Analytical and Bioanalytical Chemistry (v.405, #27)
Angels’ share challenge by Reinhard Meusinger (8685-8689).
Solution to the pycnometer challenge by Olaf Rienitz; Axel Pramann; Carola Pape (8691-8692).
Linking analytical chemistry Master’s programs: the Franco (Pau)—Spanish (Oviedo) case by Jorge Ruiz Encinar; Brice Bouyssiere; Florence Pannier (8693-8698).
has been an associate professor at the University of Oviedo (Spain) since 2010. His research interests are mass spectrometry and stable isotope-based methods, mostly applied to environment, speciation, and heteroatom-tagged proteomics, and the development of new biological applications of functionalized nanoparticles. In 2007 he was awarded the Young Chemist Prize by the Spanish Royal Society of Chemistry. He is currently the coordinator of the International Master in Analytical and Bioanalytical Sciences at the University of Oviedo. has been a professor at the University of Pau since 2013. Before this he performed post-doctoral research with the Group of Trace Element and Element Species Analysis (GKSS, Geesthacht, Germany). His research interests are mass spectrometry methods applied to speciation analysis in petroleum samples and to study of metabolism of platinum anticancer drugs during treatment. Dr Bouyssiere was the Erasmus coordinator at the University of Pau from 2008 to 2012. has been a professor at the University of Pau et des Pays de l’Adour (France) since 2008. Her research interests include the development of analytical methods based on coupled techniques, including HPLC or GC coupled with AFS, ICP–MS or MS, for use in speciation analysis of metalloids and, particularly, selenium. In this area, her main research field is determination of unknown species in a variety of matrices of toxicological or ecotoxicological concern. She is currently the head of the Master of Chemistry and coordinator of the International Master in Analytical Sciences for Life and Environment at the University of Pau.
Morpho-spectral imaging in the biosciences by Cyril Petibois; Yeukuang Hwu (8699-8700).
Assistant Professor of Biochemistry at the University of Bordeaux, France, is involved in the development of imaging methodology for biosample analysis (Inserm U1029 research unit; “Biophysics of vascular plasticity” group). His research interests involve the development of multimodal imaging methods for full characterization of biosamples, from synthetic models to individual cells and small animals, by combining elemental, morphological, molecular, and chemical techniques. His laboratory is also engaged in the development of high-performance imaging techniques, with advanced industrial collaboration with companies that are leaders in the field. is a Professor of physics at the Institute of Physics, Academia Sinica, Taiwan, in charge of the NanoX laboratory. He has published more than 150 scientific papers and registered several patents related to X-ray imaging and its applications. X-ray imaging (radiology) is the most widely used X-ray technique and his main research interest is the development of effective methods for characterization of biological objects, notably the microvascular network, in organs and tissues. In recent years, because of the excellent performance of X-ray sources boosted by synchrotron radiation, X-ray microscopic imaging reached a new level of performance in resolution and contrast which, in turn, has led to breakthroughs in the nano-characterization of biosamples, from the individual cell to small animals.
The role of asbestos morphology on their cellular toxicity: an in vitro 3D Raman/Rayleigh imaging study by Yao Seydou; Hsiang-Hsin Chen; Etienne Harte; Giancarlo Della Ventura; Cyril Petibois (8701-8707).
Amphiboles caused cohorts of deaths in exposed workers, leading to some of the largest class actions in the industry. Once inhaled, these inorganic fibers are thought to be both chemically and morphologically toxic, and their biopersistence in the lungs over decades lead to progressive pathologies, mesothelioma, and asbestosis. However, this exceptionally long chronicity for human pathologies suggests that chemical toxicity is certainly low, suggesting that morphological parameters could be more relevant in the pathology. Here, we developed a 3D Raman/optical imaging methodology in vitro to characterize both morphological and chemical parameters of cell/fiber interactions. We determined that lung cells could vesiculate amphiboles with length below 5 μm or could embed those not exceeding 15 μm in their fibrous extracellular matrix. Lung cells can thus develop defense strategies for handling the biopersistence of inorganic species, which may thus have major impact for biosafety issues related to nanomaterials.
Keywords: Bioanalytical methods; Cell systems/single cell analysis; Nanoparticles/nanotechnology; Raman imaging; Asbestos; Inorganic compounds/trace inorganic compounds
Characterization of the structure of human skin substitutes by infrared microspectroscopy by Marie Leroy; Michel Lafleur; Michèle Auger; Gaétan Laroche; Roxane Pouliot (8709-8718).
The skin acts mainly as a protective barrier from the external environment, thanks to the stratum corneum which is the outermost layer of the skin. As in vitro tests on skin are essential to elaborate new drugs, the development of skin models closer to reality becomes essential. It is now possible to produce in vitro human skin substitutes through tissue engineering by using the self-assembly method developed by the Laboratoire d’Organogénèse Expérimentale. In the present work, infrared microspectroscopy imaging analyses were performed to get in-depth morpho-spectral characterization of the three characteristic layers of human skin substitutes and normal human skin, namely the stratum corneum, living epidermis, and dermis. An infrared spectral analysis of the skin is a powerful tool to gain information on the order and conformation of the lipid chains and the secondary structure of proteins. On one hand, the symmetric stretching mode of the lipid methylene groups (2,850 cm−1) is sensitive to the acyl chain conformational order. The evolution profile of the frequency of this vibrational mode throughout the epidermis suggests that lipids in the stratum corneum are more ordered than those in the living epidermis. On the other hand, the frequencies of the infrared components underneath the envelop of the amide I band provide information about the overall protein conformation. The analysis of this mode establishes that the proteins essentially adopt an α-helix conformation in the epidermis, probably associated with the presence of keratin, while modifications of the protein content are observed in the dermis (extracellular matrix made of collagen). Finally, the lipid organization, as well as the protein composition in the different layers, is similar for human skin substitutes and normal human skin, confirming that the substitutes reproduce essential features of real skin and are appropriate biomimetics.
Keywords: Infrared microspectroscopy; Skin; Skin substitutes; Lipids; Proteins
Hyperspectral unmixing of Raman micro-images for assessment of morphological and chemical parameters in non-dried brain tumor specimens by Norbert Bergner; Anna Medyukhina; Kathrin D. Geiger; Matthias Kirsch; Gabriele Schackert; Christoph Krafft; Jürgen Popp (8719-8728).
Hyperspectral unmixing is an unsupervised algorithm to calculate a bilinear model of spectral endmembers and abundances of components from Raman images. Thirty-nine Raman images were collected from six glioma brain tumor specimens. The tumor grades ranged from astrocytoma WHO II to glioblastoma multiforme WHO IV. The abundance plots of the cell nuclei were processed by an image segmentation procedure to determine the average nuclei size, the number of nuclei, and the fraction of nuclei area. The latter two morphological parameters correlated with the malignancy. A combination of spectral unmixing and non-negativity constrained linear least squares fitting is introduced to assess chemical parameters. First, endmembers of the most abundant and most dissimilar components were defined that represent all data sets. Second, the content of the obtained components’ proteins, nucleic acids, lipids, and lipid to protein ratios were determined in all Raman images. Except for the protein content, all chemical parameters correlated with the malignancy. We conclude that the morphological and chemical information offer new ways to develop Raman-based classification approaches that can complement diagnosis of brain tumors. The role of non-linear Raman modalities to speed-up image acquisition is discussed. Figure Raman images provide morphological details about cell nuclei that are automatically processed by image segmentation procedures.
Keywords: Raman microscopy; Brain tumor; Astrocytoma; Hyperspectral unmixing; Nuclei segmentation
FTIR spectro-imaging of collagen scaffold formation during glioma tumor development by Razia Noreen; Chia-Chi Chien; Hsiang-Hsin Chen; Vladimir Bobroff; Michel Moenner; Sophie Javerzat; Yeukuang Hwu; Cyril Petibois (8729-8736).
Evidence has recently emerged that solid and diffuse tumors produce a specific extracellular matrix (ECM) for division and diffusion, also developing a specific interface with microvasculature. This ECM is mainly composed of collagens and their scaffolding appears to drive tumor growth. Although collagens are not easily analyzable by UV-fluorescence means, FTIR imaging has appeared as a valuable tool to characterize collagen contents in tissues, specially the brain, where ECM is normally devoid of collagen proteins. Here, we used FTIR imaging to characterize collagen content changes in growing glioma tumors. We could determine that C6-derived solid tumors presented high content of triple helix after 8–11 days of growth (typical of collagen fibrils formation; 8/8 tumor samples; 91 % of total variance), and further turned to larger α-helix (days 12–15; 9/10 of tumors; 94 % of variance) and β-turns (day 18–21; 7/8 tumors; 97 % of variance) contents, which suggest the incorporation of non-fibrillar collagen types in ECM, a sign of more and more organized collagen scaffold along tumor progression. The growth of tumors was also associated to the level of collagen produced (P < 0.05). This study thus confirms that collagen scaffolding is a major event accompanying the angiogenic shift and faster tumor growth in solid glioma phenotypes.
Keywords: Collagens; FTIR imaging; Glioma tumor growth; Solid tumor phenotype
Erratum to: The role of asbestos morphology on their cellular toxicity: an in vitro 3D Raman/Rayleigh imaging study by Seydou Yao; Hsiang-Hsin Chen; Etienne Harte; Giancarlo Della Ventura; Cyril Petibois (8737-8737).
Synchrotron-based photoelectron spectroscopy provides evidence for a molecular bond between calcium and mineralizing organic phases in invertebrate calcareous skeletons by Jean-Pierre Cuif; Azzedine Bendounan; Yannicke Dauphin; Julius Nouet; Fausto Sirotti (8739-8748).
was appointed Professor of Palaeontology at the University Paris Sud after obtaining a doctoral thesis at the National Museum of Natural History. He has conducted extensive investigations on structural and chemical properties of calcareous biominerals with special attention to environmental and aquacultural applications is a permanent scientist at the TEMPO XPS beamline at SOLEIL, the French synchrotron radiation source. He previously worked as a researcher at Würzburg University (Germany) and the Paul Scherrer Institut (Switzerland). His interest lies in understanding the nature of chemical reactions in complex systems with special focus on the electronic structure of molecular systems interacting with solid surfaces is assistant professor at UPMC (P. and M. Curie, Paris). She is a founding member of the “Biominerals” research team at the Geosciences laboratory of the Université Paris-Sud. Experimental data on structure and biogeochemical composition of modern and fossil mollusc shells, corals, and vertebrate skeletons are used for evolutionary history, palaeoenvironmental reconstructions, and aquaculture is Research engineer at IDES geoscience laboratory, Université de Paris-Sud, Orsay, France. Working in the “Biominerals” team his research is focused on the microstructural organization and biochemical composition of biogenic carbonates, especially in modern and fossil mollusc shells is Research Director at the CNRS and responsible for the TEMPO beamline at SOLEIL, the French synchrotron radiation source. He has been working on the development of new synchrotron radiation-based instrumentation dedicated to time-dependent photoelectron spectroscopy. His main scientific interest is surface magnetism and surface chemistry, with applications to data storage and nanotechnology Organic compounds have been extracted from calcium carbonate skeletons produced by three invertebrate species belonging to distinct phyla. The soluble parts of these skeleton matrices were isolated and analysed by synchrotron-based X-ray spectroscopy (XPS). The presence of calcium associated with these organic materials was revealed in every sample studied, with important variations in Ca 2p binding energy from species to species. Measured Ca 2p binding energy values are more related to compositional diversity of the mineralizing matrices of the skeletons, whose taxonomic dependence has long been established, than to the Ca carbonate polymorph selected to build the skeletal units. This suggests a physical bond between species-specific mineralizing organic assemblages and the associated calcium. Remarkably, the binding energy of 2p electrons in calcium associated with mineralizing matrices is consistently higher than Ca 2p values obtained in purely mineral carbonate (both calcite and aragonite). The ability both to identify and measure the effect of organic matrices on their mineral counterpart in calcareous biominerals opens a new perspective for a functional approach to the biomineralization process.
Keywords: Photo-emission; Biominerals; Ca carbonates; Mineralizing matrices
Non-destructive elemental analysis of large meteorite samples by prompt gamma-ray neutron activation analysis with the internal mono-standard method by Sk. A. Latif; Y. Oura; M. Ebihara; H. Nakahara (8749-8759).
Prompt gamma-ray neutron activation analysis (PGNAA) using the internal mono-standard method was tested for its applicability to analyzing large solid samples including irregularly shaped meteorite samples. For evaluating the accuracy and precision of the method, large quantities of the Geological Survey of Japan standardized rock powders (JB-1a, JG-1a, and JP-1) were analyzed and 12 elements (B, Na, Mg, Al, Cl, K, Ca, Ti, Mn, Fe, Sm, and Gd) were determined by using Si as an internal standard element. Analytical results were mostly in agreement with literature values within 10 %. The precision of the method was also shown to be within 10 % (1σ) for most of these elements. The analytical procedure was then applied to four stony meteorites (Allende, Kimble County, Leedey, Lake Labyrinth) and four iron meteorites (Canyon Diablo, Toluca (Mexico), Toluca (Xiquipilco), Squaw Creek) consisting of large chunks or single slabs. For stony meteorites, major elements (Mg, Al, Si, S, Ca, and Ni), minor elements (Na and Mn) and trace element (B, Cl, K, Ti, Co, and Sm) were determined with adequate accuracy. For iron meteorites, results for the Co and Ni mass fractions determined are all consistent with corresponding literature values. After the analysis, it was confirmed that the residual radioactivity remaining in the sample after PGNAA was very low and decreased down to the background level. This study shows that PGNAA with the internal mono-standard method is highly practical for determining the elemental composition of large, irregularly shaped solid samples including meteorites.
Keywords: Prompt gamma-ray neutron activation analysis (PGNAA); Large samples; Meteorites; Internal monostandard method; Neutron activation analysis
Fast and accurate determination of K, Ca, and Mg in human serum by sector field ICP-MS by Lee L. Yu; W. Clay Davis; Yoana Nuevo Ordonez; Stephen E. Long (8761-8768).
Electrolytes in serum are important biomarkers for skeletal and cellular health. The levels of electrolytes are monitored by measuring the Ca, Mg, K, and Na in blood serum. Many reference methods have been developed for the determination of Ca, Mg, and K in clinical measurements; however, isotope dilution thermal ionization mass spectrometry (ID-TIMS) has traditionally been the primary reference method serving as an anchor for traceability and accuracy to these secondary reference methods. The sample matrix must be separated before ID-TIMS measurements, which is a slow and tedious process that hindered the adoption of the technique in routine clinical measurements. We have developed a fast and accurate method for the determination of Ca, Mg, and K in serum by taking advantage of the higher mass resolution capability of the modern sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). Each serum sample was spiked with a mixture containing enriched 44Ca, 26Mg, and 41K, and the 42Ca+:44Ca+, 24Mg+:26Mg+, and 39K+:41K+ ratios were measured. The Ca and Mg ratios were measured in medium resolution mode (m/Δm ≈ 4 500), and the K ratio in high resolution mode (m/Δm ≈ 10 000). Residual 40Ar1H+ interference was still observed but the deleterious effects of the interference were minimized by measuring the sample at K > 100 ng g−1. The interferences of Sr++ at the two Ca isotopes were less than 0.25 % of the analyte signal, and they were corrected with the 88Sr+ intensity by using the Sr++:Sr+ ratio. The sample preparation involved only simple dilutions, and the measurement using this sample preparation approach is known as dilution-and-shoot (DNS). The DNS approach was validated with samples prepared via the traditional acid digestion approach followed by ID-SF-ICP-MS measurement. DNS and digested samples of SRM 956c were measured with ID-SF-ICP-MS for quality assurance, and the results (mean ± expanded uncertainty in mg dL−1 unit) for Ca (DNS = 10.14 ± 0.13, digested = 10.11 ± 0.10), Mg (DNS = 2.093 ± 0.008, digested = 2.098 ± 0.007), and K (DNS = 15.48 ± 0.11, digested = 15.50 ± 0.28) were in good agreement with the certified values (Ca = 10.17 ± 0.06, Mg = 2.084 ± 0.023, K = 15.55 ± 0.13). Major sources of uncertainty are sample measurement, spike calibration, and instrument factor including mass discrimination of the spectrometer and the detector deadtime.
Keywords: Ca; Mg; K; Electrolyte; Serum; Isotope dilution; High resolution; Sector field; ICP-MS
Mass spectrometric monitoring of Sr-enriched bone cements—from in vitro to in vivo by Marcus Rohnke; Anja Henss; Julia Kokesch-Himmelreich; Matthias Schumacher; Seemun Ray; Volker Alt; Michael Gelinsky; Juergen Janek (8769-8780).
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a well-established technique in materials science, but is now increasingly applied also in the life sciences. Here, we demonstrate the potential of this analytical technique for use in the development of new bone implant materials. We tracked strontium-enriched calcium phosphate cements, which were developed for the treatment of osteoporotic bone, from in vitro to in vivo. Essentially, the spatial distribution of strontium in two different types of strontium-modified calcium phosphate cements is analysed by SIMS depth profiling. To gain information about the strontium release kinetics, the cements were immersed for 3, 7, 14 and 21 days in α-MEM and tris(hydroxymethyl)-aminomethane solution and analysed afterwards by ToF-SIMS depth profiling. For cements stored in α-MEM solution an inhibited strontium release was observed. By using principal component analysis to evaluate TOF-SIMS surface spectra, we are able to prove the adsorption of proteins on the cement surface, which inhibit the release kinetics. Cell experiments with human osteoblast-like cells cultured on the strontium-modified cements and subsequent mass spectrometric analysis of the mineralised extracellular matrix (mECM) prove clearly that strontium is incorporated into the mECM of the osteoblast-like cells. Finally, in an animal experiment, the strontium-doped cements are implanted into the femur of osteoporotic rats. After 6 weeks, only a slight release of strontium was found in the vicinity of the implant material. By using ToF-SIMS, it is proven that strontium is localised in regions of newly formed bone but also within the pre-existing tissue. Figure Schematic illustration of the performed measurements.
Keywords: Sr-enriched calcium phosphate cements; Time-of-flight secondary ion mass spectrometry; Osteoporosis; Bone imaging; Bone implant interface
Innovative technique for the direct determination of proteins in calcified aortic valves by Lucie Coufalova; Stepanka Kuckova; Martina Velcovska; Antonin Zeman; Michal Smid; Martina Havelcova; Radovan Hynek (8781-8787).
Aortal valve mineralization very frequently causes a genesis of aortic stenosis, which is the most often surgically treated heart disease. Hydroxyapatite deposits have been identified as one of the causes leading to the loss of elasticity of the aortic valves. It is known that phosphates/calcium is accumulated in valve tissues during mineralization, but the mechanism of this process remains unclear. The work is focused mainly on the study of protein composition of mineralized aortic valves by nano-liquid chromatography electrospray ionization in a quadrupole orthogonal acceleration time-of-flight mass spectrometry. New methodological approach based on direct enzymatic digestion of proteins contained in hydroxyapatite deposits was developed for the study of pathological processes connected with osteogenesis. Our objectives were to simplify the traditional analytical protocols of sample preparation and to analyze the organic components of the explanted aortic valves for significant degenerative aortic stenosis. The study of aortic valve mineralization on the molecular level should contribute to understanding this process, which should consequently lead to effective prevention as well as to new ways of treatment of this grave disease. Figure The photo of explanted calcification of human aortal valve.
Keywords: Aortic stenosis; Degenerative (calcification-sclerotic) changes; Enzymatic cleavage; Mass spectrometry; Proteins
An analytical workflow for the molecular dissection of irreversibly modified fluorescent proteins by Vivien Berthelot; Vincent Steinmetz; Luis A. Alvarez; Chantal Houée-Levin; Fabienne Merola; Filippo Rusconi; Marie Erard (8789-8798).
Owing to their ability to be genetically expressed in live cells, fluorescent proteins have become indispensable markers in cellular and biochemical studies. These proteins can undergo a number of covalent chemical modifications that may affect their photophysical properties. Among other mechanisms, such covalent modifications may be induced by reactive oxygen species (ROS), as generated along a variety of biological pathways or through the action of ionizing radiations. In a previous report , we showed that the exposure of cyan fluorescent protein (ECFP) to amounts of •OH that mimic the conditions of intracellular oxidative bursts (associated with intense ROS production) leads to observable changes in its photophysical properties in the absence of any direct oxidation of the ECFP chromophore. In the present work, we analyzed the associated structural modifications of the protein in depth. Following the quantified production of •OH, we devised a complete analytical workflow based on chromatography and mass spectrometry that allowed us to fully characterize the oxidation events. While methionine, tyrosine, and phenylalanine were the only amino acids that were found to be oxidized, semi-quantitative assessment of their oxidation levels showed that the protein is preferentially oxidized at eight residue positions. To account for the preferred oxidation of a few, poorly accessible methionine residues, we propose a multi-step reaction pathway supported by data from pulsed radiolysis experiments. The described experimental workflow is widely generalizable to other fluorescent proteins, and opens the door to the identification of crucial covalent modifications that affect their photophysics. Figure Barrel structure of ECFP: residues that were found to be oxidized by .OH radicals are highlighted
Keywords: Protein oxidation; Mass spectrometry; •OH radicals; Cyan fluorescent protein (ECFP); γ- and pulsed radiolysis
Encapsulation of silica nano-spheres with polymerized ionic liquid for selective isolation of acidic proteins by Lu Han; Yang Shu; Xiaofeng Wang; Xuwei Chen; Jianhua Wang (8799-8806).
A nanocomposite is prepared by encapsulating silica nano-spheres with polymerized ionic liquid in aqueous medium without use of any organic solvents. Vinyl groups are covalently introduced on to the surface of silica nano-spheres, which are then encapsulated by copolymerization of 1-vinyl-3-ethylimidazolium bromide (monomer) and 1,4-butanediyl-3,3′-bis-l-vinylimidazolium dibromide (cross-linker) at room temperature. The derived nanocomposite, PIL@SiO2, provides a green adsorbent for protein sorption. PIL@SiO2 is selective toward acidic proteins, and its selectivity can be controlled via varying the amount of monomer used in the copolymerization process. At pH 6.0, use of 5 mg PIL@SiO2 nanocomposite results in a sorption efficiency of up to 95 % for 200 mg L−1 ovalbumin in 1 mL sample solution. Electrostatic and hydrophobic interactions between PIL@SiO2 and protein species dominate the adsorption process. The ovalbumin adsorption behavior is consistent with the Langmuir model, giving a sorption capacity of 333.3 mg g−1. The retained ovalbumin is recovered by elution with 0.2 % SDS solution. Circular dichroism spectra reveal virtually no change to the α-helix content of ovalbumin after elimination of SDS by use of dialysis. In summary, high-purity ovalbumin is isolated from chicken egg-white by use of the PIL@SiO2 nanocomposite as adsorbent.
Keywords: Polymeric ionic liquid; Silica nano-sphere; Acidic protein; Isolation
In vitro metabolic study of Rhizoma coptidis extract using liver microsomes immobilized on magnetic nanoparticles by Ying Xue; Jing Xiong; Hai-Li Shi; Yi-Ming Liu; Lin-Sen Qing; Xun Liao (8807-8817).
Although metabolic study of individual active compounds isolated from herbal plants has been intensive, it cannot truly reflect the fate of herbs because the herbal extracts in use have many constituents. To address this problem, whole extracts of herbs should be investigated. Microsomes have been heavily used in the in vitro metabolic study of drugs, and various materials have been used to immobilize microsomes to develop highly effective and reusable bioreactors in this field. In this work, rat liver microsomes were immobilized on magnetic nanoparticles (LMMNPs) to develop a highly active and recoverable nanoparticle bioreactor. Using this bioreactor, we investigated the in vitro metabolism of Rhizoma coptidis extract. Incubation of berberine, a major active ingredient of R. coptidis, with LMMNPs for 20 min produced two metabolites, i.e., demethyleneberberine and thalifendine, at high levels. From a comparison of the time courses of thalifendine formation obtained by ultraperformance liquid chromatography–mass spectrometry analysis, it was found that LMMNPs had a higher biological activity than free liver microsomes in metabolizing berberine. Further, the activity of LMMNPs remained almost unchanged after six consecutive uses in the incubation tests. Metabolism of R. coptidis extracts by LMMNPs was studied. The same two metabolites of berberine, i.e., demethyleneberberine and thalifendine, were detected. After a thorough study seeking support for this observation, it was found that demethyleneberberine was the common metabolite of five protoberberine-type alkaloids present in R. coptidis extract, including palmatine, jatrorrhizine, columbanine, epiberberine, and berberine. Figure A highly effective and reusable bioreactor was developed by immobilizing liver microsomes on magnetic nanoparticles, and it was used to investigate the metabolism of the whole extract of a Chinese herb Rhizoma coptidis
Keywords: Magnetic nanoparticles; Liver microsomes; In vitro metabolism; Electrospray ionization tandem mass spectrometry; Rhizoma coptidis
Batch profiling calibration for robust NMR metabonomic data analysis by Anne Fages; Clément Pontoizeau; Elodie Jobard; Pierre Lévy; Birke Bartosch; Bénédicte Elena-Herrmann (8819-8827).
Metabonomic studies involve the analysis of large numbers of samples to identify significant changes in the metabolic fingerprints of biological systems, possibly with sufficient statistical power for analysis. While procedures related to sample preparation and spectral data acquisition generally include the use of independent sample batches, these might be sources of systematic variation whose effects should be removed to focus on phenotyping the relevant biological variability. In this work, we describe a grouped-batch profile (GBP) calibration strategy to adjust nuclear magnetic resonance (NMR) metabolomic data-sets for batch effects either introduced during NMR experiments or samples work-up. We show how this method can be applied to data calibration in the context of a large-scale NMR epidemiological study where quality control samples are available. We also illustrate the efficiency of a batch profile correction for NMR metabonomic investigation of cell extracts, where GBP can significantly improve the predictive power of multivariate statistical models for discriminant analysis of the cell infection status. The method is applicable to a broad range of NMR metabolomic/metabonomic cohort studies.
Keywords: NMR; Metabonomics; Metabolomics; Batch calibration; Batch profiling
Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction by Réjane Morand; Massimiliano Donzelli; Manuel Haschke; Stephan Krähenbühl (8829-8836).
Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine. Figure A method is presented for the analysis of carnitine and acylcarnitines in urine which includes a precipitation step, on-column extraction and LC-MS/MS. The run time is 8 minutes and the method was validated for carnitine, acetylcarnitine, octanoylcarnitine and palmitoylcarnitine. Analysis of a patient sample with medium-chain acyl-CoA dehydrogenase deficiency is shown.
Keywords: Liquid chromatography/mass spectrometry; Online solid-phase extraction; Carnitine; Acylcarnitines
Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column by Nilusha L. T. Padivitage; Milan K. Dissanayake; Daniel W. Armstrong (8837-8848).
A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van’t Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.
Keywords: Cyclofructan 6; Hydrophilic interaction chromatography (HILIC); Nucleotides; FRULIC-N
New approach in RNA quantification using arginine-affinity chromatography: potential application in eukaryotic and chemically synthesized RNA by R. Martins; J. A. Queiroz; F. Sousa (8849-8858).
The knowledge of RNA’s role in biological systems and the recent recognition of its potential use as a reliable biotherapeutic tool increase the demand for development and validation of analytical methods for accurate analysis of RNA. Affinity chromatography is a unique technique because of the versatility of applications reliant on the affinity ligand used. Recently, an arginine-based matrix has been effectively applied in the purification of RNA because of the specific recognition mechanism for RNA molecules. This interaction is suggested to be due to the length of arginine side chain and its ability to produce good hydrogen bonding geometries, which promote multi-contact with RNA backbone or RNA bases, based on RNA folding. Thus, this work presents the development and validation of an analytical method with ultraviolet detection for the quantification of RNA using affinity chromatography with arginine amino acid as immobilized ligand. The method was validated according to International and European legislation for bioanalytical methods. The results revealed that the proposed method is suitable for the reliable detection, separation, and quantification of RNA, showing that the method is precise and accurate for concentrations up to 200 ng/μL of RNA. Furthermore, the versatility of the methodology was demonstrated by its applicability in the quantification of RNA from different eukaryotic cells and in crude samples of chemically synthesized RNA. Therefore, the proposed method demonstrates a potential multipurpose applicability in molecular biology RNA-based analysis and RNA therapeutics. Figure Proposed interactions occurring between arginine–agarose matrix and RNA molecules. Given the multiplicity of arginine side-chain interactions and depending upon RNA folding state, arginine will preferably bind to phosphate groups of RNA backbone or RNA bases.
Keywords: Affinity; Arginine; Chromatography; RNA; Transcription
Optimization of global DNA methylation measurement by LC-MS/MS and its application in lung cancer patients by Chiung-Wen Hu; Huei Lee; Jian-Lian Chen; Yi-Jie Li; Mu-Rong Chao (8859-8869).
Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2′-deoxycytidine (5-medC) and 2′-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated (N7-methylguanine (N7-meG), O 6-methylguanine (O 6-meG), and N3-methyladenine (N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16–4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O 6-meG and N3-meA were however found to be undetectable in all lung tissue samples. Figure Optimization of global DNA methylation detection
Keywords: DNA methylation; LC-MS/MS; DNA digestion; Human lung; DNA lesions
Determination of acetamiprid partial-intercalative binding to DNA by use of spectroscopic, chemometrics, and molecular docking techniques by Yue Zhang; Guowen Zhang; Xiaoyue Zhou; Yu Li (8871-8883).
Acetamiprid (ACT) is an insecticide widely used for controlling a variety of insect pests. The binding mode associated with calf thymus DNA (ctDNA) upon interaction with ACT was determined using spectroscopic, chemometrics, and molecular docking techniques to clarify the interaction mechanism at the molecular level. Fluorescence titration suggested that the fluorescence quenching of ACT by ctDNA is a static procedure. The binding constants between ACT and ctDNA at different temperatures were calculated to be of the order 103−104 L mol−1. The positive values of enthalpy and entropy change suggested that the binding process is primarily driven by hydrophobic interactions. Multivariate curve resolution−alternating least squares (MCR−ALS), a chemometrics approach, was used to resolve the expanded UV–visible spectral data matrix. The concentration profiles and the spectra for the three reaction components (ACT, ctDNA, and ACT−ctDNA complex) of the system, which formed a highly overlapping composite response, were then successfully obtained and used to evaluate the progress of ACT interacting with ctDNA. The results of the single-stranded ctDNA and iodide quenching experiments, ctDNA-melting investigations, and viscosity measurements indicated that ACT binds to ctDNA by means of a partial intercalation. Molecular docking studies showed that the specific binding site is mainly located between the ACT and G–C base pairs of ctDNA. This docking prediction was confirmed by use of Fourier-transform infrared (FT-IR) spectral analysis. Results from circular dichroism (CD) spectroscopy revealed that ACT induced a conformational change from the B–ctDNA form to the A–ctDNA form. Figure Acetamiprid partialintercalative binding to ctDNA
Keywords: Acetamiprid; Calf thymus DNA; Binding mode; Multivariate curve resolution−alternating least squares (MCR−ALS); Spectroscopy; Molecular docking
An analytical investigation of 24 oxygenated-PAHs (OPAHs) using liquid and gas chromatography–mass spectrometry by Steven G. O’Connell; Theodore Haigh; Glenn Wilson; Kim A. Anderson (8885-8896).
We developed two independent approaches for separation and quantitation of 24 oxygenated polycyclic aromatic hydrocarbons (OPAHs) using both liquid chromatography-atmospheric pressure chemical ionization/mass spectrometry (LC-APCI/MS) and gas chromatography-electron impact/mass spectrometry (GC-EI/MS). Building on previous OPAH research, we examined laboratory stability of OPAHs, improved existing method parameters, and compared quantification strategies using standard addition and an internal standard on an environmental sample. Of 24 OPAHs targeted in this research, 19 compounds are shared between methods, with 3 uniquely quantitated by GC-EI/MS and 2 by LC-APCI/MS. Using calibration standards, all GC-EI/MS OPAHs were within 15 % of the true value and had less than 15 % relative standard deviations (RSDs) for interday variability. Similarly, all LC-APCI/MS OPAHs were within 20 % of the true value and had less than 15 % RSDs for interday variability. Instrument limits of detection ranged from 0.18 to 36 ng mL−1 on the GC-EI/MS and 2.6 to 26 ng mL−1 on the LC-APCI/MS. Four standard reference materials were analyzed with each method, and we report some compounds not previously published in these materials, such as perinaphthenone and xanthone. Finally, an environmental passive sampling extract from Portland Harbor Superfund, OR was analyzed by each method using both internal standard and standard addition to compensate for potential matrix effects. Internal standard quantitation resulted in increased precision with similar accuracy to standard addition for most OPAHs using 2-fluoro-fluorenone-13C as an internal standard. Overall, this work improves upon OPAH analytical methods and provides some considerations and strategies for OPAHs as focus continues to expand on this emerging chemical class. Figure OPAH chromatograms from GC-EI/MS and LC-APCI/MS methods for NIST SRM 1650b (diesel particulate matter)
Keywords: LCMS; GCMS; OPAH; Oxy-PAH; Stability; Standard addition
Gas chromatography–tandem mass spectrometry analysis of 52 monohydroxylated metabolites of polycyclic aromatic hydrocarbons in hairs of rats after controlled exposure by Nathalie Grova; Guillaume Salquèbre; Brice M. R. Appenzeller (8897-8911).
A method based on gas chromatography–tandem mass spectrometry after derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide was developed for the analysis of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in hair. The method focused on 52 target compounds corresponding to two- to six-ring monohydroxylated metabolites of polycyclic aromatic hydrocarbons (PAHs). The limits of quantification ranged from 0.2 to 50 pg mg−1. The method was then applied to the analysis of hair samples collected from rats exposed to 12 PAHs at 0.01, 0.1, and 1 mg kg−1, by intraperitoneal injection, for 28 days. The results of this study confirm that these metabolites can be incorporated in hair after intraperitoneal administration of the corresponding parent compound. Only 20 of the 52 metabolites were actually detected in hair samples and corresponded to nine parent PAHs. The mean concentrations of OH-PAHs in rat hair samples exposed to PAHs at 1 mg kg−1 ranged from 0.6 ± 0.2 pg mg−1 for 8-hydroxybenzo[b]fluoranthene to 6.7 ± 1.0 pg mg−1 for 1-hydroxypyrene. The results also demonstrated that hair pigmentation has no influence on the concentration of most OH-PAHs. This animal experiment confirmed the incorporation of PAH metabolites in hair and demonstrated that the method was sufficiently sensitive to detect low levels of exposure to PAHs. These results confirmed the usefulness of hair analysis in the biomonitoring of human exposure to PAHs. Figure Analysis of 52 monohydroxylated polyccyclic aromatic hydrocarbons in a supplemented hair sample by GC-EI-MS/MS
Keywords: Hair; Monohydroxylated polycyclic aromatic hydrocarbons; Analysis; Polycyclic aromatic hydrocarbons; Biomonitoring; Gas chromatography–electron ionization tandem mass spectrometry
Determination of 13 estrogenic endocrine disrupting compounds in atmospheric particulate matter by pressurised liquid extraction and liquid chromatography-tandem mass spectrometry by N. Salgueiro-González; M. López de Alda; S. Muniategui-Lorenzo; D. Prada-Rodríguez; Damià Barceló (8913-8923).
This paper describes the optimisation of an analytical method for the simultaneous determination of up to 13 estrogenic endocrine disrupting compounds (EDCs), including natural and synthetic estrogens, alkylphenols, bisphenol A and phthalate esters, in atmospheric particulate matter. This methodology is based on pressurised liquid extraction and liquid chromatography-tandem mass spectrometry and is the first method described for analysis of estrogens in air. Samples were collected with high-volume samplers equipped with quartz microfiber filters. Two selected reaction monitoring transitions were chosen for identification and confirmation of the target compounds. Quantitation was performed by the internal standard method with the use of deuterated surrogate standards. The analytical features of the method were satisfactory: absolute recoveries were around 45 % for all compounds, except estrogens (∼29 %), because of matrix effects. Repeatability was in all cases below 15 %. Quantitation limits of the method ranged from 5 pg/m3 (for diethylphthalate) to 83 pg/m3 (for 17-α-ethynylestradiol). As a part of the validation procedure, the developed method was applied to the analysis of eight air particulate samples (fine grain-size particles, PM2.5) collected in industrial, urban and non-polluted suburban areas of A Coruña (NW of Spain). Results evidenced the presence of dibutylphthalate, diethylhexylphthalate and nonylphenol in all samples. The highest concentration corresponded to dibutylphthalate (54.7 ng/m3) in an industrial area. Daily exposure to these EDCs in terms of estradiol equivalents (3 pg/day for toddlers and 5 pg/day for adults) is not expected to pose a risk to human health.
Keywords: Atmospheric particulate matter; Steroid sex hormones; Phthalate esters; Alkylphenols; Bisphenol A; Estrogenicity
Development of a direct ELISA based on carboxy-terminal of penicillin-binding protein BlaR for the detection of β-lactam antibiotics in foods by Juan Peng; Guyue Cheng; Lingli Huang; Yulian Wang; Haihong Hao; Dapeng Peng; Zhenli Liu; Zonghui Yuan (8925-8933).
β-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of β-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of β-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by β-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual β-lactam was known. The IC50 values of 15 β-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 μg L−1. Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected β-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 μg kg−1, respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 β-lactams in edible tissues, among which 11 β-lactams controlled by European Union could be detected below maximum residue limits. Figure The receptor-based ELISA for blank sample (negative samples, left) and sample containing β-lactam antibiotics (positive samples, right)
Keywords: β-lactam; Receptor; BlaR-CTD; Purification; ELISA
Chiral separation of racemic mandelic acids by use of an ionic liquid-mediated imprinted monolith with a metal ion as self-assembly pivot by Li-Hong Bai; Xiu-Xiu Chen; Yan-Ping Huang; Qing-Wei Zhang; Zhao-Sheng Liu (8935-8943).
A new chiral stationary phase based on molecularly imprinted polymers (MIP) was prepared in ionic liquid by use of the metal pivot concept. Imprinted monoliths were synthesized by use of a mixture of R-mandelic acid (template), 4-vinylpyridine, ethylene glycol dimethacrylate, and several metal ions as pivot between the template and functional monomer. A ternary mixture of dimethyl sulfoxide–dimethylformamide–[BMIM]BF4 containing metal ions was used as the porogenic system. Separation of the enantiomers of rac-mandelic acid was successfully achieved on the MIP thus obtained, with resolution of 1.87, whereas no enantiomer separation was observed on the imprinted monolithic column in the absence of metal ions. The effects of polymerization conditions, including the nature of the metal ion and the ratios of template to metal ions and template to functional monomer, on the chiral separation of mandelic acid were investigated. The results reveal that use of metal ions as a pivot, in combination with ionic liquid, is an effective method for preparation of a highly efficient MIP stationary phase for chiral separation. Figure A new chiral stationary phase based on molecularly imprinted polymers (MIP) was prepared in ionic liquid by use of the metal pivot concept
Keywords: R-Mandelic acid; Monolith; Molecularly imprinted polymers; Chiral separation; Metal ion; Pivot
Determination of nitration degrees for the birch pollen allergen Bet v 1 by Kathrin Selzle; Chloé Ackaert; Christopher J. Kampf; Anna T. Kunert; Albert Duschl; Gertie J. Oostingh; Ulrich Pöschl (8945-8949).
Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitate further investigations, an efficient method to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of protein nitration. Figure Illustration of the photometry of tyrosine and nitrotyrosine
Keywords: Protein tyrosine nitration; Bet v 1.0101; HPLC-DAD; Nitration degree
A native electrophoretic technique to study oligomerization and activity of cytosolic 5′-nucleotidase II by Daniela Nicole Filoni; Rossana Pesi; Simone Allegrini; Marcella Camici; Maria Grazia Tozzi (8951-8954).
The analysis of the oligomeric active state of a native protein usually requires the application of at least two analytical methods such as gel filtration and analytical ultracentrifugation. Both methods require a substantial amount of protein, time and/or expensive equipment. We here describe a native electrophoretic method for the identification of the native molecular weight of the recombinant wild-type cytosolic 5′-nucleotidase (cN-II) and of its mutants in subunit interfaces Y115A, F36R, K311A and G319Q. The protein was stained both with protein dye and with an activity staining method. Our results demonstrated that purified recombinant protein preparations contained substantial amounts of nucleic acids and misfolded, inactive protein. Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer.
Keywords: Native electrophoresis; 5′-Nucleotidase; Oligomeric state; Activity staining