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Analytical and Bioanalytical Chemistry (v.403, #9)

Master’s degree: from analytical science to process analytical technology by Jérôme Randon (pp. 2459-2460).

is Professor of Analytical Chemistry at the Analytical Science Institute at the University of Lyon. He is the team leader of the research group in separation science (monolithic columns, lab on chip, LCxLC). Jérôme Randon is also deeply involved in education in analytical chemistry: former director of the PRACTICE, director of the master degree in Analytical Sciences http://master-analyse-controle.univ-lyon1.fr/ , and member of the board of Teaching and Education Division of the French Society of Chemistry.

Master’s degree: from analytical science to process analytical technology by Jérôme Randon (pp. 2459-2460).

Standard addition challenge by Olaf Rienitz; Anna-Lisa Hauswaldt; Reinhard Jährling (pp. 2461-2462).

Solution to the Bavarois challenge by Hervé This (pp. 2463-2463).

Thomas O. Metz (Ed.): Metabolic profiling. Methods and protocols by Eric Chun Yong Chan (pp. 2465-2466).

Khalid Shah (Ed.): Molecular imaging. Methods and protocols by Elisa Michelini (pp. 2467-2468).

Analytical Challenges in Environmental and Geosciences by Christian Zwiener (pp. 2469-2470).

is Professor of Environmental Analytical Chemistry at the Eberhard Karls University, Tübingen, Germany. His main interest is in the occurrence and fate of organic micropollutants and their transformation products in water treatment and in the aquatic environment, with a focus on method development and application of advanced mass-spectrometric techniques.

Analytical Challenges in Environmental and Geosciences by Christian Zwiener (pp. 2469-2470).

Current challenges in compound-specific stable isotope analysis of environmental organic contaminants by Martin Elsner; Maik A. Jochmann; Thomas B. Hofstetter; Daniel Hunkeler; Anat Bernstein; Torsten C. Schmidt; Arndt Schimmelmann (pp. 2471-2491).

Compound-specific stable-isotope analysis (CSIA) has greatly facilitated assessment of sources and transformation processes of organic pollutants. Multielement isotope analysis is one of the most promising applications of CSIA because it even enables distinction of different transformation pathways. This review introduces the essential features of continuous-flow isotope-ratio mass spectrometry (IRMS) and highlights current challenges in environmental analysis as exemplified for the isotopes of nitrogen, hydrogen, chlorine, and oxygen. Strategies and recent advances to enable isotopic measurements of polar contaminants, for example pesticides or pharmaceuticals, are discussed with special emphasis on possible solutions for analysis of low concentrations of contaminants in environmental matrices. Finally, we discuss different levels of calibration and referencing and point out the urgent need for compound-specific isotope standards for gas chromatography–isotope-ratio mass spectrometry (GC–IRMS) of organic pollutants. Figure Compound-specific isotope analysis of environmental contaminants: chromatographic separation is followed by online conversion to a suitable measurement gas (M) and subsequent isotope ratio mass spectrometry. Current challenges in the field concern the analysis of multiple elements (C, H, N, O, Cl) in polar compounds, at low concentrations and in the presence of matrix interferences. An urgent need exists for contaminant-specific reference materials.

Keywords: Gas chromatography-isotope-ratio mass spectrometry; Isotope fractionation; Groundwater contamination; Pollution source; Transformation pathways; Isotope standard

Is nontarget screening of emerging contaminants by LC-HRMS successful? A plea for compound libraries and computer tools by Marco Zedda; Christian Zwiener (pp. 2493-2502).

This review focuses on the possibilities and limits of nontarget screening of emerging contaminants, with emphasis on recent applications and developments in data evaluation and compound identification by liquid chromatography–high-resolution mass spectrometry (HRMS). The general workflow includes determination of the elemental composition from accurate mass, a further search for the molecular formula in compound libraries or general chemical databases, and a ranking of the proposed structures using further information, e.g., from mass spectrometry (MS) fragmentation and retention times. The success of nontarget screening is in some way limited to the preselection of relevant compounds from a large data set. Recently developed approaches show that statistical analysis in combination with suspect and nontarget screening are useful methods to preselect relevant compounds. Currently, the unequivocal identification of unknowns still requires information from an authentic standard which has to be measured or is already available in user-defined MS/MS reference databases or libraries containing HRMS spectral information and retention times. In this context, we discuss the advantages and future needs of publicly available MS and MS/MS reference databases and libraries which have mostly been created for the metabolomic field. A big step forward has been achieved with computer-based tools when no MS library or MS database entry is found for a compound. The numerous search results from a large chemical database can be condensed to only a few by in silico fragmentation. This has been demonstrated for selected compounds and metabolites in recent publications. Still, only very few compounds have been identified or tentatively identified in environmental samples by nontarget screening. The availability of comprehensive MS libraries with a focus on environmental contaminants would tremendously improve the situation.

Keywords: Liquid chromatography–high resolution mass spectrometry; Accurate mass; Molecular formula; Emerging contaminant; Nontarget analysis; Database; Computer tools; In silico fragmentation

Is nontarget screening of emerging contaminants by LC-HRMS successful? A plea for compound libraries and computer tools by Marco Zedda; Christian Zwiener (pp. 2493-2502).

Keywords: Liquid chromatography–high resolution mass spectrometry; Accurate mass; Molecular formula; Emerging contaminant; Nontarget analysis; Database; Computer tools; In silico fragmentation

Is nontarget screening of emerging contaminants by LC-HRMS successful? A plea for compound libraries and computer tools by Marco Zedda; Christian Zwiener (pp. 2493-2502).

Keywords: Liquid chromatography–high resolution mass spectrometry; Accurate mass; Molecular formula; Emerging contaminant; Nontarget analysis; Database; Computer tools; In silico fragmentation

Artificial sweeteners—a recently recognized class of emerging environmental contaminants: a review by Frank T. Lange; Marco Scheurer; Heinz-J. Brauch (pp. 2503-2518).

An overview is given of existing trace analytical methods for the determination of seven popular artificial sweeteners [acesulfame (ACE), aspartame, cyclamate (CYC), neotame, neohesperidine dihydrochalcone, saccharin (SAC), and sucralose (SUC)] from aqueous environmental samples. Liquid chromatography–electrospray ionization tandem mass spectrometry and liquid chromatography–electrospray ionization high-resolution mass spectrometry are the methods most widely applied, either directly or after solid-phase extraction. Limits of detection and limits of quantification down to the low nanogram per liter range can be achieved. ACE, CYC, SAC, and SUC were detected in wastewater treatment plants in high microgram per liter concentrations. Per capita loads of individual sweeteners can vary within a wide range depending on their use in different countries. Whereas CYC and SAC are usually degraded by more than 90 % during wastewater treatment, ACE and SUC pass through wastewater treatment plants mainly unchanged. This suggests their use as virtually perfect markers for the study of the impact of wastewater on source waters and drinking waters. In finished water of drinking water treatment plants using surface-water-influenced source water, ACE and SUC were detected in concentrations up to 7 and 2.4 μg/L, respectively. ACE was identified as a precursor of oxidation byproducts during ozonation, resulting in an aldehyde intermediate and acetic acid. Although the concentrations of ACE and SUC are among the highest measured for anthropogenic trace pollutants found in surface water, groundwater, and drinking water, the levels are at least three orders of magnitude lower than organoleptic threshold values. However, ecotoxicology studies are scarce and have focused on SUC. Thus, further research is needed both on identification of transformation products and on the ecotoxicological impact of artificial sweeteners and their transformation products.

Keywords: Artificial sweeteners; Acesulfame; Sucralose; Wastewater; Surface water; Groundwater; Drinking water; Ozonation

Artificial sweeteners—a recently recognized class of emerging environmental contaminants: a review by Frank T. Lange; Marco Scheurer; Heinz-J. Brauch (pp. 2503-2518).

Keywords: Artificial sweeteners; Acesulfame; Sucralose; Wastewater; Surface water; Groundwater; Drinking water; Ozonation

Artificial sweeteners—a recently recognized class of emerging environmental contaminants: a review by Frank T. Lange; Marco Scheurer; Heinz-J. Brauch (pp. 2503-2518).

Keywords: Artificial sweeteners; Acesulfame; Sucralose; Wastewater; Surface water; Groundwater; Drinking water; Ozonation

Highly sensitive and specific determination of mercury(II) ion in water, food and cosmetic samples with an ELISA based on a novel monoclonal antibody by Yuzhen Wang; Hong Yang; Michael Pschenitza; Reinhard Niessner; Yuan Li; Dietmar Knopp; Anping Deng (pp. 2519-2528).

Mercury is one of the most toxic heavy metals present in the environment. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the determination of Hg²⁺ was developed. A new bifunctional ligand, 6-mercaptonicotinic acid (MNA), which contains a pyridine ring bearing a carboxylic group and a mercapto group, was selected for the preparation of immunogen. After immunization of mice and performing the hybridoma technique, the obtained mAb was characterized for its binding affinity and selectivity for Hg²⁺. Based on this novel mAb, an ELISA was established. At optimal experimental conditions, the standard curve of the ELISA for Hg²⁺ was constructed in concentration range of 0.1–100 ng mL⁻¹. The values of IC₅₀ and LOD of the assay were found to be 1.12 and 0.08 ng mL⁻¹. The cross-reactivity was lower than 2 % with MNA, CH₃Hg, and CH₃Hg-MNA and was 11.5 % and 4.4 % for Hg⁺ and Au³⁺, respectively. No cross-reactivity was found with other metal ions such as Cu²⁺, Sn²⁺, Ni²⁺, Mn²⁺, Pb²⁺, Zn²⁺, Cd²⁺, Fe²⁺, Co²⁺, Mg²⁺, Ca²⁺, and anions such as Cl⁻, NO ₃ ⁻ , NO ₂ ⁻ , HCO ₃ ⁻ , F⁻, and SO ₄ ²⁻ , indicating that the assay displays not only high sensitivity but also high selectivity. Different kinds of samples including water, milk, green vegetable, kelp, facial cleanser, and night cream were spiked with Hg²⁺ and the extracts were analyzed by ELISA. Acceptable recovery rates of 80.0–113.0 % and coefficients of variation of 1.9–18.6 % were obtained. A linear relationship between ELISA and cold-vapor atomic fluorescence spectroscopy (CV-AFS) as indicated by a correlation coefficient of 0.97 for liquid samples (water samples) and 0.98 for other samples was obtained. The proposed mAb-based ELISA provides a feasible analytical method for highly sensitive and specific, fast, simple, and accurate determination of uncomplexed trace Hg²⁺ in environmental and food samples.

Keywords: Mercury; Enzyme-linked immunosorbent assay; Monoclonal antibody; Bifunctional ligand; 6-Mercaptonicotinic acid

Development of antibody-labelled superparamagnetic nanoparticles for the visualisation of benzo[a]pyrene in porous media with magnetic resonance imaging by Martin Rieger; Gabriele E. Schaumann; Yamuna Kunhi Mouvenchery; Reinhard Niessner; Michael Seidel; Thomas Baumann (pp. 2529-2540).

Biogeochemical interfaces in soil are dynamic in the spatial and temporal domain and require advanced visualisation and quantification tools to link in vitro experiments with natural systems. This study presents the development, characterization and application of functional nanoparticles coated with monoclonal antibodies to visualise the distribution of benzo[a]pyrene in porous media using magnetic resonance imaging. The labelled particles are 450 nm in diameter and interact with benzo[a]pyrene covalently bound to silanized silica gel. They did not bind to benzo[a]pyrene adsorbed to plain silica gel. Although unspecific filtration was low, washing steps are required for visualisation. The ability to visualise benzo[a]pyrene is inversely correlated to the heterogeneity of the soil materials. There are access restrictions to narrow pore spaces which allow the visualisation of only those pathways which are also accessible to bacteria and hydrocolloids. The production of the particles is applicable to other antibodies which extends the range of potential target contaminants.

Keywords: PAH; Magnetic resonance imaging (MRI); MRI label; NMR relaxometry; Anti-B[a]P antibody; Biogeochemical interface

Occurrence of residual water within disk-based solid-phase extraction and its effect on GC-MS measurement of organic extracts of environmental samples by Christine Erger; Peter Balsaa; Friedrich Werres; Torsten C. Schmidt (pp. 2541-2552).

Solid-phase extraction (SPE) is a widespread and powerful sample preparation technique in many analytical areas. Many of the used methods reduce residual water during sample preparation by drying the phase material. Despite the importance of this step, hardly any study deals specifically with the drying process, and if so, only few aspects are mentioned. The present study is the first systematic investigation of the drying process using SPE disks, including the influence of process parameters on the amount of residual water and its consequences for subsequent elution and gas chromatography–mass spectrometry (GC-MS) analysis. The following points were investigated in detail: (1) the change of pressure and volume flow during the drying process, (2) the remaining amount of water at different drying times for different SPE materials, (3) the influence of suspended particulate matter on the drying process and (4) the effects of the residual water on the elution step by using different organic solvents. The study shows that the volume of residual water in the SPE disk is affected by the fixation of the sorbent, the phase material, the amount of sorbent, the pumping settings and the duration of the drying process. Furthermore, systematic investigations demonstrate the influence of residual water on the GC-MS analysis and show analytical interferences only for a few of the investigated analytes. All results suggest that more problems in SPE GC-MS methods are caused by residual water than previously assumed.

Keywords: Solid-phase extraction disk; Drying; Residual water; GC-MS; Priority pollutants

A new analytical approach for the comprehensive characterization of polar xenobiotic organic compounds downgradient of old municipal solid waste (MSW) landfills by A. Preiss; E. Berger-Preiss; M. Elend; S. Gerling; S. Kühn; S. Schuchardt (pp. 2553-2561).

Groundwater samples collected downgradient from a former municipal solid waste landfill near Berlin, Germany, were analyzed by GC-MS, HPLC-MS, and HPLC-NMR hyphenated techniques to comprehensively characterize the xenobiotic organic compounds (XOCs). The focus thereby was on the detection and identification of the polar XOCs which were analyzed in the extract obtained after separation of the unpolar components by pre-extraction. HPLC-NMR and HPLC-MS runs were used to identify polar XOCs on-line or to obtain preliminary structure information on the other XOCs. These compounds were then isolated by HPLC fractionation and their structures elucidated by off-line NMR and MS investigations. A variety of polar XOCs, products of the dye industry, degradation products of polyethylene glycol, and some heterocyclic compounds could be identified. Furthermore, a semi-quantitative estimation of the identified polar compounds is given. Figure Analytical approach for a comprehensive characterization of XOCs downgradient of old MSW landfills

Keywords: MSW landfill; Leachate; Polar XOCs; Groundwater contamination; Non-target analysis; GC-MS; HPLC-NMR; HPLC-MS; ¹H NMR; 2D NMR; MSⁿ

Improving detection power in trace analysis using wavelet transform by Simon Prikler; Jürgen W. Einax (pp. 2563-2567).

Environmental analysis most often is trace analysis. Therefore, the concentrations are commonly in the lower working range near the limit of detection of the corresponding analytical method. However, whenever the instrument’s analytical noise is too large, it dominates the signal curves and analytes cannot be detected anymore. Furthermore, the evaluation of peaks with defined baselines is hindered very much. One possibility for de-noising is wavelet transform which is presented in this work. Different wavelet functions are applied and Symlet4 is suggested as the most powerful for analytical peaks that resemble Gaussian distribution curves, as it improves limits of detection by factors 6 to 7. The comparison of different wavelet functions has been carried out for two modern analytical scopes. At first, chromatograms are de-noised for the speciation of four arsenic compounds via the coupling of HPLC and ICP-MS. Secondly, the determination of cadmium is shown by HR-CS AAS, which is one of the most recently developed devices in atomic absorption spectrometry and allows the registration of three-dimensional spectra in order to investigate the spectral vicinity of analytical lines. On the basis of these investigations, we recommend using wavelet transform with Symlet4 for all analytical techniques which are resulting in similar signal curves.

Keywords: Atomic absorption spectrometry; Chromatography; Inductively coupled plasma; Signal-to-noise ratio; Wavelet transform

Improving detection power in trace analysis using wavelet transform by Simon Prikler; Jürgen W. Einax (pp. 2563-2567).

Keywords: Atomic absorption spectrometry; Chromatography; Inductively coupled plasma; Signal-to-noise ratio; Wavelet transform

Improving detection power in trace analysis using wavelet transform by Simon Prikler; Jürgen W. Einax (pp. 2563-2567).

Keywords: Atomic absorption spectrometry; Chromatography; Inductively coupled plasma; Signal-to-noise ratio; Wavelet transform

Evaluation of hair roots for detection of methamphetamine and 3,4-methylenedioxymethamphetamine abuse by use of an HPLC–chemiluminescence method by Mitsuhiro Wada; Yuko Ochi; Kumi Nogami; Rie Ikeda; Naotaka Kuroda; Kenichiro Nakashima (pp. 2569-2576).

We describe the use of hair roots as a matrix for detection of methamphetamine (MP) and 3,4-methylenedioxymethamphetamine (MDMA) abuse. The concentration of drugs was determined in rat hair roots, hair shafts, and plasma after a single administration of MP or MDMA, by use of an HPLC–peroxyoxalate chemiluminescence (PO-CL) method involving column switching. Plasma and hair roots and shafts were collected from male Wistar rats before and after administration of MP (10 mg kg⁻¹, i.p.). In addition, the roots and shafts of pigmented and non-pigmented hair of male Lister hooded rats were collected after administration of MDMA (10 mg kg⁻¹, i.p.). The concentrations of MP and MDMA in plasma and hair were determined by use of the HPLC–PO-CL method, with satisfactory sensitivity and reproducibility. The concentration of MP in hair roots 1–14 days after administration ranged from 0.038 to 0.115 ng mg⁻¹ (n = 3). By use of the HPLC–PO-CL method, MP could be detected in hair roots for longer (up to 14 days) than it could be detected in conventional biological specimens, for example plasma (~1 day), and MDMA was detected in hair roots from 1 to 10 days after administration. The AUC_1–10 (ng day mg⁻¹) for MDMA in roots of non-pigmented and pigmented hair was comparable (4.93 ± 2.09 vs. 6.67 ± 1.28, n = 3), whereas AUC_1–14 for hair shafts differed significantly (1.86 ± 0.93 vs. 4.58 ± 0.63, P < 0.05, n = 3). The window for detecting MP (or MDMA) in hair roots under our conditions was 1–14 (or 1–10) days.

Keywords: Hair root; Detection window; Methamphetamine; MDMA; HPLC–peroxyoxalate chemiluminescence detection

A quick, convenient and economical method for the reliable determination of methylglyoxal in millimolar concentrations: the N-acetyl-l-cysteine assay by Rebekka Wild; Lezanne Ooi; Velandai Srikanth; Gerald Münch (pp. 2577-2581).

The determination of methylglyoxal (MG) concentrations in vivo is gaining increasing importance as high levels of MG are linked to various health impairments including complications of diabetes. In order to standardize the measurements of MG in body fluids, it is necessary to precisely determine the concentration of MG stock solutions used as analytical standards. The “gold standard” method for the determination of MG concentration in the millimolar range is an enzyme-catalyzed endpoint assay based on the glyoxalase I catalyzed formation of S-lactoylglutathione. However, as this assay used purified glyoxalase I enzyme, it is quite expensive. Another method uses a derivation reaction with 2,4-dinitrophenylhydrazine, but this substance is explosive and needs special handling and storage. In addition, precipitation of the product methylglyoxal-bis-2,4-dinitrophenylhydrozone during the reaction limits the reliability of this method. In this study, we have evaluated a new method of MG determination based on the previously published fast reaction between MG and N-acetyl-l-cysteine at room temperature which yields an easily detectable condensation product, N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine. When comparing these three different assays for the measurement of MG concentrations, we find that the N-acetyl-l-cysteine assay is the most favorable, providing an economical and robust assay without the need for the use of hazardous or expensive reagents. Figure Comparison of the three different spectrophotometrical methods of MG determination

Keywords: Methylglyoxal; Glyoxalase I; 2,4-Dinitrophenylhydrazine; N-acetyl-l-cysteine; Spectrophotometric measurements

Determination of non-steroidal anti-inflammatory drugs in urine by the combination of stir membrane liquid–liquid–liquid microextraction and liquid chromatography by S. Riaño; M. C. Alcudia-León; R. Lucena; S. Cárdenas; M. Valcárcel (pp. 2583-2589).

A stir membrane liquid phase microextraction procedure working under the three-phase mode is proposed for the first time for the determination of six anti-inflammatory drugs in human urine. The target compounds are isolated and preconcentrated using a special device that integrates the extractant and the stirring element. An alkaline aqueous solution is used as extractant phase while 1-octanol is selected as supported liquid membrane solvent. After the extraction, all the analytes are determined by liquid chromatography (LC) with ultraviolet detection (UV). The analytical method is optimized considering the main involved variables (e.g., pH of donor and acceptor phases, extraction time, stirring rate) and the results indicate that the determination of anti-inflammatory drugs at therapeutic and toxic levels is completely feasible. The limits of detection are in the range from 12.6 (indomethacin) to 30.7 μg/L (naproxen). The repeatability of the method, expressed as relative standard deviation (RSD, n = 5) varies between 3.4 % (flurbiprofen) and 5.7 % (ketoprofen), while the enrichment factors are in the range from 35.0 (naproxen) to 72.5 (indomethacin).

Keywords: Stir membrane liquid–liquid microextraction; Non steroidal anti-inflammatory drugs; Urine; Liquid chromatography

Rapid reagentless quantification of alginate biosynthesis in Pseudomonas fluorescens bacteria mutants using FT-IR spectroscopy coupled to multivariate partial least squares regression by Elon Correa; Håvard Sletta; David I. Ellis; Sunniva Hoel; Helga Ertesvåg; Trond E. Ellingsen; Svein Valla; Royston Goodacre (pp. 2591-2599).

Alginate is an important medical and commercial product and currently is isolated from seaweeds. Certain microorganisms also produce alginate and these polymers have the potential to replace seaweed alginates in some applications, mainly because such production will allow much better and more reproducible control of critical qualitative polymer properties. The research conducted here presents the development of a new approach to this problem by analysing a transposon insertion mutant library constructed in an alginate-producing derivative of the Pseudomonas fluorescens strain SBW25. The procedure is based on the non-destructive and reagent-free method of Fourier transform infrared (FT-IR) spectroscopy which is used to generate a complex biochemical infrared fingerprint of the medium after bacterial growth. First, we investigate the potential differences caused by the growth media fructose and glycerol on the bacterial phenotype and alginate synthesis in 193 selected P. fluorescens mutants and show that clear phenotypic differences are observed in the infrared fingerprints. In order to quantify the level of the alginate we also report the construction and interpretation of multivariate partial least squares regression models which were able to quantify alginate levels successfully with typical normalized root-mean-square error in predictions of only approximately 14 %. We have demonstrated that this high-throughput approach can be implemented in alginate screens and we believe that this FT-IR spectroscopic methodology, when combined with the most appropriate chemometrics, could easily be modified for the quantification of other valuable microbial products and play a valuable screening role for synthetic biology. Figure PLS regression coefficients (top) and FT-IR spectra of pure fructose, pure glycerol, and pure alginate (bottom)

Keywords: Pseudomonas fluorescens ; Alginate; Alginate acetylation; FT-IR spectroscopy; Partial least squares regression

Fast quantitative determination of platinum in liquid samples by laser-induced breakdown spectroscopy by Flory-Anne Barreda; Florian Trichard; Sophie Barbier; Nicole Gilon; Laurent Saint-Jalmes (pp. 2601-2610).

The potential of laser-induced breakdown spectroscopy (LIBS) for the rapid determination of platinum in liquid silicone oils has been evaluated in the framework of on-line process control. A comparison of LIBS sensitivity between three setups designed for liquid analysis (static, liquid jet and flowing liquid) was performed using a 266 nm Nd/YAG laser irradiation. Best results were obtained using the flowing liquid setup and a similar limit of detection was obtained using the liquid jet. The effect of different buffer gases (Ar, He, N₂, etc.) on the signal sensitivity was studied in liquid jet analysis and best values were obtained with a nitrogen sheath gas. Detection limits were in the 100 mg/kg range for both setups. Quantitative determination of platinum in real liquid samples was also investigated using both liquid jet and flowing liquid setups. Calibration curves were plotted for Pt with the liquid jet and the flowing liquid setups under optimised temporal acquisition parameters (delay time and gate width). A normalisation using a silicon line was applied and recovery ranged from 3 to 15 % for Pt in catalyst samples with both setups showing that LIBS is a sensitive and accurate method for on-line applications. Figure Emission spectrum from 200 to 800 nm with a zoom on the area of interest for Pt and Si detection

Keywords: Laser-induced breakdown spectroscopy; Platinum; Silicone; Liquid analysis; On-line process control; Laser ablation

Use of ¹H and ³¹P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies by Vicent Esteve; Bernardo Celda; M. Carmen Martínez-Bisbal (pp. 2611-2625).

Quantitative multinuclear high-resolution magic angle spinning was performed in order to determine the tissue pH values of and the absolute metabolite concentrations in 33 samples of human brain tumour tissue. Metabolite concentrations were quantified by 1D ¹H and ³¹P HRMAS using the electronic reference to in vivo concentrations (ERETIC) synthetic signal. ¹H–¹H homonuclear and ¹H–³¹P heteronuclear correlation experiments enabled the direct assessment of the ¹H–³¹P spin systems for signals that suffered from overlapping in the 1D ¹H spectra, and linked the information present in the 1D ¹H and ³¹P spectra. Afterwards, the main histological features were determined, and high heterogeneity in the tumour content, necrotic content and nonaffected tissue content was observed. The metabolite profiles obtained by HRMAS showed characteristics typical of tumour tissues: rather low levels of energetic molecules and increased concentrations of protective metabolites. Nevertheless, these characteristics were more strongly correlated with the total amount of living tissue than with the tumour cell contents of the samples alone, which could indicate that the sampling conditions make a significant contribution aside from the effect of tumour development in vivo. The use of methylene diphosphonic acid as a chemical shift and concentration reference for the ³¹P HRMAS spectra of tissues presented important drawbacks due to its interaction with the tissue. Moreover, the pH data obtained from ³¹P HRMAS enabled us to establish a correlation between the pH and the distance between the N(CH₃)₃ signals of phosphocholine and choline in ¹H spectra of the tissue in these tumour samples. Figure ¹H-³¹P HSQC (upper) and TOCSY (lower) spectra with the corresponding 1D ¹H and ³¹P spectra for one GBM sample. The paths for homo and heteronuclear assignment are drawn as dashed lines (in blue for GPC, in red for GPE, in green for PC and in yellow for PE). The signals can be observed in the corresponding 1D ³¹P spectrum by the right margin of the HSQC spectrum, but not in the crowded 1D ¹H spectrum above the HSQC spectrum

Keywords: ¹H and ³¹P spectroscopy; Human tumour biopsies; Metabolite concentration quantification

Analytical performances of a DNA-ligand system using time-resolved fluorescence for the determination of ochratoxin A in wheat by Annalisa De Girolamo; Linda Le; Gregory Penner; Roberto Schena; Angelo Visconti (pp. 2627-2634).

The analytical performances of a novel DNA-ligand system using the time-resolved fluorescence (TRF) response of ochratoxin A (OTA)–terbium–DNA aptamer interaction were tested for the quantitative determination of OTA in wheat. Wheat was extracted with acetonitrile/water (60:40, v/v) followed by clean-up through affinity columns containing a DNA-aptamer-based oligosorbent. Then, OTA was detected by TRF spectroscopy after reaction with a terbium fluorescent solution containing the DNA-aptamer probe. The entire procedure was performed in less than 30 min, including sample preparation, and allowed analysis of several samples simultaneously with a 96-well microplate reader. The average recovery from samples spiked with 2.5-25 μg kg⁻¹ OTA was 77 %, with a relative standard deviation lower than 6 % and a quantification limit of 0.5 μg kg⁻¹. Comparative analyses of 29 naturally contaminated (up to 14 μg kg^-1) wheat samples using the aptamer-affinity column/TRF method or the immunoaffinity column/high-performance liquid chromatography method showed good correlation (r = 0.985) in the range tested. The trueness of the aptamer-based method was additionally assessed by analysis of two quality control wheat materials for OTA. The DNA-ligand system is innovative, simple and rapid, and can be used to screen large quantities of samples for OTA contamination at levels below the EU regulatory limit with analytical performances satisfying EU criteria for method acceptability.

Keywords: Ochratoxin A; DNA aptamer; Aptamer-affinity columns; Time-resolved fluorescence; Terbium; Wheat

Stacked and continuous helical self-assemblies of guanosine monophosphates detected by vibrational circular dichroism by Iryna Goncharova; Jana Novotná; Marie Urbanová (pp. 2635-2644).

The aim of this study was to characterize self-assembled structures of guanosine derivatives in aqueous solutions by vibrational circular dichroism (VCD) and electronic circular dichroism (ECD). Three guanosine derivatives were studied [5′-guanosine monophosphate (GMP), diphosphate (GDP), and triphosphate (GTP)] using a broad range of concentrations and various metal/guanosine ratios. VCD was used for the first time in this field and showed itself to be a powerful method for obtaining specific structural information in solution. It can also help to determine the impact that the cations have, when added to the solution, on the versatile structures of guanine derivatives in terms of their association and disassociation. Based on the markedly different intensities and signs of the VCD signals observed for different concentrations of guanosine derivatives, we propose various structures based on guanine quartets for high guanosine concentrations and high K⁺/guanosine ratios (i.e., columnar helical organization of the quartets, which are rearranged into a continuous helix). We performed a degenerate coupled oscillator (DCO) calculation to interpret the VCD spectra obtained and how they vary during the assembly of guanosine derivatives. The calculations correctly predicted the VCD spectra and enabled us to identify the structures of the metal cation/guanosine monophosphate aggregates. ECD in the ultraviolet region was used as a diagnostic tool to characterize the studied systems and as a contact point between the previously defined structures of the guanine derivative assemblies and the molecular systems studied here. These studies revealed that the VCD technique is a powerful new method for determining the structures of optically active guanosine motifs. Figure Proposed geometries of the guanosine adducts, the corresponding spectra calculated by the degenerate coupled oscillator method, and experimental vibrational circular dichroism spectra

Keywords: Vibrational circular dichroism; Degenerate coupled oscillator calculation; Self-assembly; G-quartet

Stacked and continuous helical self-assemblies of guanosine monophosphates detected by vibrational circular dichroism by Iryna Goncharova; Jana Novotná; Marie Urbanová (pp. 2635-2644).

Keywords: Vibrational circular dichroism; Degenerate coupled oscillator calculation; Self-assembly; G-quartet

Stacked and continuous helical self-assemblies of guanosine monophosphates detected by vibrational circular dichroism by Iryna Goncharova; Jana Novotná; Marie Urbanová (pp. 2635-2644).

Keywords: Vibrational circular dichroism; Degenerate coupled oscillator calculation; Self-assembly; G-quartet

Two-dimensional flow magnetophoresis of microparticles by Makoto Kawano; Hitoshi Watarai (pp. 2645-2653).

A new two-dimensional micro-flow magnetophoresis device was constructed in a superconducting magnet (10 T) using triangular shaped pole pieces, which could apply a magnetic strength, B(dB/dx), in the range of ca. 0–14,000 T² m⁻¹ across a capillary cell. Polystyrene particles with diameters of 1, 3, and 6 μm were used as test samples in a paramagnetic medium of 1 M MnCl₂ to evaluate the performance of this method. Microparticles migrated across the capillary along the edge of the pole pieces, and then flowed through the gap in the pole piece at a position defined as the migration distance, depending on the magnetic susceptibility and the size of particles as well as the flow rate. The most effective flow rate to exhibit the largest resolution among the particles was theoretically predicted and experimentally confirmed. By this method, the magnetic susceptibilities of individual deoxygenated and non-deoxygenated red blood cells were measured from the relative migration distance. Figure Two-dimensional flow magnetophoresis of microparticles, e.g., red blood cells

Keywords: Magnetophoresis; Magnetic fractionation; Microparticle; Red blood cell; Magnetic susceptibility

Two-dimensional flow magnetophoresis of microparticles by Makoto Kawano; Hitoshi Watarai (pp. 2645-2653).

Keywords: Magnetophoresis; Magnetic fractionation; Microparticle; Red blood cell; Magnetic susceptibility

Two-dimensional flow magnetophoresis of microparticles by Makoto Kawano; Hitoshi Watarai (pp. 2645-2653).

Keywords: Magnetophoresis; Magnetic fractionation; Microparticle; Red blood cell; Magnetic susceptibility

Synthesis of a monolithic, micro-immobilised enzyme reactor via click-chemistry by Bekir Çelebi; Aslıhan Bayraktar; Ali Tuncel (pp. 2655-2663).

An immobilised enzyme reactor (IMER) in the form of capillary monolith was developed for a micro-liquid chromatography system. The plain monolith was obtained by in situ thermal copolymerisation of glycidyl methacrylate and ethylene dimethacrylate in a fused silica capillary (200 × 0.53 mm ID) by using n-propanol/1,4-butanediol as porogen. The enzyme, α-chymotrypsin (CT), was covalently attached onto the monolith via triazole ring formation by click-chemistry. For this purpose, the monolithic support was treated with sodium azide and reacted with the alkyne carrying enzyme derivative. CT was covalently linked to the monolith by triazole-ring formation. The activity behaviour of monolithic IMER was investigated in a micro-liquid chromatography system by using benzoyl-l-tyrosine ethyl ester (BTEE) as synthetic substrate. The effects of mobile-phase flow rate and substrate feed concentration on the final BTEE conversion were investigated under steady-state conditions. In the case of monolithic IMER, the final substrate conversion increased with increasing feed flow rate and increasing substrate feed concentration. Unusual behaviour was explained by the presence of convective diffusion in the macropores of monolith. The results indicated that the monolithic-capillary IMER proposed for micro-liquid chromatography had significant advantages with respect to particle-based conventional high-performance liquid chromatography–IMERs. Figure The variation of DAD signal and final BTEE conversion with the flow rate of substrate solution.

Keywords: Immobilised enzyme reactor; Micro-liquid chromatography; Enzyme immobilisation; HPLC; Protein hydrolysis; Pre-column derivatisation

Synthesis of a monolithic, micro-immobilised enzyme reactor via click-chemistry by Bekir Çelebi; Aslıhan Bayraktar; Ali Tuncel (pp. 2655-2663).

Keywords: Immobilised enzyme reactor; Micro-liquid chromatography; Enzyme immobilisation; HPLC; Protein hydrolysis; Pre-column derivatisation

Synthesis of a monolithic, micro-immobilised enzyme reactor via click-chemistry by Bekir Çelebi; Aslıhan Bayraktar; Ali Tuncel (pp. 2655-2663).

Keywords: Immobilised enzyme reactor; Micro-liquid chromatography; Enzyme immobilisation; HPLC; Protein hydrolysis; Pre-column derivatisation

Enantiomeric separations of chiral polychlorinated biphenyls on three polysaccharide-type chiral stationary phases by supercritical fluid chromatography by Anping Zhang; Weiliang Gao; Binbin Ma; Lixia Jin; Chunmian Lin (pp. 2665-2672).

Enantiomeric separations of 18 chiral polychlorinated biphenyls (PCBs) were investigated on three polysaccharide-type chiral stationary phases (CSPs; Sino-Chiral OJ, Chiralpak IB, and Chiralcel OD) by supercritical fluid chromatography (SFC). With these commonly used polysaccharide CSPs, 17 PCBs except PCB 135 (R _S = 0.81) were well resolved (R _S > 1.5) under appropriate mobile phases and temperatures. Using Sino-Chiral OJ, 14 PCBs could be baseline-separated, while only one and nine PCBs could be completely separated using Chiralpak IB and Chiralcel OD, respectively. The influence of column temperature was studied for the optimization of resolution, as well as for the type and percentage of organic modifier in the mobile phase. The resolution decreased as the temperature increased in the range of 26–40 °C in which the enantiomeric separations were an enthalpy-driven process. The addition of modifiers in the mobile phase decreased the resolution of the PCB enantiomers, but it clearly shortened their retention time. These separation results indicate that SFC is a promising chromatographic technique for chiral separation and enantiopure standard preparation. Figure

Keywords: Chiral polychlorinated biphenyls; Enantiomeric separation; Supercritical fluid chromatography; Sino-Chiral OJ

Mass spectral evaluation of column bleeding for imidazolium-based ionic liquids as GC liquid phases by M. V. Shashkov; V. N. Sidelnikov (pp. 2673-2682).

Mass spectra were obtained to evaluate the use of numerous single-cation and dicationic ionic liquids as stationary liquid phases in GC/MS at high temperature. Background mass spectra and product ion mass spectra of several ions in the background spectrum were obtained. Fragmentation mechanisms were propounded, including the detailed fragmentation pathway of the 1,2-dimethyl-3-propylimidazole cation. The relation between temperature and the main signals in the mass spectra of ILs was studied.

Keywords: GC/MS; Ionic liquids; Capillary column; MS-MS

Mass spectral evaluation of column bleeding for imidazolium-based ionic liquids as GC liquid phases by M. V. Shashkov; V. N. Sidelnikov (pp. 2673-2682).

Keywords: GC/MS; Ionic liquids; Capillary column; MS-MS

Mass spectral evaluation of column bleeding for imidazolium-based ionic liquids as GC liquid phases by M. V. Shashkov; V. N. Sidelnikov (pp. 2673-2682).

Keywords: GC/MS; Ionic liquids; Capillary column; MS-MS

Large-scale profiling of diterpenoid glycosides from Stevia rebaudiana using ultrahigh performance liquid chromatography/tandem mass spectrometry by Behnaz Shafii; Ramin Vismeh; Randy Beaudry; Ryan Warner; A. Daniel Jones (pp. 2683-2690).

The plant Stevia rebaudiana accumulates a suite of diterpenoid metabolites that are natural sweeteners finding increased use as sugar substitutes. To guide breeding of stevia plants that accumulate substances with desirable flavor in high yield, rapid and accurate methods are needed to profile these substances in plant populations. This report describes an 8-min ultrahigh performance liquid chromatography–tandem mass spectrometry method for separation and quantification of seven stevia glycosides including steviolbioside; stevioside; rebaudiosides A, B, and C; rubusoside; and dulcoside as well as aglycones steviol and isosteviol. This negative mode electrospray ionization/multiple reaction monitoring method yielded low limits of detection <1 ng/mL for steviol, 6 ng/mL for isosteviol, and <15 ng/mL for all stevia glycosides. Stevioside and Reb A, B, and C were quantified in more than 1,100 extracts from stevia leaves as part of a large-scale profiling exercise. Leaf tissue levels in this population spanned about two orders of magnitude for stevioside (2–125 mg/g dry weight), Reb A (2.5–164 mg/g), Reb B (0.5–50 mg/g), and Reb C (1.5–125 mg/g), but levels of individual metabolites exhibited independent variation. The wide spread of metabolite levels highlights the utility and importance of performing targeted metabolic profiling for large plant populations.

Keywords: Stevia glycosides; Diterpenoids; Ultrahigh performance liquid chromatography; Tandem mass spectrometry; Large-scale metabolite profiling; Natural products

Rational design of core–shell molecularly imprinted polymer based on computational simulation and Doehlert experimental optimization: application to the separation of tanshinone IIA from Salvia miltiorrhiza Bunge by Xianjun Jia; Hong Li; Jing Luo; Qing Lu; Yan Peng; Liying Shi; Liping Liu; Shuhu Du; Guijun Zhang; Lina Chen (pp. 2691-2703).

Computational simulation and Doehlert experimental optimization were done for the rational design of a core–shell molecularly imprinted polymer (CS-MIP) for use in the highly selective separation of Tanshinone IIA (TSIIA) from the crude extracts of Salvia miltiorrhiza Bunge (SMB). The functional monomer layer of the polymer shells directed the selective occurrence of imprinting polymerization at the surface of silica through the copolymerization of vinyl end groups with functional monomers and also drove TSIIA templates into the formed polymer shells through the charge–transfer complex interactions between TSIIA and the functional monomer layer. As a result, the maximum rebinding capacity was achieved with the use of optimal grafting ratio by the Doehlert design. The CS-MIP exhibited high recognition selectivity and binding affinity to TSIIA. When the imprinted particles were used as dispersive solid phase extraction sorbents, the recovery yield of TSIIA reached 93 % by a one-step extraction from the crude extracts of SMB, and the purity of TSIIA was larger than 98 % by HPLC analysis. These results show the possibility of a highly selective separation and enrichment of TSIIA from the SMB using the TSIIA-imprinted core–shell molecularly imprinted polymers.

Keywords: Core–shell molecularly imprinted polymer; Tanshinone IIA; Molecular modeling; Doehlert design; Separation

Applicability of multisyringe chromatography coupled to on-line solid-phase extraction to the simultaneous determination of dicamba, 2,4-D, and atrazine by C. A. Chávez-Moreno; J. L. Guzmán-Mar; L. Hinojosa-Reyes; A. Hernández-Ramírez; L. Ferrer; V. Cerdà (pp. 2705-2714).

Simultaneous determination of three herbicides (dicamba, 2,4-D, and atrazine) has been achieved by on-line solid-phase extraction (SPE) coupled to multisyringe chromatography (MSC) with UV detection. The preconcentration conditions were optimized; a preconcentration flow rate of 0.5 mL min⁻¹ and elution at 0.8 mL min⁻¹ were the optimum conditions. A C₁₈ (8 mm i.d.) membrane extraction disk conditioned with 0.3 mol L⁻¹ HCl in 0.5 % MeOH was used. A 3-mL sample was preconcentrated, then eluted with 0.43 mL 40:60 water–MeOH. A C₁₈ monolithic column (25 mm × 4.6 mm) was used for chromatographic separation. Separation of the three compounds was achieved in 10 min by use of 0.01 % aqueous acetic acid–MeOH (60:40) as mobile phase at a flow rate of 0.8 mL min⁻¹. The limits of detection (LOD) were 13, 57, and 22 μg L⁻¹ for dicamba, 2,4-D, and atrazine, respectively. The sampling frequency was three analyses per hour, and each analysis consumed only 7.3 mL solvent. The method was applied to spiked water samples, and recovery between 85 and 112 % was obtained. Recovery was significantly better than in the conventional HPLC–UV method. These results indicated the reliability and accuracy of this flow-based method. This is the first time this family of herbicides has been simultaneously analyzed by on-line SPE–MSC using a monolithic column. Figure On-line solid phase extraction coupled to multisyringe chromatography

Keywords: Herbicides; Multisyringe chromatography; On-line solid-phase extraction; Monolithic column; Environmental analysis

Development and validation of a mass spectrometric detection method of peginesatide in dried blood spots for sports drug testing by Ines Möller; Andreas Thomas; Hans Geyer; Wilhelm Schänzer; Mario Thevis (pp. 2715-2724).

As recently reported, dried blood spot (DBS) analysis is an advantageous technique for doping control purposes due to the minimal invasive sample collection, the simple and economic manner, as well as the low susceptibility to manipulation. Its general applicability to the sports drug testing arena has been shown for analytes of various substance classes, all of which comprise exclusively low molecular mass compounds. The aim of the present study was to investigate whether the technique of DBS analysis is applicable also to (pegylated) peptides with relevance for doping controls. As target analyte, peginesatide (Omontys, Hematide), a recently approved pegylated erythropoietin-mimetic peptide of approximately 45 kDa, tested for the treatment of anaemia in patients with renal failure, was chosen, which has been prohibited in elite sports due to its assumed endurance enhancing effects. Therefore, a detection method for peginesatide employing DBS was developed based on extraction, proteolytic digestion and cation-exchange purification followed by liquid chromatography–tandem mass spectrometry analysis. Eventually, the assay was validated for qualitative purposes and proved to be specific, sensitive (limit of detection, 10 ng/mL) and precise (relative standard deviations below 18 %), demonstrating the general suitability of DBS analysis in sports drug testing also for (pegylated) peptides.

Keywords: Peginesatide; Omontys; Hematide; Doping; Erythropoiesis-stimulating agents; Q Exactive

Novel peptide–protein assay for identification of antimicrobial peptides by fluorescence quenching by K. Dobslaff; T. Kreisig; N. Berthold; R. Hoffmann; T. Zuchner (pp. 2725-2731).

The specific interaction of peptides with proteins is often a key factor which determines biological activities. The determination of K _d values of such interactions is commonly performed with fluorescence polarization. However, fluorescence polarization assays are prone to false-positive results due to the potential for non-specific interactions and only afford very low signal-to-background ratios. Here, we present as an alternative a fluorescence resonance energy transfer based quenching assay to measure peptide–protein interactions in solution. In a test setup where antimicrobial peptides were tested for their affinity towards the protein DnaK, the assay provided high specificity and good reproducibility and correlated with the results obtained by fluorescence polarization methods. Furthermore, we established a fast prescreening method which will allow a highly efficient screening of peptide libraries by reducing the amount of sample by 98 % compared to conventional fluorescence polarization assays.

Keywords: K _d values; Quenching assay; Antimicrobial peptides

Novel peptide–protein assay for identification of antimicrobial peptides by fluorescence quenching by K. Dobslaff; T. Kreisig; N. Berthold; R. Hoffmann; T. Zuchner (pp. 2725-2731).

Keywords: K _d values; Quenching assay; Antimicrobial peptides

Novel peptide–protein assay for identification of antimicrobial peptides by fluorescence quenching by K. Dobslaff; T. Kreisig; N. Berthold; R. Hoffmann; T. Zuchner (pp. 2725-2731).

Keywords: K _d values; Quenching assay; Antimicrobial peptides

Structural elucidation of the tetrasaccharide pool in enoxaparin sodium by Jennifer Ozug; Steve Wudyka; Nur Sibel Gunay; Daniela Beccati; Jonathan Lansing; Jing Wang; Ishan Capila; Zachary Shriver; Ganesh V. Kaundinya (pp. 2733-2744).

Low-molecular-weight heparins (LMWHs) are produced from heparin by various depolymerization strategies, which result in a reduction of the average molecular weight of the polysaccharide chains, a reduction of the anti-factor IIa activity (and a concomitant increase in the anti-factor Xa/anti-factor IIa ratio), and introduction of process-related structural signatures. Numerous techniques have been developed to characterize LMWHs and to measure the type and extent of structural modifications that are introduced as a function of the depolymerization process. We present here an analysis of the tetrasaccharide pool of enoxaparin sodium, a LMWH produced by chemical β-elimination of heparin benzyl ester. We identify the predominant sequences present within the tetrasaccharide pool and demonstrate that this pool provides a sensitive, specific readout of the physicochemical process conditions used to generate enoxaparin sodium.

Keywords: Low-molecular-weight heparin; Enoxaparin sodium; Tetrasaccharide; Heparinases; Nuclear magnetic resonance; Mass spectrometry

Rapid nanoscale quantitative analysis of plant sphingolipid long-chain bases by GC-MS by Jean-Luc Cacas; Su Melser; Frédéric Domergue; Jérôme Joubès; Brice Bourdenx; Jean-Marie Schmitter; Sébastien Mongrand (pp. 2745-2755).

In eukaryotic organisms, sphingolipids are major structural lipids of biological membranes and perform additional essential functions as signalling molecules. While long-chain bases (LCB), the common precursor to all sphingolipid classes, is represented by only one major molecular species in animals and fungi, up to nine LCB have been found in plants. In the absence of genuine plant sphingolipid references required for proper quantification, we have reinvestigated and optimized a protocol destined to the quantification of total plant LCB that relies on the use of gas chromatography-mass spectrometry (GC-MS). This rapid three-step protocol sequentially involves (1) the release of LCB from biological samples using barium hydroxide solution, (2) their oxidation into aldehydes by metaperiodate, and (3) the subsequent identification/quantification of these aldehydes by GC-MS. It is simple and reliable and enables separation of aldehydes upon their stero-specificity. It further enables the quantification of total LCB from a wide variety of samples including yeast and animal cell cultures. Figure Rationale for rapid LCB analysis by GC-MS

Keywords: GC; Bioanalytical methods; Biological samples

High-capacity and high-intensity DNA microarray spots using oxygen-plasma nanotextured polystyrene slides by K. Tsougeni; G. Koukouvinos; P. S. Petrou; A. Tserepi; S. E. Kakabakos; E. Gogolides (pp. 2757-2764).

Commercially available polystyrene (PS) slides were plasma nanotextured (nano-roughened) through treatment in oxygen plasma discharges to create substrates with increased surface area for microarray applications. Conditions of plasma treatment were determined for maximum and uniform oligonucleotide immobilization on these nanotextured PS slides. Oligonucleotides were immobilized onto the surface in the form of biotinylated oligonucleotide/streptavidin conjugates to take advantage of increased protein binding capacity of the substrate. It was found that the amount of oligonucleotides that could be immobilized was increased up to ten times on plasma treated as compared with untreated slides. The sensitivity of detection of labelled hybridized probes was improved by a factor of 20. Optimized nanotextured PS slides were subsequently used to develop a microarray for the detection of three deleterious BRCA1 gene mutations by immobilizing oligonucleotides corresponding to wild and mutant-type sequences. The microarray developed on the nanotextured PS slides provided higher specific hybridization signal and discrimination ratios as compared with flat untreated PS slides. Figure The process of O₂ plasma nanotexturing and biomolecule immobilization is shown. An image from the slide scanner showing bright spots is presented together with an SEM image of the slide.

Keywords: PS slides; Plasma nanotexturing; DNA microarrays

Determination of glyoxylic acid in urine by liquid chromatography with fluorescence detection, using a novel derivatization procedure based on the Petasis reaction by Kohki Chihara; Naoya Kishikawa; Kaname Ohyama; Kenichiro Nakashima; Naotaka Kuroda (pp. 2765-2770).

The Petasis reaction is the multi-component reaction of a carbonyl compound, amine, and arylboronic acid to form an α-amino acid or a β-aminoalcohol. In this work, as the first analytical application of the Petasis reaction, a high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for determination of glyoxylic acid. The glyoxylic acid was derivatized with 1-pyreneboronic acid, as fluorescent arylboronic acid, in the presence of N-methylbutylamine, as amine, to give a fluorescent α-amino acid. HPLC separation of the fluorescent derivative was performed within 30 min on an octyl column eluted with a gradient prepared from acetonitrile and 50 mmol L⁻¹ acetate buffer (pH 4.0). The detection limit (S/N=3) for glyoxylic acid was 5.0 nmol L⁻¹ (20 fmol/injection). The method can be used to determine the concentration of glyoxylic acid in human urine without interference from biological components.

Keywords: Petasis reaction; Fluorescence derivatization; Glyoxylic acid; Fluorescent arylboronic acid

Improved fluoroquinolone detection in ELISA through engineering of a broad-specific single-chain variable fragment binding simultaneously to 20 fluoroquinolones by Kai Wen; Greta Nölke; Stefan Schillberg; Zhanhui Wang; Suxia Zhang; Congming Wu; Haiyang Jiang; Hui Meng; Jianzhong Shen (pp. 2771-2783).

Fluoroquinolones (FQs) are a group of synthetic, broad-spectrum antibacterial agents. Due to its extensive use in animal industry and aquaculture, residues of these antibiotics and the emergence of bacteria resistant to FQs have become a major public health issue. To prepare a generic antibody capable of recognizing nearly all FQs, a single-chain variable fragment (scFv) was generated from the murine hybridoma cells C49H1 producing a FQ-specific monoclonal antibody. This scFv was characterized by indirect competitive enzyme-linked immunosorbent assay (ciELISA), and it showed identical binding properties to parental monoclonal antibody: it was capable of recognizing 17 of 20 targeted FQs below maximum residue limits, except for sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO) which are highly concerned members in the FQs family. In order to broaden the specificity of this scFv to SAR and its analogues (DIF and TRO), protein homology modeling and antibody-ligands docking analysis were employed to identify the potential key amino acid residues involved in hapten antibody. A mutagenesis phage display library was generated by site directed mutagenesis randomizing five aminoacid residues in the third heavy-chain complementarity determining region. After one round of panning against biotinylated norfloxacin (NOR) and four rounds of panning against biotinylated SAR, scFv variants we screened showed up to 10-fold improved IC₅₀ against SAR, DIF, and TRO in ciELISA while the specificity against other FQs was fully retained. Figure Modeling of the interaction between scFvC4A9H1 and FQs. a Cα ribbon diagram of homology model of Fv domain; b interactions between NOR and scFv binding site; c the superimposition of NOR-scFvC4A9H1 and SAR-scFvC4A9H1 complexes. CDR H1, H2, and H3 were shown as solvent accessible surface in yellow, orange, and red, respectively, and CDR L1, L2, and L3 were shown in light blue, sky blue, and deep blue, respectively, the NOR and SAR molecules were shown as sticks in red and blue, respectively; d interactions between SAR and scFvC4A9H1 binding site. The predicted interactions are shown as straight lines

Keywords: Fluoroquinolones; Molecular modeling; Phage display; scFv; Site-directed mutagenesis

Letter to the Editor regarding “GC–MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia” by Petr Husek (pp. 2785-2786).

Response to Letter to the Editor regarding “GC-MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia” by Xiaojiao Zheng; Mingming Su; Yunping Qiu; Wei Jia (pp. 2787-2789).

Erratum to: Preparation and certification of Hijiki reference material, NMIJ CRM 7405-a, from the edible marine algae hijiki (Hizikia fusiforme) by Tomohiro Narukawa; Kazumi Inagaki; Yanbei Zhu; Takayoshi Kuroiwa; Izumi Narushima; Koichi Chiba; Akiharu Hioki (pp. 2791-2793).

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Analytical and Bioanalytical Chemistry (v.403, #9)