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Analytical and Bioanalytical Chemistry (v.402, #8)


Analytical science in Switzerland and ANAKON 2011 by Petra Dittrich; Detlef Günther; Gérard Hopfgartner; Renato Zenobi (pp. 2483-2484).
has been Assistant Professor for Bioanalysis in the Department of Chemistry and Applied Biosciences since July 2008. Her research on lab-on-a-chip technology focuses on miniaturization of high-sensitivity devices for chemical and biological analysis, and on microfluidics-aided organization of materials. She earned her PhD in 2003 at the Max Planck Institute for Biophysical Chemistry (MPI Göttingen, Germany). After another year at MPI Göttingen, as a postdoctoral fellow, from 2004–2008 she worked with Andreas Manz at the Institute for Analytical Sciences (ISAS Dortmund, Germany), where she developed microfluidic techniques for cell analysis and lipid vesicle formation. She has also performed research at Cornell University (2002) and the University of Tokyo (2005). In 2008 she obtained an ERC Starting Grant for research on biomimetic membrane formation and on micro and nanofluidic manipulation. She was awarded the “Analytica Forschungspreis” in 2010. is Full Professor for Trace Element and Micro Analysis in the Laboratory of Inorganic Chemistry at ETH Zurich and is currently Chair of the Department for Chemistry and Applied Biosciences. His research program focuses on fundamental and applied studies in inductively coupled plasma–mass spectrometry (ICP–MS) and laser ablation–inductively coupled plasma–mass spectrometry (LA–ICP–MS), which includes studies on laser–sample interaction, aerosol transport, and plasma-related excitation processes. His fundamental understanding of UV-ns and UV-fs laser ablation in combination with Q–ICP–MS, SF–ICP–MS, ICP–TOFMS, and also MC–ICP–MS, and alternative excitation sources has been demonstrated in a wide variety of applications, e.g., analysis of fluid inclusions, gemstones, metals, minerals, ceramics, and other industrial materials. is Full Professor of Analytical Pharmaceutical Chemistry and Mass Spectrometry in the Laboratory of Life Sciences Mass Spectrometry at the School of Pharmaceutical Sciences of the University of Lausanne and Geneva. His scientific interests focus on the development and application of novel mass spectrometry approaches (from QqQLIT to FT-ICR, including ion mobility) with and without chromatography, in the life sciences. His research is based on novel qualitative and quantitative methods for low-molecular-weight compounds, peptides, and proteins in analytical metabolomics, proteomics, mass spectrometry imaging, drug metabolism, and toxicology. is Professor of Analytical Chemistry at the Organic Chemistry Laboratory of the Swiss Federal Institute of Technology (ETH), Zurich. He was also one of the founders and directors of the center of excellence in analytical chemistry (CEAC, 1995–2007) at ETH Zurich. In 2010 he was appointed Associate Editor of Analytical Chemistry (American Chemical Society). Zenobi’s research includes laser-based analytical chemistry, electrospray and laser-assisted mass spectrometry, laser–surface interactions, and near-field optical microscopy and spectroscopy. He has made important contributions to understanding the mechanism of ion formation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, to ambient ionization methods, and to the development of tools for nanoscale analysis. Renato Zenobi has received many honors for his scientific work, among them the Ruzicka Prize and the Heinrich Emanuel Merck Prize, and honorary professorships from several places including the Chinese Academy of Sciences (Changchun).

Direct analysis of dried blood spots coupled with mass spectrometry: concepts and biomedical applications by Julien Déglon; Aurélien Thomas; Patrice Mangin; Christian Staub (pp. 2485-2498).
Because of the emergence of dried blood spots (DBS) as an attractive alternative to conventional venous plasma sampling in many pharmaceutical companies and clinical laboratories, different analytical approaches have been developed to enable automated handling of DBS samples without any pretreatment. Associated with selective and sensitive MS–MS detection, these procedures give good results in the rapid identification and quantification of drugs (generally less than 3 min total run time), which is desirable because of the high throughput requirements of analytical laboratories. The objective of this review is to describe the analytical concepts of current direct DBS techniques and to present their advantages and disadvantages, with particular focus on automation capacity and commercial availability. Finally, an overview of the different biomedical applications in which these concepts could be of major interest will be presented. Figure Direct analysis of dried blood spots

Keywords: Dried blood spots; Direct LC–MS–MS analysis; Ambient MS; Automated on-line DBS


Wipe sampling procedure coupled to LC–MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces by Susanne Nussbaumer; Laurent Geiser; Farshid Sadeghipour; Denis Hochstrasser; Pascal Bonnabry; Jean-Luc Veuthey; Sandrine Fleury-Souverain (pp. 2499-2509).
A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC–MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm−2) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy. Figure Wipe sampling procedure for the determination of cytotoxic drugs

Keywords: Cytotoxic; Antineoplastic drugs; Surface contamination; Environmental monitoring; LC–MS/MS; Wipe sampling


What determines MALDI ion yields? A molecular dynamics study of ion loss mechanisms by Richard Knochenmuss; Leonid V. Zhigilei (pp. 2511-2519).
Ion recombination in matrix-assisted laser desorption/ionization (MALDI) is as important as any ion formation process in determining the quantity of ions observed but has received comparatively little attention. Molecular dynamics simulations are used here to investigate some models for recombination, including a Langevin-type model, a soft threshold model and a tunneling model. The latter was found to be superior due to its foundations in a widespread physical phenomenon, and its lack of excessive sensitivity to parameter choice. Tunneling recombination in the Marcus inverted region may be a major reason why MALDI is a viable analytical method, by allowing ion formation to exceed ion loss on the time scale of the plume expansion. Ion velocities, photoacoustic transients and pump-probe measurements might be used to investigate the role of recombination in different MALDI matrices, and to select new matrices. Figure Ablation of a thin MALDI sample as simulated by molecular dynamics. Yellow represents high density, black is low. The laser pulse was incident on the right side

Keywords: MALDI ionisation; Recombination; Molecular dynamics


On the utility of predictive chromatography to complement mass spectrometry based intact protein identification by Marina L. Pridatchenko; Tatyana Yu. Perlova; Hisham Ben Hamidane; Anton A. Goloborodko; Irina A. Tarasova; Alexander V. Gorshkov; Victor V. Evreinov; Yury O. Tsybin; Mikhail V. Gorshkov (pp. 2521-2529).
The amino acid sequence determines the individual protein three-dimensional structure and its functioning in an organism. Therefore, “reading” a protein sequence and determining its changes due to mutations or post-translational modifications is one of the objectives of proteomic experiments. The commonly utilized approach is gradient high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry. While serving as a way to simplify the protein mixture, the liquid chromatography may be an additional analytical tool providing complementary information about the protein structure. Previous attempts to develop “predictive” HPLC for large biomacromolecules were limited by empirically derived equations based purely on the adsorption mechanisms of the retention and applicable to relatively small polypeptide molecules. A mechanism of the large biomacromolecule retention in reversed-phase gradient HPLC was described recently in thermodynamics terms by the analytical model of liquid chromatography at critical conditions (BioLCCC). In this work, we applied the BioLCCC model to predict retention of the intact proteins as well as their large proteolytic peptides separated under different HPLC conditions. The specific aim of these proof-of-principle studies was to demonstrate the feasibility of using “predictive” HPLC as a complementary tool to support the analysis of identified intact proteins in top-down, middle-down, and/or targeted selected reaction monitoring (SRM)-based proteomic experiments. Figure Intact protein LC retention time prediction assists protein identification in top- and middle-down proteomics

Keywords: Proteins; Liquid chromatography; Retention time prediction; Mass spectrometry; Sequence variants


Online monitoring of coffee roasting by proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS): towards a real-time process control for a consistent roast profile by Flurin Wieland; Alexia N. Gloess; Marco Keller; Andreas Wetzel; Stefan Schenker; Chahan Yeretzian (pp. 2531-2543).
A real-time automated process control tool for coffee roasting is presented to consistently and accurately achieve a targeted roast degree. It is based on the online monitoring of volatile organic compounds (VOC) in the off-gas of a drum roaster by proton transfer reaction time-of-flight mass spectrometry at a high time (1 Hz) and mass resolution (5,500 m/Δm at full width at half-maximum) and high sensitivity (better than parts per billion by volume). Forty-two roasting experiments were performed with the drum roaster being operated either on a low, medium or high hot-air inlet temperature (= energy input) and the coffee (Arabica from Antigua, Guatemala) being roasted to low, medium or dark roast degrees. A principal component analysis (PCA) discriminated, for each one of the three hot-air inlet temperatures, the roast degree with a resolution of better than ±1 Colorette. The 3D space of the three first principal components was defined based on 23 mass spectral profiles of VOCs and their roast degree at the end point of roasting. This provided a very detailed picture of the evolution of the roasting process and allowed establishment of a predictive model that projects the online-monitored VOC profile of the roaster off-gas in real time onto the PCA space defined by the calibration process and, ultimately, to control the coffee roasting process so as to achieve a target roast degree and a consistent roasting. Figure Online monitoring of coffee roasting by real-time analysis of the roaster off-gas using PTR-ToF-MS. In a first phase, 42 calibration experiments were conducted at three different roasting temperatures and to three final roast degrees, to generate the 3D space defined by the three first principle components PC1, PC2 and PC3. Inverted triangles mark the dark roast degree, square medium and circle light, respectively. The hot-air inlet temperature is marked as follows: high (black), medium (grey), low (white). The different hot-air inlet temperatures and roast degrees are clearly separated. In a second phase, an online monitored PTR-ToF-MS spectrum of a roasting process was projected onto the 3D space, allowing following in real-time the roasting process and halting the roasting with a precision better that ± 1 Colorette roast degree.

Keywords: Process analysis; Foods; Beverages; Gas sensors; Quality assurance/control; Sampling; Agriculture


Measuring multiple neurochemicals and related metabolites in blood and brain of the rhesus monkey by using dual microdialysis sampling and capillary hydrophilic interaction chromatography–mass spectrometry by Juan Li; Veronika von Pföstl; Daniel Zaldivar; Xiaozhe Zhang; Nikos Logothetis; Alexander Rauch (pp. 2545-2554).
In vivo measurement of multiple functionally related neurochemicals and metabolites (NMs) is highly interesting but remains challenging in the field of basic neuroscience and clinical research. We present here an analytical method for determining five functionally and metabolically related polar substances, including acetylcholine (quaternary ammonium), lactate and pyruvate (organic acids), as well as glutamine and glutamate (amino acids). These NMs are acquired from samples of the brain and the blood of non-human primates in parallel by dual microdialysis, and subsequently analyzed by a direct capillary hydrophilic interaction chromatography (HILIC)–mass spectrometry (MS) based method. To obtain high sensitivity in electrospray ionization (ESI)–MS, lactate and pyruvate were detected in negative ionization mode whereas the other NMs were detected in positive ionization mode during each HILIC-MS run. The method was validated for linearity, the limits of detection and quantification, precision, accuracy, stability and matrix effect. The detection limit of acetylcholine, lactate, pyruvate, glutamine, and glutamate was 150 pM, 3 μM, 2 μM, 5 nM, and 50 nM, respectively. This allowed us to quantitatively and simultaneously measure the concentrations of all the substances from the acquired dialysates. The concentration ratios of both lactate/pyruvate and glutamine/glutamate were found to be higher in the brain compared to blood (p < 0.05). The reliable and simultaneous quantification of these five NMs from brain and blood samples allows us to investigate their relative distribution in the brain and blood, and most importantly paves the way for future non-invasive studies of the functional and metabolic relation of these substances to each other. Figure Measuring multiple polar multiple neurochemicals and related metabolites using HILIC-ESI/MS in combination with dual brain and blood sampling

Keywords: Neurochemicals; HILIC-MS; Microdialysis; Rhesus monkey; Brain; Blood


Real-time 2D separation by LC × differential ion mobility hyphenated to mass spectrometry by Emmanuel Varesio; J. C. Yves Le Blanc; Gérard Hopfgartner (pp. 2555-2564).
The liquid chromatography–mass spectrometry (LC-MS) analysis of complex samples such as biological fluid extracts is widespread when searching for new biomarkers as in metabolomics. The success of this hyphenation resides in the orthogonality of both separation techniques. However, there are frequent cases where compounds are co-eluting and the resolving power of mass spectrometry (MS) is not sufficient (e.g., isobaric compounds and interfering isotopic clusters). Different strategies are discussed to solve these cases and a mixture of eight compounds (i.e., bromazepam, chlorprothixene, clonapzepam, fendiline, flusilazol, oxfendazole, oxycodone, and pamaquine) with identical nominal mass (i.e., m/z 316) is taken to illustrate them. Among the different approaches, high-resolution mass spectrometry or liquid chromatography (i.e., UHPLC) can easily separate these compounds. Another technique, mostly used with low resolving power MS analyzers, is differential ion mobility spectrometry (DMS), where analytes are gas-phase separated according to their size-to-charge ratio. Detailed investigations of the addition of different polar modifiers (i.e., methanol, ethanol, and isopropanol) into the transport gas (nitrogen) to enhance the peak capacity of the technique were carried out. Finally, a complex urine sample fortified with 36 compounds of various chemical properties was analyzed by real-time 2D separation LC×DMS-MS(/MS). The addition of this orthogonal gas-phase separation technique in the LC-MS(/MS) hyphenation greatly improved data quality by resolving composite MS/MS spectra, which is mandatory in metabolomics when performing database generation and search.

Keywords: Real-time 2D separation; Differential ion mobility spectrometry; Liquid chromatography; Mass spectrometry


Tunable fragmentation of organic molecules in laser ablation glow discharge time-of-flight mass spectrometry by G. Lotito; D. Günther (pp. 2565-2576).
A DC-pulsed glow discharge (GD) has distinct temporal regimes which are characterized by “softer” or “harder” ionization of analytes introduced into the discharge. It is thus possible to obtain both molecular weight and structural fragment information from the same spectra. In order to extend the capabilities of this technique a laser ablation (LA) sampling system was coupled to a DC-pulsed GD and to a time-of-flight (TOF) mass spectrometer (MS) for characterizing organic samples such as oleic acid, reserpine, two different peptides, and a polymer. Both hard and soft ionization regimes were studied. These LAGD-TOFMS results were compared to matrix-assisted laser desorption ionization (MALDI) spectra using the same compounds (i.e., analytes, concentration, and matrix). It was found that LAGD offers tunable ionization and provides a reduced matrix dependence. However, the sensitivity achieved by the prototype LAGD-TOFMS was significantly lower when compared with commercially available MALDI-TOFMS instrumentation. Since LAGD-TOFMS is rather new, some technical details to increase its sensitivity are discussed. Figure Schematics of molecular and structural information of oleic acid molecule during the different temporal regimes of a pulsed GD.

Keywords: Pulsed glow discharge; MALDI; Laser ablation; Fragmentation; Chemical speciation


Monitoring induced gene expression of single cells in a multilayer microchip by C. Hanke; S. Waide; R. Kettler; P. S. Dittrich (pp. 2577-2585).
We present a microfluidic system that facilitates long-term measurements of single cell response to external stimuli. The difficulty of addressing cells individually was overcome by using a two-layer microfluidic device. The top layer is designed for trapping and culturing of cells while the bottom layer is employed for supplying chemical compounds that can be transported towards the cells in defined concentrations and temporal sequences. A porous polyester membrane that supports transport and diffusion of compounds from below separates the microchannels of both layers. The performance and potential of the device are demonstrated using human embryonic kidney cells (HEK293) transfected with an inducible gene expression system. Expression of a fluorescent protein (ZsGreen1-DR) is observed while varying the concentration and exposure time of the inducer tetracycline. The study reveals the heterogeneous response of the cells as well as average responses of tens of cells that are analyzed in parallel. The microfluidic platform enables systematic studies under defined conditions and is a valuable tool for general single cell studies to obtain insights into mechanisms and kinetics that are not accessible by conventional macroscopic methods. Figure A two-layer microfluidic device is presented that facilitates measurements of single cell response to external stimuli

Keywords: Single cell analysis; Microfluidics; Multilayer soft lithography; Fluorescent protein; Gene expression


High-resolution mass spectrometry for integrated qualitative and quantitative analysis of pharmaceuticals in biological matrices by Gérard Hopfgartner; David Tonoli; Emmanuel Varesio (pp. 2587-2596).
Quantitative and qualitative high-resolution (HR) dependent and independent acquisition schemes on a QqTOF MS (with resolving power 20,000–40,000) were investigated for the analysis of pharmaceutical compounds in biological fluids. High-resolution selected reaction monitoring (HR-SRM) was found to be linear over three orders of magnitude for quantitative analysis of paracetamol in human plasma, offering a real alternative to triple quadrupole LC–SRM/MS. Metabolic stability of talinolol in microsomes was characterized by use of three different acquisition schemes: (i) information-dependent acquisition (IDA) with a TOF MS experiment as survey scan and product-ion scan as dependent scan; (ii) MSALL by collecting TOF mass spectra with and without fragmentation by alternating the collision energy of the collision cell between a low (i.e., 10 eV) and high setting (i.e., 40 eV); and (iii) a novel independent acquisition mode referred to as “sequential window acquisition of all theoretical fragment-ion spectra” (SWATH) or “global precursor ions scan mode” (GPS) in which sequential precursor ions windows (typically 20 u) are used to collect the same spectrum precursor and fragment ions using a collision energy range. SWATH or GPS was found to be superior to IDA or MSALL in combination with UHPLC for qualitative analysis but requires a rapidly acquiring mass spectrometer. Finally, the GPS concept was used for QUAL/QUAN analysis (i.e. integration of qualitative and quantitative analysis) of bosentan and its metabolites in urine over a concentration range from 5 to 2,500 ng mL−1.

Keywords: Mass spectrometry; High resolution; Fast chromatography; Metabolism; QUAL/QUAN


Double-pulse laser-induced breakdown spectroscopy for analysis of molten glass by A.-M. Matiaske; I. B. Gornushkin; U. Panne (pp. 2597-2606).
A mobile double-pulse laser-induced breakdown spectroscopy system for industrial environments is presented. Its capabilities as a process analytical technique for the recovery of metals from molten inorganic wastes are investigated. Using low-melting glass doped with different amounts of additives as a model system for recycling slags, the optimum number of shots, laser inter-pulse and acquisition delay times are optimized for solid and liquid (1200 °C) glass. Limits of detection from 7 ppm (Mn) to 194 ppm (Zn) are achieved working at a distance of 75 cm from the sample. To simplify the quantification of molten samples in an industrial furnace, the possibility is examined of using solid standards for analysis of molten material.

Keywords: Laser-induced breakdown spectroscopy, LIBS; Double-pulse LIBS; Molten glass; Recycling


Marker peptide selection for the determination of hazelnut by LC–MS/MS and occurrence in other nuts by Parisa Ansari; Norbert Stoppacher; Sabine Baumgartner (pp. 2607-2615).
The aim of this work was identifying and selecting hazelnut marker peptides and subsequently developing a complementary method of common immunoassay for the detection of hazelnut. For this purpose, at first, an in silico digestion of three major hazelnut allergens (Cor a 8, Cor a 9 and Cor a 11) was performed to get information about expected peptides. After extraction and trypsin digestion of hazelnut proteins, the samples were measured with tandem mass spectrometry (MS/MS) by direct infusion, which led to identification of 14 peptides. Eight of them with the highest MS signal were synthesized and used as standards for developing a liquid chromatography (LC)–MS/MS method in selected reaction monitoring (SRM) mode. Since almost all food allergens derived from nuts belong to the seed storage protein family and have homologue structure, a Basic Local Alignment Search Tool (BLAST) search was performed to identify the hazelnut specificity of the developed method. According to BLAST, only one peptide occurs in three other nuts, and the remaining seven selected peptides are hazelnut specific. Additionally to hazelnut, the eight other listed nuts in Directive 2003/89/EC as allergen were extracted, digested and measured with the developed method to prove the BLAST results. The analytical data confirmed that six peptides are hazelnut specific, on the contrary to anti-hazelnut antibodies, which showed cross-reactivities to all other nut extracts. Comparing these results, it could be shown that with this LC–MS/MS method in SRM mode, the specific detection of hazelnut is possible.

Keywords: Nut allergens; Cor a 8; Cor a 9; Cor a 11; Hazelnut; LC–MS/MS


Fast counter-electroosmotic capillary electrophoresis–time-of-flight mass spectrometry of hyaluronan oligosaccharides by Marco Grundmann; Martin Rothenhöfer; Günther Bernhardt; Armin Buschauer; Frank-Michael Matysik (pp. 2617-2623).
Fast capillary electrophoresis–mass spectrometry measurements under counter-electroosmotic analyte migration conditions are presented. Efficient separations of a homologous series of six hyaluronan oligosaccharides (comprising 1–6 hyalobiuronic acid moieties) could be completed in 65 s. Separations were achieved in short-length fused silica capillaries under high electric field strengths of up to 1.25 kV·cm−1. Capillary inner diameters ranging from 5 to 50 μm were investigated, resulting in an optimal value of 15 μm. The influence of capillary dimensions and buffer composition on separation efficiency and sensitivity are discussed. Optimal separations were achieved using a 28 cm × 15 μm capillary, a separation high voltage of 35 kV, a background electrolyte of 25 mM ammonium acetate adjusted to pH 8.5, and negative ionization mode. The optimized method was successfully applied to a bovine testicular hyaluronidase digest of hyaluronan. Only minimal sample pretreatment for protein-containing samples is required. The simple manual injection procedure and fast separations allow for a sample throughput of 35 samples per hour.

Keywords: Capillary electrophoresis; Counter-electroosmotic; Time-of-flight mass spectrometry; High electric field strength; Hyaluronan; Bovine testicular hyaluronidase


Fluorescence dye as novel label molecule for quantitative SERS investigations of an antibiotic by Anne März; Sabine Trupp; Petra Rösch; Gerhard J. Mohr; Jürgen Popp (pp. 2625-2631).
Within this contribution, the proof-of-principle for a new concept for indirect surface-enhanced Raman spectroscopy (SERS) detection is presented. The fluorescence dye FR-530 is applied as a label molecule for the antibiotic erythromycin. The antibiotic binds directly to the label molecule. Changes within the SERS spectrum of the fluorescence dye appearing with the presence of the antibiotic are utilized for the detection and quantitative investigations of erythromycin. With the new concept of binding the label molecule directly to the analyte molecule, the application of linkage compounds like antibodies or any other recognition molecules becomes dispensable.

Keywords: SERS; Label molecules; Antibiotic; Microfluidic


On the mechanism of extractive electrospray ionization (EESI) in the dual-spray configuration by Rui Wang; Arto Juhani Gröhn; Liang Zhu; Rolf Dietiker; Karsten Wegner; Detlef Günther; Renato Zenobi (pp. 2633-2643).
Dual-spray extractive electrospray ionization (EESI) mass spectrometry as a versatile analytical technique has attracted much interest due to its advantages over conventional electrospray ionization (ESI). The crucial difference between EESI and ESI is that in the EESI process, the analytes are introduced in nebulized form via a neutral spray and ionized by collisions with the charged droplets from an ESI source formed by spraying pure solvent. However, the mechanism of the droplet–droplet interactions in the EESI process is still not well understood. For example, it is unclear which type of droplet–droplet interaction is dominant: bounce, coalescence, disruption, or fragmentation? In this work, droplet–droplet interaction was investigated in detail based on a theoretical model. Phase Doppler anemometry (PDA) was employed to investigate the droplet behavior in the EESI plume and provide the experimental data (droplet size and velocity) necessary for theoretical analysis. Furthermore, numerical simulations were performed to clarify the influence of the sheath gas flow on the EESI process. No coalescence between the droplets in the ESI spray and the droplets in the sample spray was observed using various geometries and sample flow rates. Theoretical analysis, together with the PDA results, suggests that droplet fragmentation may be the dominant type of droplet–droplet interaction in the EESI. The interaction time between the ESI droplet and the sample droplet was estimated to be <5 μs. This work gives a clear picture of droplet–droplet interactions in the dual-spray EESI process and detailed information for the optimization of this method for future applications that require higher sensitivity.

Keywords: Mass spectrometry; Extractive electrospray; Ionization mechanisms


Total serum IgE quantification by microfluidic ELISA using magnetic beads by Gaëlle Proczek; Anne-Laure Gassner; Jean-Marc Busnel; Hubert H. Girault (pp. 2645-2653).
The present work reports on the quantification of total IgE in human serum using a microanalytical device whose fluidics is driven by gravity and capillary forces only. Thanks to the eight parallel microchannels in each microchip, calibration and sample analysis are performed simultaneously. A mixture of magnetic bead/analyte/second antibody is incubated off-line and then percolated through the channels where magnetic beads are trapped, enabling the separation of the solid phase from the excess reagents. The entire assay is performed in less than 1 h, and thanks to the miniaturized format, only a small volume of serum is required. Non-specific adsorption was first investigated and a blocking agent compatible with this allergy-based test was chosen. Then, the assay was optimized by determining the best magnetic bead and labelled antibody concentrations. After achievement of a calibration curve with a reference material, the protocol was applied to total IgE quantification of a patient serum sample that showed results in good accordance with those obtained by ImmunoCap® and Immunoaffinity capillary electrophoresis measurements. A detection limit of 17.5 ng ml−1 was achieved and good reproducibility (RSD < 10%) inter- and intra-chip was observed. Figure Off-line incubation of the patient sample with anti-IgE grafted magnetic beads and ALP-labelled anti-IgE is carried out in an Eppendorff. Detection is then performed with the GRAVI®-Cell device from DiagnoSwiss, where fluidics is driven by gravity and capillary forces only.

Keywords: Immunoassay; Allergy; Microfluidics; Magnetic particles


Fragmentation methods on the balance: unambiguous top–down mass spectrometric characterization of oxaliplatin–ubiquitin binding sites by Samuel M. Meier; Yury O. Tsybin; Paul J. Dyson; Bernhard K. Keppler; Christian G. Hartinger (pp. 2655-2662).
The interaction between oxaliplatin and the model protein ubiquitin (Ub) was investigated in a top–down approach by means of high-resolution electrospray ionization mass spectrometry (ESI-MS) using diverse tandem mass spectrometric (MS/MS) techniques, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD). To the best of our knowledge, this is the first time that metallodrug–protein adducts were analyzed for the metal-binding site by ETD-MS/MS, which outperformed both CID and HCD in terms of number of identified metallated peptide fragments in the mass spectra and the localization of the binding sites. Only ETD allowed the simultaneous and exact determination of Met1 and His68 residues as binding partners for oxaliplatin. CID-MS/MS experiments were carried out on orbitrap and ion cyclotron resonance (ICR)-FT mass spectrometers and both instruments yielded similar results with respect to number of metallated fragments and the localization of the binding sites. A comparison of the protein secondary structure with the intensities of peptide fragments generated by collisional activation of the [Ub + Pt-(chxn)] adduct [chxn = (1R,2R)-cyclohexanediamine] revealed a correlation with cleavages in solution phase random coil areas, indicating that the N-terminal β-hairpin and α-helix structures are retained in the gas phase. Figure CID, HCD and ETD were used to determine the binding site of the anticancer agent oxaliplatin on ubiquitin in a top-down approach

Keywords: Anticancer metallodrugs; Tandem mass spectrometry; Electron transfer dissociation; Oxaliplatin; Ubiquitin


A flow-through microarray cell for the online SERS detection of antibody-captured E. coli bacteria by Maria Knauer; Natalia P. Ivleva; Reinhard Niessner; Christoph Haisch (pp. 2663-2667).
We present an immunoassay microarray flow-through system for the surface-enhanced Raman scattering (SERS) analysis of bacteria. The system has been constructed to support and automatize the nondestructive in situ analysis of different microorganisms in aqueous environment. After the immobilization of the desired antibodies to an activated PEG-coated surface, the chip is placed into the flow cell which is then flushed with the contaminated sample. Finally, colloidal metal nanoparticles are added and the cells are detected label-free by SERS. Here, we introduce the successful imaging of single microorganisms in the flow cell as well as the quantification of microorganisms in water by SERS mapping with a linear range between 4.3 × 103 to 4.3 × 105 cells/mL. The method has potential for routine application, e.g. for drinking water control.

Keywords: Flow cell; SERS; Microorganisms; Microarray


Fast online emission monitoring of volatile organic compounds (VOC) in wastewater and product streams (using stripping with direct steam injection) by Alexander Schocker; Bert Lißner (pp. 2669-2674).
Open-loop stripping analysis (also referred to as dynamic headspace) is a very flexible and robust technology for online monitoring of volatile organic compounds in wastewater or coolant. However, the quality and reliability of the analytical results depend strongly on the temperature during the stripping process. Hence, the careful and constant heating of the liquid phase inside the stripping column is a critical parameter. In addition, this stripping at high temperatures extends the spectrum of traceable organics to less volatile and more polar compounds with detection limits down to the ppm-level. This paper presents a novel and promising approach for fast, efficient, and constant heating by the direct injection of process steam into the strip medium. The performance of the system is demonstrated for temperatures up to 75 °C and traces of various hydrocarbons in water (e.g., tetrahydrofuran, methanol, 1-propanol, n-butanol, ethylbenzene).

Keywords: Online process analysis; Dynamic headspace technique; Direct steam injection; Volatile organic compounds
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