on-line method, using APPI as a source and toluene as a dopant, is beneficial as it saves time due to on-line extraction and reduces interference and matrix effects using gas-phase photoionization. Groundwater, wastewater, runoff, and soil samples were collected and analyzed using the method indicating that traces of several androgens, estrogens, and other steroid-like compounds do occur in samples impacted by animal waste. Figure Sensitive and simplified analysis of natural and synthetic steroids in water and solids using on-line solid phase extraction and microwave-assisted solvent extraction coupled to liquid chromatography tandem mass spectrometry atmospheric pressure photoionization

Keywords: Steroid hormones; Environmental; On-line solid-phase extraction; Atmospheric pressure photoionization; Mass spectrometry

New matrix-free reference material for ethene in the form of optical fibres by Marta Słomińska; Piotr Konieczka; Jacek Namieśnik (pp. 1773-1778).

Reference materials are indispensable in the quality control and quality assurance of analytical measurements. One novel approach to the generation of standard gaseous mixtures of toxic, reactive, volatile, labile, and malodorous substances involves thermal decomposition or rearrangement, under defined temperature conditions, of compounds immobilized, by chemical bonding, on the surface of an appropriate carrier to release specific amounts of a volatile compound. In this technique the type of support used to immobilize the compound is extremely important, because the amount of analyte released depends directly on the choice of material. In this paper we report the novel preparation of a matrix-free ethene standard in the form of glass fibres coated with a thin layer of aluminium, the surface of which is modified by reaction with a specific compound. As a result of thermal decomposition of this compound, gaseous ethene is formed. In this paper we present the results obtained from tests of stability and homogeneity, two properties of crucial importance in the preparation of reference materials, by comparison of a series of results obtained for randomly chosen samples of the reference material. Interlaboratory comparative studies resulted in determination of a reference value for the ethene formed after thermal decomposition of the surface compound ((2.12 ± 0.14) ng per fibre).

Keywords: Air; Gases; Reference materials; Quality assurance; Control

Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay by Mario Cappiello; Eleonora Peroni; Ambra Lepore; Roberta Moschini; Antonella Del Corso; Francesco Balestri; Umberto Mura (pp. 1779-1785).

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG. Fig Colorimetric method for the specific measurement of glutathione. γ-glutamyltransferase (γ-GT) transfers the γ-glutamyl moiety from glutathione to an acceptor (Gly-Gly), with the formation of γ-glutamyl-Gly-Gly and Cys-Gly. The latter dipeptide is hydrolized by leucyl-aminopeptidase (LAP) to form cysteine, which can be easily measured using a colorimetric assay at 560 nm

Keywords: Glutathione; Gamma-glutamyltransferase; Lens; Glutathione reductase; Astrocytoma; Oxidative stress

Erratum to: Characterization of nanochannel delivery membrane systems for the sustained release of resveratrol and atorvastatin: new perspectives on promoting heart health by Juliana Sih; Shyam S. Bansal; Stefano Filippini; Silvia Ferrati; Kunal Raghuwansi; Erika Zabre; Eugenia Nicolov; Daniel Fine; Mauro Ferrari; Ganesh Palapattu; Alessandro Grattoni (pp. 1787-1787).

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Analytical and Bioanalytical Chemistry (v.405, #5)