Analytical and Bioanalytical Chemistry (v.408, #23)

ABC’s spotlight on the nanoworld by Günter Gauglitz (6235-6237).
is Senior Professor at the Eberhard Karls University of Tübingen working in analytical and physical chemistry. He was chairman of the GDCh Division of Analytical Chemistry and chaired the Europt(r)ode VIII meeting. For more than 15 years, his main scientific interests have centered on research and development in chemical and biochemical sensors with special focus on the characterization of interfaces of polymers and biomembrane surfaces, spectroscopic techniques, use of spectral interferometry to monitor changes in optical thickness of thin layers, and effects of Fresnel reflectivity at interfaces. He has been Editor of Analytical and Bioanalytical Chemistry (ABC) since 2002.

Some ruminations on graduate students by Apryll M. Stalcup (6239-6243).
is currently Professor of Chemical Sciences at Dublin City University in Ireland. Her research interests emphasize chromatographic and electrophoretic separation mechanisms, linear solvation energy relationships, exploring new separation methods, and characterizing complex carbohydrates. Her group pioneered the use of sulfated-β-cyclodextrin, heparin, and quinine as chiral additives in capillary electrophoresis, and demonstrated the wide range of separation modes (reversed phase, normal phase, ion exchange, and ion exclusion) obtainable with surface-confined ionic liquids (SCIL) liquid chromatographic stationary phases. Prof. Stalcup is the author of over 100 publications, reviews, book chapters, and one patent. She is a Fellow of the Institute of Chemistry of Ireland and the Royal Society of Chemistry, a member of the American Chemical Society, the American Association of the Advancement of Science, Sigma Xi, the University of Cincinnati Graduate Fellows, and a Charter member of the University of Cincinnati Chapter’s National Academy of Inventors. She was the recipient of the 2015 American Microchemical Society A. A. Benedetti-Pichler Award and the 2011 Cincinnati Section of the American Chemical Society Cincinnati Chemist of the Year Award. She serves as Co-Chair (with Professor Jeremy Glennon, University College Cork) of the 31st International Symposium on Chromatography in 2016.

is a Research Professor at DOM Lab at Sejong University in Seoul, South Korea. She has been working for a decade on the dynamics of DOM with multiple methodologies in diverse aquatic ecosystems from headwaters to oceans, including the Han River in South Korea, the Florida coastal Everglades, the East Sea, and the Arctic Ocean. is an Associate Professor at Kyungpook National University in South Korea. Dr. Kim’s research is focused on understanding chemical compositions of crude oils and humic substance by high resolution mass spectrometry, ion mobility mass spectrometry statistical analysis, NMR, and chromatographic separation. is a researcher at Korea Basic Science Institute-Mass Spectrometry Research Center in South Korea. She is interested in understanding the characteristics of DOM in various aquatic environments including freshwater and wastewater via high resolution mass spectrometry. is a researcher at DOM Lab at Sejong University in South Korea. He has been working on improving solid-phase extraction of DOM with various origins and on enhancing oxidation processes of natural organic matter for total organic carbon measurements. is a Professor at the Department of Environment and Energy at Sejong University in South Korea, leading DOM Lab. His research focuses on characterizing DOM of various sources via many advanced technologies including EEM-PARAFAC, SEC-OCD, and FT-ICR-MS to seek answers to many fundamental questions on how DOM changes in given natural and engineered systems and how DOM influences the environments. Although PPL-based solid-phase extraction (SPE) has been widely used before dissolved organic matter (DOM) analyses via advanced measurements such as ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), much is still unknown about the structural and compositional changes in DOM pool through SPE. In this study, selected DOM from various sources were tested to elucidate the differences between before and after the SPE utilizing multiple analytical tools including fluorescence spectroscopy, FT-ICR-MS, and size exclusion chromatography with organic carbon detector (SEC-OCD). The changes of specific UV absorbance indicated the decrease of aromaticity after the SPE, suggesting a preferential exclusion of aromatic DOM structures, which was also confirmed by the substantial reduction of fluorescent DOM (FDOM). Furthermore, SEC-OCD results exhibited very low recoveries (1–9 %) for the biopolymer fraction, implying that PPL needs to be used cautiously in SPE sorbent materials for treating high molecular weight compounds (i.e., polysaccharides, proteins, and amino sugars). A careful examination via FT-ICR-MS revealed that the formulas lost by the SPE might be all DOM source-dependent. Nevertheless, the dominant missing compound groups were identified to be the tannins group with high O/C ratios (>0.7), lignins/carboxyl-rich alicyclic molecules (CRAM), aliphatics with high H/C >1.5, and heteroatomic formulas, all of which were prevailed by pseudo-analogous molecular formula families with different methylene (−CH2) units. Our findings shed new light on potential changes in the compound composition and the molecular weight of DOM upon the SPE, implying precautions needed for data interpretation. Graphical Abstract Tracking the characteristics of DOM from various origins upon PPL-based SPE utilizing EEMPARAFAC, SEC-OCD, and FT-ICR-MS
Keywords: Solid-phase extraction; DOM sources; EEM-PARAFAC; FT-ICR-MS; SEC-OCD; Molecular weight

IR-MALDI ion mobility spectrometry by José Villatoro; Martin Zühlke; Daniel Riebe; Jens Riedel; Toralf Beitz; Hans-Gerd Löhmannsröben (6259-6268).
is a PhD fellow at the University of Potsdam, Institute of Chemistry, and at the Zuse Institute Berlin. His research is currently financed by the German Excellence Initiative-funded School of Analytical Sciences Adlershof (SALSA) and BAM, the German Federal Institute for Materials Research and Testing. His research interests include IR-MALDI for the analysis of biologically relevant molecules with ion mobility spectrometry (IMS), novel ionization source design for IMS, and conformational dynamics studies of peptides. is a PhD student in the Physical Chemistry group at the University of Potsdam. After his master thesis dealing with the combination of infrared-matrix assisted laser desorption and ionization (IR-MALDI) and ion mobility spectrometry (IMS), he has been working in the field of electrospray ionization (ESI)-IMS as a HPLC detector for the separation and analysis of complex mixtures. received his Dipl-Chem from the University of Potsdam, where he is currently working on his PhD thesis. His research is concerned with the design and construction of novel ion mobility mass spectrometry instrumentation and the implementation of different ionization sources as well as the theoretical calculation of collision cross sections. Additionally, he has been working on gas-phase spectroscopic techniques, such as LIF and LIBS. is currently responsible for designing ambient ionization instrumentation at BAM, the Federal Institute for Materials Research and Testing, in Berlin. After receiving his PhD from the Christian Albrechts University in Kiel, he worked at the Institute for Atomic and Molecular Sciences in Taipei and the Fritz Haber Institute of the Max Planck Society in Berlin. Besides the development of spectroscopic and spectrometric devices, his major research interest lies in gas-phase dynamics and effects at the liquid-gas boundary. is a Senior Research Scientist in the Physical Chemistry group at the University of Potsdam. He has been working on the characterization of different ionization sources (ESI, IR-MALDI, REMPI) in mass and ion mobility spectrometry. His further research interests are experimental and theoretical investigations of ion–molecule interactions in ion mobility spectrometry. obtained his PhD in physical chemistry in 1985 from the University of Göttingen. He was Senior Research Scientist and Lecturer at the Technical University of Braunschweig, and held Professorships at the University of Applied Science in Emden and at the University of Erlangen-Nürnberg. Since 2000 he has been Professor for physical chemistry at Potsdam University, funding member and Director of the Center for Innovation Competence innoFSPEC Potsdam (since 2008), awardee in the German Excellence Initiative with the Graduate School of Analytical Sciences Adlershof (SALSA), and member of SALSA Board (since 2012). His research interests include photophysics, photochemistry, laser spectroscopy, and optical sensing as well as ion mobility spectrometry and gas-phase reaction kinetics. The novel combination of infrared matrix-assisted laser dispersion and ionization (IR-MALDI) with ion mobility (IM) spectrometry makes it possible to investigate biomolecules in their natural environment, liquid water. As an alternative to an ESI source, the IR-MALDI source was implemented in an in-house-developed ion mobility (IM) spectrometer. The release of ions directly from an aqueous solution is based on a phase explosion, induced by the absorption of an IR laser pulse (λ = 2.94 μm, 6 ns pulse width), which disperses the liquid as nano- and micro-droplets. The prerequisites for the application of IR-MALDI-IM spectrometry as an analytical method are narrow analyte ion signal peaks for a high spectrometer resolution. This can only be achieved by improving the desolvation of ions. One way to full desolvation is to give the cluster ions sufficient time to desolvate. Two methods for achieving this are studied: the implementation of an additional drift tube, as in ESI-IM-spectrometry, and the delayed extraction of the ions. As a result of this optimization procedure, limits of detection between 5 nM and 2.5 μM as well as linear dynamic ranges of 2–3 orders of magnitude were obtained for a number of substances. The ability of this method to analyze simple mixtures is illustrated by the separation of two different surfactant mixtures.
Keywords: Ion mobility spectrometry; IR-MALDI; Laser

Bacteria or their protein and peptide entity enrichment using biomolecules-functionalized magnetic nanoparticles, and analysis by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) is a promising technique to analyze microorganisms. High and low molecular weight proteins like penicillin-binding proteins are responsible for final step synthesis of peptidoglycan biosynthesis; those are the target of lactam antibiotics. In this paper, we synthesized magnetic nanoparticles (mag-NPs) and further modified them with 3-aminopropyltriethoxysilane, and then the β-lactam antibiotic amoxicillin was covalently linked to their surface. β-Lactam group attributes as penicillin binding proteins (PBPs) in bacteria. Staphylococcus aureus and Escherichia coli were used as model bacteria for enrichment based on the β-lactam affinity of magnetic nanoparticles, and then the bacteria were easily separated by an external magnet. Several high molecular weight penicillin binding proteins (PBPs) were detected by MALDI MS containing 104 and 103 colony-forming unit (cfu) per milileter (mL) of S. aureus and E. coli, respectively. In the case of E. coli, higher molecular weight PBPs were observed at 20 to 55 kDa in MALDI mass spectra. However, S. aureus bacteria resulted with femAB operon-based proteins, with molecular weight of 49570.4 Da, by MALDI MS after using amoxicillin functionalized-mag-NPs. The current approach provides an effective bacteria detection and preconcentration method that has high potential in the near future for fast and sensitive diagnosis of pathogenic bacteria infection. Graphical Abstract Schematic for large proteins analysis by MALDI TOF MS (a) mag-NPs and bacterial interaction (b) Penicillin binding proteins trapping by Amox-mag-NPs
Keywords: Amoxicillin; Magnetite nanoparticles; β-Lactam affinity; Bacteria enrichment; Matrix assisted laser desorption/ionization time of flight mass spectrometry

Desomorphine is an opioid misused as “crocodile”, a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.
Keywords: Desomorphine; Crocodile; LC-HR-MS/MS; Metabolism; Human liver preparation; Hepatocyte cell cultures

Cigarette smoke can increase oxidative DNA damage. The main component in cigarette smoke is nicotine. Nicotine is metabolized to cotinine, which can be regarded as a biomarker for measuring exposure to tobacco smoke. A sensitive, simple, and robust method based on on-line solid-phase extraction liquid chromatography with tandem mass spectrometry (on-line SPE LC-MS/MS) has been developed and validated for the simultaneous determination of 8-OHdG and cotinine. The matrix effects of 8-OHdG and cotinine were measured at 97.1 and 91.7 %, with values for CV at 4.4 and 4.2 %, respectively. The limits of detection of 8-OHdG and cotinine were 10.0 and 5.5 pg mL−1, and the limits of quantification were 40.0 and 20.0 pg mL−1, respectively. The total run time was 12 min. We quantified 8-OHdG and cotinine in the urine of 80 male subjects. The results showed the levels of 8-OHdG and cotinine in smokers were significantly higher than that in non-smokers. Furthermore, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide conjugate (defined as total NNAL) are the nitrosation metabolites of nicotine. In this study, urinary levels of 8-OHdG and cotinine were well correlated with urinary levels of total NNAL. This is also the first study to focus on the future risk of oxidative stress from exposure to cigarette smoke based on the relationship between 8-OHdG levels, cotinine levels, and total NNAL concentrations in the urine of humans. Graphical Abstract On-line SPE LC-MS/MS for the simultaneous determination of 8-OHdG and cotinine in human urine
Keywords: 8-OHdG; Cotinine; NNAL; On-line SPE LC-MS/MS; Oxidative stress

Metabolomics profiling of the free and total oxidised lipids in urine by LC-MS/MS: application in patients with rheumatoid arthritis by Junzeng Fu; Johannes C. Schoeman; Amy C. Harms; Herman A. van Wietmarschen; Rob J. Vreeken; Ruud Berger; Bart V. J. Cuppen; Floris P. J. G. Lafeber; Jan van der Greef; Thomas Hankemeier (6307-6319).
Oxidised lipids, covering enzymatic and auto-oxidation-synthesised mediators, are important signalling metabolites in inflammation while also providing a readout for oxidative stress, both of which are prominent physiological processes in a plethora of diseases. Excretion of these metabolites via urine is enhanced through the phase-II conjugation with glucuronic acid, resulting in increased hydrophilicity of these lipid mediators. Here, we developed a bovine liver-β-glucuronidase hydrolysing sample preparation method, using liquid chromatography coupled to tandem mass spectrometry to analyse the total urinary oxidised lipid profile including the prostaglandins, isoprostanes, dihydroxy-fatty acids, hydroxy-fatty acids and the nitro-fatty acids. Our method detected more than 70 oxidised lipids biosynthesised from two non-enzymatic and three enzymatic pathways in urine samples. The total oxidised lipid profiling method was developed and validated for human urine and was demonstrated for urine samples from patients with rheumatoid arthritis. Pro-inflammatory mediators PGF and PGF and oxidative stress markers iPF- IV, 11-HETE and 14-HDoHE were positively associated with improvement of disease activity score. Furthermore, the anti-inflammatory nitro-fatty acids were negatively associated with baseline disease activity. In conclusion, the developed methodology expands the current metabolic profiling of oxidised lipids in urine, and its application will enhance our understanding of the role these bioactive metabolites play in health and disease.
Keywords: LC-MS/MS; Metabolomics; Oxidized lipids; Urine; β-glucuronidase

Altered plasma levels of decanoic acid in colorectal cancer as a new diagnostic biomarker by Sara Crotti; Elisa Agnoletto; Gabriella Cancemi; Valerio Di Marco; Pietro Traldi; Salvatore Pucciarelli; Donato Nitti; Marco Agostini (6321-6328).
Colorectal cancer (CRC) is one of the most common tumors in developed countries. The five-year survival rate decreases depending on how advanced the CRC is when first diagnosed. Screening has been proven to greatly reduce mortality from colorectal cancer, but an ideal screening tool is far from being established. Here, we aimed to discover and validate early CRC biomarkers by means of an untargeted/targeted metabolomic approach. A preliminary untargeted analysis of plasma lipids performed on a small patient cohort (30 plasma samples) revealed some alterations that occurred in the presence of this tumor. In particular, medium-chain fatty acids with between six and twelve carbon atoms (C6–C12) were found to be the lipid class that showed the most marked changes upon the development of CRC. In order to evaluate the utility of this lipid class as diagnostic CRC biomarkers, a further study based on a wider cohort of patients (117 plasma samples) was performed. Using a targeted approach, these fatty acids were quantified in plasma samples by means of fast gas chromatography coupled to a time-of-flight analyzer. Plasma samples from patients with CRCs at different tumor stages were analyzed and compared to those from healthy subjects, ulcerative colitis patients, high-grade dysplasia adenoma patients, and breast cancer patients in order to test the specificity and sensitivity of these possible biomarkers. Results revealed significant differences among the considered groups in terms of their C6, C8, C10, and C12 fatty acid plasma concentrations. In particular, receiver operating characteristic (ROC) curves obtained for the C10 fatty acid gave an area under the curve of 0.8195 along with a sensitivity of 87.8 % and a specificity of 80 %, strongly suggesting that it could be a valuable early diagnostic biomarker of CRC.
Keywords: Colorectal cancer; Biomarkers; Free fatty acids; GC-MS

On the possibility of ephedrine detection: time-resolved fluorescence resonance energy transfer (FRET)-based approach by Antonio Varriale; Vincenzo Manuel Marzullo; Stefano Di Giovanni; Andrea Scala; Alessandro Capo; Adelia Majoli; Angela Pennacchio; Maria Staiano; Sabato D’Auria (6329-6336).
Ephedrine is one of the main precursor compounds used in the illegal production of amphetamines and related drugs. Actually, conventional analytical methods such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and gas chromatography–mass spectrometry (GC–MS) are used for the detection of ephedrine; sadly, these methods require qualified personnel and are time-consuming and expensive. In order to overcome these problems, in recent years, different methods have been developed based on the surface plasmon resonance (SPR) and electrochemical method. In this work, we present a simple, rapid, and effective method to detect the presence of ephedrine in solution, based on competitive fluorescence resonance energy transfer (FRET) assay. The antibody anti-ephedrine and ephedrine derivative were produced and labeled respectively, with two different fluorescent probes (donor and acceptor). The change in FRET signal intensity between donor and acceptor ephedrine compounds gives the possibility of detecting ephedrine traces of at least 0.81 ± 0.04 ppm (LOD). Graphical abstract A new Time-resolved Fluorescence Resonance Energy Transfer (FRET) assay for ephedrine detection
Keywords: Fluorescence resonance energy transfer (FRET); Biosensors; Antibody; Ephedrine detection

Metaproteomic analysis of atmospheric aerosol samples by Fobang Liu; Senchao Lai; Kathrin Reinmuth-Selzle; Jan Frederik Scheel; Janine Fröhlich-Nowoisky; Viviane R. Després; Thorsten Hoffmann; Ulrich Pöschl; Christopher J. Kampf (6337-6348).
Metaproteomic analysis of air particulate matter provides information about the abundance and properties of bioaerosols in the atmosphere and their influence on climate and public health. We developed and applied efficient methods for the extraction and analysis of proteins from glass fiber filter samples of total, coarse, and fine particulate matter. Size exclusion chromatography was applied to remove matrix components, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied for protein fractionation according to molecular size, followed by in-gel digestion and LC-MS/MS analysis of peptides using a hybrid Quadrupole-Orbitrap MS. Maxquant software and the Swiss-Prot database were used for protein identification. In samples collected at a suburban location in central Europe, we found proteins that originated mainly from plants, fungi, and bacteria, which constitute a major fraction of primary biological aerosol particles (PBAP) in the atmosphere. Allergenic proteins were found in coarse and fine particle samples, and indications for atmospheric degradation of proteins were observed. Graphical abstract Workflow for the metaproteomic analysis of atmospheric aerosol samples
Keywords: Metaproteomics; Atmospheric aerosols; Bioanalytical methods; HPLC; Mass spectrometry

Cadaver-detection dogs are a preferred search tool utilised by law enforcement agencies for the purposes of locating victim remains due to their efficiency and minimal disturbance to the crime scene. In Australia, a specific group of these canines are blood-detection dogs, which are trained to detect and locate blood evidence and search potential crime scenes in cases where a cadaver may not be present. Their role sometimes requires searches to be carried out after considerable time has passed since the crime occurred, and this is important for developing effective training protocols. This study aimed to investigate the volatile organic compounds (VOCs) produced from fresh and aged human blood on various surfaces. Solid phase microextraction (SPME) was used to extract VOCs from the headspace of dried blood samples aged and sampled periodically over 12 months from a non-porous (i.e. aluminium) and porous (i.e. cotton) surface. Samples were analysed using comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC-TOFMS). Fresh blood produced distinctively different VOC patterns compared to blood aged longer than 1 week with the overall profile differing between the two surface types, and a large subset of the VOC profile found to be responsible for these differences. When analysing the various functional groups present in the samples, a common pattern between ages and surface types was observed with no specific chemical class dominating the overall profile. The results highlight the importance of evaluating training aids for scent-detection canines to ensure the greatest efficacy during training and subsequently at crime scene searches.
Keywords: Blood; Ageing; Volatile organic compounds (VOCs); Scent-detection canines; Solid phase microextraction (SPME); Comprehensive two-dimensional gas chromatography (GC×GC)

Synthesis and electrochemical detection of a thiazolyl-indole natural product isolated from the nosocomial pathogen Pseudomonas aeruginosa by Alyah Buzid; Eoin Ó Muimhneacháin; F. Jerry Reen; Phyllis E. Hayes; Leticia M. Pardo; Fengjun Shang; Fergal O’Gara; Jonathan Sperry; John H. T. Luong; Jeremy D. Glennon; Gerard P. McGlacken (6361-6367).
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, capable of surviving in a broad range of natural environments and quickly acquiring resistance. It is associated with hospital-acquired infections, particularly in patients with compromised immunity, and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa is also of nosocomial importance on dairy farms and veterinary hospitals, where it is a key morbidity factor in bovine mastitis. P. aeruginosa uses a cell-cell communication system consisting of signalling molecules to coordinate bacterial secondary metabolites, biofilm formation, and virulence. Simple and sensitive methods for the detection of biomolecules as indicators of P. aeruginosa infection would be of great clinical importance. Here, we report the synthesis of the P. aeruginosa natural product, barakacin, which was recently isolated from the bovine ruminal strain ZIO. A simple and sensitive electrochemical method was used for barakacin detection using a boron-doped diamond (BDD) and glassy carbon (GC) electrodes, based on cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The influence of electrolyte pH on the peak potential and peak currents was also investigated. At pH 2.0, the peak current was linearly dependent on barakacin concentration (in the range used, 1–10 μM), with correlation coefficients greater than 0.98 on both electrodes. The detection limit (S/N = 3) on the BDD electrode was 100-fold lower than that obtained on the GC electrode. The optimized method using the BDD electrode was extended to bovine (cow feces) and human (sputum of a CF patient) samples. Spiked barakacin was easily detected in these matrices at a limit of 0.5 and 0.05 μM, respectively. Graphical abstract Electrochemical detection of barakacin
Keywords: Natural product; Biomarker; Pseudomonas aeruginosa ; Electrochemical detection; Bovine; Cystic fibrosis

The aim of the present study was to optimize a protocol for extracting extracellular polymeric substances (EPS) from biofilms on rocky substrata, as the EPS matrix is considered key to understanding the biofilm mode of life. For this purpose, we tested the extraction efficacy of NaOH and H2SO4 at different concentrations, temperatures and times for obtaining EPS from multi-species subaerial biofilms grown on granite blocks under laboratory conditions. Two experimental designs (Box-Behnken design and full factorial design) were used in testing each extractant. The extraction efficiency was determined by analysing the carbohydrate, protein and DNA contents of the extracts obtained. H2SO4 proved unsuitable as an extractant as it caused excessive cell lysis. However, response surface optimization of NaOH-mediated extraction enabled cell lysis to be minimized. Confirmation experiments were performed under the optimal conditions established and a protocol for extracting EPS is proposed, yielding the first quantitative data on EPS extracted from subaerial biofilms developed on rocky substrata. Graphical abstract Development of a method for extracting EPS from subaerial biofilms on rocky substrata
Keywords: Subaerial biofilms; Extracellular polymeric substances (EPS); Response surface methodology; Extraction protocol

This report describes, for the first time, the simultaneous enantioselective determination of proton-pump inhibitors (PPIs-omeprazole, lansoprazole, pantoprazole, and rabeprazole) in environmental water matrices based on solid-phase extraction combined with dispersive liquid-liquid microextraction (SPE-DLLME) and chiral liquid chromatography-tandem mass spectrometry. The optimized results of SPE-DLLME were obtained with PEP-2 column using methanol-acetonitrile (1/1, v/v) as elution solvent, dichloroethane, and acetonitrile as extractant and disperser solvent, respectively. The separation and determination were performed using reversed-phase chromatography on a cellulose chiral stationary phase, a Chiralpak IC (250 mm × 4.6 mm, 5 μm) column, under isocratic conditions at 0.6 mL min−1 flow rate. The analytes were detected in multiple reaction monitoring (MRM) mode by triple quadrupole mass spectrometry. Isotopically labeled internal standards were used to compensate matrix interferences. The method provided enrichment factors of around 500. Under optimal conditions, the mean recoveries for all eight enantiomers from the water samples were 89.3–107.3 % with 0.9–10.3 % intra-day RSD and 2.3–8.1 % inter-day RSD at 20 and 100 ng L−1 levels. Correlation coefficients (r 2) ≥ 0.999 were achieved for all enantiomers within the range of 2–500 μg L−1. The method detection and quantification limits were at very low levels, within the range of 0.67–2.29 ng L−1 and 2.54–8.68 ng L−1, respectively. This method was successfully applied to the determination of the concentrations and enantiomeric fractions of the targeted analytes in wastewater and river water, making it applicable to the assessment of the enantiomeric fate of PPIs in the environment. Graphical Abstract Simultaneous enantioselective determination of representative proton-pump inhibitors in water samples
Keywords: Chiral LC-MS/MS; Solid-phase extraction combined with dispersive liquid-liquid microextraction; Proton-pump inhibitors; Chiralpak IC; Environmental water matrices

Solid phase microextraction and gas chromatography–mass spectrometry methods for residual solvent assessment in seized cocaine and heroin by Pamela Cabarcos; Paloma Herbello-Hermelo; Iván Álvarez-Freire; Antonio Moreda-Piñeiro; María Jesús Tabernero; Ana María Bermejo; Pilar Bermejo-Barrera (6393-6402).
A simple sample pre-treatment method based on solid phase microextraction (SPME) and gas chromatography–mass spectrometry (GC-MS) has been optimized and validated for the assessment of 15 residual solvents (2-propanol, 2-methylpentane, 3-methylpentane, acetone, ethyl acetate, benzene, hexane, methylcyclohexane, methylcyclopentane, m-xylene, propyl acetate, toluene, 1,2,4-trimethylbenzene, dichloromethane, and ethylbenzene) in seized illicit cocaine and heroin. DMSO and DMF as sample diluents were found to offer the best residual solvent transference to the head space for further adsorption onto the SPME fiber, and the developed method therefore showed high sensitivity and analytical recovery. Variables affecting SPME were fully evaluated by applying an experimental design approach. Best conditions were found when using an equilibration time of 5 min at 70 °C and headspace sampling of residual solvents at the same temperature for 15 min. Method validation, performed within the requirements of international guidelines, showed excellent sensitivity, as well as intra- and inter-day precision and accuracy. The proposed methodology was applied to 96 cocaine samples and 14 heroin samples seized in Galicia (northwestern Spain) within 2013 and 2014.
Keywords: Residual solvents; Seized cocaine; Seized heroin; GC-MS; SPME

Quantitative determination and classification of energy drinks using near-infrared spectroscopy by Anita Rácz; Károly Héberger; Marietta Fodor (6403-6411).
Almost a hundred commercially available energy drink samples from Hungary, Slovakia, and Greece were collected for the quantitative determination of their caffeine and sugar content with FT-NIR spectroscopy and high-performance liquid chromatography (HPLC). Calibration models were built with partial least-squares regression (PLSR). An HPLC-UV method was used to measure the reference values for caffeine content, while sugar contents were measured with the Schoorl method. Both the nominal sugar content (as indicated on the cans) and the measured sugar concentration were used as references. Although the Schoorl method has larger error and bias, appropriate models could be developed using both references. The validation of the models was based on sevenfold cross-validation and external validation. FT-NIR analysis is a good candidate to replace the HPLC-UV method, because it is much cheaper than any chromatographic method, while it is also more time-efficient. The combination of FT-NIR with multidimensional chemometric techniques like PLSR can be a good option for the detection of low caffeine concentrations in energy drinks. Moreover, three types of energy drinks that contain (i) taurine, (ii) arginine, and (iii) none of these two components were classified correctly using principal component analysis and linear discriminant analysis. Such classifications are important for the detection of adulterated samples and for quality control, as well. In this case, more than a hundred samples were used for the evaluation. The classification was validated with cross-validation and several randomization tests (X-scrambling). Graphical Abstract The way of energy drinks from cans to appropriate chemometric models
Keywords: Energy drink; Caffeine; Sugar; Classification; PCA; LDA; FT-NIR

The program HypCal has been developed to provide a means for the simultaneous determination, from data obtained by isothermal titration calorimetry, of both standard enthalpy of reaction and binding constant values. The chemical system is defined in terms of species of given stoichiometry rather than in terms of binding models (e.g., independent or cooperative). The program does not impose any limits on the complexity of the chemical systems that can be treated, including competing ligand systems. Many titration curves may be treated simultaneously. HypCal can also be used as a simulation program when designing experiments. The use of the program is illustrated with data obtained with nicotinic acid (niacin, pyridine-3 carboxylic acid). Preliminary experiments were used to establish the rather different titration conditions for the two sets of titration curves that are needed to determine the parameters for protonation of the carboxylate and amine groups.
Keywords: Calorimetric titration; Standard enthalpy of reaction; Binding constant; Experiment design

Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3). Then, the protein was refolded by stepwise urea gradient dialysis and biotinylated in vitro by E. coli biotin ligase (BirA). Subsequently, the scFv-BAD protein was purified from the biotinylated system with high yield (66.7 mg L−1) and confirmed by SDS-PAGE and Western blot. Based on the biotinylated scFv-BAD, a sensitive and broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for detection of OPs was developed. The cross-reactivity (CR) studies demonstrated that the ciELISA described here exhibited the broadest detection spectrum for OPs up to now, and 30 OPs could be determined with 50 % inhibition value (IC50) values ranging from 19.4 to 515.2 ng mL−1. Moreover, the developed ciELISA was used for the recovery study of the spiked samples and showed satisfactory recoveries. Graphical Abstract Schematic diagram of the development of biotinylated broad-specificity single-chain variable fragment antibody-based immunoassay for organophosphorus pesticides
Keywords: Phage display; ScFv; Refolding; Biotinylation; Organophosphorus pesticides

Analytics of nonpeptidic erythropoietin mimetic agents in sports drug testing employing high-resolution/high-accuracy liquid chromatography-mass spectrometry by Matthias Vogel; Josef Dib; Laura Tretzel; Thomas Piper; Andreas Thomas; Wilhelm Schänzer; Mario Thevis (6431-6442).
Since its release as anti-anemic drug, recombinant erythropoietin (rEPO) gradually entered the illicit way to sports competitions as endurance-enhancing drug. Novel modifications biopharmaceutically introduced into the rEPO molecule in the form of carbohydrate or polyethylene glycol moieties made robust and sensitive test methods vital to doping controls in order to provide the necessary tools enabling the conviction of dishonest athletes. Modern protein analysis by means of gel electrophoretic separation and western blotting represents the status quo in rEPO anti-doping analysis. However, new therapeutically promising erythropoietin receptor activating compounds have been developed that exhibit cytokine hormone-mimicking properties but lack any protein structure. Progression to evade parenteral application and substitute for rEPO by low molecular mass and orally available compounds is still one of the major objectives in pharmaceutical research. In this approach, four promising in-house synthesized nonpeptidic erythropoietin mimetic agents, namely compound 129, compound 163, A1B10C1, and A5B10C4 were thoroughly evaluated by employing high-resolution/high-accuracy liquid chromatography tandem mass spectrometry experiments. Characteristic product ions were determined supporting the identification of these drugs and putative metabolites as well as related compounds in future doping controls. Test methods employing direct urine injection and receptor affinity purification strategies were assessed, which demonstrated that EPO receptor purification is of limited utility for nonpeptidic EPOR agonists while direct urine injection allowed for comprehensive method characterization. Thereby, achieved limits of detection were 1 ng/mL for compounds 129/163 and 5 ng/mL for A1B10C1/A5B10C4.
Keywords: Nonpeptidic erythropoietin mimetic agents; EPO; Liquid chromatography tandem mass spectrometry; Cytokine receptor agonists; Sports drug testing

Hybridoma as a specific, sensitive, and ready to use sensing element: a rapid fluorescence assay for detection of Vibrio cholerae O1 by Parichehr Zamani; Reza H. Sajedi; Saman Hosseinkhani; Mehdi Zeinoddini (6443-6451).
Over the last decade, isolation and purification of monoclonal antibodies, for diagnostic analysis, have been carried out using the hybridoma expression system. The present study describes a novel example of a detection system using hybridoma cells containing antibody against O1 antigen directly for V. cholerae diagnosis, which is a major health problem in many parts of the world, especially in developing countries. This method has advantages such as simplicity, ease of process, and it does not require manipulation of hybridoma cell. For this approach, an efficient amount of fluorescence calcium indicator, fura 2-AM, was utilized, which emitted light when the intracellular calcium concentration increased as result of antigen binding to specific antibody. More reliable results are obtained via this method and it is considerably faster than other methods, which has the response time of less than 45 s for detection of V. Cholerae O1. Also, the limit of detection was computed to be 50 CFU/mL (<13 CFU per assay). In addition, no significant responses were observed in the presence of other bacteria with specific hybridoma or other cell lines exposed to V. cholerae O1. Furthermore, this method was successfully applied to V. cholerae O1 detection in spiked environmental samples, including water and stool samples without any pretreatment. All results reveal that hybridoma cells can provide a valuable, simple, and ready to use tool for rapid detection of other pathogenic bacteria, toxins, and analytes.
Keywords: Vibrio cholerae O1; Hybridoma; Fura 2-AM; Calcium increasing; Fluorescence emission; Monoclonal antibody

Direct and precise length measurement of single, stretched DNA fragments by dynamic molecular combing and STED nanoscopy by Namdoo Kim; Hyung Jun Kim; Younggyu Kim; Kyung Suk Min; Seong Keun Kim (6453-6459).
A combination of DNA stretching method and super-resolution nanoscopy allows an accurate and precise measurement of the length of DNA fragments ranging widely in size from 117 to 23,130 bp. BstEII- and HindIII-treated λDNA fragments were stained with an intercalating dye and then linearly stretched on a coverslip by dynamic molecular combing. The image of individual DNA fragments was obtained by stimulated emission depletion nanoscopy. For DNA fragments longer than ∼1000 bp, the measured lengths of DNA fragments were consistently within ∼0.5 to 1.0 % of the reference values, raising the possibility of this method in a wide range of applications including facile detection for copy number variations and trinucleotide repeat disorder.
Keywords: DNA; Dynamic molecular combing; Length measurement; STED nanoscopy

Simultaneous quantification of 11 cannabinoids and metabolites in human urine by liquid chromatography tandem mass spectrometry using WAX-S tips by Maria Andersson; Karl B. Scheidweiler; Cristina Sempio; Allan J. Barnes; Marilyn A. Huestis (6461-6471).
A comprehensive cannabinoid urine quantification method may improve clinical and forensic result interpretation and is necessary to support our clinical research. A liquid chromatography tandem mass spectrometry quantification method for ∆9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), ∆9-tetrahydrocannabinolic acid (THCAA), cannabinol (CBN), cannabidiol (CBD), cannabigerol (CBG), ∆9-tetrahydrocannabivarin (THCV), 11-nor-9-carboxy-THCV (THCVCOOH), THC-glucuronide (THC-gluc), and THCCOOH-glucuronide (THCCOOH-gluc) in urine was developed and validated according to the Scientific Working Group on Toxicology guidelines. Sample preparation consisted of disposable pipette extraction (WAX-S) of 200 μL urine. Separation was achieved on a Kinetex C18 column using gradient elution with flow rate 0.5 mL/min, mobile phase A (10 mM ammonium acetate in water), and mobile phase B (15 % methanol in acetonitrile). Total run time was 14 min. Analytes were monitored in both positive and negative ionization modes by scheduled multiple reaction monitoring. Linear ranges were 0.5–100 μg/L for THC and THCCOOH; 0.5–50 μg/L for 11-OH-THC, CBD, CBN, THCAA, and THC-gluc; 1–100 μg/L for CBG, THCV, and THCVCOOH; and 5–500 μg/L for THCCOOH-gluc (R 2 > 0.99). Analytical biases were 88.3–113.7 %, imprecisions 3.3–14.3 %, extraction efficiencies 42.4–81.5 %, and matrix effect −10 to 32.5 %. We developed and validated a comprehensive, simple, and rapid LC-MS/MS cannabinoid urine method for quantification of 11 cannabinoids and metabolites. This method is being used in a controlled cannabis administration study, investigating urine cannabinoid markers documenting recent cannabis use, chronic frequent smoking, or route of drug administration and potentially improving urine cannabinoid result interpretation.
Keywords: Cannabinoid; Urine; LC-MS/MS; DPX; THC

Nicotine (Nic) distribution in human fluids and tissues has a deleterious effect on human health. In addition to its poisoning profile, Nic may contribute to the particular impact of smoking on human reproduction. Although present in seminal fluid, still nobody knows whether nicotine is available in sperm or not. Herein, we developed and validated a new bioanalytical method, for simultaneous determination of Nic, cotinine (Cot), and nicotine N′-oxide (Nox) in human plasma, semen, and sperm by LC-ESI-orbitrap-MS. Blood and semen samples were collected from 12 healthy smoking volunteers in this study. Sperm bodies were then separated quantitatively from 1 mL of semen samples by centrifugation. The developed method was fully validated for plasma following European and American guidelines for bioanalytical method validation, and partial validation was applied to semen analysis. Plasma, semen, and sperm samples were treated by trichloroacetic acid solution for protein direct precipitation in single extraction step. The established calibration range for Nic and Nox in plasma and semen was linear between 5 and 250 ng/mL, and for Cot between 10 and 500 ng/mL. Nic and Cot were detected in human sperm at concentrations as high as in plasma. In addition, Nox was present in semen and sperm but not in plasma. Graphical abstract Nicotine correlation between plasma and semen a; Nicotine correlation between semen and sperm c; Cotinine correlation between plasma and semen b; Cotinine correlation between semen and sperm d.
Keywords: Cotinine; Nicotine; Blood; Sperm; Semen

Native concentrations of α-ionone, β-ionone, and β-damascenone were studied in various authentic and commercial wines. In addition, the enantiomeric distribution of α-ionone was determined and its merits as a potential marker for aroma adulteration in wine were discussed. For extraction of volatiles, headspace solid-phase microextraction (HS-SPME) was applied, followed by heart-cut multidimensional gas chromatography coupled to tandem mass spectrometric detection for trace-level analysis. The enantioselective analysis of α-ionone was achieved with octakis(2,3-di-O-pentyl-6-O-methyl)-γ-cyclodextrin as the chiral selector in the separation column for gas chromatography (GC). In all the authentic wines studied, α-ionone showed a high enantiomeric ratio in favor of the (R)-enantiomer. Since an illegal addition of α-ionone in a racemic form changes the enantiomeric ratio, this ratio may serve as an adulteration marker. Concentrations varied between
Keywords: Heart-cut multidimensional gas chromatography; Tandem mass spectrometry; C13-Norisoprenoids; Adulteration; Authenticity; Enantiodifferentiation; Odor threshold determination

Plasma lipidomics reveals potential lipid markers of major depressive disorder by Xinyu Liu; Jia Li; Peng Zheng; Xinjie Zhao; Chanjuan Zhou; Chunxiu Hu; Xiaoli Hou; Haiyang Wang; Peng Xie; Guowang Xu (6497-6507).
Major depressive disorder (MDD) is a grave debilitating mental disease with a high incidence and severely impairs quality of life. Therefore, its physiopathological basis study and diagnostic biomarker discovery are extremely valuable. In this study, a non-targeted lipidomics strategy using ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to reveal differential lipids between MDD (n = 60) and healthy controls (HCs, n = 60). Validation of changed lipid species was performed in an independent batch including 75 MDD and 52 HC using the same lipidomic method. Pronouncedly changed lipid species in MDD were discovered, which mainly were lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), 1-O-alkyl-2-acyl-PE (PE O), 1-O-alkyl-2-acyl-PC (PC O), sphingomyelin (SM), diacylglycerol (DG), and triacylglycerol (TG). Among these lipid species, LPC, LPE, PC, PE, PI, TG, etc. remarkably increased in MDD and showed pronounced positive relationships with depression severity, while 1-O-alkyl-2-acyl-PE and SM with odd summed carbon number significantly decreased in MDD and demonstrated negative relationships with depression severity. A combinational lipid panel including LPE 20:4, PC 34:1, PI 40:4, SM 39:1, 2, and TG 44:2 was defined as potential diagnostic biomarker with a good sensitivity and specificity for distinguishing MDD from HCs. Our study brings insights into lipid metabolism disorder in MDD and provides a specific potential biomarker for MDD diagnose.
Keywords: Major depressive disorder; Non-targeted lipidomics; Diagnostic biomarker; UPLC-MS

Erratum to: Multiplex detection of food allergens and gluten by Chung Y. Cho; William Nowatzke; Kerry Oliver; Eric A. E. Garber (6509-6510).