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Analytical and Bioanalytical Chemistry (v.405, #8)

Competition in structural analysis—old wine in new skins by Jürgen Popp; Manfred Reichenbächer (pp. 2411-2414).

studied chemistry at the universities of Erlangen and Würzburg. After completing his PhD degree in chemistry, he joined Yale University for postdoctoral work. He subsequently returned to Würzburg University, where he completed his habilitation in 2002. Since 2002 he has held a chair for physical chemistry at Friedrich Schiller University Jena. Since 2006 he has also been the scientific director of the Institute of Photonic Technology, Jena. His research interests are mainly concerned with biophotonics. is the scientific organizer of the competition in structural analysis. He has expert knowledge of exercises, practical courses and lectures in structural analysis over more than 30 years.

Competition in structural analysis—old wine in new skins by Jürgen Popp; Manfred Reichenbächer (pp. 2411-2414).

Paul van der Heide: X-ray photoelectron spectroscopy: An introduction to principles and practices by Peter M. A. Sherwood (pp. 2415-2416).

A microfluidic device for open loop stripping of volatile organic compounds by Benjamin Z. Cvetković; Petra S. Dittrich (pp. 2417-2423).

The detection of volatile organic compounds is of great importance for assessing the quality of water. In this contribution, we describe a miniaturized stripping device that allows fast online detection of organic solvents in water. The core component is a glass microfluidic chip that facilitates the creation of an annular-flowing stream of water and nitrogen gas. Volatile compounds are transferred efficiently from the water into the gas phase along the microfluidic pathway at room temperature within less than 5 s. Before exiting the microchip, the liquid phase is separated from the enriched gas phase by incorporating side capillaries through which the hydrophilic water phase is withdrawn. The gas phase is conveniently collected at the outlet reservoir by tubing. Finally, a semiconductor gas sensor analyzes the concentration of (volatile) organic compounds in the nitrogen gas. The operation and use of the stripping device is demonstrated for the organic solvents THF, 1-propanol, toluene, ethylbenzene, benzaldehyde, and methanol. The mobile, inexpensive, and continuously operating system with liquid flow rates in the low range of microliters per minute can be connected to other detectors or implemented in chemical production line for process control. Figure We present a microfluidic device for highly efficient analytical stripping of organic solvents from water/solvent mixtures at room temperature

Keywords: Microfluidics; Open loop stripping; Water monitoring; Volatile organic compounds; Process analytical technology

Offline and online capillary electrophoresis enzyme assays of β-N-acetylhexosaminidase by Tomáš Křížek; Veronika Doubnerová; Helena Ryšlavá; Pavel Coufal; Zuzana Bosáková (pp. 2425-2434).

Enzyme assays of β-N-acetylhexosaminidase from Aspergillus oryzae using capillary electrophoresis in the offline and online setup have been developed. The pH value and concentration of the borate-based background electrolyte were optimized in order to achieve baseline separation of N,N′,N″-triacetylchitotriose, N,N′-diacetylchitobiose, and N-acetyl-d-glucosamine. The optimized method using 25 mM tetraborate buffer, pH 10.0, was evaluated in terms of repeatability, limits of detection, quantification, and linearity. The method was successfully applied to the offline enzyme assay of β-N-acetylhexosaminidase, which was demonstrated by monitoring the hydrolysis of N,N′,N″-triacetylchitotriose. The presented method was also utilized to study the pH dependence of enzyme activity. An online assay with N,N′-diacetylchitobiose as a substrate was developed using the Transverse Diffusion of Laminar Flow Profiles model to optimize the injection sequence and in-capillary mixing of substrate and enzyme plugs. The experimental results were in good agreement with predictions of the model. The online assay was successfully used to observe the inhibition effect of N,N′-dimethylformamide on the activity of β-N-acetylhexosaminidase with nanoliter volumes of reagents used per run and a high degree of automation. After adjustment of background electrolyte pH, an online assay with N,N′,N″-triacetylchitotriose as a substrate was also performed. Figure Electropherograms resulting from online enzyme assays of β-N-acetylhexosaminidase for chitobiose as a substrate with 10-min (red line), 5-min (blue line) and 0-min (black line) reaction time. Peak identification: 1 chitobiose, 2 N-acetylglucosamine

Keywords: β-N-Acetylhexosaminidase; Capillary electrophoresis; Chitobiose; Chitotriose; Electrophoretically mediated microanalysis; Enzyme activity

Epigenotoxicity of environmental pollutants evaluated by a combination of DNA methylation inhibition and capillary electrophoresis–laser-induced fluorescence immunoassay by Xiaoli Wang; Hailin Wang (pp. 2435-2442).

A variety of environmental pollutants may cause abnormal DNA methylation, which further disturb gene expression. In this work, we developed a rapid and sensitive method for the characterization and identification of the epigenotoxicity of environmental pollutants in terms of DNA methylation. The method combines in vitro inhibition reactions of a model DNA methyltransferase (DNMT) with rapid and sensitive capillary electrophoresis–laser-induced fluorescence (CE-LIF) immunoassays. This method was first assessed using two known DNMT inhibitors, (–)-epigallocatechin-3-gallate and RG108, and then applied to epigenotoxic evaluation of four aldehydes and six benzo-1,4-quinones. It was found that all these electrophilic chemicals could inhibit DNMT activity, probably due to their interactions with the active sites of DNMT. Interestingly, benzo-1,4-quinones displayed more inhibitory effects on DNMT activity than aldehydes. Among the tested six benzo-1,4-quinones, halogenated benzo-1,4-quinone showed higher inhibitory activity than non-halogenated p-benzo-1,4-quinone. Owing to its speed and sensitivity, our method will be potentially applicable for fast epigenotoxic screening of environmental pollutants and mechanistic study of environmental epigenetics. Figure Epigenotoxicity evaluation of environmental pollutants

Keywords: Environmental pollutants; Epigenotoxicity; DNA methylation; DNA methyltransferase; Capillary electrophoresis–laser-induced fluorescence immunoassay

Screening interaction between ochratoxin A and aptamers by fluorescence anisotropy approach by Xia Geng; Dapeng Zhang; Hailin Wang; Qiang Zhao (pp. 2443-2449).

By taking advantage of the intrinsic fluorescence of ochratoxin A (OTA), we present a fluorescence anisotropy approach for rapid analysis of the interactions between OTA and aptamers. The specific binding of OTA with a 36-mer aptamer can induce increased fluorescence anisotropy (FA) of OTA as the result of the freedom restriction of OTA and the increase of molecular volume, and the maximum FA change is about 0.160. This FA approach enables an easy way to investigate the effects of buffer compositions like metal ions on the affinity binding. FA analysis shows the interaction between OTA and aptamer is greatly enhanced by the simultaneous presence of Ca²⁺ and Na⁺, while the binding affinity of aptamer decreases more than 18-fold when only Ca²⁺ exists, and the binding is completely lost when Ca²⁺ is absent. Crucial region of the aptamer for binding can be mapped through FA analysis and aptamer mutation. The demonstrated FA approach maintains the advantages of FA in simplicity, rapidity, and robustness. This investigation will help the development of aptamer-based assays for OTA detection in optimizing the binding conditions, modification of aptamers, and rational design. Figure The free ochratoxin A (OTA) molecule tumbles rapidly and shows low fluorescence anisotropy (FA), while the bound OTA by the aptamer has increased molecular volume and restricted freedom, showing enhanced FA. FA analysis allows screening the interaction between OTA and aptamer

Keywords: Ochratoxin A; Aptamer; Fluorescence anisotropy; Fluorescence polarization; Interaction

Automated method for analysis of tryptophan and tyrosine metabolites using capillary electrophoresis with native fluorescence detection by Christopher A. Dailey; Nicolas Garnier; Stanislav S. Rubakhin; Jonathan V. Sweedler (pp. 2451-2459).

Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) detection offers the ability to characterize low levels of selected analyte classes, depending on the excitation and emission wavelengths used. Here a new automated CE-LINF system that provides deep ultraviolet (DUV) excitation (224 nm) and variable emission wavelength detection was evaluated for the analysis of small molecule tryptophan- and tyrosine-related metabolites. The optimized instrument design includes several features that increase throughput, lower instrument cost and maintenance, and decrease complexity when compared with earlier systems using DUV excitation. Sensitivity is enhanced by using an ellipsoid detection cell to increase the fluorescence collection efficiency. The limits of detection ranged from 4 to 30 nmol/L for serotonin and tyrosine, respectively. The system demonstrated excellent linearity over several orders of magnitude of concentration and intraday precision from 1–11 % relative standard deviation (RSD). The instrument’s performance was validated via tryptophan and serotonin characterization using tissue extracts from the mammalian brain stem, with RSDs of less than 10 % for both metabolites. The flexibility and sensitivity offered by DUV laser excitation and tunable emission enables a broad range of small-volume measurements.

Keywords: Capillary electrophoresis; Laser-induced native fluorescence; Serotonin; Automation; High throughput

Quantitative, solution-phase profiling of multiple transcription factors in parallel by Betul Bilgin; Li Liu; Christina Chan; S. Patrick Walton (pp. 2461-2468).

Transcription factors are regulatory proteins that bind to specific sites of chromosomal DNA to enact responses to intracellular and extracellular stimuli. Transcription factor signalling networks are branched and interconnected so that any single transcription factor can activate many different genes and one gene can be activated by a combination of different transcription factors. Thus, trying to characterize a cellular response to a stimulus by measuring the level of only one transcription factor potentially ignores important simultaneous events that contribute to the response. Hence, parallel measurements of transcription factors are necessary to capture the breadth of valuable information about cellular responses that would not be obtained by measuring only a single transcription factor. We have sought to develop a new, scalable, flexible, and sensitive approach to analysis of transcription factor levels that complements existing parallel approaches. Here, we describe proof-of-principle analyses of purified human transcription factors and breast cancer nuclear extracts. Our assay can successfully quantify transcription factors in parallel with ∼10-fold better sensitivity than current techniques. Sensitivity of the assay can be further increased by 200-fold through the use of PCR for signal amplification.

Keywords: Transcription factors; Parallel; DNA binding activity; Magnetic beads separation; Breast cancer

Nanoscale reversed-phase liquid chromatography–mass spectrometry of permethylated N-glycans by Ilja Ritamo; Jarkko Räbinä; Suvi Natunen; Leena Valmu (pp. 2469-2480).

Reversed-phase liquid chromatography on the nanoscale coupled to electrospray tandem mass spectrometry was used to analyse a mixture of four commercial glycan standards, and the method was further adapted to N-glycans enzymatically released from alpha-1-acid glycoprotein and immunoglobulin gamma. Glycans were permethylated to enable their separation by reversed-phase chromatography and to facilitate interpretation of fragmentation data. Prior to derivatization of glycans by permethylation, they were reduced to cancel anomerism because, although feasible, it was not desired to separate α- and β-anomers. The effect of supplementing chromatographic solvent with sodium hydroxide to guide adduct formation was investigated. Raising the temperature in which the separation was performed improved chromatographic resolution and affected retention times as expected. It was shown by using the tetrasaccharides sialyl Lewis X and sialyl Lewis A that reversed-phase chromatography could achieve the separation of methylated isobaric glycan analytes. Isobaric glycans were detected among the N-glycans of immunoglobulin gamma and further analysed by tandem mass spectrometry.

Keywords: Mass spectrometry; Liquid chromatography; Glycan; Permethylation

Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals by Fabian Higel; Uwe Demelbauer; Andreas Seidl; Wolfgang Friess; Fritz Sörgel (pp. 2481-2493).

N-Glycosylation is a common post-translational modification of monoclonal antibodies with a potential effect on the efficacy and safety of the drugs; detailed knowledge about this glycosylation is therefore crucial. We have developed a reversed-phase liquid chromatographic–mass spectrometric method, with different fluorescent labels, for analysis of N-glycosylation, and compared the sensitivity and selectivity of the methods. Our work demonstrates that anthranilic acid as fluorescent label in combination with reversed-phase liquid chromatography–mass spectrometry is an advantageous method for identification and quantification of neutral and acidic N-glycans. Our results show that mass spectrometry-based quantification correlates with quantification by fluorescence. Chromatographic discrimination between several structural glycan isomers was achieved. The sharp peaks of the eluting anthranilic acid-labeled N-glycans enabled on-line mass spectrometric analysis of even low-abundance glycan species. The method is broadly applicable to N-glycan analysis and is an orthogonal analytical method to the widely established hydrophilic-interaction liquid chromatography of 2-aminobenzamide-labeled N-glycans for characterization of N-glycans derived from biopharmaceuticals.

Keywords: Anthranilic acid; 2-aminobenzamide; Mass spectrometry; N-glycosylation; Reversed-phase chromatography; Monoclonal antibody

Pharmacodynamic assay of thymidylate synthase activity in peripheral blood mononuclear cells by Dick Pluim; Kim A. A. Schilders; Bart A. W. Jacobs; Daniëlle Vaartjes; Jos H. Beijnen; Jan H. M. Schellens (pp. 2495-2503).

A simple, selective, and sensitive method utilizing tritium (³H) release from ³H-deoxyuridine 5′-monophosphate (dUMP) substrate for accurate and precise determination of the low basal thymidylate synthase activity (TSA) in normal healthy peripheral blood mononuclear cells (PBMCs) was developed and validated. The method is based on the removal of the remaining substrate after the TSA reaction by absorption onto activated carbon and measurement of the supernatant fluid by liquid scintillation counting. The method background was substantially decreased by using lyophilized substrate and optimized binding conditions of remaining substrate onto carbon after TSA reaction. The concentration of cofactor N ₅,N ₁₀ methylene-(6R,S)-tetrahydrofolate was increased to obtain maximal TSA. Method sensitivity was further increased by omission of ethylenediaminetetraacetic acid from the reaction mix and by using longer reaction times. The validation parameters included specificity, linearity, sensitivity, precision, and stability. The lower limit of quantification was 25 μg PBMC cytosolic lysate, which released 1.4 pmol ³H/h. TSA was stable in PBMC pellets stored for 6 months at −80 °C. The applicability of the method was demonstrated by the successful determination of TSA in PBMC cytosolic lysates from ten healthy volunteers with and without the specific TSA inhibitor FdUMP. Figure Effect of the specific thymidylate synthase inhibitor FdUMP on thymidylate synthase activity (TSA) in peripheral blood mononuclear cells (PBMC)from ten healthy volunteers in the absence (minus sign) or presence (plus sign) of 100 μM FdUMP. Results are shown as the intraindividual mean TSA for ten healthy volunteers measured in triplicate.

Keywords: Thymidylate synthase activity; ³H-release; PBMC; Pharmacodynamic

Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole by A. Miodek; A. Poturnayová; M. Šnejdárková; T. Hianik; H. Korri-Youssoufi (pp. 2505-2514).

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP^C) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP^C interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP^C with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP^C and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part. Fig Aptasensors for kinetic evaluation and detection of prions by SPR

Keywords: Prions; Aptamers; Kinetics; SPR; Polypyrrole

Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity by Petr Tomek; Brian D. Palmer; Jack U. Flanagan; Sai-Parng S. Fung; David J. A. Bridewell; Joanne F. Jamie; Lai-Ming Ching (pp. 2515-2524).

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme whose expression by a broad range of clinical tumors is associated with immunosuppression and poor patient outcome. Here we describe a new fluorescence assay for measuring IDO1 activity suitable for high-throughput screening of compound libraries for novel IDO1 inhibitors. This assay is easy to perform, requiring the addition of only one reagent prior to readout. In place of measuring kynurenine, it uses the in situ formation of an N-formylkynurenine-derived fluorophore (NFKPIP) measured at an excitation wavelength of 400 nm and an emission wavelength of 500 nm. The fluorescence intensity of the NFKPIP formed is directly related to the amount of enzyme activity, and the signal is stable over 8 h. This assay has a lower limit of detection, equating to 153 nM N-formylkynurenine, which is over 30-fold lower than the limits of detection of existing assays for IDO1 activity. When we compared the performance of the new assay with that of the published colorimetric absorbance assay in screening the National Cancer Institute Diversity Set III of 1,597 compounds for IDO1 inhibitors, we obtained an identical list of the 25 most active compounds in the two assays. Although 93 compounds (aldehydes, ketones, and aromatic amines) in the library interfered with the absorbance readout, only 18 compounds (conjugated systems and fused cycles) interfered with the readout of the new fluorescence assay. IC₅₀ values determined using the new assay for three known IDO1 inhibitors—1,4-naphthoquinone, 4-amino-N-(3-chloro-4-fluorophenyl)-N’-hydroxy-1,2,5-oxadiazole-3-carboximidamide and 4-phenyl-1H-imidazole—were consistent with their literature values, further validating the new assay for measuring IDO1 activity. Figure Formation of an N-formylkynurenine-derived fluorophore for measuring indoleamine 2,3-dioxygenase 1 activity

Keywords: Indoleamine 2,3-dioxygenase; N-Formylkynurenine; Enzymatic assays; High-throughput screening

Preparation of an amperometric sensor for norfloxacin based on molecularly imprinted grafting photopolymerization by Zhihua Wang; Jinshu Li; Xiaole Liu; Jianming Yang; Xiaoquan Lu (pp. 2525-2533).

A sensitive amperometric sensor for norfloxacin (NF) was introduced. The receptor layer was prepared by molecularly imprinted photopolymerization of acrylamide and trimethylolpropane trimethacrylate on the surface of a gold electrode. The binding mechanism of the molecularly imprinted polymer was explored by ultraviolet (UV) and infrared (IR) spectroscopy. The chemosensor was characterized by cyclic voltammetry (CV), differential pulse voltammetry (DPV), electrochemical impedance (EI), and scanning electron microscopy (SEM). The electrode prepared by photopolymerization has a better recognition ability to template molecules than that of electropolymerization and NIP. Some parameters affecting sensor response were optimized. Norfloxacin was detected by measurements of an amperometric i–t curve. The linear relationships between current and logarithmic concentration are obtained from 1.0 × 10⁻⁹ to 1.0 × 10⁻³ mol L⁻¹. The detection limit of the sensor was 1.0 × 10⁻¹⁰ mol L⁻¹. The proposed method is sensitive, simple, and cheap, and is applied to detect NF in human urine successfully. Figure Amperometric i-t curves of MIPs electrode

Keywords: Molecularly imprinted polymer; Norfloxacin; Photopolymerization; Sensor

An evaluation of the capability of a biolayer interferometry biosensor to detect low-molecular-weight food contaminants by Terry F. McGrath; Katrina Campbell; Terry L. Fodey; Richard O’Kennedy; Christopher T. Elliott (pp. 2535-2544).

The safety of our food is an essential requirement of society. One well-recognised threat is that of chemical contamination of our food, where low-molecular-weight compounds such as biotoxins, drug residues and pesticides are present. Low-cost, rapid screening procedures are sought to discriminate the suspect samples from the population, thus selecting only these to be forwarded for confirmatory analysis. Many biosensor assays have been developed as screening tools in food contaminant analysis, but these tend to be electrochemical, fluorescence or surface plasmon resonance based. An alternative approach is the use of biolayer interferometry, which has become established in drug discovery and life science studies but is only now emerging as a potential tool in the analysis of food contaminants. A biolayer interferometry biosensor was assessed using domoic acid as a model compound. Instrument repeatability was tested by simultaneously producing six calibration curves showing replicate repeatability (n = 2) ranging from 0.1 to 6.5 % CV with individual concentration measurements (n = 12) ranging from 4.3 to 9.3 % CV, giving a calibration curve midpoint of 7.5 ng/ml (2.3 % CV (n = 6)). Reproducibility was assessed by producing three calibration curves on different days, giving a midpoint of 7.5 ng/ml (3.4 %CV (n = 3)). It was further shown, using assay development techniques, that the calibration curve midpoint could be adjusted from 10.4 to 1.9 ng/ml by varying assay parameters before the simultaneous construction of three calibration curves in matrix and buffer. Sensitivity of the assay compared favourably with previously published biosensor data for domoic acid.

Keywords: Biolayer interferometry (BLI); Label-free optical biosensor; Low-molecular-weight food contaminants; Concentration analysis; Shellfish analysis; Domoic acid

Development of a stable biosensor based on a SiO₂ nanosheet–Nafion–modified glassy carbon electrode for sensitive detection of pesticides by Long Yang; Guang-Can Wang; Yong-Jun Liu; Jing-Jing An; Min Wang (pp. 2545-2552).

SiO₂ nanosheets (SNS) have been prepared by a chemical method using montmorillonite as raw material and were characterized by scanning electron microscopy and X-ray diffraction. SiO₂ nanosheet–Nafion nanocomposites with excellent conductivity, catalytic activity, and biocompatibility provided an extremely hydrophilic surface for biomolecule adhesion. Chitosan was used as a cross-linker to immobilize acetylcholinesterase (AChE), and Nafion was used as a protective membrane to efficiently improve the stability of the AChE biosensor. The AChE biosensor showed favorable affinity for acetylthiocholine chloride and catalyzed the hydrolysis of acetylthiocholine chloride with an apparent Michaelis–Menten constant of 134 μM to form thiocholine, which was then oxidized to produce a detectable and fast response. Based on the inhibition by pesticides of the enzymatic activity of AChE, detection of the amperometric response from thiocholine on the biosensor is a simple and effective way to biomonitor exposure to pesticides. Under optimum conditions, the biosensor detected methyl parathion, chlorpyrifos, and carbofuran at concentrations ranging from 1.0 × 10⁻¹² to 1 × 10⁻¹⁰ M and from 1.0 × 10⁻¹⁰ to 1 × 10⁻⁸ M. The detection limits for methyl parathion, chlorpyrifos, and carbofuran were 5 × 10⁻¹³ M. The biosensor developed exhibited good sensitivity, stability, reproducibility, and low cost, thus providing a new promising tool for analysis of enzyme inhibitors. Figure Performances and detection pesticides of a SiO₂ nanosheet biosensor

Keywords: SiO₂ nanosheets; Acetylcholinesterase; Chitosan; Nafion; Amperometric biosensor

Effect-directed analysis of endocrine-disrupting compounds in multi-contaminated sediment: identification of novel ligands of estrogen and pregnane X receptors by Nicolas Creusot; Hélène Budzinski; Patrick Balaguer; Saïd Kinani; Jean-Marc Porcher; Selim Aït-Aïssa (pp. 2553-2566).

Effect-directed analysis (EDA)-based strategies have been increasingly used in order to identify the causative link between adverse (eco-)toxic effects and chemical contaminants. In this study, we report the development and use of an EDA approach to identify endocrine-disrupting chemicals (EDCs) in a multi-contaminated river sediment. The battery of in vitro reporter cell-based bioassays, measuring estrogenic, (anti)androgenic, dioxin-like, and pregnane X receptor (PXR)-like activities, revealed multi-contamination profiles. To isolate active compounds of a wide polarity range, we established a multi-step fractionation procedure combining: (1) a primary fractionation step using normal phase-based solid-phase extraction (SPE), validated with a mixture of 12 non-polar to polar standard EDCs; (2) a secondary fractionation using reversed-phase-based high-performance liquid chromatography (RP-HPLC) calibrated with 33 standard EDCs; and (3) a purification step using a recombinant estrogen receptor (ER) affinity column. In vitro SPE and HPLC profiles revealed that ER and PXR activities were mainly due to polar to mid-polar compounds, while dioxin-like and anti-androgenic activities were in the less polar fractions. The overall procedure allowed final isolation and identification of new environmental PXR (e.g., di-iso-octylphthalate) and ER (e.g., 2,4-di-tert-butylphenol and 2,6-di-tert-butyl-α-methoxy-p-cresol) ligands by using gas chromatography coupled with mass spectrometry with full-scan mode acquisition in mid-polar fractions. In vitro biological activity of these chemicals was further confirmed using commercial standards, with di-iso-octylphthalate identified for the first time as a potent hPXR environmental agonist.

Keywords: EDCs; Emerging pollutants; Multi-contaminated sediment; Multi-step fractionation

Determination of oil–water partition coefficients of polar compounds: silicone membrane equilibrator vs. SPME passive sampler by Luise Oemisch; Kai-Uwe Goss; Satoshi Endo (pp. 2567-2574).

Experimental determination of oil-water partition coefficients often poses difficulties associated with emulsion formation. The aim of this work was to find an appropriate technique for determination of oil–water partition coefficients of polar, nonvolatile compounds. Two different methods were tested. The first method used a “silicone membrane equilibrator.” For the second method, solid-phase microextraction (SPME) fibers with a polyacrylate (PA) coating were used as a passive sampler. With both methods, oil–water partition coefficients for 14 compounds with polar functional groups were determined at 37 °C with good repeatability (standard deviation 0.11 log units or lower). The partition coefficients determined with the silicone membrane equilibrator method ranged from 0.50 to 3.49 log units. The oil–water partition coefficients obtained with the PA-SPME passive sampling approach were significantly higher than those obtained with the silicone membrane equilibrator method for nine of 14 compounds. The differences were up to 0.39 log units (i.e., a factor of 2.5). Additional experiments suggested that this difference occurred because the sorption properties of the PA fibers used were influenced by the surrounding phase, e.g., through swelling of the polymer phase. Therefore, the SPME passive sampling method using PA fibers seems to be less reliable, whereas the silicone membrane equilibrator method was found to be a convenient technique for the determination of oil–water partitioning.

Keywords: Lipophilicity; Bioaccumulation; Solid-phase microextraction; Passive sampling; Polar compounds; Pesticides

Electromembrane extraction (EME)—an easy, novel and rapid extraction procedure for the HPLC determination of fluoroquinolones in wastewater samples by María Ramos-Payán; Mercedes Villar-Navarro; Rut Fernández-Torres; Manuel Callejón-Mochón; Miguel Ángel Bello-López (pp. 2575-2584).

For the first time, an electromembrane extraction combined with a HPLC procedure using diode array and fluorescence detection has been developed for the determination of seven widely used fluoroquinolones (FQs): marbofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, gatifloxacin and grepafloxacin. The drugs were extracted from acid aqueous sample solutions (pH 5), through a supported liquid membrane consisting of 1-octanol impregnated in the walls of a S6/2 Accurel® polypropylene hollow fiber, to an acid (pH 2) aqueous acceptor solution inside the lumen of the hollow fiber. The main operational parameters were optimized, and extractions were carried out in 15 min using a potential of 50 V. Enrichment factors of 40–85 have been obtained using only 15 min of extraction time versus 330 min used in a previously proposed hollow-fiber liquid-phase microextraction procedure. The procedure allows low detection and quantitation limits of 0.005–0.07 and 0.007–0.15 μg L⁻¹, respectively. The proposed method was successfully applied to the FQs analysis in urban wastewaters. Figure EME for HPLC determination of fluoroquinolones in wastewater

Keywords: Electromembrane extraction; EME; Fluoroquinolones; Urban wastewater; HPLC

Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry by Miranda G. M. Kok; Marco M. A. Ruijken; Jonathan R. Swann; Ian D. Wilson; Govert W. Somsen; Gerhardus J. de Jong (pp. 2585-2594).

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.

Keywords: Capillary electrophoresis; Mass spectrometry; Anionic metabolic profiling; Rat urine; Gut microbial depletion

Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography–mass spectrometry by Wai Him Kwok; Emmie N. M. Ho; Ming Yip Lau; Gary N. W. Leung; April S. Y. Wong; Terence S. M. Wan (pp. 2595-2606).

In recent years, there has been an ongoing focus for both human and equine doping control laboratories on developing detection methods to control the misuse of peptide therapeutics. Immunoaffinity purification is a common extraction method to isolate peptides from biological matrices and obtain sufficient detectability in subsequent instrumental analysis. However, monoclonal or polyclonal antibodies for immunoaffinity purification may not be commercially available, and even if available, such antibodies are usually very costly. In our study, a simple mixed-mode anion exchange solid-phase extraction cartridge was employed for the extraction of seven target peptides (GHRP-1, GHRP-2, GHRP-6, ipamorelin, hexarelin, CJC-1295, and N-acetylated LKKTETQ (active ingredient of TB-500)) and their in vitro metabolites from horse plasma. The final extract was subject to ultra-high-performance liquid chromatographic separation and analysed with a hybrid high-resolution mass spectrometer. The limits of detection for all seven peptides were estimated to be less than 50 pg/mL. Method validation was performed with respect to specificity, precision, and recovery. The applicability of this multi-analyte method was demonstrated by the detection of N-acetylated LKKTETQ and its metabolite N-acetylated LK from plasma samples obtained after subcutaneous administration of TB-500 (10 mg N-acetylated LKKTETQ) to two thoroughbred geldings. This method could easily be modified to cover more bioactive peptides, such as dermorphin, β-casomorphin, and desmopressin. With the use of high-resolution mass spectrometry, the full-scan data acquired can also be re-processed retrospectively to search for peptides and their metabolites that have not been targeted at the time of analysis. To our knowledge, this is the first identification of in vitro metabolites of all the studied peptides other than TB-500 in horses. Figure Product-ion scans and mass spectral assignments of the fragment ions of the seven target peptides

Keywords: Bioactive peptides; Horse plasma; Doping control; Liquid chromatography–mass spectrometry.

Quantification of 31 illicit and medicinal drugs and metabolites in whole blood by fully automated solid-phase extraction and ultra-performance liquid chromatography–tandem mass spectrometry by Marie Kjærgaard Bjørk; Kirsten Wiese Simonsen; David Wederkinck Andersen; Petur Weihe Dalsgaard; Stella Rögn Sigurðardóttir; Kristian Linnet; Brian Schou Rasmussen (pp. 2607-2617).

An efficient method for analyzing illegal and medicinal drugs in whole blood using fully automated sample preparation and short ultra-high-performance liquid chromatography–tandem mass spectrometry (MS/MS) run time is presented. A selection of 31 drugs, including amphetamines, cocaine, opioids, and benzodiazepines, was used. In order to increase the efficiency of routine analysis, a robotic system based on automated liquid handling and capable of handling all unit operation for sample preparation was built on a Freedom Evo 200 platform with several add-ons from Tecan and third-party vendors. Solid-phase extraction was performed using Strata X-C plates. Extraction time for 96 samples was less than 3 h. Chromatography was performed using an ACQUITY UPLC system (Waters Corporation, Milford, USA). Analytes were separated on a 100 mm × 2.1 mm, 1.7 μm Acquity UPLC CSH C₁₈ column using a 6.5 min 0.1 % ammonia (25 %) in water/0.1 % ammonia (25 %) in methanol gradient and quantified by MS/MS (Waters Quattro Premier XE) in multiple-reaction monitoring mode. Full validation, including linearity, precision and trueness, matrix effect, ion suppression/enhancement of co-eluting analytes, recovery, and specificity, was performed. The method was employed successfully in the laboratory and used for routine analysis of forensic material. In combination with tetrahydrocannabinol analysis, the method covered 96 % of cases involving driving under the influence of drugs. The manual labor involved in preparing blood samples, solvents, etc., was reduced to a half an hour per batch. The automated sample preparation setup also minimized human exposure to hazardous materials, provided highly improved ergonomics, and eliminated manual pipetting. Figure Robotic setup for fully automated solid-phase extraction of whole blood

Keywords: SPE; Tecan robot; Automation; Whole blood; Illicit and medicinal drugs; UPLC-MS/MS

In vivo and in vitro metabolism of a novel β₂-adrenoceptor agonist, trantinterol: metabolites isolation and identification by LC-MS/MS and NMR by Kunjie Li; Feng Qin; Lijuan Jing; Famei Li; Xingjie Guo (pp. 2619-2634).

Trantinterol is a novel β₂-adrenoceptor agonist used for the treatment of asthma. The aim of this study is to identify the metabolites of trantinterol using liquid chromatography tandem mass spectrometry (LC-MS/MS), to isolate the main metabolites, and confirm their structures by nuclear magnetic resonance (NMR). Urine, feces, bile, and blood samples of rats were obtained and analyzed. Reference standards of six metabolites were achieved with the combination of chemical synthesis, microbial transformation, and the model systems of rats. Moreover, in order to investigate the phase I metabolism of trantinterol in humans and to study the species differences between rats and humans, incubations with liver microsomes were performed. The biotransformation by a microbial model Cunninghamella blakesleana AS 3.970 was also studied. A total of 18 metabolites were identified in vivo and in vitro together, 13 of which were newly detected. Three phase I metabolites were detected in vivo and in vitro as well as in the microbial model, including the arylhydroxylamine (M1), the tert-butyl hydroxylated trantinterol (M2) and the 1-carbonyltrantinterol (M3). Another important pathway in rats is glutathione conjugation and further catabolism and oxidation to form consecutive derivatives (M4 through M10). Other metabolites include glucuronide, glucoside, and sulfate conjugates. The results of in vitro experiments indicate no species difference exists among rats, humans, and C. blakesleana AS 3.970 on the phase I metabolism of trantinterol. Our study provided the most comprehensive picture for trantinterol in vivo and in vitro metabolism to this day, and may predict its metabolism in humans.

Keywords: Trantinterol; Metabolism; Mass spectrometry; Nuclear magnetic resonance; In vitro; In vivo

Ratios of biliary glutathione disulfide (GSSG) to glutathione (GSH): a potential index to screen drug-induced hepatic oxidative stress in rats and mice by Lei Cao; Daniel Waldon; Yohannes Teffera; John Roberts; Mary Wells; Meghan Langley; Zhiyang Zhao (pp. 2635-2642).

Hepatotoxicity of drug candidates is one of the major concerns in drug screening in early drug discovery. Detection of hepatic oxidative stress can be an early indicator of hepatotoxicity and benefits drug selection. The glutathione (GSH) and glutathione disulfide (GSSG) pair, as one of the major intracellular redox regulating couples, plays an important role in protecting cells from oxidative stress that is caused by imbalance between prooxidants and antioxidants. The quantitative determination of the GSSG/GSH ratios and the concentrations of GSH and GSSG have been used to indicate oxidative stress in cells and tissues. In this study, we tested the possibility of using the biliary GSSG/GSH ratios as a biomarker to reflect hepatic oxidative stress and drug toxicity. Four compounds that are known to alter GSH and GSSG levels were tested in this study. Diquat (diquat dibromide monohydrate) and acetaminophen were administered to rats. Paraquat and tert-butyl hydroperoxide were administered to mice to induce changes of biliary GSH and GSSG. The biliary GSH and GSSG were quantified using calibration curves prepared with artificial bile to account for any bile matrix effect in the LC–MS analysis and to avoid the interference of endogenous GSH and GSSG. With four examples (in rats and mice) of drug-induced changes in the kinetics of the biliary GSSG/GSH ratios, this study showed the potential for developing an exposure response index based on biliary GSSG/GSH ratios for predicting hepatic oxidative stress.

Keywords: Biliary; GSH/GSSG; Oxidative stress; Hepatotoxicity; LC–MS; Quantitation

Determination of isoflavones in rat serum using liquid chromatography–tandem mass spectrometry with a highly efficient core–shell column by Jennilee M. A. Gavina; Jessica Priem; Carla M. Wood; Chao Wu Xiao; Yong-Lai Feng (pp. 2643-2651).

Consumption and nutritional supplementation of soy and soy-based products have been linked to health benefits such as lower cholesterol and triglyceride levels, and decreased incidence of cardiovascular disease and diabetes. In this study, we have developed a sensitive, specific, and robust method using high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) for determination of serum isoflavones. A new highly efficient pentafluorophenyl phase core–shell column was first used to separate all isoflavones within 3 min, a separation time which is comparable to ultra-pressure liquid chromatography (UPLC) and micro-HPLC. A two-enzyme hydrolysis system with sulfatase and β-glucuronidase has also been developed to improve the efficiency of deconjugation of conjugated isoflavones in serum. The corresponding conjugated isoflavones were used to evaluate recoveries. In addition to duplicates, the method of standard addition was also applied in sample analysis for quality control. The developed method was applied to the analysis of 32 serum samples and was shown to be specific, sensitive and reproducible.

Keywords: Isoflavones; Liquid chromatography–tandem mass spectrometry (HPLC-MS/MS); Core–shell column; Dual enzyme hydrolysis; Serum

A sensitive and specific ELISA for determining a residue marker of three quinoxaline antibiotics in swine liver by Linli Cheng; Jianzhong Shen; Zhanhui Wang; Wenxiao Jiang; Suxia Zhang (pp. 2653-2659).

Methyl-3-quinoxaline-2-carboxylic acid (MQCA) is a possible residue marker for three quinoxaline veterinary medicines (olaquindox, mequindox, and quinocetone). The wide application of mequindox/quinocetone or the illegal use of olaquindox leads to MQCA residue in animal’s original food, thereby threatening the safety of human food. The indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) with a specific coating antigen and monoclonal antibody (MAB) was established and optimized for detecting MQCA in swine liver. Samples were acidified with 2 mol l⁻¹ hydrochloric acid, extracted with ethyl acetate–hexane–isopropanol (8 + 1 + 1, v/v/v) and then detected by IC-ELISA. The logarithm correlation of standards to OD values ranged from 0.2 to 200 μg l⁻¹, with IC₅₀ of 6.46 μg l⁻¹. Negligible cross-reactivity happened to five quinoxaline antibiotics (olaquindox, mequindox, quinocetone, carbadox, and cyadox) and the metabolite of carbadox and cyadox (quinoxaline-2-carboxylic acid). When spiked with 1 to 100 μg kg⁻¹ of MQCA, the recoveries ranged from 85.44 to 100.02 %, with the intra-assay coefficient of variation (CV) of 6.64–10.57 % and inter-assay CV of 7.29–10.88 %. The limit of detection for MQCA was 1.0 μg kg⁻¹ in swine liver. Furthermore, incurred samples were detected by the IC-ELISA and then conformed by a reported LC/MS/MS method, it shown that there was good correlation between the two methods. All these results indicated that the IC-ELISA method is appropriate for surveillance MQCA residue in animal tissues. Figure Synthesis route of 2-acrylic-1,4-binitrogen-quinoline combined to BSA(OVA) by active ester method

Keywords: Monoclonal antibody; Methyl-3-quinoxaline-2-carboxylic acid; ELISA; Swine liver

Oxidized single-walled carbon nanohorns as sorbent for porous hollow fiber direct immersion solid-phase microextraction for the determination of triazines in waters by Juan Manuel Jiménez-Soto; Soledad Cárdenas; Miguel Valcárcel (pp. 2661-2669).

This paper evaluates the potential of oxidized single-walled carbon nanohorns (o-SWNHs) immobilized on the pores of a hollow fiber (HF) for the direct immersion solid-phase microextraction of triazines from waters. The fabrication of the device requires the oxidation of the nanoparticles by means of microwave irradiation in order to obtain a homogeneous dispersion in methanol. Then, a porous hollow fiber is immersed in the methanolic dispersion of the o-SWNHs under ultrasound stirring. This procedure permits the immobilization of the o-SWNHs in the pores of the hollow fiber. For the extraction, a stainless steel wire was introduced inside the fiber to allow the vertical immersion of the o-SWNHs-HF in the aqueous standard/water sample. The triazines were preconcentrated on the immobilized o-SWNHs and further eluted using 150 μL of methanol. The solvent was evaporated and the residue reconstituted in 10 μL of methanol for sensitivity enhancement. Gas chromatography–mass spectrometry was selected as instrumental technique. The limits of detection were between 0.05 and 0.1 μg L⁻¹ with an excellent precision (expressed as relative standard deviation) between runs (below 10.2 %) and between fibers (below 12.8 %). Finally, the method was applied to the determination of the triazines in fortified waters, an average recovery value of 90 % being obtained.

Keywords: Oxidized single-walled carbon nanohorns; Hollow fiber; Solid-phase microextraction; Triazines; Waters

Mass spectrometry-based metabolomic fingerprinting for screening cold tolerance in Arabidopsis thaliana accessions by Lukas Vaclavik; Anamika Mishra; Kumud B. Mishra; Jana Hajslova (pp. 2671-2683).

The availability of rapid and reliable tools for monitoring of plants’ cold tolerance is a prerequisite for research aimed at breeding of cold-tolerant crop plants. Therefore, we have tested the capacity of metabolomics-based methods employing ultra-high-performance liquid chromatography (UHPLC)–mass spectrometry and direct analysis in real time–mass spectrometry for high-throughput screening of cold tolerance in eight differentially cold-tolerant accessions of Arabidopsis thaliana. Metabolomic fingerprinting of leaf tissues was performed in methanolic extracts for (1) 6-week-old non-acclimated (NAC) plants grown at room temperature, (2) NAC plants cold-acclimated (ACC) at 4 °C for 2 weeks, and (3) cold-acclimated plants given sub-zero-temperature treatments by slow cooling at −4 °C for 8 h. The generated chromatograms and mass spectra were processed with the use of multivariate statistical analysis employing principal component analysis (PCA) and linear discriminant analysis. The PCA of metabolomic fingerprints classified the investigated A. thaliana accessions into three categories with low, intermediate, and high cold tolerance for both the cold-acclimated and the sub-zero-temperature-treated plants. This indicates the potential application of metabolomics-based fingerprinting for measuring cold tolerance in the cold-acclimated state, i.e., without treating plants at freezing temperatures that is required by currently available methods. Furthermore, we employed UHPLC coupled to the quadrupole-time-of-flight mass spectrometry to identify characteristic metabolites in ACC state and found the abundance of gluconapin and flavon-3-ol glycosides, respectively, in the cold-sensitive and the cold-tolerant accessions.

Keywords: Cold tolerance; Arabidopsis thaliana ; Metabolomic fingerprinting; LC–MS; DART–MS; Chemometric analysis

Synchrotron FTIR microspectroscopy of Escherichia coli at single-cell scale under silver-induced stress conditions by Claire Saulou; Frédéric Jamme; Laurence Girbal; Claude Maranges; Isabelle Fourquaux; Muriel Cocaign-Bousquet; Paul Dumas; Muriel Mercier-Bonin (pp. 2685-2697).

The present work was focused on elucidating biochemical changes in the model bacterium Escherichia coli exposed to ionic silver mediated stress, at a single-cell scale. In order to achieve this, in situ synchrotron Fourier-transform infrared (sFTIR) microspectroscopy was performed, for the first time, on individual cells by attenuated total reflectance (ATR) combined with the use of zinc-selenide hemisphere for high spatial resolution. In a first part, the potential of the method was evaluated on bacteria subjected to a lethal 100 μM AgNO₃ concentration for 2 h compared to untreated 100 % viable cells. Differences in cell composition were assessed for the C–H stretching and protein spectral regions, indicating that the inhibitory action was targeted against both fatty acids and proteins. Transmission electron microscopy (TEM) confirmed morphological damages of the cell ultrastructure. The relevance of ATR-sFTIR microspectroscopy for highlighting the heterogeneity in Ag⁺-mediated effects within a given bacterial population was also pointed out. In a second part, cells were exposed to sub-lethal Ag⁺ concentrations (<10 μM AgNO₃) tested under “dynamic” growth mode: early addition vs. pulse in the mid-exponential phase, and compared to simultaneously batch-grown untreated bacteria or cells sampled just before the pulse, respectively. sFTIR microspectroscopy and TEM imaging were performed in close relation with growth kinetics characterization. No significant effect of the Ag⁺ pulses was detected, in accordance with macrokinetics data. For early-treated cells, effects on fatty acid composition were shown, although no major alteration of protein secondary structure was noticed. These partial effects were consistent with TEM observations and growth kinetics. Figure a FTIR raw spectra in the 4,000–800-cm⁻¹ region recorded at a single-cell scale on Escherichia coli viable (V) and dead (D) cells after a lethal 2-h exposure to 100 μM AgNO₃. b Microscopic image of E. coli cells (group D) deposited on a zinc-selenide hemisphere and analysed by in situ synchrotron FTIR microspectroscopy performed by attenuated total reflectance (sFTIR-ATR)

Keywords: Ionic silver; Escherichia coli ; Antibacterial effect; Synchrotron FTIR-ATR microspectroscopy; Single-cell scale

In-situ identification of copper-based green pigments on paintings and manuscripts by reflection FTIR by D. Buti; F. Rosi; B. G. Brunetti; C. Miliani (pp. 2699-2711).

In this work non-invasive reflection infrared spectroscopy was used to discriminate between different Cu-based green pigments (namely, malachite, verdigris, chrysocolla, emerald green and phthalo green). The pigments, chosen because of their historical widespread use in artworks, were investigated as pure powders and in situ on painted models by reflection FTIR spectroscopy. The distortion arising as a result of the specular and diffuse component of reflected radiation was evaluated as function of the optical and surface properties of the investigated mock-ups. Use of the Kramers–Kronig (KK) algorithm to correct for the distortion arising from the surface reflection gave k index maxima shifted by more than 20 cm⁻¹ relative to those obtained in conventional transmission mode. These findings stress the need to carefully manage use of the KK algorithm on reflection spectra to avoid erroneous assignment. On the other hand, combination and overtone bands (which are enhanced by the diffuse reflection) were proved to enable reliable and sensitive identification of most of the pigments studied. Knowledge acquired by study of pure compounds and painted models was subsequently applied to interpret the spectra acquired from paintings and manuscripts.

Keywords: Cu-based green pigments; Mid and near IR reflection; Kramers–Kronig conversion; Combination and overtone bands; Non-invasive analysis

Non-destructive spectroscopic investigation on historic Yemenite scriptorial fragments: evidence of different degradation and recipes for iron tannic inks by Marina Bicchieri; Michela Monti; Giovanna Piantanida; Armida Sodo (pp. 2713-2721).

Six fragments of different Yemenite manuscripts (three on parchment and three on paper) were analyzed by means of attenuated total reflectance Fourier transform infrared, micro-Raman and X-ray fluorescence spectroscopies. The combination of molecular and elemental techniques allowed the characterization of the conservation state of all the fragments, the identification of sizing agents and salts on the supports and the identification of the composition all red and black writing media. In particular, analysis of black inks provided interesting insights: independent of the substrate, all inks seemed to have identical composition, corresponding to rather well preserved high-quality iron tannic inks to which carbon black had not been added. However, in some samples, the most intense Raman peak of the ink was clearly shifted with respect to the typical bands of iron gall ink. Starting from the hypothesis that the shift could have been produced by the use of sources of tannin other than gallnuts, research was undertaken by preparing and characterizing seven different tannic inks. The experimental results confirmed the hypothesis of different tannin sources.

Keywords: Micro-Raman; Attenuated total reflectance Fourier transform infrared; X-ray fluorescence; Tannic inks

Spectroscopic studies on bilirubin aggregate at liquid/liquid interface by Zhi-Yan Xiao; Jian-Hua Yin (pp. 2723-2728).

Bilirubin (BR) aggregating at liquid/liquid interface was firstly detected by Fourier transform infrared (FTIR) imaging/spectroscopy combining with ultraviolet-visible (UV/Vis) absorption spectra. In the UV/Vis absorption spectra of BR aggregate, a new shoulder appeared at 474 nm, and BR absorption maximum underwent red shift from 450 nm to a longer wavelength at 497 nm, which indicates that BR aggregate was formed at the interface. Meanwhile, the BR molecule structure changed or conformation torsion, that is, the increase in orbit overlap or dihedral angle and the enhancement of exciton coupling. In the study of FTIR imaging/spectroscopy, the hydrogen bond-sensitive infrared bands of BR aggregate showed remarkable changes in band shift and intensity compared with those of BR powder, suggesting that the intramolecular hydrogen bonds broke out and internal structure changed. These new findings will be helpful for understanding of the BR molecular interaction, transportation, complex with serum albumin and metal ions, and the effect of BR aggregating on biomembrane and human tissues. Figure Schematic representation of the FTIR spectroscopic imaging for interfacial BR aggregation

Keywords: Bilirubin; Molecule aggregate; Infrared imaging/spectroscopy; Liquid/liquid interface

Magnetically confined hydrophobic nanoparticles for the microextraction of endocrine-disrupting phenols from environmental waters by M. C. Alcudia-León; R. Lucena; S. Cárdenas; M. Valcárcel (pp. 2729-2734).

In this article, a solid-phase extraction approach, which takes advantage of the good extraction capabilities of hydrophobic magnetic nanoparticles (MNPs), is presented. The new approach involves the deposition of a thin layer of MNPs in a dedicated stirring unit based on the dual function of a mini-magnet. The system allows the extraction of the analytes in a simple and efficient way. The approach, which reduces the negative effect of the aggregation tendency of hydrophobic MNPs, is characterized for the resolution of a model analytical problem: the determination of some endocrine-disrupting phenols in water by liquid chromatography–photometric detection. All the variables involved in the extraction process have been clearly identified and optimized. The new extraction mode allows the determination of these compounds with limits of detection in the range from 0.15 μg/L (for 4-tert-octylphenol) to 2.7 μg/L (for 4-tert-butylphenol) with a relative standard deviation lower than 5.3 % (for 4-tert-butylphenol).

Keywords: Magnetically confined nanoparticles; Octadecyl groups; Dispersive solid-phase extraction; Extraction–stirring integration; Endocrine-disrupting phenols; Waters

Metal labeling for accurate multiplexed peptide quantification via matrix-assisted laser desorption/ionization mass spectrometry by Barbara Gregorius; Thomas Jakoby; Dirk Schaumlöffel; Andreas Tholey (pp. 2735-2741).

Two peptide quantification strategies, the isobaric tags for relative or absolute quantitation (iTRAQ) labeling methodology and a metal-chelate labeling approach, were compared using matrix-assisted laser desorption/ionization-TOF/TOF MS and MS/MS analysis. Amino- and cysteine-directed labeling using the rare earth metal chelator 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) were applied for relative quantification of single peptides and a six-protein mixture. For analyte ratios close to one, iTRAQ and amino-directed DOTA labeling delivered overall comparable results regarding accuracy and reproducibility. In contrast, the MS-based quantification via amino-directed lanthanide-DOTA tags was more accurate for analyte ratios ≥5 and offered an extended dynamic range of three orders of magnitude. Our results show that the amino-directed DOTA labeling is an alternative relative quantification tool offering advantages like flexible multiplexing possibilities and, in particular, large dynamic ranges, which should be useful in sophisticated, targeted issues, where the accurate determination of extremely different protein or peptide concentration becomes relevant.

Keywords: Relative peptide quantification; Quantitative MALDI; DOTA-NHS-ester; Lanthanides

Quartz microfluidic chip for tumour cell identification by Raman spectroscopy in combination with optical traps by Sebastian Dochow; Claudia Beleites; Thomas Henkel; Günter Mayer; Jens Albert; Joachim Clement; Christoph Krafft; Jürgen Popp (pp. 2743-2746).

Three important technical innovations are reported here towards Raman-activated cell sorting. Firstly, a microfluidic chip made of quartz is introduced which integrates injection of single cells, trapping by laser fibres and sorting of cells. Secondly, a chip holder was designed to provide simple, accurate and stable adjustment of chips, microfluidic connections and the trapping laser fibres. The new setup enables to the collection of Raman spectra of single cells at 785 nm excitation with 10 s exposure time. Lastly, a new type of modelling the various background contributions is described, improving Raman-based cell identification by the classification algorithm linear discriminant analysis. Mean sensitivity and specificity determined by iterated 10-fold cross validation were 96 and 99 %, respectively, for the distinction of leucocytes extracted from blood, breast cancer cells BT-20 and MCF-7, and leukaemia cells OCI-AML3.

Keywords: Chemometrics/statistics; IR spectroscopy/Raman spectroscopy; Microfluidics/microfabrication; Single cell analysis; Flow cytometry

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Analytical and Bioanalytical Chemistry (v.405, #8)

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Analytical and Bioanalytical Chemistry (v.405, #8)