Analytical and Bioanalytical Chemistry (v.409, #6)
Challenges and successes in implementing active learning laboratory experiments for an undergraduate analytical chemistry course by Anna G. Cavinato (1465-1470).
Barry K. Lavine, Steven D. Brown, Karl S. Booksh (Eds.): 40 years of chemometrics—from Bruce Kowalski to the future by Romà Tauler (1471-1472).
Ivan Pelant and Jan Valenta: Luminescence spectroscopy of semiconductors by Ying Liu; Huangxian Ju (1473-1474).
Selective improvement of peptides imaging on tissue by supercritical fluid wash of lipids for matrix-assisted laser desorption/ionization mass spectrometry by Shoko Matsushita; Noritaka Masaki; Kohei Sato; Takahiro Hayasaka; Eiji Sugiyama; Shu-Ping Hui; Hitoshi Chiba; Nobuyuki Mase; Mitsutoshi Setou (1475-1480).
There is a high analytical demand for improving the detection sensitivity for various peptides in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) because exhaustive distribution analyses of various peptides could help to reveal the function of peptides in vivo. To improve the sensitivity of peptide detection, we used supercritical fluid of CO2 (scCO2) as washing solvent for a pretreatment to remove lipids. We evaluated whether our wash method using scCO2 with an entrainer improved the detection of peptides and suppressed lipid detection in MALDI-IMS. Our analysis revealed that the signal intensities of peptides such as m/z 3339.8, 3530.9, 4233.3, 4936.7, and 4963.7 were increased in scCO2-washed samples. The greatest improvement in the signal-to-noise ratio (S/N) was found at m/z 4963.7, which was identified as thymosin β4, with the S/N reaching almost 190-fold higher than the control. Additionally, all of the improved signals were associated with the morphologic structure. Our method allows us to analyze the distribution of molecules, especially in the region of m/z 3000–5200. For these improvements, the polarity difference between scCO2 and the matrix solution used was considered as a key. A wider variety of molecules can be analyzed in the future due to this improvement of the detection sensitivity by optimizing the polarity of scCO2 with various entrainers. Graphical Abstract Mass spectra of m/z 4900-5000 obtained from a scCO2-washed tissue (upper, blue) and a control tissue (lower, red). Ion distribution of the signals at m/z 4936.7 and m/z 4963.7 specifically ditected from scCO2-washed samples
Keywords: Imaging mass spectrometry; Supercritical fluid of CO2 ; Peptides; Pretreatment; Matrix-assisted laser desorption/ionization
Quantitative determination of the neurotoxin β-N-methylamino-l-alanine (BMAA) by capillary electrophoresis–tandem mass spectrometry by Elliott S. Kerrin; Robert L. White; Michael A. Quilliam (1481-1491).
is an MSc student at Dalhousie University and a technical officer with the Measurement Science and Standards portfolio of the National Research Council Canada in Halifax, Nova Scotia. His thesis research focuses on development of analytical methods for quantitation of BMAA in complex environmental and biological samples. is Associate Professor in the Department of Chemistry at Dalhousie University. His research interests focus on the investigation of metabolic processes in anaerobic bacteria and mass spectrometry, particularly the mechanisms of ion fragmentation reactions initiated by collision induced dissociation. is Principal Research Officer with the Measurement Science and Standards portfolio of the National Research Council Canada and Adjunct Professor of Chemistry at Dalhousie University in Halifax, Nova Scotia. His research at NRCC has focused on analytical chemistry of environmental and food contaminants, particularly biotoxins, and on the production of certified reference materials (CRMs). Recent reports of the widespread occurrence of the neurotoxin β-N-methylamino-l-alanine (BMAA) in cyanobacteria and particularly seafood have raised concerns for public health. LC–MS/MS is currently the analytical method of choice for BMAA determinations but incomplete separation of isomeric and isobaric compounds, matrix suppression and conjugated forms are plausible limitations. In this study, capillary electrophoresis (CE) coupled with MS/MS has been developed as an alternative method for the quantitative determination of free BMAA. Using a bare fused silica capillary, a phosphate buffer (250 mM, pH 3.0) and UV detection, it was possible to separate BMAA from four isomers, but the limit of detection (LOD) of 0.25 μg mL−1 proved insufficient for analysis of typical samples. Coupling the CE to a triple quadrupole MS was accomplished using a custom sheath-flow interface. The best separation was achieved with a 5 M formic acid in water/acetonitrile (9:1) background electrolyte. Strong acid hydrolysis of lyophilized samples was used to release BMAA from conjugated forms. Field-amplified stacking after injection was achieved by lowering sample ionic strength with a cation-exchange cleanup procedure. Quantitation was accomplished using isotope dilution with deuterium-labelled BMAA as internal standard. An LOD for BMAA in solution of 0.8 ng mL−1 was attained, which was equivalent to 16 ng g−1 dry mass in samples using the specified extraction procedure. This was comparable with LC–MS/MS methods. The method displayed excellent resolution of amino acid isomers and had no interference from matrix components. The presence of BMAA in cycad, mussel and lobster samples was confirmed by CE–MS/MS, but not in an in-house cyanobacterial reference material, with quantitative results agreeing with those from LC–MS/MS. Graphical Abstract CE-MS separation and detection of BMAA, its isomers and the internal standard BMAA-d3
Keywords: β-N-Methylamino-l-alanine; BMAA; Capillary electrophoresis; Mass spectrometry; Shellfish; Neurotoxin
Capillary coating as an important factor in optimization of the off-line and on-line MEKC assays of the highly hydrophobic enzyme chlorophyllase by Paweł Mateusz Nowak; Michał Woźniakiewicz; Maciej Michalik; Leszek Fiedor; Paweł Kościelniak (1493-1501).
is employed in the Department of Analytical Chemistry, Faculty of Chemistry, the Jagiellonian University in Kraków. In September 2016 he defended his PhD thesis devoted to the development of new bioanalytical methods based on the capillary electrophoresis technique. His major scientific interest embraces various aspects of capillary electrophoresis (enhancing repeatability of qualitative and quantitative analysis, separations of structurally similar molecules, analytical utilization of electrophoretic mobilities, determination of equilibrium constants), as well as supramolecular chemistry, biochemistry, and thermodynamics. He is a principal investigator of two research grants and a co-author of 17 scientific articles published since 2013. graduated with an MSc degree (in 2003) and a PhD degree (with honors, in 2008) in analytical and forensic chemistry from the Jagiellonian University in Kraków. He is an assistant professor and heads the Laboratory for Forensic Chemistry at the Jagiellonian University. His scientific efforts are focused on exploring various aspects of capillary electrophoresis, and the application of modern analytical techniques for forensic purposes. He has actively participated in many research and educational projects. He is a member of the Polish Chemical Society and a participant of the Eurolecturer Label. is a PhD candidate in the Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, the Jagiellonian University in Kraków. His research is focused on the mechanisms of solar energy conversion in light-harvesting complexes of higher plants and purple photosynthetic bacteria. graduated in 1987 from the Department of Organic Chemistry, Faculty of Chemistry, the Jagiellonian University in Kraków. In the same year he was appointed to an assistant position in the Institute of Molecular Biology at the Jagiellonian University, where he continues to be employed as a professor. In 1989 he was awarded a Kennedy Family Scholarship, and as a PhD student on leave he joined the group of Avigdor Scherz at the Weizmann Institute of Science, Rehovot, Israel. In 1994 he received his PhD degree in chemistry from the Feinberg Graduate School of the Weizmann Institute, under the supervision of Avigdor Scherz. In 1997 he received a postdoctoral fellowship from the AvH Foundation, and for 3 years joined the group of Hugo Scheer (Botanisches Institut, Ludwig–Maximilian University of Munich, Munich, Germany). Then he continued his postdoctoral studies with Yasushi Koyama (Kwansei Gakuin University, Sanda, Japan), sponsored by the Japan Society for the Promotion of Science. His main scientific interests lie in the (bio)chemistry of photosynthetic pigments, the primary photosynthetic processes, and the development of photodynamic therapy. He received his PhD degree in 1981 from the Faculty of Chemistry, the Jagiellonian University in Kraków. He is a full professor (since 2000) and Head of the Department of Analytical Chemistry at the Jagiellonian University (since 1997). He is a member of the Committee of Analytical Chemistry of the Polish Academy of Sciences. He has published more than 200 scientific articles, reviews, and book chapters. His main scientific interests are flow techniques (including capillary electrophoresis) applied to forensic chemistry and environmental analysis. The choice between bare and coated capillaries is a key decision in the development and use of any methods based on capillary electrophoresis. In this work several permanently and dynamically coated capillaries were successfully implemented in a previously developed micellar electrokinetic chromatography (MEKC) assay of the plant membrane enzyme chlorophyllase. The results obtained demonstrate the rationale behind the use of capillary coating, which is crucial for successful optimization of both the off-line mode and the on-line/electrophoretically mediated microanalysis assay mode. The application of an amine permanently coated capillary (eCAP) is a simple way to significantly increase the repeatability of migration times and peak areas, and to ensure a strong electroosmotic flow that considerably decreases the overall analysis time. A dynamic coating (CEofix) allows one to apply an on-line incubation to control the reaction progress inside the capillary, and to increase the signal-to-noise ratio and peak efficiency. The dynamic coating is possible with use of both the normally applied uncoated silica capillary and the precoated amine capillary, which ensures more repeatable migration times. The strong points of the uncoated silica capillary are its attractive price and wide range of pH that can be applied. The characteristics presented may simplify the choice of capillary modification, especially in the case of hydrophobic analytes, MEKC-based separations, and other enzymatic assays.
Keywords: Capillary electrophoresis; Capillary coating; Chlorophyll; Chlorophyllase; Electrophoretically mediated microanalysis; Micellar electrokinetic chromatography
Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification by Sabine Fuchs-Telka; Susanne Fister; Patrick-Julian Mester; Martin Wagner; Peter Rossmanith (1503-1511).
is a research associate in the Christian Doppler Laboratory for Monitoring of Microbial Contaminants at the University of Veterinary Medicine in Vienna, Austria. She is working on the development of new cell lysis and DNA extraction methods for isolation of DNA from prokaryotic cells based on ionic liquids. is a postdoctoral research fellow in the Christian Doppler Laboratory for Monitoring of Microbial Contaminants at the University of Veterinary Medicine in Vienna, Austria. She is working with phages and viruses relevant in the food industry (phages P100 and MS2, feline and murine noroviruses), and studies the use of ionic liquids for the development of new methods and applications in (food) microbiology. is a postdoctoral research fellow in the Christian Doppler Laboratory for Monitoring of Microbial Contaminants at the University of Veterinary Medicine in Vienna, Austria. His main research interests are studying the interaction and possible application of ionic liquids in the field of biotechnology and microbiology, as well as the development of novel detection methods for foodborne pathogens. is Head of the Institute for Milk Hygiene, Milk Technology, and Food Science in the Department for Farm Animals and Veterinary Public Health at the University of Veterinary Medicine in Vienna, Austria. His focus is the differentiation, detection, and phylogenetic analyses of microbial pathogens along the food chain. is Head of the Christian Doppler Laboratory for Monitoring of Microbial Contaminants in the Department for Farm Animals and Veterinary Public Health at the University of Veterinary Medicine in Vienna, Austria. His focus is on the development of methods for detection and analysis of microbes and the integration of ionic liquids in molecular biology. DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120–150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr + ][Ntf 2 - ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR
Keywords: Ionic liquids; DNA extraction; Quantitative PCR; Salmonella Typhimurium; Escherichia coli ; Solvent extraction; Two-phase system
A simple analytical method of determining 1-hydroxypyrene glucuronide in human urine by isotope dilution with ultra performance liquid chromatography-tandem mass spectrometry by Mingxin Li; Qian Wang; Jing Zhu; Nana Li; Xiaoli Zou (1513-1518).
The glucuronide conjugate of 1-hydroxypyrene (1-OHP-G) is a sensitive and reliable index biomarker for assessing low exposure to polycyclic aromatic hydrocarbons (PAHs). A simple method for determining 1-OHP-G in human urine with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established and applied to evaluate the exposure level of PAHs of pregnant women in a large sample size. After the urine sample was extracted with ethyl acetate, 0.2 mL of the aqueous phase was diluted to 1.0 mL with 5 mmol/L ammonium acetate before injection. The chromatographic separation was performed on a C18 column with a gradient elution and identification was conducted on a tandem mass spectrometry with electrospray ionization in negative mode. 1-OHP-d9-G was used as an internal standard to improve precision. The method was validated and good linearity was obtained in the range of 0.1∼2.0 ng/mL. The limit of detection (LOD) and the limit of quantification (LOQ) of 1-OHP-G were 0.015 and 0.051 ng/mL. Intra-day and inter-day precision were 4.3 and 6.7 %, respectively. The spiked recoveries were 79.4∼106 % for urine samples. This method was rapid, sensitive, and very suitable for batch analysis of urine. Six hundred seventy-five urine samples of pregnant women from the cities of Fuzhou, Shenzhen, and Nanning of P.R. China were analyzed with the proposed method. The medians of 1-OHP-G concentration were 0.27 μg/g.cr (n = 201), 0.30 μg/g.cr (n = 212), and 0.51 μg/g.cr (n = 262) for the cities of Fuzhou, Shenzhen, and Nanning, respectively. 1-OHP-G concentrations in urine samples of pregnant women from the cities of Fuzhou and Shenzhen in coastal areas were both significantly lower than that of Nanning City in inland region (p < 0.001). Graphical Abstract The internal standard 1-OHP-d9-G and 2.0 mL of ethyl acetate were added to 1.0 mL of urine sample, after vortex vibration and centrifugation the aqueous phase was removed and diluted, and 5 μl of aliquot was analyzed by UPLC-MS/MS
Keywords: 1-Hydroxypyrene glucuronide; Liquid-liquid extraction; Isotope dilution; Ultra performance liquid chromatography-tandem mass spectrometry; Urine sample
Visible and red emissive molecular beacons for optical temperature measurements and quality control in diagnostic assays utilizing temperature-dependent amplification reactions by Zerrin Fidan; Andy Wende; Ute Resch-Genger (1519-1529).
Quality control requirements imposed on assays used in clinical diagnostics and point-of-care-diagnostic testing (POCT), utilizing amplification reactions performed at elevated temperatures of 35 to 95 °C are very stringent. As the temperature of a reaction vessel has a large impact on the specificity and sensitivity of the amplification reaction, simple tools for local in situ temperature sensing and monitoring are required for reaction and assay control. We describe here a platform of stem-and-loop structured DNA hairpins (molecular beacons, MBs), absorbing and emitting in the visible and red spectral region, rationally designed for precise temperature measurements in microfluidic assays for POCT, and their application for temperature measurements in a common DNA-based molecular biological assay utilizing thermophilic helicase-dependent amplification (tHDA). Spectroscopic studies of these MBs, rationally designed from DNA sequences of different thermal stabilities, chosen not to interact with the DNA probes applied in the nucleic acid amplification assay, and temperature-dependent fluorescence measurements of MB-assay mixtures revealed the suitability of these MBs for temperature measurements directly in such an assay with a temperature resolution of about 0.5 °C without interferences from assay components. Combining two spectrally distinguishable MBs provides a broader response range and an increase in temperature sensitivity up to 0.1 °C. This approach will find future application for temperature monitoring and quality control in commercialized diagnostics assays using dried reagents and microfluidic chips as well as assays read out with tube and microplate readers and PCR detection systems for temperature measurements in the range of 35 to 95 °C. Graphical abstract Molecular beacon platform for optical temperature measurements and quality control in diagnostic assays
Keywords: Fluorescence molecular beacon; Temperature sensing; Microfluidics; Quality assurance; Diagnostic assay; DNA amplification
High transport efficiency of nanoparticles through a total-consumption sample introduction system and its beneficial application for particle size evaluation in single-particle ICP-MS by Shin-ichi Miyashita; Hiroaki Mitsuhashi; Shin-ichiro Fujii; Akiko Takatsu; Kazumi Inagaki; Toshiyuki Fujimoto (1531-1545).
In order to facilitate reliable and efficient determination of both the particle number concentration (PNC) and the size of nanoparticles (NPs) by single-particle ICP-MS (spICP-MS) without the need to correct for the particle transport efficiency (TE, a possible source of bias in the results), a total-consumption sample introduction system consisting of a large-bore, high-performance concentric nebulizer and a small-volume on-axis cylinder chamber was utilized. Such a system potentially permits a particle TE of 100 %, meaning that there is no need to include a particle TE correction when calculating the PNC and the NP size. When the particle TE through the sample introduction system was evaluated by comparing the frequency of sharp transient signals from the NPs in a measured NP standard of precisely known PNC to the particle frequency for a measured NP suspension, the TE for platinum NPs with a nominal diameter of 70 nm was found to be very high (i.e., 93 %), and showed satisfactory repeatability (relative standard deviation of 1.0 % for four consecutive measurements). These results indicated that employing this total consumption system allows the particle TE correction to be ignored when calculating the PNC. When the particle size was determined using a solution-standard-based calibration approach without an NP standard, the particle diameters of platinum and silver NPs with nominal diameters of 30–100 nm were found to agree well with the particle diameters determined by transmission electron microscopy, regardless of whether a correction was performed for the particle TE. Thus, applying the proposed system enables NP size to be accurately evaluated using a solution-standard-based calibration approach without the need to correct for the particle TE.
Keywords: Nanoparticle; spICP-MS; Transport efficiency; Total-consumption sample introduction system; Particle number concentration; Particle size
1,2-Dimethylimidazole-4-sulfonyl chloride (DMISC), a novel derivatization strategy for the analysis of propofol by LC-ESI-MS/MS by Alexandra Maas; Christoph Maier; Beate Michel-Lauter; Burkhard Madea; Cornelius Hess (1547-1554).
Analysis of the anesthetic agent propofol in biological samples by LC-MS/MS is a great challenge due to weak fragmentation and poor ionization efficacy of propofol resulting in weak signal intensities. Improvements of the ionization and fragmentation efficacy can be achieved by conversion of propofol to its dimethylimidazolesulfonyl (DMIS) derivative by a derivatization reaction using 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC). This DMIS derivative produced intense [M + H]+ ions in positive-ion LC-ESI-MS/MS with the dimethylimidazole moieties representing the most abundant product ions. Derivatization of serum samples is achieved by direct conversion of the acetonitrile supernatant of a protein precipitation with DMISC followed by a double liquid-liquid extraction using n-hexane. Reliability of the method was confirmed under consideration of the validation parameters selectivity, linearity, accuracy and precision, analytical limits, and processed sample stability. Linearity was demonstrated over the whole calibration range from 5 to 1000 ng/ml with the use of a 1/x 2 weighting. Stability of the processed samples was verified for a time period of up to 25 h. Due to its high sensitivity, appropriate quantification and detection limits (LLoQ = 5 ng/ml, LoD = 0.95 ng/ml) for toxicological propofol analyses could be achieved. Applicability of the method to biological samples could be verified by analysis of a human serum sample collected after propofol-induced sedation. Graphical abstract A novel derivatization strategy using 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) was developed to improve the ionization and fragmentation efficacy of propofol for LC-ESI-MS/MS analysis.
Keywords: Propofol; 1,2-Dimethylimidazole-4-sulfonyl chloride (DMISC); Derivatization; LC-MS/MS
Surface-enhanced Raman spectroscopy introduced into the International Standard Organization (ISO) regulations as an alternative method for detection and identification of pathogens in the food industry by Evelin Witkowska; Dorota Korsak; Aneta Kowalska; Monika Księżopolska-Gocalska; Joanna Niedziółka-Jönsson; Ewa Roźniecka; Weronika Michałowicz; Paweł Albrycht; Marta Podrażka; Robert Hołyst; Jacek Waluk; Agnieszka Kamińska (1555-1567).
We show that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) can serve as a fast, reliable, and easy method for detection and identification of food-borne bacteria, namely Salmonella spp., Listeria monocytogenes, and Cronobacter spp., in different types of food matrices (salmon, eggs, powdered infant formula milk, mixed herbs, respectively). The main aim of this work was to introduce the SERS technique into three ISO (6579:2002; 11290–1:1996/A1:2004; 22964:2006) standard procedures required for detection of these bacteria in food. Our study demonstrates that the SERS technique is effective in distinguishing very closely related bacteria within a genus grown on solid and liquid media. The advantages of the proposed ISO-SERS method for bacteria identification include simplicity and reduced time of analysis, from almost 144 h required by standard methods to 48 h for the SERS-based approach. Additionally, PCA allows one to perform statistical classification of studied bacteria and to identify the spectrum of an unknown sample. Calculated first and second principal components (PC-1, PC-2) account for 96, 98, and 90% of total variance in the spectra and enable one to identify the Salmonella spp., L. monocytogenes, and Cronobacter spp., respectively. Moreover, the presented study demonstrates the excellent possibility for simultaneous detection of analyzed food-borne bacteria in one sample test (98% of PC-1 and PC-2) with a goal of splitting the data set into three separated clusters corresponding to the three studied bacteria species. The studies described in this paper suggest that SERS represents an alternative to standard microorganism diagnostic procedures. Graphical Abstract New approach of the SERS strategy for detection and identification of food-borne bacteria, namely S. enterica, L. monocytogenes, and C. sakazakii in selected food matrices
Keywords: Salmonella Typhimurium; SERS; ISO methods; Food; Bacteria detection; PCA
Structure-performance relationships of phenyl cinnamic acid derivatives as MALDI-MS matrices for sulfatide detection by Suparna Tambe; Henning Blott; Annabelle Fülöp; Nils Spang; Dirk Flottmann; Stefan Bräse; Carsten Hopf; Hans-Dieter Junker (1569-1580).
A key aspect for the further development of matrix-assisted laser desorption ionization (MALDI)-mass spectrometry (MS) is a better understanding of the working principles of MALDI matrices. To address this issue, a chemical compound library of 59 structurally related cinnamic acid derivatives was synthesized. Potential MALDI matrices were evaluated with sulfatides, a class of anionic lipids which are abundant in complex brain lipid mixtures. For each matrix relative mean S/N ratios of sulfatides were determined against 9-aminoacridine as a reference matrix using negative ion mass spectrometry with 355 and 337 nm laser systems. The comparison of matrix features with their corresponding relative mean S/N ratios for sulfatide detection identified correlations between matrix substitution patterns, their chemical functionality, and their MALDI-MS performance. Crystal structures of six selected matrices provided structural insight in hydrogen bond interactions in the solid state. Principal component analysis allowed the additional identification of correlation trends between structural and physical matrix properties like number of exchangeable protons at the head group, MW, logP, UV-Vis, and sulfatide detection sensitivity. Graphical abstract Design, synthesis and mass spectrometric evaluation of MALDI-MS matrix compound libraries allows the identification of matrix structure - MALDI-MS performance relationships using multivariate statistics as a tool
Keywords: MALDI mass spectrometry; MALDI matrix; Sulfatide; Structure-performance relationships; Principal component analysis; X-ray crystal structure
Development of a mass spectrometry immunoassay for unambiguous detection of egg allergen traces in wines by Rosa Pilolli; Ravindra Chaudhari; Francesco Palmisano; Linda Monaci (1581-1589).
A mass spectrometry immunoassay (MSIA) specifically designed for the detection of egg allergens in wines is described. MSIA is based on an immunoaffinity enrichment procedure combined with targeted MS/MS detection of selected egg peptide markers. Polyclonal antibodies raised against native ovalbumin, chosen as the target protein tracing for egg powder, were immobilized onto low backpressure monolithic MSIA customized disposable tips. Ovalbumin-free wine samples were fortified with standard protein at different concentrations in the low microgram-per-milliliter range. A simple protocol was devised consisting of a 1:4 dilution of the wine sample with a basic solution for pH adjustment, followed by a semi-automated purification/enrichment step on MSIA customized disposable tips fitted on a multichannel electronic pipette. Among the main figures of merit, LOD and LOQ values as low as 0.01 and 0.03 μg/mL, respectively, and within-day precision of 18% should be noticed. Noteworthy, the developed assay outperformed current MS-based methods for the detection of allergenic protein in wine matrices, thanks to the immunoaffinity enrichment. In addition, compared to other immunoassays, the present approach boasts the unquestionable advantage of providing an unambiguous identification of the target protein by simultaneous detection of three unique peptide markers each giving three specific MS/MS transitions.
Keywords: Egg allergen; Wine; MSIA; Immunoassay; Mass spectrometry; Ovalbumin
Metabolic profiling of ob/ob mouse fatty liver using HR-MAS 1H-NMR combined with gene expression analysis reveals alterations in betaine metabolism and the transsulfuration pathway by Mikheil Gogiashvili; Karolina Edlund; Kathrin Gianmoena; Rosemarie Marchan; Alexander Brik; Jan T. Andersson; Jörg Lambert; Katrin Madjar; Birte Hellwig; Jörg Rahnenführer; Jan G. Hengstler; Roland Hergenröder; Cristina Cadenas (1591-1606).
Metabolic perturbations resulting from excessive hepatic fat accumulation are poorly understood. Thus, in this study, leptin-deficient ob/ob mice, a mouse model of fatty liver disease, were used to investigate metabolic alterations in more detail. Metabolites were quantified in intact liver tissues of ob/ob (n = 8) and control (n = 8) mice using high-resolution magic angle spinning (HR-MAS) 1H-NMR. In addition, after demonstrating that HR-MAS 1H-NMR does not affect RNA integrity, transcriptional changes were measured by quantitative real-time PCR on RNA extracted from the same specimens after HR-MAS 1H-NMR measurements. Importantly, the gene expression changes obtained agreed with those observed by Affymetrix microarray analysis performed on RNA isolated directly from fresh-frozen tissue. In total, 40 metabolites could be assigned in the spectra and subsequently quantified. Quantification of lactate was also possible after applying a lactate-editing pulse sequence that suppresses the lipid signal, which superimposes the lactate methyl resonance at 1.3 ppm. Significant differences were detected for creatinine, glutamate, glycine, glycolate, trimethylamine-N-oxide, dimethylglycine, ADP, AMP, betaine, phenylalanine, and uridine. Furthermore, alterations in one-carbon metabolism, supported by both metabolic and transcriptional changes, were observed. These included reduced demethylation of betaine to dimethylglycine and the reduced expression of genes coding for transsulfuration pathway enzymes, which appears to preserve methionine levels, but may limit glutathione synthesis. Overall, the combined approach is advantageous as it identifies changes not only at the single gene or metabolite level but also deregulated pathways, thus providing critical insight into changes accompanying fatty liver disease. Graphical abstract A Evaluation of RNA integrity before and after HR-MAS 1H-NMR of intact mouse liver tissue. B Metabolite concentrations and gene expression levels assessed in ob/ob (steatotic) and ob/+ (control) mice using HR-MAS 1H-NMR and qRT-PCR, respectively.
Keywords: HR-MAS 1H-NMR spectroscopy; NAFLD; Fatty liver; ob/ob mice; One-carbon metabolism; Lactate editing
Highly selective SiO2–NH2@TiO2 hollow microspheres for simultaneous enrichment of phosphopeptides and glycopeptides by Dongpo Xu; Guoquan Yan; Mingxia Gao; Chunhui Deng; Xiangmin Zhang (1607-1614).
Glycosylation and phosphorylation as the commonest and most important posttranslational modifications of proteins play critical roles in biological processes. Specific and sensitive strategies have been developed to identify the glycosylation and phosphorylation of proteins by mass spectrometry but most of these methods have mainly focused on enriching glycopeptides or phosphopeptides separately, and few of them can enrich both of them. In this work, highly selective SiO2–NH2@TiO2 hollow microspheres were designed and synthesized for the simultaneous selective enrichment of both phosphopeptides and glycopeptides. Because of the bifunctionalized property of the titanium dioxide and the amino group, the hollow microspheres were successfully applied for the simultaneous enrichment of phosphopeptides and glycopeptides. We evaluated the enrichment selectivity of the SiO2–NH2@TiO2 hollow microspheres by capturing phosphopeptides and glycopeptides from a peptide mixture of bovine β-casein, horseradish peroxidase, and bovine serum albumin in a molar ratio of 1:1:500 (1.7 × 10−12 mol β-casein and horseradish peroxidase, 8.5 × 10−10 mol bovine serum albumin in 100 μL). Graphical Abstract Workflow of the simultaneous enrichment strategy for phosphopeptides and glycopeptides by SiO2–NH2@TiO2 hollow microspheres
Keywords: Hollow microspheres; Hydrophilic; Glycopeptides; Phosphopeptides; Simultaneous enrichment; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Combination of microbiological, spectroscopic and molecular docking techniques to study the antibacterial mechanism of thymol against Staphylococcus aureus: membrane damage and genomic DNA binding by Lang-Hong Wang; Zhi-Hong Zhang; Xin-An Zeng; De-Ming Gong; Man-Sheng Wang (1615-1625).
Thymol (2-isopropyl-5-methylphenol) is a natural ingredient used as flavor or preservative agent in food products. The antibacterial mechanism of thymol against Gram-positive, Staphylococcus aureus was investigated in this work. A total of 15 membrane fatty acids were identified in S. aureus cells by gas chromatography–mass spectrometry. Exposure to thymol at low concentrations induced obvious alterations in membrane fatty acid composition, such as decreasing the proportion of branched 12-methyltetradecanoic acid and 14-methylhexadecanoic acid (from 22.4 and 17.3% to 7.9 and 10.3%, respectively). Membrane permeability assay and morphological image showed that thymol at higher concentrations disrupted S. aureus cell membrane integrity, which may decrease cell viability. Moreover, the interaction of thymol with genomic DNA was also investigated using multi-spectroscopic techniques, docking and atomic force microscopy. The results indicated that thymol bound to the minor groove of DNA with binding constant (K a) value of (1.22 ± 0.14) × 104 M−1, and this binding interaction induced a mild destabilization in the DNA secondary structure, and made DNA molecules to be aggregated. Graphical Abstract Thymol exerts its antibacterial effect throught destruction of bacterial cell membrane and binding directly to genomic DNA
Keywords: Thymol; Staphylococcus aureus ; Gas chromatography–mass spectrometry; Membrane integrity; Atomic force microscopy
Evaluation of the antibacterial activity of a cationic polymer in aqueous solution with a convenient electrochemical method by Yihong Wang; Qing Guo; Huafu Wang; Kun Qian; Liang Tian; Chen Yao; Wei Song; Weixia Shu; Ping Chen; Jinxu Qi (1627-1633).
Quaternized chitosan is a cationic biopolymer with good antibacterial activity, biocompatibility, and biodegradability, and it has been widely applied in many fields. We have developed a convenient method to evaluate the antibacterial activity of hydroxypropyltrimethylammonium chloride chitosan (HACC) with a nonionic surfactant poloxamer in aqueous solution by monitoring the change of the oxidation peak current in cyclic voltammetry. Increasing values of the oxidation peak current were positively correlated with the antibacterial activity of HACC–poloxamer solutions. Optical microscope images, the zeta potential, and fluorescence spectroscopy showed that the aggregation state of HACC–poloxamer was related to the ratio of the two polymers and also to the antibacterial activity and oxidation peak current. At an HACC-to-poloxamer ratio of 1:0.75, the maximum surface charge density and the smooth edge of HACC–poloxamer aggregates can accelerate diffusion in aqueous solution. It is expected that this convenient method can be applied for a quick evaluation of the antibacterial activity of cationic biopolymers in aqueous solution. Graphical Abstract The cyclic voltammograms of MB in HACC/poloxamer solution, and the antibacterial efficiency against S. aureus after incubated with HACC (a) and 1/0.75 of HACC/poloxamer (b)
Keywords: Quaternized chitosan; Hydroxypropyltrimethylammonium chloride chitosan–poloxamer; Cyclic voltammetry; Antibacterial activity
Coordination-induced decomposition of luminescent gold nanoparticles: sensitive detection of H2O2 and glucose by Xiaoqi Lai; Fengxian Luo; Yaping Wang; Xuxian Su; Jinbin Liu (1635-1641).
The surface inert of luminescent gold nanoparticles (AuNPs) toward biomolecules set a challenge to further exploit their bioanalytical applications using the direct luminescence response. Herein, we report a novel approach to induce significant luminescence quenching of the AuNPs upon the interaction with a metal coordination ligand tris(2-carboxyethyl)phosphine (TCEP), providing a strategy for the detection of H2O2 with a limit of detection (LOD) of 14 nM through the reaction between H2O2 and TCEP to protect the luminescence quenching of the AuNPs. Furthermore, this strategy is also extended for sensitive and selective detection of glucose with a LOD of 1.1 μM based on monitoring the production of H2O2 catalyzed from the oxidation of glucose. The highly extendable feature of this strategy can have great potential in the sensitive detection of other biomolecules. Graphical Abstract A facile and extendable strategy has been developed for the sensitive detection of H2O2 and glucose based on the interaction between luminescent gold nanoparticles and TCEP, a metal coordination ligand.
Keywords: Luminescent gold nanoparticle; Coordination; Decomposition; H2O2 ; Glucose
HPLC–MS/MS methods for the determination of 52 perfluoroalkyl and polyfluoroalkyl substances in aqueous samples by Christoph Gremmel; Tobias Frömel; Thomas P. Knepper (1643-1655).
Two quantitative methods using high-performance liquid chromatography (HPLC) combined with triple quadrupole tandem mass spectrometry (MS/MS) were developed to determine perfluoroalkyl and polyfluoroalkyl substances (PFASs) in aqueous samples. The first HPLC–MS/MS method was applied to 47 PFASs of 12 different substance classes with acidic characteristics such as perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkane sulfonic acids (PFSAs), as well as precursor substances and biotransformation intermediates (e.g., unsaturated fluorotelomer carboxylic acids). In addition, 25 13C-, 18O-, and 2H-labeled PFASs were used as internal standards in this method. The second HPLC–MS/MS method was applied to fluorotelomer alcohols (FTOHs) and perfluorooctane sulfonamidoethanols as these compounds have physicochemical properties different from those of the previous ones. Accuracy between 82% and 110% and a standard deviation in the range from 2% to 22% depending on the substances were determined during the evaluation of repeatability and precision. The method quantification limit after solid-phase extraction ranged from 0.3 to 199 ng/L depending on the analyte and matrix. The HPLC–MS/MS methods developed were suitable for the determination of PFASs in aqueous samples (e.g., wastewater treatment plant effluents or influents after solid-phase extraction). These methods will be helpful in monitoring campaigns to evaluate the relevance of precursor substances as indirect sources of perfluorinated substances in the environment. In one exemplary application in an industrial wastewater treatment plant, FTOHs were found to be the major substance class in the influent; in particular, 6:2-FTOH was the predominant compound in the industrial samples and accounted for 74% of the total PFAS concentration. The increase in the concentration of the transformation products of FTOHs in the corresponding effluent, such as fluorotelomer carboxylic acids, unsaturated fluorotelomer carboxylic acids, n:3 polyfluorinated saturated carboxylic acids (n indicates the number of nonfluorinated carbon atoms), and PFCAs, indicated biotransformation of FTOHs or their derivatives during wastewater treatment. However, only 33 mol% of the total amount of PFASs present in the influent was quantified in the corresponding effluent. Graphical abstract Method development of an HPLC-MS/MS multi-method for the determination of PFASs in aqueos samples
Keywords: Perfluoroalkyl and polyfluoroalkyl substances; Wastewater treatment plants; High-performance liquid chromatography–electrospray ionization tandem mass spectrometry; Precursor compounds of perfluoroalkyl acids
Muramic and dipicolinic acids in atmospheric particulate matter as biomarkers of bacteria and bacterial spores by Patrizia Di Filippo; Donatella Pomata; Carmela Riccardi; Francesca Buiarelli; Daniela Uccelletti; Elena Zanni (1657-1666).
Airborne bacteria are components of the atmospheric aerosol particles and can be responsible of allergic disease, regardless of their viability. In this paper, we report a method for the determination of total (viable and nonviable) bacterial content in airborne particles, using muramic and dipicolinic acids as biomarkers of bacteria and bacterial spores, respectively. The analytical procedure was optimized with bacteria and spores of Bacillus subtilis. After extraction and purification, the two biomarkers were analyzed by HPLC-ESI-MS/MS and their percentage was evaluated to be used as conversion factor. The present method for the determination of the total bacterial content was then applied to environmental samples, after a proper collection in an urban site. Thanks to the use of a low pressure impactor, capable of fractionating particles into the range of 0.03–10 μm, it was also possible to study the bacterial content in ultrafine, fine, and coarse particulate matter. The results from this study showed that muramic and dipicolinic acids can be determined together in one chromatographic run in reversed phase ion pair chromatography. Bacteria were more abundant than bacterial spores in the urban atmosphere, both showing a higher concentration in the coarse fraction of particles, although bacteria and bacterial spore amounts per unit mass of ultrafine particles were higher than in fine and coarse particles.
Keywords: Bioaerosol; Muramic acid; Dipicolinic acid; Bacteria; Bacterial spores; Conversion factors
Metabolic characterization of (1-(5-fluoropentyl)-1H-indol-3-yl)(4-methyl-1-naphthalenyl)-methanone (MAM-2201) using human liver microsomes and cDNA-overexpressed cytochrome P450 enzymes by Tae Yeon Kong; Ju-Hyun Kim; Won Gu Choi; Joo Young Lee; Hee Seung Kim; Jin Young Kim; Moon Kyo In; Hye Suk Lee (1667-1680).
MAM-2201 is a synthetic cannabinoid that is increasingly found in recreational drug abusers and cases of severe intoxication. Thus, characterization of the metabolic pathways of MAM-2201 is necessary to predict individual pharmacokinetics and toxicity differences, and to avoid toxic drug-drug interactions. Collectively, 19 phase 1 metabolites of MAM-2201 were identified using liquid chromatography–Orbitrap mass spectrometry following human liver microsomal incubations in the presence of NADPH: 7 hydroxy-MAM-2201 (M1–M7), 4 dihydroxy-MAM-2201 (M8–M11), dihydrodiol-MAM-2201 (M12), N-(5-hydroxypentyl)-MAM-2201 (M13), hydroxy-M13 (M14), N-dealkyl-MAM-2201 (M15), 2 hydroxy-M15 (M16, M17), MAM-2201 N-pentanoic acid (M18), and hydroxy-M18 (M19). On the basis of intrinsic clearance values in human liver microsomes, hydroxy-MAM-2201 (M1), N-(5-hydroxypentyl)-MAM-2201 (M13), and hydroxy-M13 (M14) were the major metabolites. Based on an enzyme kinetics study using human cDNA-expressed cytochrome P450 (CYP) enzymes and an immunoinhibition study using selective CYP antibodies in human liver microsomes, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes were responsible for MAM-2201 metabolism. The CYP3A4 enzyme played a prominent role in MAM-2201 metabolism, and CYP1A2, CYP2B6, CYP2C8, and CYP2C9 enzymes played major roles in the formation of some metabolites. MAM-2201 is extensively metabolized by multiple CYP enzymes, indicating that MAM-2201 and its metabolites should be used as markers of MAM-2201 abuse and toxicity. Graphical abstract In vitro metabolic pathways of MAM-2201 were characterized in human liver microsomes and recombinant CYPs using LC-HRMS analysis. Total 19 phase I metabolites were identified with predominant contribution of CYP3A4
Keywords: MAM-2201; In vitro metabolism; Cytochrome P450 characterization
Biotransformation and detectability of the new psychoactive substances N,N-diallyltryptamine (DALT) derivatives 5-fluoro-DALT, 7-methyl-DALT, and 5,6-methylenedioxy-DALT in urine using GC-MS, LC-MSn, and LC-HR-MS/MS by Julian A. Michely; Simon D. Brandt; Markus R. Meyer; Hans H. Maurer (1681-1695).
Derivatives of N,N-diallyltryptamine (DALT) can be classified as new psychoactive substances. Biotransformation and detectability of 5-fluoro-DALT (5-F-DALT), 7-methyl-DALT (7-Me-DALT), and 5,6-methylenedioxy-DALT (5,6-MD-DALT) are described here. Their metabolites detected in rat urine and pooled human liver microsomes were identified by liquid chromatography (LC)–high resolution (HR)–tandem mass spectrometry (MS/MS). In addition, the human cytochrome-P450 (CYP) isoenzymes involved in the main metabolic steps were identified and detectability tested in urine by the authors’ urine screening approaches using GC-MS, LC-MSn, or LC-HR-MS/MS. Aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations could be proposed for all compounds as main pathways. Carboxylation after initial hydroxylation of the methyl group could also be detected for 7-Me-DALT and O-demethylenation was observed for 5,6-MD-DALT. All phase I metabolites were extensively glucuronidated or sulfated. Initial phase I reactions were catalyzed by CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5. Rat urine samples were analyzed following two different low-dose administrations. GC-MS was not able to monitor consumption reliably, but all three compounds are predicted to be detectable in cases of overdose. The LC-MSn and LC-HR-MS/MS approaches were suitable for detecting an intake of all three compounds mainly via their metabolites. However, after the lowest dose, a reliable monitoring could only be achieved for 5-F-DALT via LC-MSn and LC-HR-MS/MS and for 7-Me-DALT via LC-HR-MS/MS. The most abundant targets in both LC-MS screenings were one of two hydroxy-aryl metabolites and both corresponding glucuronides for 5-F-DALT, one N-deallyl hydroxy-aryl, the carboxy, and one dihydroxy-aryl metabolite for 7-Me-DALT, and the demethylenyl metabolite, its oxo metabolite, and glucuronide for 5,6-MD-DALT.
Keywords: Tryptamine derivatives; 5-F-DALT; 7-Me-DALT; 5,6-MD-DALT; Metabolism; Screening
Spatially resolved metabolic distribution for unraveling the physiological change and responses in tomato fruit using matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI) by Junya Nakamura; Tomomi Morikawa-Ichinose; Yoshinori Fujimura; Eisuke Hayakawa; Katsutoshi Takahashi; Takanori Ishii; Daisuke Miura; Hiroyuki Wariishi (1697-1706).
Information on spatiotemporal metabolic behavior is indispensable for a precise understanding of physiological changes and responses, including those of ripening processes and wounding stress, in fruit, but such information is still limited. Here, we visualized the spatial distribution of metabolites within tissue sections of tomato (Solanum lycopersicum L.) fruit using a matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI) technique combined with a matrix sublimation/recrystallization method. This technique elucidated the unique distribution patterns of more than 30 metabolite-derived ions, including primary and secondary metabolites, simultaneously. To investigate spatiotemporal metabolic alterations during physiological changes at the whole-tissue level, MALDI–MSI was performed using the different ripening phenotypes of mature green and mature red tomato fruits. Although apparent alterations in the localization and intensity of many detected metabolites were not observed between the two tomatoes, the amounts of glutamate and adenosine monophosphate, umami compounds, increased in both mesocarp and locule regions during the ripening process. In contrast, malate, a sour compound, decreased in both regions. MALDI–MSI was also applied to evaluate more local metabolic responses to wounding stress. Accumulations of a glycoalkaloid, tomatine, and a low level of its glycosylated metabolite, esculeoside A, were found in the wound region where cell death had been induced. Their inverse levels were observed in non-wounded regions. Furthermore, the amounts of both compounds differed in the developmental stages. Thus, our MALDI–MSI technique increased the understanding of the physiological changes and responses of tomato fruit through the determination of spatiotemporally resolved metabolic alterations. Graphical abstract ᅟ
Keywords: Localization; MALDI–MSI; Metabolic alterations; Physiological changes and responses; Tomato fruit (Solanum lycopersicum L.)
Characterization of phthalocyanine functionalized quantum dots by dynamic light scattering, laser Doppler, and capillary electrophoresis by Gonzalo Ramírez-García; David O. Oluwole; Siphesihle Robin Nxele; Fanny d’Orlyé; Tebello Nyokong; Fethi Bedioui; Anne Varenne (1707-1715).
In this work, we characterized different phtalocyanine-capped core/shell/shell quantum dots (QDs) in terms of stability, ζ-potential, and size at various pH and ionic strengths, by means of capillary electrophoresis (CE), and compared these results to the ones obtained by laser Doppler electrophoresis (LDE) and dynamic light scattering (DLS). The effect of the phthalocyanine metallic center (Zn, Al, or In), the number (one or four), and nature of substituents (carboxyphenoxy- or sulfonated-) of functionalization on the phthalocyanine physicochemical properties were evaluated. Whereas QDs capped with zinc mono-carboxyphenoxy-phtalocyanine (ZnMCPPc-QDs) remained aggregated in the whole analyzed pH range, even at low ionic strength, QDs capped with zinc tetracarboxyphenoxy phtalocyanine (ZnTPPc-QDs) were easily dispersed in buffers at pH equal to or higher than 7.4. QDs capped with aluminum tetrasulfonated phthalocyanine (AlTSPPc-QDs) and indium tetracarboxyphenoxy phthalocyanines (InTCPPc-QDs) were stable in aqueous suspension only at pH higher than 9.0 due to the presence of functional groups bound to the metallic center of the phthalocyanine. The ζ-potential values determined by CE for all the samples decreased when ionic strength increased, being well correlated with the aggregation of the nanoconjugates at elevated salt concentrations. The use of electrokinetic methodologies has provided insights into the colloidal stability of the photosensitizer-functionalized QDs in physiological relevant solutions and thereby, its usefulness for improving their design and applications for photodynamic therapy. Graphical Abstract Schematic illustration of the phthalocyanine capped QDs nanoconjugates and the capillary electrophoresis methods applied for size and ζ-potential characterization
Keywords: Colloidal stability; Quantum dots; Capillary electrophoresis; Photodynamic therapy; Phthalocyanines; Photosensitizers
Erratum to: “Inherently chiral” thiophene-based electrodes at work: a screening of enantioselection ability toward a series of pharmaceutically relevant phenolic or catecholic amino acids, amino esters, and amine by Serena Arnaboldi; Tiziana Benincori; Roberto Cirilli; Sara Grecchi; Laura Santagostini; Francesco Sannicolò; Patrizia R. Mussini (1717-1718).