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Analytical and Bioanalytical Chemistry (v.400, #8)

Analytical sciences in Austria by Günter Allmaier; Wolfgang Buchberger; Kevin Francesconi (pp. 2273-2274).

is currently Full Professor of Analytical Chemistry and Head of the Research Division of Instrumental Analytical Chemistry as well as Group Leader of Bio- and Polymer Analysis at the Institute of Chemical Technologies and Analytics of Vienna University of Technology (TU Wien) in Vienna. His scientific interests lie in “omics” technologies and their application to bioprocess technology, human diseases (e.g. biomarker discovery related to kidney diseases) and microbiology, characterization of biopolymers (e.g. coagulation proteins), technical polymers and lipids by mass spectrometry (MS), intact cell MS for microorganism identification, gas-phase electrophoretic molecular mass analyses of functional biocomplexes, viruses and vaccines, nanobioparticle detection/characterization, and instrumentation such as capillary electrophoresis on a chip and MS ion sources. is Full Professor of Analytical Chemistry and Head of the Institute of Analytical Chemistry at Johannes Kepler University, Linz. His main research interests are in the fields of organic analytical sciences and organic trace analysis with applications in the areas of environmental analytical chemistry as well as method development for industrial chemistry, including the characterization of polymeric materials. His current projects are mainly based on coupling of analytical high-performance separation techniques such as chromatography and capillary electrophoresis with advanced and novel mass-spectrometric detection tools. Currently, he is a member of the editorial boards of several analytical journals, and he represents Austria in the Division of Analytical Chemistry within EuCheMS. is a graduate from Curtin University of Technology (B.Appl.Sci.) and from the University of Western Australia (Ph.D., organic chemistry). He worked for 20 years at the Western Australian Marine Research Laboratories, first as an analytical chemist and then as a research scientist in environmental chemistry. In 1996, he moved to the Ecotoxicology Group at the University of Southern Denmark before moving to Graz University in 2002, where he is presently Professor of Analytical Chemistry. His research has focused on the development and application of chemical and analytical methods for studying fundamental processes of biotransformation of metals in the environment and in organisms, including humans.

Brief history of the Austrian Society of Analytical Chemistry by Hans Malissa Jr. (pp. 2275-2280).

was Professor of Chemistry at the University of Salzburg until 2007. He taught general and analytical chemistry in the study programs of biology, earth, and material sciences. His research activities were related to the development of methods for measurement of traces of organic pollutants, particularly pesticides and PAHs, in water, air, and biological matrices, the design of multiresidue methods, implementation of microseparation systems, and air sampling techniques

Matrix-free and material-enhanced laser desorption/ionization mass spectrometry for the analysis of low molecular weight compounds by Matthias Rainer; Muhammad Nasimullah Qureshi; Günther Karl Bonn (pp. 2281-2288).

The application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for the analysis of low molecular weight (LMW) compounds, such as pharmacologically active constituents or metabolites, is usually hampered by employing conventional MALDI matrices owing to interferences caused by matrix molecules below 700 Da. As a consequence, interpretation of mass spectra remains challenging, although matrix suppression can be achieved under certain conditions. Unlike the conventional MALDI methods which usually suffer from background signals, matrix-free techniques have become more and more popular for the analysis of LMW compounds. In this review we describe recently introduced materials for laser desorption/ionization (LDI) as alternatives to conventionally applied MALDI matrices. In particular, we want to highlight a new method for LDI which is referred to as matrix-free material-enhanced LDI (MELDI). In matrix-free MELDI it could be clearly shown, that besides chemical functionalities, the material’s morphology plays a crucial role regarding energy-transfer capabilities. Therefore, it is of great interest to also investigate parameters such as particle size and porosity to study their impact on the LDI process. Especially nanomaterials such as diamond-like carbon, C₆₀ fullerenes and nanoparticulate silica beads were found to be excellent energy-absorbing materials in matrix-free MELDI.

Keywords: Matrix-assisted laser desorption/ionization; Laser desorption/ionization; Material-enhanced laser desorption/ionization; Matrix-free; Nanotechnology; Porosity

Porous polymer monoliths for small molecule separations: advancements and limitations by Ivo Nischang; Ian Teasdale; Oliver Brüggemann (pp. 2289-2304).

Porous polymer monoliths are considered to be one of the major breakthroughs in separation science. These materials are well known to be best suited for the separation of large molecules, specifically proteins, an observation most often explained by convective mass transfer and the absence of small pores in the polymer scaffold. However, this conception is not sufficient to explain the performance of small molecules. This review focuses in particular on the preparation of (macro)porous polymer monoliths by simple free-radical processes and the key events in their formation. There is special focus on the fluid transport properties in the heterogeneous macropore space (flow dispersion) and on the transport of small molecules in the swollen, and sometimes permanently porous, globule-scale polymer matrix. For small molecule applications in liquid chromatography, it is consistently found in the literature that the major limit for the application of macroporous polymer monoliths lies not in the optimization of surface area and/or modification of the material and microscopic morphological properties only, but in the improvement of mass transfer properties. In this review we discuss the effect of resistance to mass transfer arising from the nanoscale gel porosity. Gel porosity induces stagnant mass transfer zones in chromatographic processes, which hamper mass transfer efficiency and have a detrimental effect on macroscopic chromatographic dispersion under equilibrium (isocratic) elution conditions. The inherent inhomogeneity of polymer networks derived from free-radical cross-linking polymerization, and hence the absence of a rigid (meso)porous pore space, represents a major challenge for the preparation of efficient polymeric materials for the separation of small molecules.

Keywords: Chromatographic performance; Cross-linking; Free-radical polymerization; Gel porosity; Mass transfer; Nanoscale heterogeneity; Plate height; Porous polymers; Spatial inhomogeneity

Enantioseparation by chromatographic and electromigration techniques using ligand-exchange as chiral separation principle by Martin G. Schmid; Gerald Gübitz (pp. 2305-2316).

This article gives a short overview of the application of the principle of chiral ligand-exchange in HPLC, CE, and CEC. Since its introduction by Davankov, more than thousand articles have appeared in this field. Citing all these papers would extend the scope of this review—it would fill several books. Therefore only some milestones are mentioned in this article and it will focus on our own activities in this field. Some new developments are mentioned, and selected biochemical and biomedical application are presented.

Keywords: Ligand-exchange; Chiral selectors; Chemically bonded phases; Dynamically coated phases; Monolithic phases; HPLC; Capillary electrophoresis; Capillary electrochromatography

Rapid identification and semi-quantitative determination of polymer additives by desorption electrospray ionization/time-of-flight mass spectrometry by Silke M. Reiter; Wolfgang Buchberger; Christian W. Klampfl (pp. 2317-2322).

A fast and simple method for the direct qualitative and semi-quantitative determination of a set of four polymer additives in plastic samples by desorption electrospray ionization time-of-flight mass spectrometry (DESI-TOF-MS) is presented. After evaluation of crucial DESI parameters such as composition of spray solutions and spray voltages, a series of lab-made polypropylene samples containing Chimassorb 81 (2-hydroxy-4-n-octoxybenzophenone), Tinuvin 328 (2-(2-hydroxy-3, 5-ditert-pentylphenyl)-benzotriazole), Tinuvin 326 (2-(2-hydroxy-3-tert-butyl-5-methylphenyl)-5-chloro benzotriazole), and Tinuvin 770 (bis(2,2,6,6,-tetramethyl-4-piperidyl)sebaceate) in concentrations between 0.02% and 0.2% were analyzed, resulting in calibration graphs with R ² better than 0.994. To demonstrate the applicability of the developed method for the investigation of real samples, liners for in-ground swimming pools and polypropylene granules were analyzed with respect to their content in the selected polymer additives. Two alternative methods, both well established in the fields of polymer additive analysis, namely HPLC with UV detection (after previous extraction) and thermodesorption gas chromatography/mass spectrometry have been employed for evaluation of the results from the DESI experiments.

Keywords: Desorption electrospray ionization; Polymer additives; Time-of-flight mass spectrometry

Quantitative determination of small selenium species in human serum by HPLC/ICPMS following a protein-removal, pre-concentration procedure by Sabine Kokarnig; Doris Kuehnelt; Michael Stiboller; Ulrike Hartleb; Kevin A. Francesconi (pp. 2323-2327).

Protein precipitation was incorporated into a sample preparation method for the quantitative determination of small “non-protein” selenium species in human serum by high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC/ICPMS). The advantages of cleaner matrix and concomitant concentration of the small compounds result in quantification limits in the native serum at the sub-micrograms Se per litre level. Spiking experiments with methyl 2-acetamido-2-deoxy-1-seleno-β-d-galactopyranoside (selenosugar 1), trimethylselenonium ion, selenomethionine, methylselenocysteine (MeSeCys) and selenate yielded recoveries from 73% to 103%. Selenite had a low recovery (44%), possibly owing to protein binding. The validated method was applied to serum samples from two volunteers before and after ingestion of a selenium food supplement. HPLC/ICPMS analysis showed, besides ingested selenate, the presence of selenosugar 1 and trace amounts of MeSeCys and methyl 2-amino-2-deoxy-1-seleno-β-d-galactopyranoside, which have not been reported in human serum before. Figure A protein-removal pre-concentration procedure facilitates the determination of low concentrations of small selenium compounds in human serum by HPLC/ICPMS

Keywords: Selenium metabolism; Sample preparation; HPLC/ICPMS; Human serum

A novel approach for an automated liquid/liquid extraction system—principle and application for the determination of several trace contaminants in highly alloyed steels and base alloys by Helmar Wiltsche; Karl Prattes; Günter Knapp (pp. 2329-2338).

A novel automated liquid/liquid extraction system was developed for the determination of trace contaminants in unalloyed, alloyed and highly alloyed steels and super alloys. In the presented batch extraction system the aqueous phase and the non-water miscible organic phase were brought into close phase contact by high-speed stirring with a magnetic stir bar. Iodide complexes of Ag, Bi, Cd, Pb, Sb, Sn, Tl, and Zn were extracted from aqueous steel digests into 4-methylpentan-2-one (MIBK) containing 20 g L⁻¹ trioctylphosphine oxide. Ag, Bi, Cd, Pb, and Tl were extracted quantitatively whereas the extraction yields of Sb, Sn, and Zn were 83%, 61% and 75% respectively. Using high resolution continuum source flame AAS (HR-CS-FAAS) for analyte quantification the method was validated using 21 certified steel reference materials (CRMs). Figure

Keywords: AAS; ICPOES; Process analysis; Pre-concentration; Steel; Automated batch extraction system

Altered Mascot search results by changing the m/z range of MS/MS spectra: analysis and potential applications by Thomas Köcher; Peter Pichler; Michael Mazanek; Remco Swart; Karl Mechtler (pp. 2339-2347).

The Mascot search algorithm is one of the most commonly used tools for protein identification. Tandem mass spectrometry data searched against a protein sequence database is utilized for identifying peptides and proteins, each reported with a score. Higher Mascot scores are associated with lower chances of random hits. The process of peak selection performed by the search engine prior to the search is a critical aspect of the process. Here, we show that Mascot divides the MS/MS spectrum into fixed m/z regions for peak selection, starting at the lowest m/z value of the peak list. Therefore, modifying the m/z range of the peak lists by insertion of a dummy peak with low m/z value changes the ensemble of peaks used for searching. As a consequence, Mascot peptide scores and search results are altered significantly and a different subset of the peptides present in the sample is identified after processing. We further show that the effect can be exploited and additional proteins and peptides can be identified by repeating the search with a combined set of differently processed files, even when applying identical false-positive rates. Figure An extreme case of the effects of spectrum processing is shown. After insertion of a dummy peak at m/z 5 in an ion trap-generated CID spectrum the peptide score increased from 22 to 39.6.

Keywords: Mass spectrometry; Tandem mass spectrometry; Protein identification; Search engine

Performance evaluation of Mimetic Ligand™ B14-triazole-FractoAIMs adsorbents for the capture of human monoclonal immunoglobulin G from cell culture feed by Jeannie Horak; Stefan Hofer; Chris Sadler; Sharon Williams; Wolfgang Lindner (pp. 2349-2359).

The new affinity-type Mimetic Ligand™ B14 was coupled with a 1,2-diaminoethane spacer (2LP) and a [1,2,3]-triazole spacer (TRZ) to three different support media. In addition to the agarose-based PuraBead and the polymethacrylate-type Fractogel, three new polymeric support media were introduced, the FractoAIMs 1, 2, and 3 (FA1, FA2, and FA3). These new FA supports differ in pore size as well as density of epoxide groups. The immobilization of the B14-ligand onto an azide-group-modified surface was performed with a copper (I)-mediated Click reaction. The IgG capture performance was tested for various ligand–spacer support combinations using cell culture feed containing human immunoglobulin G₁ (hIgG₁). The most promising adsorbent, B14-TRZ-FA3, was further optimized by improving the surface chemistry through a triple endcapping concept employing an improved Click reaction protocol. This new technique enabled the most efficient deactivation of residual azide groups. In a direct comparison with a commercially available Protein A media, B14-TRZ-FA3 3× ec provided superior results at fast flow-rates and low bed-height. Dynamic binding capacities of 11.4 g/L for 10% breakthrough of hIgG₁, elution capacities of 16.0 g/L hIgG₁ and a recovery of 86% were achieved. The same results were obtained for a dialyzed and pre-purified feed solution, which is a clear indicator that triple-endcapped affinity support surfaces are practically inert to the non-specific binding of host cell proteins.

Keywords: Click chemistry; Antibody purification; Affinity chromatography; Protein A; Size exclusion chromatography; Dynamic binding

CYP2D6 genotyping by liquid chromatography-electrospray ionization mass spectrometry by Beate Beer; Robert Erb; Florian Pitterl; Harald Niederstätter; Olalla Maroñas; Alejandro Gesteira; Angel Carracedo; Irina Piatkov; Herbert Oberacher (pp. 2361-2370).

Genetic polymorphisms can significantly affect the enzyme activity of the drug metabolizing enzyme Cytochrome P450 2D6 (CYP2D6; OMIM 124030). Accordingly, CYP2D6 genotyping is considered as a valid approach to predict the individual CYP2D6 metabolizing status. We introduce ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) as method for the characterization of single base variants, small deletions, and insertions in the CYP2D6 gene. A two-step polymerase chain reaction (PCR) was developed for the simultaneous amplification of nine polymorphic regions within the CYP2D6 gene. Cleanup, separation, and denaturation of PCR amplicons were achieved by high-performance liquid chromatography. High-performance molecular mass measurements provided nucleotide composition profiles that principally enable the resolution of 37 reported CYP2D6 alleles. The developed assay was applied to the genotyping of 93 unrelated Austrian individuals. For validation, a selected number of samples and polymorphic sites were retyped by alternative genotyping technologies. The PCR-ICEMS assay turned out to be an accurate, robust, and cost-effective CYP2D6 genotyping strategy. Figure Outline of the principal steps of the PCR-ICEMS assay developed for CYP2D6 genotyping. A two-step PCR is used for the simultaneous amplification of nine polymorphic regions within the CYP2D6 gene. PCR amplicons are analyzed by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS). High-performance molecular mass measurements provide nucleotide composition profiles that enable the identification of CYP2D6 alleles

Keywords: Mass spectrometry; Genotyping; CYP2D6

Development and validation of a LC-MS/MS method based on a new 96-well Hybrid-SPE^™-precipitation technique for quantification of CYP450 substrates/metabolites in rat plasma by Karin Ardjomand-Woelkart; Manfred Kollroser; Li Li; Hartmut Derendorf; Veronika Butterweck; Rudolf Bauer (pp. 2371-2381).

A rapid and selective high-throughput HESI-LC-MS/MS method for determining eight cytochrome P450 probe drugs in one-step extraction and single run was developed and validated. The four specific probe substrates midazolam, dextromethorphan, tolbutamide, theophylline and their metabolites 1-hydroxymidazolam, dextrorphan, hydroxyl(methyl)tolbutamide, 1,3-dimethyluric acid, together with the deuterated internal standards, were extracted from rat plasma using a novel 96-well Hybrid-SPE™-precipitation technique. The bioanalytical assay was based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using selected reaction monitoring for drug (-metabolite) quantification. All analytes were separated simultaneously in a single run that lasted less than 11 min. The intra- and inter-day precisions for all eight substrates/metabolites were 1.62–12.81% and 2.09–13.02%, respectively, and the relative errors (accuracy) for the eight compounds ranged from −9.62% to 7.48% and −13.84% to 8.82%. Hence, the present method provides a robust, fast and reproducible analytical tool for the evaluation of four major drug metabolising cytochrome P450 (3A4, 2C9, 1A2 and 2D6) activities with a cocktail approach in rats to clarify herb–drug interactions. The method can be used as a basic common validated high-throughput analytical assay for in vivo interaction studies. Figure The new used 96 well Hybrid-SPE™-precipitation plate for plasma extraction.

Keywords: Hybrid-SPE™ precipitation; Validation; Phospholipids; CYP450

Mitochondrial ROS production under cellular stress: comparison of different detection methods by Andrey V. Kuznetsov; Ingeborg Kehrer; Andrey V. Kozlov; Martina Haller; Heinz Redl; Martin Hermann; Michael Grimm; Jakob Troppmair (pp. 2383-2390).

Reactive oxygen species (ROS) are involved in the regulation of many physiological processes. However, overproduction of ROS under various cellular stresses results in cell death and organ injury and thus contributes to a broad spectrum of diseases and pathological conditions. The existence of different cellular sources for ROS and the distinct properties of individual ROS (their reactivity, lifetime, etc.) require adequate detection methods. We therefore compared different models of cellular stress and various ROS-sensitive dyes—2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), MitoSOX™, and MitoTracker® red CM-H₂XRos—using a confocal fluorescent imaging approach, which has the advantage of not only detecting but also of localizing intracellular sources for ROS. Confocal acquisition of DCF-DA fluorescence can be combined with ROS detection by the mitochondria-specific probes MitoSOX™ and MitoTracker® red CM-H₂XRos. Specificity was controlled using various antioxidants such as Trolox and N-acetylcysteine. Using different fluorescent ROS-sensitive probes, we detected higher ROS production equally under cell starvation (IL-3 or serum depletion), hypoxia–reoxygenation, or treatment of cells with prooxidants. The detected increase in ROS was approximately threefold in IL-3-depleted 32D cells, approximately 3.5-fold in serum-deprived NIH cells, and 2.5-fold to threefold in hypoxic HL-1 cells, and these findings agree well with previously published spectrofluorometric measurements. In some cases, electron spin resonance (ESR) spectroscopy was used for the validation of results from confocal fluorescent imaging. Our data show that confocal fluorescent imaging and ESR data are in good agreement. Under cellular stress, mitochondrial ROS are released into the cytoplasm and may participate in many processes, but they do not escape from the cell. Online abstract Mitochondrial ROS production under cellular stress

Keywords: Confocal fluorescent imaging; Cell stress; Electron spin resonance; Laser scanning microscopy; Mitochondria; Reactive oxygen species

Morphology and efficiency of poly(styrene-co-divinylbenzene)-based monolithic capillary columns for the separation of small and large molecules by Jens H. Mohr; Remco Swart; Christian G. Huber (pp. 2391-2402).

The morphology of organic monolithic stationary phases based on poly(styrene-divinylbenzene) was modified by changing the ratio of monomers to microporogen in order to make them also suitable for small molecule separations. The morphology of the columns was characterized by high-resolution scanning electron micrography, showing larger primary globules and larger macropores, as well as no mesopores >20 nm in the monolithic skeleton. The permeability of the modified monoliths was approximately three times higher than that of columns which have been optimized for large molecule separations, enabling operation of a 30 cm long column at pressures below 250 bar. In the isocratic separation of dansylated amino acids, plate counts of 50000–107000 m⁻¹ were achievable, which are equivalent to efficiencies obtained with 3.1 μm porous particles. The separation performance for small molecules in gradient elution was investigated using mixtures of dansylated amino acids, β-lactam antibiotics, and thyroid hormones. Finally, the modified monolithic capillary columns also proved to be highly efficient in the separation of biopolymers such as peptides and proteins, enabling peak width at half height of 3–8 s and peak capacities of 110–180 in 15–30 min gradient runs.

Keywords: Monolithic columns; Capillary columns; Poly(styrene-divinylbenzene); Stationary phases; Peptides; Proteins; β-Lactam antibiotics; Dansylated amino acids; Thyroid hormones; Monolithic morphology; HPLC

Fast wheat variety classification by capillary gel electrophoresis-on-a-chip after single-step one-grain high molecular weight glutenin extraction by Martina Marchetti-Deschmann; Angela Lehner; Verena Peterseil; Friedrich Sövegjarto; Rupert Hochegger; Günter Allmaier (pp. 2403-2414).

Wheat variety identification based on one-step single-grain wheat extraction and fast capillary gel electrophoresis-on-a-chip (CGE-on-a-chip) analyses was evaluated for 15 different wheat varieties grown in Austria. The results of the capillary-based separation system were compared to the internationally accepted method from the International Union for the Protection of New Varieties of Plants which is based on time-consuming sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Comparable protein patterns were observed making the CGE-on-a-chip system a promising tool for high-throughput analysis in food control. For the development of a robust method protein extraction, shelf life of wheat extracts and the instrument’s variability were evaluated. It turned out that a one-step single-grain wheat extraction allowed the sample to be stored at 4 °C for up to 4 weeks without losing any valuable protein information. Furthermore, the technical variation of the whole method is very low making the biological variation of the selected wheat grains the only uncertain factor. Additionally, two unsupervised statistical methods (hierarchical cluster analysis and principal component analysis) were used for variety identification. Identification was successful for a reduced data set of 14 samples from five different wheat varieties making the combination of CGE-on-a-chip analysis of one-step single-grain extraction in combination with automatic data evaluation a promising tool for fast wheat differentiation (within a day).

Keywords: Wheat variety classification; Single grain extraction; Glutenin subunits; Capillary gel electrophoresis-on- a-chip; Electrophoresis; Microfluidics

Solid-phase extraction and GC-MS analysis of potentially genotoxic cleavage products of β-carotene in primary cell cultures by G. Martano; C. Vogl; E. Bojaxhi; N. Bresgen; P. Eckl; H. Stutz (pp. 2415-2426).

A validated method for the simultaneous determination of prominent volatile cleavage products (CPs) of β-carotene in cell culture media has been developed. Target CPs comprised β-ionone (β-IO), cyclocitral (CC), dihydroactinidiolide (DHA), and 1,1,6-trimethyltetraline (TMT). CPs were extracted by solid-phase extraction applying a phenyl adsorbent, eluted with 10% (v/v) tetrahydrofuran in n-hexane, and identified and quantified by gas chromatography-mass spectrometry with electron impact ionization. Method validation addressed linearity confirmation over two application ranges and homoscedasticity testing. Recoveries from culture media were between 71.7% and 95.7% at 1.0 μg/ml. Precision of recoveries determined in intra-day (N = 5) and inter-day (N = 15) assays were <2.0% and <4.8%, respectively. Limit of detection and limit of quantification of the analysis method were <18.0 and <53.0 ng/ml for β-IO, CC, and TMT, whereas 156 and 474 ng/ml were determined for DHA, respectively. Although extractions of blank matrix proved the absence of interfering peaks, statistical comparison between slopes determined for instrumental and total method linearity revealed significant differences. The method was successfully applied in selecting an appropriate solvent for the fortification of culture media with volatile CPs, including the determination of their availability over the incubation period. For the first time, quantification of volatile CPs in treatment solutions and culture media for primary cells becomes accessible by this validated method. Figure Cultured primary rat hepatocytes in phase contrast after nuclea staining with DAPI including a chromatogram (GC-MS) of volatile cleavage products of b-carotene, which are presumed to exert genotoxic effects on hepatocytes and pneumocytes

Keywords: β-carotene; Volatile cleavage products; Cell culture media; SPE GC-MS; Validation

Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches by Josephine Grass; Martin Pabst; Martina Chang; Manfred Wozny; Friedrich Altmann (pp. 2427-2438).

Recombinant human follicle stimulating hormone is an important drug in reproductive medicine. Thorough analysis of the heterodimeric heavily glycosylated protein is a prerequisite for the evaluation of production batches as well as for the determination of “essential similarity” of new biosimilars. The concerted application of different liquid chromatography-mass spectrometry methods enabled the complete depiction of the primary structure of this pituitary hormone. Sequence coverage of 100% for the α- as well as the β-chain was achieved with tryptic peptides. Most of these peptides could be verified by tandem mass spectrometry. Site-specific analysis of all four glycosylation sites was, however, not possible with tryptic but with chymotryptic peptides. Quantification of the glycoforms of each glycopeptide was accomplished with the software MassMap®. Both protein subunits gave interpretable mass spectra upon S-alkylation and separation on a C5 reversed-phase column. Glycan isomer patterns were depicted by separation on porous graphitic carbon, using mass spectrometric detection for the evaluation of the glycopeptide liquid chromatography-electrospray ionization data. The currently marketed product Gonal-f™ and a potential biosimilar were compared with the help of these procedures. Figure Schematic depiction of the glycoprotein nature of human follicle-stimulating hormone with the alfa chain in blue and the beta chain in purple and a mass spectrum of the alfa chain at the bottom.

Keywords: Follicle-stimulating hormone; Follitropin; Glycoprotein; Biosimilar

Stand-off Raman spectroscopy: a powerful technique for qualitative and quantitative analysis of inorganic and organic compounds including explosives by Bernhard Zachhuber; Georg Ramer; Alison Hobro; Engelene t. H. Chrysostom; Bernhard Lendl (pp. 2439-2447).

A pulsed stand-off Raman system has been built and optimised for the qualitative and quantitative analysis of inorganic and organic samples including explosives. The system consists of a frequency doubled Q-switched Nd:YAG laser (532 nm, 10 Hz, 4.4 ns pulse length), aligned coaxially with a 6″ Schmidt–Cassegrain telescope for the collection of Raman scattered light. The telescope was coupled via a fibre optic bundle to an Acton standard series SP-2750 spectrograph with a PI-MAX 1024RB intensified CCD camera equipped with a 500-ps gating option for detection. Gating proved to be essential for achieving high signal-to-noise ratios in the recorded stand-off Raman spectra. In some cases, gating also allowed suppression of disturbing fluorescence signals. For the first time, quantitative analysis of stand-off Raman spectra was performed using both univariate and multivariate methods of data analysis. To correct for possible variation in instrumental parameters, the nitrogen band of ambient air was used as an internal standard. For the univariate method, stand-off Raman spectra obtained at a distance of 9 m on sodium chloride pellets containing varying amounts of ammonium nitrate (0–100%) were used. For the multivariate quantification of ternary xylene mixtures (0–100%), stand-off spectra at a distance of 5 m were used. The univariate calibration of ammonium nitrate yielded R ² values of 0.992, and the multivariate quantitative analysis yielded root mean square errors of prediction of 2.26%, 1.97% and 1.07% for o-, m- and p-xylene, respectively. Stand-off Raman spectra obtained at a distance of 10 m yielded a detection limit of 174 μg for NaClO₃. Furthermore, to assess the applicability of stand-off Raman spectroscopy for explosives detection in “real-world” scenarios, their detection on different background materials (nylon, polyethylene and part of a car body) and in the presence of interferents (motor oil, fuel oil and soap) at a distance of 20 m was also investigated. Figure Stand-off Raman spectroscopy

Keywords: Raman; Stand-off; Quantification; Remote; Explosive

Determination of the xanthate group distribution on viscose by liquid-state ¹H NMR spectroscopy by Andreas Schwaighofer; Gerhard Zuckerstätter; Judith Schlagnitweit; Herbert Sixta; Norbert Müller (pp. 2449-2456).

An analytical method for determination of the xanthate group distribution on viscoses based on liquid-state NMR spectroscopy was developed. Sample preparation involves stabilization of the xanthate group by allylation followed by derivatization of the remaining free hydroxyl groups at the glucose unit. The method was applied for studying (1) the γ-value (number of xanthate groups per 100 glucose units) of viscose, (2) the distribution of the xanthate groups on the anhydroglucose unit (AGU), and (3) changes of the xanthate group distribution during ripening. Results of the γ-value determination are well comparable with reference methods. Elucidation of the xanthate group distribution on the AGU gives the percentage at the C-6 position and a cumulative share of the positions C-2 and C-3. During ripening, xanthate groups at C-2 and C-3 degrade first, while xanthates at C-6 decompose at a slower rate.

Keywords: ¹H NMR; Viscose; Xanthate group distribution; Gamma value

QCM gas phase detection with ceramic materials—VOCs and oil vapors by Usman Latif; Andreas Rohrer; Peter A. Lieberzeit; Franz L. Dickert (pp. 2457-2462).

Titanate sol–gel layers imprinted with carbonic acids were used as sensitive layers on quartz crystal microbalance. These functionalized ceramics enable us detection of volatile organic compounds such as ethanol, n-propanol, n-butanol, n-hexane, n-heptane, n-/iso-octane, and n-decane. Variation of the precursors (i.e., tetrabutoxy titanium, tetrapropoxy titanium, tetraethoxy titanium) allows us to tune the sensitivity of the material by a factor of 7. Sensitivity as a function of precursors leads to selective inclusion of n-butanol vapors down to 1 ppm. The selectivity of materials is optimized to differentiate between isomers, e.g., n- and iso-octane. The results can be rationalized by correlating the sensor effects of hydrocarbons with the Wiener index. A mass-sensitive sensor based on titanate layer was also developed for monitoring emanation of degraded engine oil. Heating the sensor by a meander avoids vapor condensation. Thus, a continuously working oil quality sensor was designed.

Keywords: Sensors; Titanate sol–gel layers; Quartz crystal microbalance (QCM); Volatile organic compounds (VOCs)

A novel planar optical sensor for simultaneous monitoring of oxygen, carbon dioxide, pH and temperature by Sergey M. Borisov; Roman Seifner; Ingo Klimant (pp. 2463-2474).

The first quadruple luminescent sensor is presented which enables simultaneous detection of three chemical parameters and temperature. A multi-layer material is realized and combines two spectrally independent dually sensing systems. The first layer employs ethylcellulose containing the carbon dioxide sensing chemistry (fluorescent pH indicator 8-hydroxy-pyrene-1,3,6-trisulfonate (HPTS) and a lipophilic tetraalkylammonium base). The cross-linked polymeric beads stained with a phosphorescent iridium(III) complex are also dispersed in ethylcellulose and serve both for oxygen sensing and as a reference for HPTS. The second (pH/temperature) dually sensing system relies on the use of a pH-sensitive lipophilic seminaphthorhodafluor derivative and luminescent chromium(III)-activated yttrium aluminum borate particles (simultaneously acting as a temperature probe and as a reference for the pH indicator) which are embedded in polyurethane hydrogel layer. A silicone layer is used to spatially separate both dually sensing systems and to insure permeation selectivity for the CO₂/O₂ layer. The CO₂/O₂ and the pH/temperature layers are excitable with a blue and a red LED, respectively, and the emissions are isolated with help of optical filters. The measurements are performed at two modulation frequencies for each sensing system and the modified Dual Lifetime Referencing method is used to access the analytical information. The feasibility of the simultaneous four-parameter sensing is demonstrated. However, the practical applicability of the material may be compromised by its high complexity and by the performance of individual indicators. Figure Cross-section of the multi-analyte sensor

Keywords: Optical sensor; Oxygen; pH; Carbon dioxide; Temperature; Luminescence

A novel planar optical sensor for simultaneous monitoring of oxygen, carbon dioxide, pH and temperature by Sergey M. Borisov; Roman Seifner; Ingo Klimant (pp. 2463-2474).

Keywords: Optical sensor; Oxygen; pH; Carbon dioxide; Temperature; Luminescence

New fluorescent perylene bisimide indicators—a platform for broadband pH optodes by Daniel Aigner; Sergey M. Borisov; Ingo Klimant (pp. 2475-2485).

Asymmetric perylene bisimide (PBI) dyes are prepared and are shown to be suitable for the preparation of fluorescence chemosensors for pH. They carry one amino-functional substituent which introduces pH sensitivity via photoinduced electron transfer (PET) while the other one increases solubility. The luminescence quantum yields for the new indicators exceed 75% in the protonated form. The new indicators are non-covalently entrapped in polyurethane hydrogel D4 and poly(hydroxyalkylmethacrylates). Several PET functions including aliphatic and aromatic amino groups were successfully used to tune the dynamic range of the sensor. Because of their virtually identical spectral properties, various PBIs with selected PET functions can easily be integrated into a single sensor with enlarged dynamic range (over 4 pH units). PBIs with two different substitution patterns in the bay position are investigated and possess variable spectral properties. Compared with their tetrachloro analogues, tetra-tert-butyl-substituted PBIs yield more long-wave excitable sensors which feature excellent photostability. Cross-sensitivity to ionic strength was found to be negligible. The practical applicability of the sensors may be compromised by the long response times (especially in case of tetra-tert-butyl-substituted PBIs).

Keywords: Perylene; pH; Sensor; Fluorescence; Photoinduced electron transfer

Time-resolved mid-IR spectroscopy of (bio)chemical reactions in solution utilizing a new generation of continuous-flow micro-mixers by Christoph Wagner; Wolfgang Buchegger; Michael Vellekoop; Martin Kraft; Bernhard Lendl (pp. 2487-2497).

A specially designed micro-mixer made of silicon, calcium fluoride, and silicone with an optical transmission path of 8 μm has been used for mid-IR spectroscopy monitoring of mixing-induced chemical reactions in the low millisecond time regime. The basic principle of the proposed continuous-flow technique is to mix two liquids introduced in two times two alternatingly stacked layers through diffusion at the entrance of a 200 μm wide, 1 cm long micro-fluidic channel also serving as measurement area. By using this special, dedicated arrangement, diffusion lengths and hence the mixing times can be significantly shortened and the overall performance improved in comparison to previous systems and alternative methods. Measurements were carried out in transmission mode using an Fourier transform infrared (FTIR) microscope, recording spectra with spot sizes of 180 × 100 μm² each at defined spots along this channel. Each of these spots corresponds to a specific reaction time: moving the measurement spot towards the entry yields shorter reaction times, moving it towards the channel’s end gives longer reaction times. This principle is generic in nature and provides a solution for accurate, chemically induced triggering of reactions requiring the mixing of two liquid reagents or reagent solutions. A typical experiment thus yields up to 85 time-coded data points, covering a time span from 1 to 80 ms at a total reagent consumption of only about 125 μL. Using the fast neutralization reaction of acetic acid with sodium hydroxide as a model, the time required for 90% mixing was determined to be around 4 ms. Additionally, first experiments on ubiquitin changing its secondary structure from native to “A-state” were carried out, illustrating the potential for time-resolved measurements of proteins in aqueous solutions. Figure

Keywords: IR spectroscopy; Microfluidics; Kinetics

Surface molecular imprints of WGA lectin as artificial receptors for mass-sensitive binding studies by Thipvaree Wangchareansak; Chak Sangma; Kiattawee Choowongkomon; Franz Dickert; Peter Lieberzeit (pp. 2499-2506).

Wheat germ agglutinin (WGA) lectin is a model compound for the interaction between viruses and cells during infection events and thus an interesting analyte for mass-sensitive sensing to study these interaction phenomena. Scanning tunneling microscopy studies reveal that surface molecular imprinting leads to cavities having the dimensions of WGA dimers. These reincorporate WGA from phosphate-buffered saline between 1 and 160 μg/ml. Whereas the quartz crystal microbalance (QCM) frequency for molecularly imprinted polymer (MIP)-coated electrodes decreases, indicating uptake of the analyte, their nonimprinted counterparts yield positive, concentration-dependent frequency shifts characteristic for slip of the analyte on the QCM surface. The MIPs achieve selectivity factors towards bovine serum albumin of roughly 4 at higher protein concentrations. Brunauer-Emmett-Teller analysis reveals that binding is favored by 29 kJ/mol until the adsorption of up to ten monolayers on the MIP, whereas above this range the value is lower. Together with the binding behavior of MIP and nonimprinted polymers, this indicates that the MIP acts as a nucleus for multilayer deposition onto the surface.

Keywords: Wheat germ agglutinin lectin; Molecular imprinting; Quartz crystal microbalance; Brunauer–Emmett–Teller analysis

Dual and tetraelectrode QCMs using imprinted polymers as receptors for ions and neutral analytes by Usman Latif; Adnan Mujahid; Adeel Afzal; Renatus Sikorski; Peter A. Lieberzeit; Franz L. Dickert (pp. 2507-2515).

Polymers as coating materials were combined with quartz crystal microbalances (QCMs) to design sensor devices for the detection of both ionic and neutral analytes in liquid phase. The design and geometry of dual and tetraelectrode QCMs have been optimized to reduce electric field interferences. An unusual Sauerbrey effect was observed while exposing potassium salt solution to 10- and 20-MHz QCMs, i.e. increase in the frequency shifts by a factor of seven, which is attributed to electro-acoustic phenomena. Non-functionalized sol-gel materials were synthesized by templating with hydrophobic salt such as tetraethyl ammonium picrate. Imprinting with these ions of low charge density leads to sensitive layers, and UV–Vis spectroscopy was used to check re-inclusion of this analyte. In the next strategy, functionalized polyurethane for potassium ions and sol-gel materials with aminopropyl group as ligand were generated to tune selectivity and sensitivity towards Ni²⁺ and Cu²⁺. Methacrylic acid polymers were optimized for the detection of atrazine by hydrogen bonding; double molecular imprinted polyurethane approach was followed for pyrene recognition. Finally, these imprinted polymers were combined with tetraelectrode QCM to develop sensor platform.

Keywords: Molecular imprinting; Polyurethane; QCM; Self-organised structures; Sol-gel materials

Multi-modal applicability of a reversed-phase/weak-anion exchange material in reversed-phase, anion-exchange, ion-exclusion, hydrophilic interaction and hydrophobic interaction chromatography modes by Michael Lämmerhofer; Raquel Nogueira; Wolfgang Lindner (pp. 2517-2530).

We recently introduced a mixed-mode reversed-phase/weak anion-exchange type separation material based on silica particles which consisted of a hydrophobic alkyl strand with polar embedded groups (thioether and amide functionalities) and a terminal weak anion-exchange-type quinuclidine moiety. This stationary phase was designed to separate molecules by lipophilicity and charge differences and was mainly devised for peptide separations with hydroorganic reversed-phase type elution conditions. Herein, we demonstrate the extraordinary flexibility of this RP/WAX phase, in particular for peptide separations, by illustrating its applicability in various chromatographic modes. The column packed with this material can, depending on the solute character and employed elution conditions, exploit attractive or repulsive electrostatic interactions, and/or hydrophobic or hydrophilic interactions as retention and selectivity increments. As a consequence, the column can be operated in a reversed-phase mode (neutral compounds), anion-exchange mode (acidic compounds), ion-exclusion chromatography mode (cationic solutes), hydrophilic interaction chromatography mode (polar compounds), and hydrophobic interaction chromatography mode (e.g., hydrophobic peptides). Mixed-modes of these chromatographic retention principles may be materialized as well. This allows an exceptionally flexible adjustment of retention and selectivity by tuning experimental conditions. The distinct separation mechanisms will be outlined by selected examples of peptide separations in the different modes. Figure A silica-based material functionalized with alkyl-strands having polar embedded groups and terminal anion-exchange site provides exceptional operational flexibility and can be used in reversed-phase, anion-exchange, ionexclusion, hydrophilic interaction and hydrophobic interaction chromatography as well as mix-modes thereof

Keywords: Mixed-mode stationary phase; Reversed-phase/weak anion-exchange material; Hydrophilic interaction chromatography (HILIC); Hydrophobic interaction chromatography (HIC); Ion-exclusion chromatography

Characterization of polyoxyethylenes according to the number of hydroxy end groups by hydrophilic interaction chromatography at critical conditions for polyethylene glycol by Shazia Abrar; Bernd Trathnigg (pp. 2531-2537).

Polyoxyethylenes with different functionality and architecture can be separated according to the number of terminal hydroxy groups on polar stationary phases in acetone water mobile phases containing 90–97% acetone. The best results were obtained on a HILIC column. Typical samples, which can be analyzed by this technique, are polyethylene glycols, their mono- and dialkyl ethers, macromonomers, the fatty esters of PEG, and ethoxylated glycerol. Figure

Keywords: PEG; Ethoxylate; Functionality; Liquid chromatography under critical conditions; HILIC

Chocolate HILIC phases: development and characterization of novel saccharide-based stationary phases by applying non-enzymatic browning (Maillard reaction) on amino-modified silica surfaces by Georg Schuster; Wolfgang Lindner (pp. 2539-2554).

Novel saccharide-based stationary phases were developed by applying non-enzymatic browning (Maillard Reaction) on aminopropyl silica material. During this process, the reducing sugars glucose, lactose, maltose, and cellobiose served as “ligand primers”. The reaction cascade using cellobiose resulted in an efficient chromatographic material which further served as our model Chocolate HILIC column. (Chocolate refers to the fact that these phases are brownish.) In this way, an amine backbone was introduced to facilitate convenient manipulation of selectivity by additional attractive or repulsive ionic solute–ligand interactions in addition to the typical HILIC retention mechanism. In total, six different test sets and five different mobile phase compositions were investigated, allowing a comprehensive evaluation of the new polar column. It became evident that, besides the so-called HILIC retention mechanism based on partition phenomena, additional adsorption mechanisms, including ionic interactions, take place. Thus, the new column is another example of a HILIC-type column characterized by mixed-modal retention increments. The glucose-modified materials exhibited the relative highest overall hydrophobicity of all grafted Chocolate HILIC columns which enabled retention of lipophilic analytes with high water content mobile phases. Proposed reaction scheme for Chocolate HILIC packings, exemplified for glucose-modified G-Choc HILIC. The brown circles represent the “Maillard Reaction” based ligands.

Keywords: HILIC; Hydrophilic interaction chromatography; Weak anion exchanger; Stationary phase; Maillard reaction

Analysis of flavonoids in honey by HPLC coupled with coulometric electrode array detection and electrospray ionization mass spectrometry by Karoline Petrus; Heidi Schwartz; Gerhard Sontag (pp. 2555-2563).

The analysis of flavonoids in unifloral honeys by high-performance liquid chromatography (HPLC) coupled with coulometric electrode array detection (CEAD) is described. The compounds were extracted by a nonionic polymeric resin (Amberlite XAD-2) and then separated on a reversed phase column using gradient elution. Quercetin, naringenin, hesperetin, luteolin, kaempferol, isorhamnetin, and galangin were detected in a coulometric electrode array detection system between +300 and +800 mV against palladium reference electrodes, and their presence was additionally confirmed by HPLC coupled with electrospray ionization mass spectrometry. The method was applied to analysis of 19 honeys of different varieties and origin. The limits of detection and quantitation ranged between 1.6 and 8.3 μg/kg and 3.9 and 27.4 μg/kg, respectively. The recoveries were above 96% in fluid and above 89% in creamy honeys. Some of these honeys (melon, pumpkin, cherry blossom, dandelion, maple, and pine tree honey) were investigated for their flavonoid content and profile for the first time. Differences between honeys were observed both in flavonoid concentrations and in the flavonoid profiles. The flavonoid concentrations ranged from 0.015 to 3.4 mg/kg honey. Galangin, kaempferol, quercetin, isorhamnetin, and luteolin were detected in all investigated honeys, whereas hesperetin occurred only in lemon and orange honeys and naringenin in lemon, orange, rhododendron, rosemary, and cherry blossom honeys. Figure Electrode array detection

Keywords: Honeys; Flavonoid profiles; HPLC-CEAD; HPLC-ESI-MS

Identification and quantification of flavonoids and ellagic acid derivatives in therapeutically important Drosera species by LC–DAD, LC–NMR, NMR, and LC–MS by Martin Zehl; Christina Braunberger; Jürgen Conrad; Marija Crnogorac; Stanimira Krasteva; Bernhard Vogler; Uwe Beifuss; Liselotte Krenn (pp. 2565-2576).

Droserae herba is a drug commonly used for treatment of convulsive or whooping cough since the seventeenth century. Because of the contribution of flavonoids and ellagic acid derivatives to the therapeutic activity of Droserae herba, an LC–DAD method has been developed for quantification of these analytes in four Drosera species used in medicine (Drosera anglica, D. intermedia, D. madagascariensis, and D. rotundifolia). During elaboration of the method 13 compounds, including three substances not previously described for Drosera species, were detected and unambiguously identified by means of extensive LC–MS and LC–NMR experiments and by off-line heteronuclear 2D NMR after targeted isolation. The most prominent component of D. rotundifolia and D. anglica, 2″-O-galloylhyperoside, with myricetin-3-O-β-glucopyranoside and kaempferol-3-O-(2″-O-galloyl)-β-galactopyranoside, were identified for the very first time in this genus. The LC–DAD method for quantification was thoroughly validated, and enables, for the first time, separation and precise analysis of these analytes in Droserae herba. Simple sample preparation and use of a narrow-bore column guarantee low cost and simplicity of the suggested system, which is excellently suited to quality control of the drug or herbal medicinal products containing this drug. Figure 2″-O-galloylhyperoside - a major compound in Drosera anglica and Drosera rotundifolia

Keywords: Drosera; Identification; Quantification; LC–DAD; LC–MS; LC–NMR

Analysis of polyethyleneoxide macromonomers by liquid chromatography along the critical adsorption line by Shazia Abrar; Bernd Trathnigg (pp. 2577-2586).

Polyoxyethylene macromonomers are analyzed by one-dimensional liquid chromatography under different conditions, depending on the required information. These samples may contain polyethylene glycol (PEG) and the corresponding di(meth)acrylate besides the desired mono(meth)acrylate. The molar mass distribution (MMD) of the PEG and the monoester can be obtained by liquid adsorption chromatography (LAC) on a reversed-phase column in acetone–water with a gradient from 10% to 20% acetone. The MMD of the diesters can be obtained with isocratic elution by liquid chromatography at critical conditions (LCCC) on a reversed-phase column in 31% acetone, or using size-exclusion conditions for PEG and LAC conditions for the end groups, which is the case in 40–55% acetone. The absolute amount of the series with different functionality can be obtained by LCCC in ternary mobile phases consisting of acetone, methanol, and water along the critical adsorption line. Under such conditions, all series elute as narrow peaks (regardless their MMD), which can easily be integrated and quantified.

Keywords: PEG-MA; Macromonomer; Functionality; Liquid chromatography under critical conditions; Critical adsorption line

LC–MS analysis of low molecular weight organic acids derived from root exudation by Leonhard Jaitz; Bernhard Mueller; Gunda Koellensperger; Daniela Huber; Eva Oburger; Markus Puschenreiter; Stephan Hann (pp. 2587-2596).

A sensitive method for quantification of citric, fumaric, malic, malonic, oxalic, trans aconitic, and succinic acid in soil- and root-related samples is presented. The method is based on a novel, fast, and simple esterification procedure and subsequent analysis via liquid chromatography–mass spectrometry. Derivatization comprises in situ generation of HCl, which catalyzes the Fischer esterification with benzyl alcohol. As a key advance, the esterification with the aromate allows reversed-phase separation and improves electrospray ionization efficiency. The method provided procedural detection limits of 1 nM for citric, 47 nM for fumaric, 10 nM for malic, 10 nM for malonic, 16 nM for oxalic, 15 nM for succinic, and 2 nM for aconitic acid utilizing 500 μL of liquid sample. The working range was 3 nM to 10 μM for citric acid, 158 nM to 10 μM for fumaric acid, 34 nM to 10 μM for malic acid, 33 nM to 10 μM for malonic acid, 53 nM to 10 μM for oxalic acid, 48 nM to 10 μM for succinic acid, and 6 nM to 10 μM for aconitic acid. Quantification of the analytes in soil-related samples was performed via external calibration of the entire procedure utilizing ¹³C-labeled oxalic and citric acid as internal standards. The robustness of the method was tested with soil extracts and samples from hydroponic experiments. The latter concerned the regulation of phosphorus solubilization via plant root exudation of citric, malic, and oxalic acid.

Keywords: Low molecular weight organic acids; Root exudates; LC–MS

Quantitative analysis of cycloartane glycosides in black cohosh rhizomes and dietary supplements by RRLC-ELSD and RRLC-qTOF-MS by Serhat S. Cicek; Anita Aberham; Markus Ganzera; Hermann Stuppner (pp. 2597-2605).

In this study, a fast and reproducible RRLC-ELSD method for the quantitative analysis of 17 cycloartane glycosides and the aglycone cimigenol in black cohosh rhizomes and dietary supplements has been developed. Separation of the 18 triterpenes was achieved within 16 min using reversed phase material and a gradient elution system consisting of water, acetonitrile and methanol. The method was validated for accuracy (recovery rates from 96.79% to 102.86%), precision (intra-day variation ≤5.98%, inter-day variation ≤3.74%), repeatability (R.S.D. ≤ 6.94%) and sensitivity, with detection limits below 4.0 μg/mL and quantification limits lower than 13.2 μg/mL. Calibration curves were established in the range from 5–1,000 μg/mL, with correlation coefficients higher than 0.998 for all constituents investigated. Peak purity and peak assignment were confirmed by means of RRLC-qTOF-MS and in comparison with reference compounds. Three different MS sources (ESI, APCI and APPI) were compared for their ionisation potential regarding cycloartane derivatives. One of the isolated black cohosh constituents, 24-O-acetylhydroshengmanol-3-O-α-l-arabinopyranoside, could be identified as new natural compound.

Keywords: Actaea racemosa ; Black cohosh; Triterpenoids; Cycloartane glycosides; RRLC-ELSD/MS; Dietary supplements

Improved SEC-FTIR method for the characterization of multimodal high-density polyethylenes by Christian Piel; Andreas Albrecht; Corinna Neubauer; Christian W. Klampfl; Jens Reussner (pp. 2607-2613).

A size-exclusion chromatography-Fourier transform infrared spectroscopy (SEC-FTIR) method for the analysis of high-density polyethylene copolymers was developed, providing superior resolution for the determination of short-chain branching as a function of time and improved repeatability by hardware adaptation and processing optimization. SEC-FTIR for characterization of polyolefins is a compromising technique. Best resolution in terms of molecular weight and molecular weight distribution requires a very low sample solution concentration in size-exclusion chromatography while best results from online infrared (IR) spectroscopy require as high concentrations as possible. The signal-to-noise ratio at the IR detector could be increased significantly after application of a bandpass filter instead of a steel mesh attenuator and furthermore influences of system instabilities could be decreased by changes in data processing. Reliable short-chain branching information in the high molecular weight section in respect to accuracy and repeatability with better chromatographic resolution could be achieved.

Keywords: Polyolefine analysis; Short-chain branching; Size-exclusion chromatography; Fourier transform infrared spectroscopy

Determination of ochratoxin A in grains by immuno-ultrafiltration and HPLC-fluorescence detection after postcolumn derivatisation in an electrochemical cell by Elisabeth Viktoria Reiter; Margit Cichna-Markl; Duck-Hwa Chung; Won-Bo Shim; Jürgen Zentek; Ebrahim Razzazi-Fazeli (pp. 2615-2622).

The paper presents a new sample clean-up method based on immuno-ultrafiltration for the analysis of ochratoxin A in cereals. In contrast to immunoaffinity chromatography, in immuno-ultrafiltration, the antibodies are used in non-immobilised form. Ochratoxin A was extracted with ACN/water (60/40, v/v), and the extract was loaded onto the ultrafiltration device. After a washing step with phosphate-buffered saline, containing 0.05% Tween 20, ochratoxin A was eluted with MeOH/acetic acid (99/1, v/v). The detection of ochratoxin A was carried out with high-performance liquid chromatography and a fluorescence detector coupled to an electrochemical cell (Coring cell). The electrochemical cell was used to eliminate matrix interferences by oxidising matrix compounds. The method was validated by repeatedly analysing spiked barley and rye samples as well as a certified wheat reference material. Recoveries and standard deviations (1 SD) were found to be 71 ± 9%, 77 ± 12% and 77 ± 8% in wheat, barley and rye, respectively. The limit of detection (S/N = 3) and limit of quantitation (S/N = 10) were determined to be 0.4 μg kg^-1 and 1 μg kg^-1. The analysis of the certified reference material resulted in ochratoxin A concentrations which were in the range assigned by the producer. Additionally, the effect of the electrochemical cell on other widely used clean-up techniques, namely the immunoaffinity clean-up and multifunctional columns (Mycosep #229), was evaluated. In all clean-up methods, an improvement of the chromatogram quality was registered.

Keywords: Ochratoxin A; Immuno-ultrafiltration; HPLC; Electrochemical cell; Sample clean-up; Grains

A versatile electrophoric derivatisation reagent for negative ion chemical ionisation mass spectrometry: o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride by Hans Jörg Leis; Werner Windischhofer (pp. 2623-2630).

The synthesis of a novel electrophoric derivatisation reagent, o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride, is described. The reagent was tested against selected primary and secondary amino compounds, as well as phenolic and aliphatic hydroxyl compounds as analytical targets. The derivatives exhibit excellent mass spectral properties under negative ion chemical ionisation, i.e. reduced fragmentation and thus high ion current for the targeted m/z during analysis. Since the reagent bears a pentafluorobenzyl ester group, resulting negative ion chemical ionisation mass spectra were expectedly dominated by dissociative resonance electron capture typically observed with these compounds, additionally showing neutral loss of carbon dioxide and ammonia (in the case of primary amines). The reagent is suitable for detecting the target compounds with high sensitivity, as exemplified for the analysis of amphetamine and methylphenidate from human plasma where chromatographic background is drastically reduced by a shift in detected m/z and retention time and lower limits of quantification at 7.8 pg/mL (amphetamine) and 4.5 pg/mL (methylphenidate) can be obtained. The choice of two or three target quantification masses allows selective detection and adjustment of lowest background interference. No carryover effect was observed for the derivatives of amphetamine and methylphenidate. Figure Structure of PBTFB-Cl

Keywords: NICI; GC-MS; Electrophore; Derivatisation

Use of proton transfer reaction time-of-flight mass spectrometry for the analytical detection of illicit and controlled prescription drugs at room temperature via direct headspace sampling by B. Agarwal; F. Petersson; S. Jürschik; P. Sulzer; A. Jordan; T. D. Märk; P. Watts; C. A. Mayhew (pp. 2631-2639).

The first reported use of proton transfer reaction time-of-flight mass spectrometry (PTR-TOF-MS) for the detection of a range of illicit and prescribed drugs is presented here. We describe the capabilities of PTR-TOF-MS to detect the following commonly used narcotics—ecstasy (N-methyl-3,4-methylenedioxyamphetamine), morphine, codeine, cocaine and heroin—by the direct sampling of the headspace above small solid quantities (approximately 50 mg) of the drugs placed in glass vials at room temperature, i.e. with no heating of the sample and no pre-concentration. We demonstrate in this paper the ability to identify the drugs, both illicit and prescribed, using PTR-TOF-MS through the accurate m/z assignment of the protonated parent molecule to the second decimal place. We have also included in this study measurements with an impure sample of heroin, containing typical substances found in “street” heroin, to illustrate the use of the technology for more “real-world” samples. Therefore, in a real-world complex chemical environment, a high level of confidence can be placed on the detection of drugs. Although the protonated parent is observed for all drugs, the reactant channel leading to this species is not the only one observed and neither is it necessarily the most dominant. Details on the observed fragmentation behaviour are discussed and compared to electrospray ionisation MSⁿ studies available in the literature.

Keywords: Drug detection; PTR-TOF-MS; Illicit drugs; Heroin; Cocaine; Ecstasy; Morphine; Codeine

Quality evaluation of tandem mass spectral libraries by Herbert Oberacher; Wolfgang Weinmann; Sebastian Dresen (pp. 2641-2648).

Tandem mass spectral libraries are gaining more and more importance for the identification of unknowns in different fields of research, including metabolomics, forensics, toxicology, and environmental analysis. Particularly, the recent invention of reliable, robust, and transferable libraries has increased the general acceptance of these tools. Herein, we report on results obtained from thorough evaluation of the match reliabilities of two tandem mass spectral libraries: the MSforID library established by the Oberacher group in Innsbruck and the Weinmann library established by the Weinmann group in Freiburg. Three different experiments were performed: (1) Spectra of the libraries were searched against their corresponding library after excluding either this single compound-specific spectrum or all compound-specific spectra prior to searching; (2) the libraries were searched against each other using either library as reference set or sample set; (3) spectra acquired on different mass spectrometric instruments were matched to both libraries. Almost 13,000 tandem mass spectra were included in this study. The MSforID search algorithm was used for spectral matching. Statistical evaluation of the library search results revealed that principally both libraries enable the sensitive and specific identification of compounds. Due to higher mass accuracy of the QqTOF compared with the QTrap instrument, matches to the MSforID library were more reliable when comparing spectra with both libraries. Furthermore, only the MSforID library was shown to be efficiently transferable to different kinds of tandem mass spectrometers, including “tandem-in-time” instruments; this is due to the coverage of a large range of different collision energy settings—including the very low range—which is an outstanding characteristics of the MSforID library. Figure Comparison of the number of false-negative search results obtained for a set of spectra acquired on different tandem mass spectrometric instruments matched to either the MSforID library or the Weinmann library

Keywords: Tandem mass spectrometry; Mass spectral library; Forensics/toxicology

Investigation of element distribution and homogeneity of TXRF samples using SR-micro-XRF to validate the use of an internal standard and improve external standard quantification by C. Horntrich; S. Smolek; A. Maderitsch; R. Simon; P. Kregsamer; C. Streli (pp. 2649-2654).

Total reflection X-ray fluorescence analysis (TXRF) offers a nondestructive qualitative and quantitative analysis of trace elements. Due to its outstanding properties TXRF is widely used in the semiconductor industry for the analysis of silicon wafer surfaces and in the chemical analysis of liquid samples. Two problems occur in quantification: the large statistical uncertainty in wafer surface analysis and the validity of using an internal standard in chemical analysis. In general TXRF is known to allow for linear calibration. For small sample amounts (low nanogram (ng) region) the thin film approximation is valid neglecting absorption effects of the exciting and the detected radiation. For higher total amounts of samples deviations from the linear relation between fluorescence intensity and sample amount can be observed. This could be caused by the sample itself because inhomogeneities and different sample shapes can lead to differences of the emitted fluorescence intensities and high statistical errors. The aim of the study was to investigate the elemental distribution inside a sample. Single and multi-element samples were investigated with Synchrotron-radiation-induced micro X-ray Fluorescence Analysis (SR-μ-XRF) and with an optical microscope. It could be proven that the microscope images are all based on the investigated elements. This allows the determination of the sample shape and potential inhomogeneities using only light microscope images. For the multi-element samples, it was furthermore shown that the elemental distribution inside the samples is homogeneous. This justifies internal standard quantification. Figure Microscope (left) and SR-μ-XRF images (right) of a multi-element sample (V, Mn, Ni, Ge; 10 ng each)

Keywords: Internal standard; External standard; TXRF; SR-μ-XRF; Homogeneity

An ultra-sensitive online SPE-LC-MS/MS method for the quantification of levonorgestrel released from intrauterine devices by Christina Moser; Andreas Gschließer; Verena Mattle; Ludwig Wildt; Andrea Griesmacher; Christoph Seger (pp. 2655-2662).

A selective and sensitive liquid chromatography-tandem mass spectrometry method for the determination of very low levonorgestrel (d-(−)-norgestrel) serum levels such as those found in patients using levonorgestrel-releasing intrauterine devices (IUDs) was developed. To achieve the sub-nanomolar sensitivity needed to measure such serum levels, a diethyl ether extraction sample preparation protocol was applied prior to the online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) assay. Analyte quantification from the selected reaction monitoring experiments relied on the use of sixfold deuterated norgestrel as internal standard. The final method was linear up to 1.50 ng/ml with a lower limit of quantification (LLOQ) of 0.05 ng/ml. It was found to be precise and accurate with imprecision <8% and bias <6% assessed at three control levels. Total analyte recovery measured in patient pools at three concentration levels was found to exceed 92%. Matrix interferences were excluded by post-column analyte infusion experiments. As a proof of concept, a set of IUD patient serum samples was screened for their levonorgestrel content. A total of 97.5% (n = 94) of the samples did show serum levels exceeding the LLOQ, proving the applicability of the assay in relevant clinical cohorts. This method must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the in vitro diagnostic directive (98/79/EG) of the European community.

Keywords: Levonorgestrel; Contraceptives; Intrauterine device; LC-MS/MS

Quantitative determination of methylphenidate in plasma by gas chromatography negative ion chemical ionisation mass spectrometry using o-(pentafluorobenzyloxycarbonyl)-benzoyl derivatives by Hans J. Leis; Helmut Schütz; Werner Windischhofer (pp. 2663-2670).

The use of a novel electrophoric derivatisation reagent, o-(pentafluorobenzyloxycarbonyl)-benzoyl chloride, for the quantitative determination of methylphenidate in plasma is described. The drug can be quantitatively measured down to 72 pg/mL plasma using only 250 μL of sample due to the extraordinary sensitivity of the derivatives under negative ion chemical ionisation mass spectrometry. Plasma samples were made alkaline with carbonate buffer and treated with extraction solvent n-hexane and reagent solution for 30 min, which, after concentration, was measured by GC-NICI-MS. The method is rapid as extraction and derivatisation occur in one single step. A stable isotope-labelled internal standard was used and its synthesis described. Full validation data are given to demonstrate the usefulness of the assay, including specificity, linearity, accuracy and precision, long-term stability, short-term stability, freeze–thaw stability, stock solution stability, autosampler stability, aliquot analysis, robustness, matrix effect, and prospective analytical batch size accuracy. The method has been successfully applied to pharmacokinetic profiling of the drug after oral application.

Keywords: Methylphenidate; GC-MS; Negative ion chemical ionisation; PBBCl; Derivatisation

Erratum to: Rapid identification and semi-quantitative determination of polymer additives by desorption electrospray ionization/time-of-flight mass spectrometry by Silke M. Reiter; Wolfgang Buchberger; Christian W. Klampfl (pp. 2671-2671).

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Analytical and Bioanalytical Chemistry (v.400, #8)

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Analytical and Bioanalytical Chemistry (v.400, #8)