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Analytical and Bioanalytical Chemistry (v.403, #2)

Electrochemistry–mass spectrometry: an emerging hyphenated technique for bioanalysis by Martin Vogel; Uwe Karst (pp. 333-334).

studied chemistry at the University of Münster from 1994 to 1998 and finished with a “Dipl.-Chem.”. In 2001, he received a Ph.D. in analytical chemistry. His thesis work, performed under the direction of Professor Karst, was directed towards the development of a derivatisation method for the determination of airborne isocyanates utilising liquid chromatography with spectroscopic and mass spectrometric detection. Following his doctoral studies, he joined the faculty of Chemical Technology at the University of Twente (the Netherlands) as an assistant professor of analytical chemistry in 2001. Since May 2006, Martin Vogel has been a member of the Department of Analytical Chemistry at the University of Münster. received his Ph.D. at the University of Münster, Germany, working in Karl Cammann’s group, and moved to the University of Colorado in Boulder for a postdoctoral fellowship with Robert E. Sievers. After finishing his habilitation in Münster, he was appointed Full Professor of Chemical Analysis at the University of Twente (the Netherlands). In 2005, he took over his current position as Chair of Analytical Chemistry in Münster. His main research interests include hyphenated techniques, focussing in particular on pharmaceutical analysis, elemental speciation and metallomics.

Utilizing the inherent electrolysis in a chip-based nanoelectrospray emitter system to facilitate selective ionization and mass spectrometric analysis of metallo alkylporphyrins by Gary J. Van Berkel; Vilmos Kertesz (pp. 335-343).

A commercially available chip-based infusion nanoelectrospray ionization system was used to ionize metallo alkylporphyrins for mass spectrometric detection and structure elucidation by mass spectrometry. Different ionic forms of model compounds (nickel (II), vanadyl (II), copper (II), and cobalt (II) octaethylporphyrin) were created by using two different types of conductive pipette tips supplied with the device. These pipette tips provide the conductive contact to solution at which the electrolysis process inherent to electrospray takes places in the device. The original unmodified, bare carbon-impregnated plastic pipette tips were exploited to intentionally electrochemically oxidize (ionize) the porphyrins to form molecular radical cations for detection. Use of modified pipette tips, with a surface coating devised to inhibit analyte mass transport to the surface or slow the kinetics of the analyte electrochemical reactions, was shown to limit the ionic species observed in the mass spectra of these porphyrins largely, but not exclusively, to the protonated molecule. Under the conditions of these experiments, the effective upper potential limit for oxidation with the uncoated pipette tip was 1.1 V or less, and the coated pipette tips effectively prevented the oxidation of analytes with redox potentials greater than about 0.25 V. Product ion spectra of either molecular ionic species could be used to determine the alkyl chain length on the porphyrin macrocycle. The utility of this electrochemical ionization approach for the analysis of naturally occurring samples was demonstrated using nickel geoporphyrin fractions isolated from Gilsonite bitumen. Acquiring neutral loss spectra as a means to improve the specificity of detection in these complex natural samples was also illustrated. Figure Cross sectional view of the chip-based nanoESI device used for selective ionization of metallo alkylporphyrins

Keywords: Electrospray; Electrochemistry; Oxidation; Porphyrins; Gilsonite

Simulation of the oxidative metabolism of diclofenac by electrochemistry/(liquid chromatography/)mass spectrometry by Helene Faber; Daniel Melles; Christine Brauckmann; Christoph Alexander Wehe; Kristina Wentker; Uwe Karst (pp. 345-354).

Diclofenac is a frequently prescribed drug for rheumatic diseases and muscle pain. In rare cases, it may be associated with a severe hepatotoxicity. In literature, it is discussed whether this toxicity is related to the oxidative phase I metabolism, resulting in electrophilic quinone imines, which can subsequently react with nucleophiles present in the liver in form of glutathione or proteins. In this work, electrochemistry coupled to mass spectrometry is used as a tool for the simulation of the oxidative pathway of diclofenac. Using this purely instrumental approach, diclofenac was oxidized in a thin layer cell equipped with a boron doped diamond working electrode. Sum formulae of generated oxidation products were calculated based on accurate mass measurements with deviations below 2 ppm. Quinone imines from diclofenac were detected using this approach. It could be shown for the first time that these quinone imines do not react with glutathione exclusively but also with larger molecules such as the model protein β-lactoglobulin A. A tryptic digest of the generated drug–protein adduct confirms that the protein is modified at the only free thiol-containing peptide. This simple and purely instrumental set-up offers the possibility of generating reactive metabolites of diclofenac and to assess their reactivity rapidly and easily.

Keywords: Diclofenac; Electrochemistry; Phase I metabolism; Protein adducts; Glutathione adducts; High-resolution mass spectrometry

Investigation of some biologically relevant redox reactions using electrochemical mass spectrometry interfaced by desorption electrospray ionization by Mei Lu; Chloe Wolff; Weidong Cui; Hao Chen (pp. 355-365).

Recently we have shown that, as a versatile ionization technique, desorption electrospray ionization (DESI) can serve as a useful interface to combine electrochemistry (EC) with mass spectrometry (MS). In this study, the EC/DESI-MS method has been further applied to investigate some aqueous phase redox reactions of biological significance, including the reduction of peptide disulfide bonds and nitroaromatics as well as the oxidation of phenothiazines. It was found that knotted/enclosed disulfide bonds in the peptides apamin and endothelin could be electrochemically cleaved. Subsequent tandem MS analysis of the resulting reduced peptide ions using collision-induced dissociation (CID) and electron-capture dissociation (ECD) gave rise to extensive fragment ions, providing a fast protocol for sequencing peptides with complicated disulfide bond linkages. Flunitrazepam and clonazepam, a class of nitroaromatic drugs, are known to undergo reduction into amines which was proposed to involve nitroso and N-hydroxyl intermediates. Now in this study, these corresponding intermediate ions were successfully intercepted and their structures were confirmed by CID. This provides mass spectrometric evidence for the mechanism of the nitro to amine conversion process during nitroreduction, an important redox reaction involved in carcinogenesis. In addition, the well-known oxidation reaction of chlorpromazine was also examined. The putative transient one-electron transfer product, the chlorpromazine radical cation (m/z 318), was captured by MS, for the first time, and its structure was also verified by CID. In addition to these observations, some features of the DESI-interfaced electrochemical mass spectrometry were discussed, such as simple instrumentation and the lack of background signal. These results further demonstrate the feasibility of EC/DESI-MS for the study of the biology-relevant redox chemistry and would find applications in proteomics and drug development research. Figure Electrochemistry coupled with mass spectrometry by desorption electrospray ionization (EC/DESI-MS) has been applied to investigate aqueous phase redox reactions of biological significance, including the reduction of peptide disulfide bonds and nitroaromatics as well as the oxidation of chlorpromazine

Keywords: Mass spectrometry; Electrochemistry; Desorption electrospray ionization; Disulfide bond reduction; Nitroreduction mechanism; Chlorpromazine radical cation

On-line electrochemistry–bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38α kinase inhibitors by David Falck; Jon S. B. de Vlieger; Martin Giera; Maarten Honing; Hubertus Irth; Wilfried M. A. Niessen; Jeroen Kool (pp. 367-375).

In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a tracer (SKF-86002) that shows fluorescence enhancement in the orthosteric binding site of the p38α kinase is the readout for bioaffinity. Parallel HR-MSⁿ experiments provided information on the identity of binders and non-binders. Finally, the data produced with this on-line system were compared to electrochemical conversion products generated off-line. The electrochemical conversion of 1-{6-chloro-5-[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazine-1-carbonyl]-3aH-indol-3-yl}-2-morpholinoethane-1,2-dione resulted in eight products, three of which showed bioaffinity in the continuous-flow p38α bioaffinity assay used. Electrochemical conversion of BIRB796 resulted, amongst others, in the formation of the reactive quinoneimine structure and its corresponding hydroquinone. Both products were detected in the p38α bioaffinity assay, which indicates binding to the p38α kinase.

Keywords: Electrochemical oxidation; p38 mitogen-activated protein kinase; High resolution mass spectrometry; Hyphenation; Bioassays

Electrochemical oxidation and protein adduct formation of aniline: a liquid chromatography/mass spectrometry study by Daniel Melles; Torsten Vielhaber; Anne Baumann; Raniero Zazzeroni; Uwe Karst (pp. 377-384).

Historically, skin sensitization tests are typically based on in vivo animal tests. However, for substances used in cosmetic products, these tests have to be replaced according to the European Commission regulation no. 1223/2009. Modification of skin proteins by electrophilic chemicals is a key process associated with the induction of skin sensitization. The present study investigates the capabilities of a purely instrumental setup to determine the potential of commonly used non-electrophilic chemicals to cause skin sensitization by the generation of electrophilic species from the parent compound. In this work, the electrophiles were generated by the electrochemical oxidation of aniline, a basic industrial chemical which may also be released from azo dyes in cosmetics. The compound is a known sensitizer and was oxidized in an electrochemical thin-layer cell which was coupled online to electrospray ionization–mass spectrometry. The electrochemical oxidation was performed on a boron-doped diamond working electrode, which is able to generate hydroxyl radicals in aqueous solutions at high potentials. Without any pretreatment, the oxidation products were identified by electrospray ionization/time-of-flight mass spectrometry (ESI-ToF-MS) using their exact masses. A mass voltammogram was generated by plotting the obtained mass spectra against the applied potential. Oligomerization states with up to six monomeric units in different redox states of aniline were observed using this setup. This approach was extended to generate adducts between the oxidation products of aniline and the tripeptide glutathione. Two adducts were identified with this trapping experiment. Protein modification was carried out subsequently: Aniline was oxidized at a constant potential and was allowed to react with β-lactoglobulin A (β-LGA) or human serum albumin (HSA), respectively. The generated adducts were analyzed by liquid chromatography coupled to ESI-ToF-MS. For both β-LGA and HSA, aniline adducts were successfully generated and identified.

Keywords: Protein modification; Electrochemistry/liquid chromatography/mass spectrometry; Aniline; Skin sensitization

Jerry Zweigenbaum (Ed.): Mass spectrometry in food safety. Methods and protocols by Simon Hird (pp. 385-386).

Karolin K. Kroening, Renee N. Easter, Douglas D. Richardson, Stuart A. Willison, Joseph A. Caruso: Analysis of chemical warfare degradation products by Tjorben Nils Posch (pp. 387-388).

10th Dresdner Sensor-Symposium—an anniversary to celebrate by Melanie Ewald; Barbara Schwarz (pp. 389-390).

Infrared spectroscopy in hemodialysis: reagent-free monitoring of patient detoxification by infrared spectroscopy by Andreas Roth; Fabian Dornuf; Oliver Klein; Daniel Schneditz; Hildegard Hafner-Gießauf; Werner Mäntele (pp. 391-399).

A method for monitoring hemodialysis based on quantitative infrared spectroscopic determination of the molecules dialyzed from patient blood is reported. The measurements are reagent-free and aim at real-time and in-line monitoring of the hemodialysis patient. A flow cell using attenuated total reflection infrared spectroscopy is coupled downstream of the dialysis filter unit. A calibration model has been developed from real hemodialysis samples analyzed by chemical reference analysis and from artificially mixed dialysis samples. The infrared monitoring of hemodialysis includes quantitative determination of urea as the lead substance, as well as glucose, lactate, and creatinine, all at a precision only limited by the chemical reference analysis. The flow cell can be fitted to all standard hemodialysis systems. Preliminary tests with hemodialysis patients have demonstrated that detoxification can be clearly monitored. Furthermore, these experiments demonstrate that a wide, real-time control of the patient’s physiological parameters is possible with this method, which could lead to increased patient safety. Figure Infrared Spectroscopy in hemodialysis: Dialysis and measuring principle

Keywords: Hemodialysis; Infrared spectroscopy; Attenuated total reflection; Fourier transform infrared spectroscopy, FT-IR; Glucose; Urea

Fast detection of triacetone triperoxide (TATP) from headspace using planar solid-phase microextraction (PSPME) coupled to an IMS detector by Wen Fan; Mimy Young; Jon Canino; James Smith; Jimmie Oxley; Jose R. Almirall (pp. 401-408).

Triacetone triperoxide (TATP) is a high explosive synthesized from easily available reactants making it accessible for illicit uses. In this study, fast detection of TATP is achieved using a novel planar solid-phase microextraction (PSPME) as a preconcentration and sampling device for headspace analysis offering improved sensitivity and reduced sampling time over the conventional fiber-based solid-phase microextraction (SPME) when followed by ion mobility spectrometer (IMS) detection. Quantitation and comparison of the retention capabilities of PSPME as compared to the commercially available SPME were determined using TATP standards and analyzed using gas chromatography–mass spectrometry for SPME analysis and a commercial IMS with no instrumental modification for PSPME. Static and dynamic headspace extractions were used and compared for PSPME extractions, in which low milligram quantities of TATP were detected within 30 s of static mode sampling and less than 5 s in the dynamic mode sampling for PSPME–IMS.

Keywords: Ion mobility spectrometer (IMS); Planar solid-phase microextraction (PSPME); Solid-phase microextraction (SPME); Triacetone triperoxide (TATP)

Detection of beryllium in digested autopsy tissues by inductively coupled plasma mass spectrometry using a high matrix interface configuration by Dominic Larivière; Mélodie Tremblay; Myriam Durand-Jézéquel; Sergei Tolmachev (pp. 409-418).

This article describes a robust methodology using the combination of instrumental design (high matrix interface—HMI), sample dilution and internal standardization for the quantification of beryllium (Be) in various digested autopsy tissues using inductively coupled plasma mass spectrometry. The applicability of rhodium as a proper internal standard for Be was demonstrated in three types of biological matrices (i.e., femur, hair, lung tissues). Using HMI, it was possible to achieve instrumental detection limits and sensitivity of 0.6 ng L⁻¹ and 157 cps L ng⁻¹, respectively. Resilience to high salt matrices of the HMI setup was also highlighted using bone mimicking solution ([Ca²⁺] = 26 to 1,400 mg L⁻¹), providing a 14-fold increase in tolerance and a 2.7-fold decrease in method detection limit compared to optimized experimental conditions obtained without the HMI configuration. Precision of the methodology to detect low levels of Be in autopsy samples was demonstrated using hair and blood certified reference materials. Be concentration ranging from 0.015 to 255 μg kg⁻¹ in autopsy samples obtained from the U.S. Transuranium and Uranium Registries were measured using the methodology presented.

Keywords: ICP-MS; Beryllium; Biological samples; High matrix; Nuclear application

Pyrolysis and combustion of tobacco in a cigarette smoking simulator under air and nitrogen atmosphere by Christian Busch; Thorsten Streibel; Chuan Liu; Kevin G. McAdam; Ralf Zimmermann (pp. 419-430).

A coupling between a cigarette smoking simulator and a time-of-flight mass spectrometer was constructed to allow investigation of tobacco smoke formation under simulated burning conditions. The cigarette smoking simulator is designed to burn a sample in close approximation to the conditions experienced by a lit cigarette. The apparatus also permits conditions outside those of normal cigarette burning to be investigated for mechanistic understanding purposes. It allows control of parameters such as smouldering and puff temperatures, as well as combustion rate and puffing volume. In this study, the system enabled examination of the effects of “smoking” a cigarette under a nitrogen atmosphere. Time-of-flight mass spectrometry combined with a soft ionisation technique is expedient to analyse complex mixtures such as tobacco smoke with a high time resolution. The objective of the study was to separate pyrolysis from combustion processes to reveal the formation mechanism of several selected toxicants. A purposely designed adapter, with no measurable dead volume or memory effects, enables the analysis of pyrolysis and combustion gases from tobacco and tobacco products (e.g. 3R4F reference cigarette) with minimum aging. The combined system demonstrates clear distinctions between smoke composition found under air and nitrogen smoking atmospheres based on the corresponding mass spectra and visualisations using principal component analysis.

Keywords: Single-photon ionisation; Mass spectrometry; Pyrolysis; Cigarette mainstream smoke; Tobacco; 3R4F

Glutathione peroxidase inhibitory assay for electrophilic pollutants in diesel exhaust and tobacco smoke by Norbert Staimer; Tran B. Nguyen; Sergey A. Nizkorodov; Ralph J. Delfino (pp. 431-441).

We developed a rapid kinetic bioassay demonstrating the inhibition of glutathione peroxidase 1 (GPx-1) by organic electrophilic pollutants, such as acrolein, crotonaldehyde, and p-benzoquinone, that are frequently found as components of tobacco smoke, diesel exhaust, and other combustion sources. In a complementary approach, we applied a high-resolution proton-transfer reaction time-of-flight mass spectrometer to monitor in real-time the generation of electrophilic volatile carbonyls in cigarette smoke. The new bioassay uses the important antioxidant selenoenzyme GPx-1, immobilized to 96-well microtiter plates, as a probe. The selenocysteine bearing subunits of the enzyme’s catalytic site are viewed as cysteine analogues and are vulnerable to electrophilic attack by compounds with conjugated carbonyl systems. The immobilization of GPx-1 to microtiter plate wells enabled facile removal of excess reactive inhibitory compounds after incubation with electrophilic chemicals or aqueous extracts of air samples derived from different sources. The inhibitory response of cigarette smoke and diesel exhaust particle extracts were compared with chemical standards of a group of electrophilic carbonyls and the arylating p-benzoquinone. GPx-1 activity was directly inactivated by millimolar concentrations of highly reactive electrophilic chemicals (including acrolein, glyoxal, methylglyoxal, and p-benzoquinone) and extracts of diesel and cigarette smoke. We conclude that the potential of air pollutant components to generate oxidative stress may be, in part, a result of electrophile-derived covalent modifications of enzymes involved in the cytosolic antioxidant defense. Figure Cu/Zn superoxide dismutase (SOD-1) and glutathione peroxidase (GPx) are linked together in the cytosolic defense against reactive oxygen and nitrogen species (RONS). Cu/Zn-SOD catalyzes the dismutation of superoxide to oxygen and hydrogen peroxide (H₂O₂). H₂O₂ and other hydroperoxides are subsequently reduced by the selenoenzyme GPx. The selenofunction is viewed as a cysteine analogue, and in comparison to other thiol enzymes, is even more vulnerable to electrophilic attack by chemicals such as acrolein at physiological conditions. Cu/Zn-SOD and GPx team up with a complex cellular antioxidant system that includes catalase, glutathione transferase and reduced glutathione (not shown). Environmental exposure to reactive electrophiles present in cigarette smoke and diesel exhaust emissions may add to the endogenous burden of oxidative stress by direct inactivation of GPx

Keywords: Air pollution; Electrophiles; Antioxidant enzymes; Oxidative stress

Automated annotation and quantification of metabolites in ¹H NMR data of biological origin by Erik Alm; Tove Slagbrand; K. Magnus Åberg; Erik Wahlström; Ingela Gustafsson; Johan Lindberg (pp. 443-455).

In ¹H NMR metabolomic datasets, there are often over a thousand peaks per spectrum, many of which change position drastically between samples. Automatic alignment, annotation, and quantification of all the metabolites of interest in such datasets have not been feasible. In this work we propose a fully automated annotation and quantification procedure which requires annotation of metabolites only in a single spectrum. The reference database built from that single spectrum can be used for any number of ¹H NMR datasets with a similar matrix. The procedure is based on the generalized fuzzy Hough transform (GFHT) for alignment and on Principal-components analysis (PCA) for peak selection and quantification. We show that we can establish quantities of 21 metabolites in several ¹H NMR datasets and that the procedure is extendable to include any number of metabolites that can be identified in a single spectrum. The procedure speeds up the quantification of previously known metabolites and also returns a table containing the intensities and locations of all the peaks that were found and aligned but not assigned to a known metabolite. This enables both biopattern analysis of known metabolites and data mining for new potential biomarkers among the unknowns.

Keywords: ¹H NMR; Alignment; Multivariate; Metabolomics; Hough transform; Urine; Quantification; Spectral profiling

Profiling changes triggered during maturation of dendritic cells: a lipidomic approach by Deolinda R. Santinha; Diane R. Marques; Elisabete A. Maciel; Cláudia S. O. Simões; Susana Rosa; Bruno M. Neves; Bárbara Macedo; Pedro Domingues; M. Teresa Cruz; M. Rosário M. Domingues (pp. 457-471).

Lipids are important in several biological processes because they act as signalling and regulating molecules, or, locally, as membrane components that modulate protein function. This paper reports the pattern of lipid composition of dendritic cells (DCs), a cell type of critical importance in inflammatory and immune responses. After activation by antigens, DCs undergo drastic phenotypical and functional transformations, in a process known as maturation. To better characterize this process, changes of lipid profile were evaluated by use of a lipidomic approach. As an experimental model of DCs, we used a foetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). The results showed that LPS treatment increased ceramide (Cer) and phosphatidylcholine (PC) levels and reduced sphingomyelin (SM) and phosphatidylinositol (PI) content. Mass spectrometric analysis of a total lipid extract and of each class of lipids revealed that maturation promoted clear changes in ceramide profile. Quantitative analysis enabled identification of an increase in the total ceramide content and enhanced Cer at m/z 646.6, identified as Cer(d18:1/24:1), and at m/z 648.6, identified as Cer(d18:1/24:0). The pattern of change of these lipids give an extremely rich source of data for evaluating modulation of specific lipid species triggered during DC maturation.

Keywords: Lipidomics; Phospholipids; Sphingolipids; Dendritic cells; Lipopolysaccharide; Mass spectrometry; Electrospray ionization

Deuterium-labelled N-acyl-l-homoserine lactones (AHLs)—inter-kingdom signalling molecules—synthesis, structural studies, and interactions with model lipid membranes by Dorota Jakubczyk; Christoph Barth; Adam Kubas; Frances Anastassacos; Patrick Koelsch; Karin Fink; Ute Schepers; Gerald Brenner-Weiß; Stefan Bräse (pp. 473-482).

N-Acyl-l-homoserine lactones (AHLs) are synthesized by Gram-negative bacteria. These quorum-sensing molecules play an important role in the context of bacterial infection and biofilm formation. They also allow communication between microorganisms and eukaryotic cells (inter-kingdom signalling). However, very little is known about the entire mechanism of those interactions. Precise structural studies are required to analyse the different AHL isomers as only one form is biologically most active. Theoretical studies combined with experimental infrared and Raman spectroscopic data are therefore undertaken to characterise the obtained compounds. To mimic interactions between AHL and cell membranes, we studied the insertion of AHL in supported lipid bilayers, using vibrational sum-frequency-generation spectroscopy. Deuterium-labelled AHLs were thus synthesized. Starting from readily available deuterated fatty acids, a two-step procedure towards deuterated N-acyl-l-homoserine lactones with varying chain lengths is described. This included the acylation of Meldrum’s acid followed by amidation. Additionally, the detailed analytical evaluation of the products is presented herein. Figure Figure Deuterium labelled N-acyl-l-homoserine lactones (AHLs) were synthesized in 2 steps. The combination of theoretical and experimental IR and Raman spectroscopy enables identification of most probable structures of AHLs. The integration of the deuterated AHLs in model lipid membranes (supported lipid bilayers) was further investigated using sum-frequency-generation (SFG) spectroscopy, to mimic interactions between AHL and cell membranes

Keywords: Inter-kingdom signalling; Deuterium-labelled AHLs; DFT modelling; Geometry optimization; Supported lipid bilayers (SLBs); Sum-frequency-generation (SFG) spectroscopy

Metabotyping of human colorectal cancer using two-dimensional gas chromatography mass spectrometry by Mainak Mal; Poh Koon Koh; Peh Yean Cheah; Eric Chun Yong Chan (pp. 483-493).

Colorectal cancer (CRC) is the fourth most common cause of death from cancer in the world. The limitations of the currently available methods and biomarkers for CRC management highlight the necessity of finding novel markers. Metabonomics can be used to search for potential markers that can provide molecular insight into human CRC. The emergence of two-dimensional gas chromatography time of flight mass spectrometry (GC × GC/TOFMS) has comprehensively enhanced the metabolic space coverage of conventional GC/MS. In this study, a GC × GC/TOFMS was developed for the tissue-based global metabonomic profiling of CRC. A Pegasus GC × GC/TOFMS (Leco Corp., St. Joseph, MI, USA) system comprising an Agilent 7890 GC and Pegasus IV TOFMS was used for this purpose. An Agilent DB-1 (30 m × 250 μm × 0.25 μm) fused silica capillary column and a Restek Rxi®-17 (1 m × 100 μm × 0.10 μm) fused silica capillary column were used as the primary and secondary columns, respectively. The method was applied for global metabonomic profiling of matched CRC and normal tissues (n = 63) obtained from 31 CRC patients during surgery. An attempt was also made to compare GC × GC/TOFMS with GC/MS and NMR in similar application. The results showed that the metabotype associated with CRC is distinct from that of normal tissue and led to the identification of chemically diverse marker metabolites. Metabolic pathway mapping suggested deregulation of various biochemical processes such as glycolysis, Krebs cycle, osmoregulation, steroid biosynthesis, eicosanoid biosynthesis, bile acid biosynthesis, lipid, amino acid and nucleotide metabolism. Fig Workflow of GCGC/TOFMS metabonomic profiling of human colorectal cancer

Keywords: Two-dimensional gas chromatography mass spectrometry; Metabonomics; Colorectal cancer; Global metabolic profiling; Endogenous metabolites

Diels–Alder derivatization for sensitive detection and characterization of conjugated linoleic acids using LC/ESI-MS/MS by Tatsuya Higashi; Mioko Takekawa; Jun Zhe Min; Toshimasa Toyo’oka (pp. 495-502).

The utility of Diels–Alder derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) for liquid chromatography/electrospray ionization tandem mass spectrometry of conjugated linoleic acids (CLAs) was examined. PTAD rapidly reacted with the CLAs, and the resulting derivatives were highly responsive in electrospray ionization mass spectrometry operating in the positive-ion mode. The derivatives produced characteristic product ions during tandem mass spectrometry, which enabled the sensitive detection [limit of detection 18 fmol (signal-to-noise ratio of 5)] and the identification of the conjugated diene position. The PTAD derivatization also significantly increased the reversed-phase liquid chromatography separation selectivity for the most biologically active CLA isomers: cis-9,trans-11-CLA and trans-10,cis-12-CLA. The PTAD derivatization was applied to analyses of food and biological samples; the major CLAs in milk and beef fat samples were successfully identified, and trace amounts of CLAs in human saliva were detected with a simple pretreatment and short analysis time.

Keywords: Conjugated linoleic acid; 4-Phenyl-1,2,4-triazoline-3,5-dione; Liquid chromatography/electrospray ionization tandem mass spectrometry; Diels–Alder derivatization; Positional isomer; Detectability

A broadly applicable approach to prepare monoclonal anti-cephalosporin antibodies for immunochemical residue determination in milk by Anna Bremus; Richard Dietrich; Lars Dettmar; Ewald Usleber; Erwin Märtlbauer (pp. 503-515).

A simple, efficient and rapid method for the synthesis of cephalosporin–protein conjugates was established. These conjugates were used as immunogens to produce monoclonal antibodies (mAbs) and as solid phase antigens in competitive indirect enzyme immunoassays (EIAs). With this generic approach, a novel set of monoclonal antibodies for cephalosporins was prepared, including ceftiofur and cephalexin as well as, reported here for the first time, cefoperazone, cefquinome and cephapirin. All 5 EIAs were highly sensitive, with standard curve IC₅₀ values of 0.7 (ceftiofur), 1.1 (cefquinome), 5.2 (cephalexin), 13.8 (cefoperazone) and 40.3 ng mL⁻¹ (cephapirin). Detection limits (IC₃₀) ranged from 0.3 (ceftiofur mAb 1D7) to 17.2 ng mL⁻¹ (cephapirin mAb 2F10). Specificity studies revealed that cephalosporin–antibody binding was strongly determined by the side chain residues of the cephem nucleus. Therefore all mAbs, to some extent, recognized other beta-lactam antibiotics with similar side chain residues. Within the group of cephalosporins approved for use in veterinary medicine, however, the final EIAs were highly selective for their respective antigen, except for the ceftiofur EIA which showed cross-reactions with cefquinome. The applicability of the five assays for drug residue testing in milk was demonstrated. In each EIA the target drug could be determined in milk with high accuracy and precision at concentrations far below the European Union maximum residue limits. Figure Structures of cephalosporins for which an MRL has been set within the EU. Monoclonal antibodies were produced against those substances shown in green lettering.

Keywords: Cephalosporins; Enzyme immunoassay; Milk; Monoclonal antibodies; Residues

A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format by Meike V. Beer; Claudia Rech; Sylvia Diederichs; Kathrin Hahn; Kristina Bruellhoff; Martin Möller; Lothar Elling; Jürgen Groll (pp. 517-526).

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems. Figure We present a hydrogel coating system for well-plates that can be covalently modified with peptides, sugars or proteins by dip coating. These coatings then allow specific interaction screening of the immobilized ligands with peptides, proteins or cells.

Keywords: Bioassays; Immunoassays/ELISA; Interaction screening; Biofunctionalization; Hydrogel coating; 96-Well plate format

An international assessment of the metrological equivalence of higher-order measurement services for creatinine in serum by Johanna E. Camara; Katrice A. Lippa; David L. Duewer; Hugo Gasca-Aragon; Blaza Toman (pp. 527-535).

The Consultative committee for amount of substance-metrology in chemistry (CCQM)-K80 Key Comparison directly assessed the equivalence of many of the world’s higher-order value-assigned materials (HOVAMs) for creatinine in human serum. This 2009 international study compared the certified values and uncertainties of the materials using measurements made under repeatability conditions. The study evaluated 17 materials submitted by 6 national metrology institutes (NMIs). The creatinine quantity in these materials ranged from 3 mg/kg to 57 mg/kg (about 0.3 mg/dL to 6 mg/dL or 30 nmol/L to 500 nmol/L). All materials were stored and prepared according the specifications provided by the participating NMIs. Samples were processed and analyzed under repeatability conditions by one analyst using isotope-dilution liquid chromatography–mass spectrometry in two measurement campaigns. The certified values and repeatability measurements were compared using uncertainty-weighted generalized distance regression. The instrumental repeatability relative standard deviation was 1.2%. The measurement design required assessment of within-unit and between-campaign variability in addition to measurement repeatability. At a 95% level of confidence, the certified values for all 17 materials agreed to within their assigned uncertainties. CCQM-K80 demonstrated the metrological equivalence of the currently available HOVAMs for creatinine in human serum and of the creatinine measurement services provided by the participating NMIs.

Keywords: Creatinine; Certified reference material; Degree of equivalence; Key comparison; Metrology; National metrology institute

A Bayesian approach to the evaluation of comparisons of individually value-assigned reference materials by Blaza Toman; David L. Duewer; Hugo Gasca Aragon; Franklin R. Guenther; George C. Rhoderick (pp. 537-548).

Several recent international comparison studies used a relatively novel experimental design to evaluate the measurement capabilities of participating organizations. These studies compared the values assigned by each participant to one or more qualitatively similar materials with measurements made on all of the materials by one laboratory under repeatability conditions. A statistical model was then established relating the values to the repeatability measurements; the extent of agreement between the assigned value(s) and the consensus model reflected the participants’ measurement capabilities. Since each participant used their own supplies, equipment, and methods to produce and value-assign their material(s), the agreement between the assigned value(s) and the model was a fairer reflection of their intrinsic capabilities than provided by studies that directly compared time- and material-constrained measurements on unknown samples prepared elsewhere. A new statistical procedure is presented for the analysis of such data. The procedure incorporates several novel concepts, most importantly a leave-one-out strategy for the estimation of the consensus value of the measurand, model fitting via Bayesian posterior probabilities, and posterior coverage probability calculation for the assigned 95% uncertainty intervals. The benefits of the new procedure are illustrated using data from the CCQM-K54 comparison of eight cylinders of n-hexane in methane.

Keywords: Bayesian analysis; Degrees of equivalence; Generalized distance regression; Leave-one-out analysis; Posterior coverage probability

Development of hemoglobin A_1c certified reference material by liquid chromatography isotope dilution mass spectrometry by Jiaming Bi; Liqing Wu; Bin Yang; Yi Yang; Jing Wang (pp. 549-554).

We report the development of a National Institute of Metrology (NIM) hemoglobin A_1c (HbA_1c) certified reference material (CRM). Each CRM unit contains about 10 μL of hemoglobin. Both hemoglobin and glycated hemoglobin were quantitatively determined by high-performance liquid chromatography (HPLC)–isotope dilution mass spectrometry (IDMS) with synthesized VHLTPE and glycated VHLTPE as standards. The mass fraction of synthesized VHLTPE or glycated VHLTPE was also quantitatively determined by HPLC-IDMS with NIM amino acid CRMs as standards. The homogeneity and stability of the CRMs were examined with a commercial HbA_1c analyzer based on the HPLC principle. Fifteen units were randomly selected for homogeneity examination, and statistical analysis showed there was no inhomogeneity. Examination of the stability showed that the CRM was stable for at least 6 months at -80 °C. Uncertainty components of the balance, amino acid purity, hydrolysis and proteolysis efficiency, method reproducibility, homogeneity, and stability were taken into consideration for uncertainty evaluation. The certified value of NIM HbA_1c CRM was expressed as the ratio of HbA_1c to total hemoglobin in moles, and was (9.6 ± 1.9)% . The CRM can be used as a calibration or validation standard for clinical diagnostics. It is expected to improve the comparability for HbA_1c measurement in China.

Keywords: Hemoglobin A_1c ; Certified reference material; Liquid chromatography isotope dilution mass spectrometry; High-performance liquid chromatography isotope dilution mass spectrometry; Diabetes

Time course of expiratory propofol after bolus injection as measured by ion molecule reaction mass spectrometry by Cyrill Hornuss; Dirk Wiepcke; Siegfried Praun; Michael E. Dolch; Christian C. Apfel; Gustav Schelling (pp. 555-561).

Propofol in exhaled breath can be detected and monitored in real time by ion molecule reaction mass spectrometry (IMR-MS). In addition, propofol concentration in exhaled breath is tightly correlated with propofol concentration in plasma. Therefore, real-time monitoring of expiratory propofol could be useful for titrating intravenous anesthesia, but only if concentration changes in plasma can be determined in exhaled breath without significant delay. To evaluate the utility of IMR-MS during non-steady-state conditions, we measured the time course of both expiratory propofol concentration and the processed electroencephalography (EEG) as a surrogate outcome for propofol effect after an IV bolus induction of propofol. Twenty-one patients scheduled for routine surgery were observed after a bolus of 2.5 mg kg⁻¹ propofol for induction of anesthesia. Expiratory propofol was measured using IMR-MS and the cerebral propofol effect was estimated using the bispectral index (BIS). Primary endpoints were time to detection of expiratory propofol and time to onset of propofol’s effect on BIS, and the secondary endpoint was time to peak effect (highest expiratory propofol or lowest BIS). Expiratory propofol and changes in BIS were first detected at 43 ± 21 and 49 ± 11 s after bolus injection, respectively (P = 0.29). Peak propofol concentrations (9.2 ± 2.4 parts-per-billion) and lowest BIS values (23 ± 4) were reached after 208 ± 57 and 219 ± 62 s, respectively (P = 0.57). Expiratory propofol concentrations measured by IMR-MS have similar times to detection and peak concentrations compared with propofol effect as measured by the processed EEG (BIS). This suggests that expiratory propofol concentrations may be useful for titrating intravenous anesthesia.

Keywords: Propofol; Expiratory; Breath monitoring; Ion molecule reaction mass spectrometry

Identification and analysis of stereoselective drug interactions with low-density lipoprotein by high-performance affinity chromatography by Matthew R. Sobansky; David S. Hage (pp. 563-571).

Columns containing immobilized low-density lipoprotein (LDL) were prepared for the analysis of drug interactions with this agent by high-performance affinity chromatography (HPAC). R/S-Propranolol was used as a model drug for this study. The LDL columns gave reproducible binding to propranolol over 60 h of continuous use in the presence of pH 7.4 0.067 M potassium phosphate buffer. Experiments conducted with this type of column through frontal analysis indicated that two types of interactions were occurring between R-propranolol and LDL, while only a single type of interaction was observed between S-propranolol and LDL. The first type of interaction, which was seen for both enantiomers, involved non-saturable binding; this interaction had an overall affinity (nK _a) of 1.9 (±0.1) × 10⁵ M⁻¹ for R-propranolol and 2.7 (±0.2) × 10⁵ M⁻¹ for S-propranolol at 37 °C. The second type of interaction was observed only for R-propranolol and involved saturable binding that had an association equilibrium constant (K _a) of 5.2 (±2.3) × 10⁵ M⁻¹ at 37 °C. Similar differences in binding behavior were found for the two enantiomers at 20 °C and 27 °C. This is the first known example of stereoselective binding of drugs by LDL or other lipoproteins. This work also illustrates the ability of HPAC to be used as a tool for characterizing mixed-mode interactions that involve LDL and related binding agents. Online abstract figure Potential routes for drug interactions with low density lipoprotein (LDL).

Keywords: Low-density lipoprotein; Propranolol; Drug interactions; High-performance affinity chromatography; Stereoselective binding

Use of NMR metabolomic plasma profiling methodologies to identify illicit growth-promoting administrations by Stewart F. Graham; Ainhoa Ruiz-Aracama; Arjen Lommen; Francesca T. Cannizzo; Bartolomeo Biolatti; Christopher T. Elliott; Mark H. Mooney (pp. 573-582).

Detection of growth-promoter use in animal production systems still proves to be an analytical challenge despite years of activity in the field. This study reports on the capability of NMR metabolomic profiling techniques to discriminate between plasma samples obtained from cattle treated with different groups of growth-promoting hormones (dexamethasone, prednisolone, oestradiol) based on recorded metabolite profiles. Two methods of NMR analysis were investigated—a Carr–Purcell–Meiboom–Gill (CPMG)-pulse sequence technique and a conventional ¹H NMR method using pre-extracted plasma. Using the CPMG method, 17 distinct metabolites could be identified from the spectra. ¹H NMR analysis of extracted plasma facilitated identification of 23 metabolites—six more than the alternative method and all within the aromatic region. Multivariate statistical analysis of acquired data from both forms of NMR analysis separated the plasma metabolite profiles into distinct sample cluster sets representative of the different animal study groups. Samples from both sets of corticosteroid-treated animals—dexamethasone and prednisolone—were found to be clustered relatively closely and had similar alterations to identified metabolite panels. Distinctive metabolite profiles, different from those observed within plasma from corticosteroid-treated animal plasma, were observed in oestradiol-treated animals and samples from these animals formed a cluster spatially isolated from control animal plasma samples. These findings suggest the potential use of NMR methodologies of plasma metabolite analysis as a high-throughput screening technique to aid detection of growth promoter use. Figure ¹H NMR metabolomic profiling of plasma identifies illegal growth-promoting administrations in cattle

Keywords: Growth promoters; Plasma; Metabolite profiling; NMR; CPMG

Determination of pharmaceuticals and antiseptics in water by solid-phase extraction and gas chromatography/mass spectrometry: analysis via pentafluorobenzylation and stable isotope dilution by Jim T. Yu; Kevin J. Bisceglia; Edward J. Bouwer; A. Lynn Roberts; Mehmet Coelhan (pp. 583-591).

A sensitive yet robust analytical method is presented for the simultaneous determination of 12 human pharmaceuticals (valproic acid, phenytoin, ibuprofen, gabapentin, acetaminophen, gemfibrozil, naproxen, ketoprofen, secobarbital, phenobarbital, 5-fluorouracil, and diclofenac) and 6 antiseptics (biosol, biphenylol, p-chloro-m-cresol, p-chloro-m-xylenol, chlorophene, and triclosan). The method employs solid-phase extraction (SPE) followed by a novel pentafluorobenzylation using a mixture of acetontrile/water (1/1, v/v). The method is simple to perform (derivatization can be completed in a single test tube) and eliminates the need for any solvent/SPE cartridge drying or blow-down. It affords excellent resolution, high sensitivity and reproducibility, and freedom from interference even for matrices as complex as untreated sewage. The method was applied to the analysis of sewage samples using 15 isotopically labeled surrogates, which resulted in the detection of 10 of the 12 pharmaceuticals and all of the antiseptics sought. Ten of 15 surrogates were synthesized from pure analytes by a simple H-D exchange reaction employing D₂O and D₂SO₄. Measured recoveries were sensitive to matrix effects and varied substantially among analytes, indicative of the limitations associated with using a single surrogate standard.

Keywords: Gas chromatography–mass spectrometry (GC-MS); PPCPs; Pharmaceuticals; Wastewater; Pentafluorobenzylation

Electrosynthesis of polypyrrole on steel fiber for solid-phase microextraction of citalopram in serum by A. Nezhadali; G. Ahmadi Bonakdar; H. Nakhaei (pp. 593-600).

A solid-phase microextraction (SPME) technique using steel fiber coated with polypyrrole (PPY) was developed for UV–Vis determination of citalopram in blood serum. The coating was prepared using a three-electrode electrochemical system from an acetonitrile/aqueous (ACN/H₂O) oxalic acid solution containing 0.01 M of pyrrole monomer by applying a potential range (0–1.3 V) for 25 min. In order to obtain an efficient film of PPY, experimental parameters related to the coating process were optimized, specifically deposition potential, concentration of monomer, and number of cycles. The effects of various parameters on the efficiency of the SPME process such as extraction time, stirring speed, adsorption and desorption temperature, desorption solvent, and pH of desorption solution were also studied. The coating was stable and adhered to the surface of the steel fiber. The method was linear over about three orders of magnitude with an average correlation coefficient of 0.97. Spiking of blank samples with 0.2 μg/mL citalopram afforded a recovery of 84% with a precision of 10.2%. A limit of detection of 0.046 μg/mL (based on S/N = 3) was obtained in the linear dynamic range of 0.046–2.0 μg/mL. The proposed method was applied to monitor citalopram in serum samples. Figure Schematic of the SPME technique developed using PPY-coated steel fiber for the UV–Vis determination of citalopram in blood serum

Keywords: Polypyrrole; Solid-phase microextraction; Citalopram; Cyclic voltammetry

Two-dimensional liquid chromatography of PDMS–PS block copolymers by Muhammad Imran Malik; Gareth W. Harding; Harald Pasch (pp. 601-611).

In this study, liquid chromatography at critical conditions of polystyrene (PS) and polydimethylsiloxane (PDMS) is used as the first dimension for the two-dimensional analysis of polydimethylsiloxane-block-polystyrene copolymers. Comprehensive two-dimensional liquid chromatography with size exclusion chromatography as the second dimension reveals information about the molar mass distributions of all separated fractions from the first dimension. Furthermore, fractions eluting at the critical conditions were collected and subjected to analysis in the second dimension at the critical adsorption point of the other block. These fractions were analyzed by Fourier transform infrared spectroscopy to determine their chemical compositions. The combination of the above approaches and the calibration of the evaporative light scattering detector for the first-dimension analysis yield deep insights into the molecular heterogeneity of the block copolymer samples. The composition of the samples and the chemical composition of the real block copolymer are also calculated by combining the results obtained at both critical conditions.

Keywords: Hybrid materials; Block copolymers; Critical adsorption point (CAP); Liquid chromatography at critical conditions (LCCC); Polydimethylsiloxane (PDMS); Polystyrene (PS); Two-dimensional liquid chromatography (2D-LC); FTIR

Ambient-ageing processes in amine self-assembled monolayers on microarray slides as studied by ToF-SIMS with principal component analysis, XPS, and NEXAFS spectroscopy by Hyegeun Min; Pierre-Luc Girard-Lauriault; Thomas Gross; Andreas Lippitz; Paul Dietrich; Wolfgang E. S. Unger (pp. 613-623).

We investigated the ageing of amine-terminated self-assembled monolayers (amine-SAMs) on different silica substrates due to exposure to different ambient gases, pressures, and/or temperatures using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with principal component analysis and complementary methods of surface analysis as X-ray photoelectron spectroscopy (XPS) and near edge X-ray absorption fine structure (NEXAFS). The goal of this study is to examine the durability of primary amine groups of amine-SAMs stored in a user laboratory prior to being used as supports for biomolecule immobilization and other applications. We prepared amine-SAMs on the native oxides of silicon wafers and glass slides using 3-aminopropyl triethoxysilane, by using optimized conditions such as anhydrous organic solvent and reaction time scale of hours to avoid multilayer growth. Selected commercial amine-SAM slides have been investigated, too. When the amine-SAMs are exposed to air, oxygen incorporation occurs, followed by formation of amide groups. The formation of oxygen species due to ageing was proved by ToF-SIMS, XPS, and NEXAFS findings such as CNO⁻ secondary ion emission at m/z 42, observation of the N 1s HNC=O component peak at 400.2–400.3 eV in XPS, and, last but not least, by formation of a π*(HNC=O) resonance at 401 eV in the N K-edge X-ray absorption spectrum. It is concluded that the used multi-method approach comprising complementary ToF-SIMS, XPS, and NEXAFS analyses is well suited for a thorough study of chemical aspects of ageing phenomena of amine-SAM surfaces.

Keywords: Amine-SAM; Ageing process; ToF-SIMS; NEXAFS; XPS

New catalytic ultrasound method for derivatization of 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid in urine, with analysis by GC-MS/MS by Vanessa de M. Prata; Elissandro S. Emídio; Haroldo S. Dorea (pp. 625-632).

A new procedure is described for the derivatization by silylation of 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) present in urine, followed by analysis using gas chromatography–tandem mass spectrometry. A conventional procedure for derivatization of the analyte was evaluated using two types of experimental design. A 2³ factorial design considered the parameters temperature, reaction time, and the solvent/derivatization agent ratio. A central composite design (CCD) was applied to optimize the values of the significant variables. The optimum conditions were a reaction temperature of 50 °C, a reaction time of 30 min, and a BSTFA/acetone ratio of 40:20. The use of imidazole as a catalyst, together with ultrasonication, reduced the reaction time to 5 min and increased the efficiency of derivatization of THCCOOH, compared with the conventional method. The operating conditions of the tandem mass spectrometer were also optimized. The method was linear in the concentration range 1–50 ng mL⁻¹ (R ² = 0.9951). Intra- and inter-day precisions were 7.7–12.3% and 11.1–13.9%, respectively, recoveries ranged between 91 ± 8% and 101 ± 12%, accuracy (as % bias) was between –11.7% and +0.7%, and limits of detection and quantification were 0.5 and 1.0 ng mL⁻¹, respectively. Fig

Keywords: Derivatization; Silylation; THCCOOH; Urine; GC-MS/MS

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Analytical and Bioanalytical Chemistry (v.403, #2)