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Analytical and Bioanalytical Chemistry (v.398, #1)

The mechanics of granting tenure: suggestions for academic departments by Apryll M. Stalcup (pp. 1-7).

is currently a Professor of Chemistry at the University of Cincinnati with over 20 years of experience in the area of intermolecular interactions, using separations as the primary interrogative tool. Both fundamental research and practical applications have been emphasized in her work. Her research group has used a variety of separation platforms, including gas chromatography, liquid chromatography, capillary electrophoresis, and preparative electrophoresis. Her research effort has focused on the development of new strategies for chiral separations and includes the introduction of sulfated β-cyclodextrin and heparin as chiral selectors for capillary electrophoresis, preparative electrophoretic chiral separations, as well as the synthesis and characterization of new chromatographic stationary phases, including surface-confined ionic liquids. She currently serves on the Cincinnati Water Quality Advisory Board, the Advisory Board of Analytical and Bioanalytical Chemistry, and the editorial board of Trends in Analytical Chemistry.

The uncooked egg challenge by Hervé This (pp. 9-10).

Solution to the Avogadro constant challenge by William B. Jensen; Juris Meija (pp. 11-12).

Dimitri Pappas: Practical cell analysis by Simone König (pp. 13-14).

Yingfu Li, Yi Lu (Eds.): Functional nucleic acids for analytical applications by Xiaohong Fang (pp. 15-16).

Scientometric analysis of national university research performance in analytical chemistry on the basis of academic publications: Italy as case study by Anna Annibaldi; Cristina Truzzi; Silvia Illuminati; Giuseppe Scarponi (pp. 17-26).

is a research associate in Analytical Chemistry at the Marche Polytechnic University, Ancona (Italy), where she teaches the course on “Analysis of pollutants”. She graduated in Chemistry at the University of Bologna in 2001 and she obtained a PhD degree in Biology and Marine Ecology in 2005. Her scientific interests have focussed principally on the field of environmental analytical chemistry, particularly on the voltammetric study of heavy metals in several environmental matrices, and on the determination of organic pollutants in food. She has contributed to the development of chemical fractionation procedures for heavy metals in atmospheric aerosols and in snow. is researcher in Analytical Chemistry at the Marche Polytechnic University in Ancona (Italy) were she teaches courses on “Instrumental analytical chemistry” and “Food chemical analysis”. She worked previously at the University of Modena in the field of analytical pharmacology and environmental toxicology, on the determination of heavy metals and the study of their toxic effects in organisms. Currently, her scientific interests are focused mainly on environmental analytical chemistry, with particular attention to the determination and speciation of trace heavy metals in different environmental matrixes with different amounts of anthropization (seawater, snow, aerosol. organisms), using voltammetric techniques (DPASV, SWASV). She is also interested in determination of organic pollutants in the environment and food by gas chromatography-mass spectrometry and in the application of environmental chemometrics to marine science. She has taken part in several oceanographic campaigns. She is a member of the Italian Chemical Society (Division of Analytical Chemistry). is a research associate in Analytical Chemistry at the Marche Polytechnic University, Ancona (Italy). She graduated in Biology at the same university in 2001 and she obtained a PhD degree in Biology and Marine Ecology in 2005. Her scientific interests have focussed principally on environmental analytical chemistry, particularly on the voltammetric study of heavy metals in different environmental and food matrices and also of biomarkers in marine organisms. She has taken part in two Italian Expeditions to Antarctica and in several oceanographic campaigns (Italy, United Kingdom), contributing to the development of a voltammetric in-situ profiler system for real time heavy metal determination in sea water. is full professor of Analytical Chemistry of the Marche Polytechnic University in Ancona (Italy). He previously worked at the Universities of Camerino, Genoa, and Venice. He is a member of the Analytical Chemistry Divisions of the American Chemical Society (since 1983) and the Italian Chemical Society (since 1975) of which he has been a member of the Governing Board since 2007. His scientific interests have focused mainly on electroanalytical chemistry, environmental analytical chemistry, and chemometrics. He has taken part in seven Italian expeditions to Antarctica and in oceanographic cruises in the Mediterranean Sea and in the Ross Sea as leader of the analytical chemistry group. He was the scientific coordinator of the Italian Antarctic Station during the 1998–1999 austral summer campaign. He is a member of the scientific committee of the European Research Course on Atmospheres (Grenoble) and he is a founding associate of the Italian Aerosol Society.

Analytical chemistry for drug discovery and development by Aldo Roda (pp. 27-28).

Recent advances of capillary electrophoresis in pharmaceutical analysis by Leena Suntornsuk (pp. 29-52).

This review covers recent advances of capillary electrophoresis (CE) in pharmaceutical analysis. The principle, instrumentation, and conventional modes of CE are briefly discussed. Advances in the different CE techniques (non-aqueous CE, microemulsion electrokinetic chromatography, capillary isotachophoresis, capillary electrochromatography, and immunoaffinity CE), detection techniques (mass spectrometry, light-emitting diode, fluorescence, chemiluminescence, and contactless conductivity), on-line sample pretreatment (flow injection) and chiral separation are described. Applications of CE to assay of active pharmaceutical ingredients (APIs), drug impurity testing, chiral drug separation, and determination of APIs in biological fluids published from 2008 to 2009 are tabulated.

Keywords: Capillary electrophoresis; Active pharmaceutical ingredient; Impurity; Chiral separation; Biological fluid

Drug–protein binding: a critical review of analytical tools by Karine Vuignier; Julie Schappler; Jean-Luc Veuthey; Pierre-Alain Carrupt; Sophie Martel (pp. 53-66).

The extent of drug binding to plasma proteins, determined by measuring the free active fraction, has a significant effect on the pharmacokinetics and pharmacodynamics of a drug. It is therefore highly important to estimate drug-binding ability to these macromolecules in the early stages of drug discovery and in clinical practice. Traditionally, equilibrium dialysis is used, and is presented as the reference method, but it suffers from many drawbacks. In an attempt to circumvent these, a vast array of different methods has been developed. This review focuses on the most important approaches used to characterize drug–protein binding. A description of the principle of each method with its inherent strengths and weaknesses is outlined. The binding affinity ranges, information accessibility, material consumption, and throughput are compared for each method. Finally, a discussion is included to help users choose the most suitable approach from among the wealth of methods presented. Figure Range of binding constants (log K_a) assessable by the main separative and non-separative analytical tools used to characterize drug-protein interactions. ED: equilibrium dialysis, UF: ultrafiltration, PAMPA: parallel artificial membrane permeability assay, HPAC/ZE: high-performance affinity chromatography/zonal elution approach, HPAC/FA: high-performance affinity chromatography/frontal analysis approach, ACE: affinity capillary electrophoresis (mobility shift assay), CE/FA: capillary electrophoresis/frontal analysis, Spectro.: spectroscopic assays, ITC: isothermal titration calorimetry, comp.: competition studies, titration: titration studies, DSC: differential scanning calorimetry, SPR: surface plasmon resonance-based assays.

Keywords: Plasma proteins; Binding affinity constants; Protein binding methods; Method selection

Recent advances in the assessment of the antioxidant capacity of pharmaceutical drugs: from in vitro to in vivo evidence by Giangiacomo Beretta; Roberto Maffei Facino (pp. 67-75).

In this review, some well-established assays and more recent markers developed for the understanding of the biological activity of pharmaceutical drugs belonging to different pharmacological classes (nonsteroidal anti-inflammatory drugs, cardiovascular drugs, and central-nervous-system-acting drugs) are considered. The results of in vitro studies are reviewed and critically compared with those available from clinical trials, and their relevance for the elucidation of the mechanism of action of the drugs is discussed. Although from this examination a positive correlation between the in vitro and in vivo data seems to emerge, the small number of clinical trials available, their low number of patients enrolled, and sometimes the arbitrary or inappropriate choice of the biomarker(s) used highlight the need for (1) more standardized protocols to allow a reliable comparison of the results from different studies and (2) the development of new and more appropriate and specific biomarkers for the evaluation of oxidative stress before and after drug intervention. Figure Drug antioxidant capacity: from in vitro testing to in vivo evidence

Keywords: Pharmaceutical drugs; Free radicals; Antioxidant capacity; In vitro assays; Biomarkers

Counterfeit drugs: analytical techniques for their identification by R. Martino; M. Malet-Martino; V. Gilard; S. Balayssac (pp. 77-92).

In recent years, the number of counterfeit drugs has increased dramatically, including not only “lifestyle” products but also vital medicines. Besides the threat to public health, the financial and reputational damage to pharmaceutical companies is substantial. The lack of robust information on the prevalence of fake drugs is an obstacle in the fight against drug counterfeiting. It is generally accepted that approximately 10% of drugs worldwide could be counterfeit, but it is also well known that this number covers very different situations depending on the country, the places where the drugs are purchased, and the definition of what constitutes a counterfeit drug. The chemical analysis of drugs suspected to be fake is a crucial step as counterfeiters are becoming increasingly sophisticated, rendering visual inspection insufficient to distinguish the genuine products from the counterfeit ones. This article critically reviews the recent analytical methods employed to control the quality of drug formulations, using as an example artemisinin derivatives, medicines particularly targeted by counterfeiters. Indeed, a broad panel of techniques have been reported for their analysis, ranging from simple and cheap in-field ones (colorimetry and thin-layer chromatography) to more advanced laboratory methods (mass spectrometry, nuclear magnetic resonance, and vibrational spectroscopies) through chromatographic methods, which remain the most widely used. The conclusion section of the article highlights the questions to be posed before selecting the most appropriate analytical approach. Figure DOSY spectrum of a counterfeit artesunate tablet containing paracetamol instead of artesunate

Keywords: Counterfeit drugs; Colorimetry; Chromatography; Mass spectrometry; Diffusion-ordered ¹H NMR spectroscopy; Vibrational spectroscopies

Practical issues of hyperspectral imaging analysis of solid dosage forms by José Manuel Amigo (pp. 93-109).

Hyperspectral imaging techniques have widely demonstrated their usefulness in different areas of interest in pharmaceutical research during the last decade. In particular, middle infrared, near infrared, and Raman methods have gained special relevance. This rapid increase has been promoted by the capability of hyperspectral techniques to provide robust and reliable chemical and spatial information on the distribution of components in pharmaceutical solid dosage forms. Furthermore, the valuable combination of hyperspectral imaging devices with adequate data processing techniques offers the perfect landscape for developing new methods for scanning and analyzing surfaces. Nevertheless, the instrumentation and subsequent data analysis are not exempt from issues that must be thoughtfully considered. This paper describes and discusses the main advantages and drawbacks of the measurements and data analysis of hyperspectral imaging techniques in the development of solid dosage forms.

Keywords: Hyperspectral imaging; Near infrared; Middle infrared; Raman; Pharmaceutical research; Solid dosage forms; Tablets; Process analytical technologies

Proteomics for quality-control processes in transfusion medicine by Angelo D’Alessandro; Lello Zolla (pp. 111-124).

Following the publication of recent retrospective and highly debated studies, transfusionists are increasingly asking for improved tools to assess the quality of transfusion-relevant products. At its dawn, proteomics emerged as a potential candidate for in-depth investigations of blood components and plasma derivatives. As its maturity is now at hand, the proteomic expertise seems to be ready to be massively transferred to the clinical setting, where it could be potentially used as a valid tool to test, from bench to bedside, the quality of collected blood components prior to or during storage. Proteomic strategies have been demonstrated to be also suited to verify the effects of the production and pathogen-reduction processes of plasma derivatives and blood components on the protein fractions, or to discover particular biomarkers readily adoptable for targeted evaluation of blood-component integrity or functionality. Although the technical background is in continuous and rapid expansion, the spread of proteomics in clinical routine practice has been hitherto hampered by high costs for dedicated facilities and specialized personnel.

Keywords: Pharmacoproteomics; Proteomics; Drug; Plasma; Red blood cell; Transfusion safety

Quality analytics of internet pharmaceuticals by B. Baert; B. De Spiegeleer (pp. 125-136).

Trading pharmaceutical products through the Internet poses several challenges related to legal responsibilities, good distribution practices, information content and patient use, financial implications, but also regarding product quality. One of the major concerns is the well-known phenomenon of counterfeited and/or substandard drugs commercialized through rogue Internet sites. Therefore, controlling and assuring the quality of those products has become an important and challenging task for the authorities. This review gives an overview of the different quality attributes that can be evaluated to have a complete understanding of the quality of the finished pharmaceutical product traded through the Internet, as well as the current analytical techniques that serve this objective. Aspects considered are labelling and packaging, physicochemical quality attributes, identification and assay of active substances and/or excipients, impurity profiling, biopharmaceutical testing and data interpretation.

Keywords: Internet; Quality; Pharmaceuticals; Counterfeit; Substandard

Process analytical technology (PAT) for biopharmaceutical products by A. S. Rathore; R. Bhambure; V. Ghare (pp. 137-154).

The “Pharmaceutical Current Good Manufacturing Practices (CGMPs) for the 21st Century—A Risk Based Approach” initiative announced by the FDA in August 2002 to improve and modernize pharmaceutical manufacturing facilitated adoption of process analytical technology (PAT) by the pharmaceutical industry. The potential for improved operational control and compliance resulting from continuous real-time quality assurance was highlighted as a likely benefit that would result from PAT implementation. A considerable amount of work has been done on this topic by academic and industrial contributors in the last decade. In this paper, we will start with a brief overview of evolution of PAT concepts and a review of their application in the wider pharmaceutical industry. The rest of the paper focuses on PAT applications for biotech processes with emphasis on developments in the last five years. It is our observation that while significant advances have been accomplished with regard to our ability to analyze/monitor key process and quality attributes in the biotech industry, much more needs to be done with regard to utilizing the collected data for subsequent control of the process, to achieve optimum yield and product quality. The latter is necessary to achieve the benefits that will result from PAT implementation. Figure Ease of PAT implementation for some of the commonly used unit operations in biotech processes

Keywords: Process analytical technology, PAT; Bioprocessing; Quality by design

Circular dichroism in drug discovery and development: an abridged review by Carlo Bertucci; Marco Pistolozzi; Angela De Simone (pp. 155-166).

Chirality plays a fundamental role in determining the pharmacodynamic and pharmacokinetic properties of drugs, and contributes significantly to our understanding of the mechanisms that lie behind biorecognition phenomena. Circular dichroism spectroscopy is the technique of choice for determining the stereochemistry of chiral drugs and proteins, and for monitoring and characterizing molecular recognition phenomena in solution. The role of chirality in our understanding of recognition phenomena at the molecular level is discussed here via several selected systems of interest in the drug discovery and development area. The examples were selected in order to underline the utility of circular dichroism in emerging studies of protein–protein interactions in biological context. In particular, the following aspects are discussed here: the relationship between stereochemistry and pharmacological activity—stereochemical characterization of new leads and drugs; stereoselective binding of leads and drugs to target proteins—the binding of drugs to serum albumins; conformational transitions of peptides and proteins of physiological relevance, and the stereochemical characterization of therapeutic peptides.

Keywords: Circular dichroism; Chiral analysis; Protein–protein interactions; Drug–protein interactions; Bioanalytical methods; Molecular recognition mechanism

Applications of fluorescence and bioluminescence resonance energy transfer to drug discovery at G protein coupled receptors by Elisa Alvarez-Curto; John D. Pediani; Graeme Milligan (pp. 167-180).

The role of G protein coupled receptors (GPCRs) in numerous physiological processes that may be disrupted or modified in disease makes them key targets for the development of new therapeutic medicines. A wide variety of resonance energy transfer (RET) techniques such as fluorescence RET and bioluminescence RET have been developed in recent years to detect protein–protein interactions in living cells. Furthermore, these techniques are now being exploited to screen for novel compounds that activate or block GPCRs and to search for new, previously undiscovered signaling pathways activated by well-known pharmacologically classified drugs. The high resolution that can be achieved with these RET methods means that they are well suited to study both intramolecular conformational changes in response to ligand binding at the receptor level and intermolecular interactions involving protein translocation in subcellular compartments resulting from external stimuli. In this review we highlight the latest advances in these technologies to illustrate general principles. Figure Acceptor photobleaching of a mCerulean-mCitrine fluorescent protein tandem. Images show HEK293 cells transfected with a mCerulean-mCitrine fluorescent protein concatamer prior to selective photo-bleaching of the acceptor in the region marked by a red square. The absence of fluorescence signal in post-bleached images within the region of interest confirms that the acceptor fluorescence has been bleached and irreversibly destroyed. Once this process has taken place the donor returns to its de-quenched state that, in turn, will result in an increase in donor fluorescence intensity being detected within the bleached region.

Keywords: G protein coupled receptor; Fluorescence resonance energy transfer; Time-resolved fluorescence resonance energy transfer; Bioluminescence resonance energy transfer; High throughput; Arrestin; Drug discovery

Fluorescent cell-based sensing approaches for toxicity testing by Michael Fritzsche; Carl-Fredrik Mandenius (pp. 181-191).

Fluorimetric cell-based sensing methods have attracted increasing interest in toxicity testing of pharmaceuticals, pathogens, environmental pollutants, and other chemicals. The objective of this review is to summarise the variety of approaches reported up to now and to present recent developments in this area. The different approaches are described in relation to their underlying mechanism and, especially, to the role of the fluorophore involved. The methods discussed include the use of fluorescent or fluorogenic indicators, fluorescence-based testing for membrane integrity, approaches based on fluorescence labelling, inducible fluorescent protein expression, and analysis of cellular autofluorescence. Several of these approaches have been shown to be advantageous in comparison with non-fluorescence methods and have potential in high-throughput screening, for example in drug discovery and safety pharmacology.

Keywords: Fluorescence; Cytotoxicity; Alternative methods; In-vitro testing; In-vitro assays

Microfabricated analytical systems for integrated cancer cytomics by Donald Wlodkowic; Jonathan M. Cooper (pp. 193-209).

Tracking and understanding cell-to-cell variability is fundamental for systems biology, cytomics and computational modelling that aids e.g. anti-cancer drug discovery. Limitations of conventional cell-based techniques, such as flow cytometry and single cell imaging, however, make the high-throughput dynamic analysis on cellular and subcellular processes tedious and exceedingly expensive. The development of microfluidic lab-on-a-chip technologies is one of the most innovative and cost-effective approaches towards integrated cytomics. Lab-on-a-chip devices promise greatly reduced costs, increased sensitivity and ultrahigh throughput by implementing parallel sample processing. The application of laminar fluid flow under low Reynolds numbers provides an attractive analytical avenue for the rapid delivery and exchange of reagents with exceptional accuracy. Under these conditions, the fluid flow has no inertia, enabling the precise dosing of drugs, both spatially and temporally. In addition, by confining the dimensions of the microfluidic structure, it is possible to facilitate the precise sequential delivery of drugs and/or functional probes into the cellular systems. As only low cell numbers and operational reagent volumes are required, high-throughput integrated cytomics on a single cell level finally appears within the reach of clinical diagnostics and drug screening routines. Lab-on-a-chip microfluidic technologies therefore provide new opportunities for the development of content-rich personalized clinical diagnostics and cost-effective drug discovery. It is largely anticipated that advances in microfluidic technologies should aid in tailoring of investigational therapies and support the current computational efforts in systems biology.

Keywords: Cytomics; Cytometry; Microfluidics; Lab-on-a-chip; Real-time cell assays; Cell sorting

Functional multineuron calcium imaging for systems pharmacology by Naoya Takahashi; Yuji Takahara; Daisuke Ishikawa; Norio Matsuki; Yuji Ikegaya (pp. 211-218).

Functional multineuron calcium imaging (fMCI) is a large-scale technique used to access brain function on a single-neuron scale. It detects the activity of individual neurons by imaging action potential-evoked transient calcium influxes into their cell bodies. fMCI has recently been used as a high-throughput research tool to examine how neuronal activity is altered in animal models of brain diseases, for example stroke, Alzheimer’s disease, and epilepsy, and to estimate how pharmacological agents act on normal and abnormal states of neuronal networks. It offers unique opportunities to discover the mechanisms underlying neurological disorders and new therapeutic targets.

Keywords: Receptors/ion channels; Pharmaceuticals; Optical sensors; Fluorescence/luminescence

The beautiful cell: high-content screening in drug discovery by Marc Bickle (pp. 219-226).

The term “high-content screening” has become synonymous with imaging screens using automated microscopes and automated image analysis. The term was coined a little over 10 years ago. Since then the technology has evolved considerably and has established itself firmly in the drug discovery and development industry. Both the instruments and the software controlling the instruments and analyzing the data have come to maturity, so the full benefits of high-content screening can now be realized. Those benefits are the capability of carrying out phenotypic multiparametric cellular assays in an unbiased, fully automated, and quantitative fashion. Automated microscopes and automated image analysis are being applied at all stages of the drug discovery and development pipeline. All major pharmaceutical companies have adopted the technology and it is in the process of being embraced broadly by the academic community. This review aims at describing the current capabilities and limits of the technology as well as highlighting necessary developments that are required to exploit fully the potential of high-content screening and analysis.

Keywords: High-content screening; Imaging; Drug discovery; Multiparametric; Cell-based assay

Cell-based assays: fuelling drug discovery by Elisa Michelini; Luca Cevenini; Laura Mezzanotte; Andrea Coppa; Aldo Roda (pp. 227-238).

It has been estimated that over a billion dollars in resources can be consumed to obtain clinical approval, and only a few new chemical entities are approved by the US Food and Drug Administration (FDA) each year. Therefore it is of utmost importance to obtain the maximum amount of information about biological activity, toxicological profile, biochemical mechanisms, and off-target interactions of drug-candidate leads in the earliest stages of drug discovery. Cell-based assays, because of their peculiar advantages of predictability, possibility of automation, multiplexing, and miniaturization, seem the most appealing tool for the high demands of the early stages of the drug-discovery process. Nevertheless, cellular screening, relying on different strategies ranging from reporter gene technology to protein fragment complementation assays, still presents a variety of challenges. This review focuses on main advantages and limitations of different cell-based approaches, and future directions and trends in this fascinating field. Figure Different cell-based strategies can improve the throughput and reliability of the first stages of the drug discovery process

Keywords: Drug monitoring/drug screening; Cell systems/single-cell analysis; Biosensors; Biochips/high-throughput screening; Bioanalytical methods

Microfluidics in macro-biomolecules analysis: macro inside in a nano world by Iuliana Oita; Hadewych Halewyck; Bert Thys; Bart Rombaut; Yvan Vander Heyden; Debby Mangelings (pp. 239-264).

Use of microfluidic devices in the life sciences and medicine has created the possibility of performing investigations at the molecular level. Moreover, microfluidic devices are also part of the technological framework that has enabled a new type of scientific information to be revealed, i.e. that based on intensive screening of complete sets of gene and protein sequences. A deeper bioanalytical perspective may provide quantitative and qualitative tools, enabling study of various diseases and, eventually, may offer support for the development of accurate and reliable methods for clinical assessment. This would open the way to molecule-based diagnostics, i.e. establish accurate diagnosis and disease prognosis based on identification and/or quantification of biomacromolecules, for example proteins or nucleic acids. Finally, the development of disposable and portable devices for molecule-based diagnosis would provide the perfect translation of the science behind life-science research into practical applications dedicated to patients and health practitioners. This review provides an analytical perspective of the impact of microfluidics on the detection and characterization of bio-macromolecules involved in pathological processes. The main features of molecule-based diagnostics and the specific requirements for the diagnostic devices are discussed. Further, the techniques currently used for testing bio-macromolecules for potential diagnostic purposes are identified, emphasizing the newest developments. Subsequently, the challenges of this type of application and the status of commercially available devices are highlighted, and future trends are noted.

Keywords: Microfluidics; Diagnostics; Proteins; Nucleic acids; PCR; Virus

Ambient mass spectrometry: bringing MS into the “real world” by Rosana M. Alberici; Rosineide C. Simas; Gustavo B. Sanvido; Wanderson Romão; Priscila M. Lalli; Mario Benassi; Ildenize B. S. Cunha; Marcos N. Eberlin (pp. 265-294).

Mass spectrometry has recently undergone a second contemporary revolution with the introduction of a new group of desorption/ionization (DI) techniques known collectively as ambient mass spectrometry. Performed in an open atmosphere directly on samples in their natural environments or matrices, or by using auxiliary surfaces, ambient mass spectrometry (MS) has greatly simplified and increased the speed of MS analysis. Since its debut in 2004 there has been explosive growth in the applications and variants of ambient MS, and a very comprehensive set of techniques based on different desorption and ionization mechanisms is now available. Most types of molecules with a large range of masses and polarities can be ionized with great ease and simplicity with the outstanding combination of the speed, selectivity, and sensitivity of MS detection. This review describes and compares the basis of ionization and the concepts of the most promising ambient MS techniques known to date and illustrates, via typical analytical and bioanalytical applications, how ambient MS is helping to bring MS analysis deeper than ever into the “real world” open atmosphere environment—to wherever MS is needed. Figure Schematic of EASI

Keywords: Mass spectrometry; Ambient ionization; Desorption; Ion sources; Direct analysis; Ionization mechanisms

Chromatographic separation of antiviral/anticancer nucleoside reverse transcriptase inhibitor drugs by H. Kalász; M. Y. Hasan; E. Adeghate; K. Tekes; A. Adem (pp. 295-312).

This paper discusses the current methods used for quantitative determination of analogues of nucleotide reverse transcriptase inhibitors (NtRTIs) in body fluids, cells, and tissues. Nucleoside reverse transcriptase inhibitors (NRTIs) prodrugs given to AIDS/herpes/cancer patients conjugate with phosphates at the site of their action. Separation of phosphorylated NRTIs is generally performed by reversed-phase chromatography. After separation, plasma NRTIs can be detected using a variety of methods, including immunoassay through monitoring of UV absorbance, fluorescence, and mass spectrometry. The most recent development in the field of detection of plasma NtRTIs shows a tendency toward the use double- or triple-focusing mass spectrometry, the most specific and sensitive monitoring technique.

Keywords: High-performance liquid chromatography; Pharmaceuticals; Antiviral drugs; Phosphate conjugation; On-site analysis

Development and validation of an analytical method for determination of 3-chloropropane-1,2-diol in rat blood and urine by gas chromatography-mass spectrometry in negative chemical ionization mode by Edith Berger-Preiß; Susanne Gerling; Elisabeth Apel; Alfonso Lampen; Otto Creutzenberg (pp. 313-318).

We have developed a highly selective and sensitive method using gas chromatography-mass spectrometry with negative chemical ionization for measuring 3-chloropropane-1,2-diol (3-MCPD) in rat blood and urine. Samples were adsorbed on silica gel, extracted with ethyl acetate, and derivatized by chemical derivatization with heptafluorobutyric acid anhydride. For quantification, matrix-based calibration curves and 3-MCPD-d ₅, as an isotope-labeled internal standard, were used. The relative recoveries of 3-MCPD were between 80 and 110% in most cases and the relative standard deviations were typically less than 10%, with some exceptions. The limit of quantification of the method was found to be about 2 ng/mL. In conclusion, a valuable, robust, and sensitive method for detection of 3-MCPD is now available for biokinetics studies. Figure Typical ion chromatogram of spiked rat urine (25 ng/mL 3-MCPD)

Keywords: Biomonitoring; 3-chloropropane-1,2-diol; Rat; Blood; Urine; Gas chromatography-mass spectrometry-negative chemical ionization

Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots by Roland J. W. Meesters; Jeroen J. A. van Kampen; Mariska L. Reedijk; Rachel D. Scheuer; Lennard J. M. Dekker; David M. Burger; Nico G. Hartwig; Albert D. M. E. Osterhaus; Theo M. Luider; Rob A. Gruters (pp. 319-328).

Kaletra® (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for therapeutic drug monitoring of lopinavir and ritonavir concentrations in whole blood and in plasma from HIV-1-infected children. Whole blood was blotted onto dried blood spot (DBS) collecting cards, and plasma was collected simultaneously. DBS collecting cards were extracted by an acetonitrile/water mixture while plasma samples were deproteinized with acetone. Drug concentrations were determined by matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (MALDI-QqQ-MS/MS). The application of DBS made it possible to measure lopinavir and ritonavir in whole blood in therapeutically relevant concentrations. The MALDI-QqQ-MS/MS plasma assay was successfully cross-validated with a commonly used high-performance liquid chromatography (HPLC)–ultraviolet (UV) assay for the therapeutic drug monitoring (TDM) of HIV-1-infected patients, and it showed comparable performance characteristics. Observed DBS concentrations showed as well, a good correlation between plasma concentrations obtained by MALDI-QqQ-MS/MS and those obtained by the HPLC-UV assay. Application of DBS for TDM proved to be a good alternative to the normally used plasma screening. Moreover, collection of DBS requires small amounts of whole blood which can be easily performed especially in (very) young children where collection of large whole blood amounts is often not possible. DBS is perfectly suited for TDM of HIV-1-infected children; but nevertheless, DBS can also easily be applied for TDM of patients in areas with limited or no laboratory facilities.

Keywords: Protease inhibitors; Dried blood spots; MALDI-QqQ-MS/MS; HIV-1; Lopinavir; Ritonavir

A fast method for screening and/or quantitation of tetrahydrocannabinol and metabolites in urine by automated SPE/LC/MS/MS by Eshwar Jagerdeo; Madeline A. Montgomery; Roman P. Karas; Martin Sibum (pp. 329-338).

Marijuana is one of the most commonly used illicit substances. The high usage of this substance results in it being commonly encountered in clinical samples throughout the USA and Europe. Due to its wide availability and use, marijuana is also commonly encountered in forensic toxicology laboratories. The proposed method utilized an automated solid phase extraction (SPE) coupled to liquid chromatography/mass spectrometry (LC/MS). The automated SPE procedure was developed using Hysphere C8-EC sorbent, and the high performance liquid chromatography (HPLC) separation was performed using an Xterra MS C₁₈ column with a total runtime of 10 min. The standard curves linearity generally fell between 6 and 500 ng/mL. The limits of detection ranged from 2 to 4 ng/mL, and the limits of quantitation ranged from 8 to 12 ng/mL. The bias and imprecision were determined using a simple analysis of variance (single factor). The results demonstrate bias as <11% and percent imprecision as <12% for all components at four quality control levels. This method has been in use for over 2 years and has been applied to numerous forensic samples. When compared to other published methods, it exceeds others in its simplicity and speed of analysis. This method takes advantage of robotics and automation for a total analysis time of 10 min, including sample preparation, separation, and detection. Figure Automated solid phase extraction unit coupled to a triple quadrupole mass spectrometer

Keywords: Automation; SPE; LC/MS; Tetrahydrocannabinol; Tetrahydrocannabinol metabolites; Urine

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin by Marla D. Swain; George P. Anderson; Dan Zabetakis; Rachael D. Bernstein; Jinny L. Liu; Laura J. Sherwood; Andrew Hayhurst; Ellen R. Goldman (pp. 339-348).

Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders. All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated selected sdAb as capture and tracer elements demonstrated that all of the sdAb were able to recognize soluble (“live”) BoNT A Tx and BoNT Ac Tx with virtually no cross-reactivity with other BoNT serotypes. The isolated sdAb did not exhibit the high degree of thermal stability often associated with these reagents; after the first heating cycle most of the binding activity was lost, but the portion of the protein that did refold and recover antigen-binding activity showed only minimal loss on subsequent heating and cooling cycles. The binding kinetics of selected binders, assessed by both an equilibrium fluid array assay as well as surface plasmon resonance (SPR) using toxoided material, gave dissociation constants (K _D ) in the range 2.2 × 10⁻¹¹ to 1.6 × 10⁻¹⁰ M. These high-affinity binders may prove beneficial to the development of recombinant reagents for the rapid detection of BoNT A, particularly in field screening and monitoring applications.

Keywords: Single-domain antibody; Botulinum neurotoxin; Camelid; Immunoassay

Integration of spore-based genetically engineered whole-cell sensing systems into portable centrifugal microfluidic platforms by Amol Date; Patrizia Pasini; Sylvia Daunert (pp. 349-356).

Bacterial whole-cell biosensing systems provide important information about the bioavailable amount of target analytes. They are characterized by high sensitivity and specificity/selectivity along with rapid response times and amenability to miniaturization as well as high-throughput analysis. Accordingly, they have been employed in various environmental and clinical applications. The use of spore-based sensing systems offers the unique advantage of long-term preservation of the sensing cells by taking advantage of the environmental resistance and ruggedness of bacterial spores. In this work, we have incorporated spore-based whole-cell sensing systems into centrifugal compact disk (CD) microfluidic platforms in order to develop a portable sensing system, which should enable the use of these hardy sensors for fast on-field analysis of compounds of interest. For that, we have employed two spore-based sensing systems for the detection of arsenite and zinc, respectively, and evaluated their analytical performance in the miniaturized microfluidic format. Furthermore, we have tested environmental and clinical samples on the CD microfluidic platforms using the spore-based sensors. Germination of spores and quantitative response to the analyte could be obtained in 2.5–3 h, depending on the sensing system, with detection limits of 1 × 10⁻⁷ M for arsenite and 1 × 10⁻⁶ M for zinc in both serum and fresh water samples. Incorporation of spore-based whole-cell biosensing systems on microfluidic platforms enabled the rapid and sensitive detection of the analytes and is expected to facilitate the on-site use of such sensing systems.

Keywords: Spores; Whole-cell sensing systems; Centrifugal microfluidic platforms; Portable analytical devices; On-site analysis; Environmental and clinical samples

Ionic liquid matrices for MALDI-TOF-MS analysis of intact glycoproteins by Estela Giménez; Fernando Benavente; José Barbosa; Victoria Sanz-Nebot (pp. 357-365).

2,5-Dihydroxybenzoic acid (DHB) has been demonstrated to be a more suitable matrix than 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) to obtain reliable molecular mass values of intact glycoproteins because it prevents sugar fragmentation. Lack of spot homogeneity during the crystallization step was prevented by drying the sample-matrix mixture under vacuum conditions. Nevertheless, this sample-matrix preparation procedure requires a specific experimental setup and may be time-consuming. In this work, we investigated the effectiveness of different ionic liquid matrices (ILMs) with SA and DHB on the ionization of a set of intact glycoproteins with several degrees of glycosylation. The obtained results demonstrate that some of the tested ILMs allow detection of the studied intact glycoproteins. Furthermore, the selected optimum conditions solve the reproducibility issue of using the DHB as a solid matrix without the vacuum drying method and, surprisingly, avoid sugar fragmentation when both SA and DHB were used as ILMs.

Keywords: Erythropoietin; Glycoprotein; Glycosylation degree; Ionic liquid; MALDI

Detection of nucleotides in positive-mode electrospray ionization mass spectrometry using multiply-charged cationic ion-pairing reagents by Edra Dodbiba; Zachary S. Breitbach; Eranda Wanigasekara; Tharanga Payagala; Xiaotong Zhang; Daniel W. Armstrong (pp. 367-376).

Nucleotides are a class of molecules that play an essential role in biological systems. A new method has been developed in the detection of nucleotides. These molecules can exist as monomers or constituents of oligomers and polymers. As such, they carry from one to several negative charges. In this study, different cationic ion-pairing reagents were used to complex with each of the 28 nucleotide monomers and nucleotide containing compounds. By using this method, this discrete set of anions was able to be detected in the positive-mode electrospray ionization mass spectrometry, as positively charged complexes. Tandem mass spectrometry experiments were also completed on the ion-pairing reagents that performed the best in the single ion monitoring (SIM) ion mode, and the sensitivity was lowered even further for most of the anions. Limits of detection for compounds such as thymidine diphosphate were improved as much as 100 times compared to the positive SIM mode, and 750 times when compared to the negative mode. A few nucleotides did not show a significant increase in sensitivity when analyzed in the positive ion mode, but in general the new method developed herein resulted in a much greater sensitivity than traditional detection in the negative mode.

Keywords: Nucleotides; Positive-mode ESI-MS; Limits of detection; Ion-pairing reagent

Part I: characterization of the extracellular proteome of the extreme thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS² by David Muddiman; Genna Andrews; Derrick Lewis; Jaspreet Notey; Robert Kelly (pp. 377-389).

The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. In the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS² analysis and prediction servers suggested probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments, all including the C₁₈ bead technique, demonstrated that two levels of sample purification were necessary to effectively desalt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described, as enzymatic and putative uncharacterized proteins, anticipate consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion, and 11 contained transmembrane domain stretches suggested by Phobius and transmembrane hidden Markov model. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study, involved acetone precipitation followed by the C₁₈ bead method in which 2.4% (63 proteins) of the predicted proteome was identified, including proteins suggested to have secretion and transmembrane moieties. Figure Experimental workflow for the evaluation of sample cleanup techniques for the secretome of the thermophilic bacterium Caldicellulosiruptor saccharolyticus with possibility to traverse other similarly grown bacterium. Several sample purification methods were assessed individually as well as in combination with a C₁₈ bead method in an effort to afford the greatest number of confidently identified proteins. Analysis of the identified proteins by prediction servers complemented the experimental secretome investigation.

Keywords: Secretome; Caldicellulosiruptor saccharolyticus ; GeLC-MS² ; Thermophile; Sample preparation

Part II: defining and quantifying individual and co-cultured intracellular proteomes of two thermophilic microorganisms by GeLC-MS² and spectral counting by David Muddiman; Genna Andrews; Derrick Lewis; Jaspreet Notey; Robert Kelly (pp. 391-404).

Probing the intracellular proteome of Thermotoga maritima and Caldicellulosiruptor saccharolyticus in pure and co-culture affords a global investigation into the machinery and mechanisms enduring inside the bacterial thermophilic cell at the time of harvest. The second of a two part study, employing GeLC-MS² a variety of proteins were confidently identified with <1% false discovery rate, and spectral counts for label-free relative quantification afforded indication of the dynamic proteome as a function of environmental stimuli. Almost 25% of the T. maritima proteome and 10% of the C. saccharolyticus proteome were identified. Through comparison of growth temperatures for T. maritima, a protein associated with chemotaxis was uniquely present in the sample cultivated at the non-optimal growth temperature. It is suspected that movement was induced due to the non-optimal condition as the organism may need to migrate in the culture to locate more nutrients. The inventory of C. saccharolyticus proteins identified in these studies and attributed to spectral counting, demonstrated that two CRISPR-associated proteins had increased expression in the pure culture versus the co-culture. Further focusing on this relationship, a C. saccharolyticus phage-shock protein was identified in the co-culture expanding a scenario that the co-culture had decreased antiviral resistance and accordingly an infection-related protein was present. Alterations in growth conditions of these bacterial thermophilic microorganisms offer a glimpse into the intricacy of microbial behavior and interaction. Figure A summary comparing the number of confidently identified proteins from the cell lysis of the thermophilic bacteria T. maritima and C. saccharolyticus in mono- and co-culture. The expression profile of identified proteins provides insight into microbial interaction and behavior.

Keywords: Thermotoga maritima ; Caldicellulosiruptor saccharolyticus ; GeLC-MS² ; Co-cultivation; Intracellular proteome; Spectral counting

Simultaneous sampling of volatile and non-volatile analytes in beer for fast fingerprinting by extractive electrospray ionization mass spectrometry by Liang Zhu; Zhong Hu; Gerardo Gamez; Wai Siang Law; HuanWen Chen; ShuiPing Yang; Konstantin Chingin; Roman M. Balabin; Rui Wang; TingTing Zhang; Renato Zenobi (pp. 405-413).

By gently bubbling nitrogen gas through beer, an effervescent beverage, both volatile and non-volatile compounds can be simultaneously sampled in the form of aerosol. This allows for fast (within seconds) fingerprinting by extractive electrospray ionization mass spectrometry (EESI-MS) in both negative and positive ion mode, without the need for any sample pre-treatment such as degassing and dilution. Trace analytes such as volatile esters (e.g., ethyl acetate and isoamyl acetate), free fatty acids (e.g., caproic acid, caprylic acid, and capric acid), semi/non-volatile organic/inorganic acids (e.g., lactic acid), and various amino acids, commonly present in beer at the low parts per million or at sub-ppm levels, were detected and identified based on tandem MS data. Furthermore, the appearance of solvent cluster ions in the mass spectra gives insight into the sampling and ionization mechanisms: aerosol droplets containing semi/non-volatile substances are thought to be generated via bubble bursting at the surface of the liquid; these neutral aerosol droplets then collide with the charged primary electrospray ionization droplets, followed by analyte extraction, desolvation, ionization, and MS detection. With principal component analysis, several beer samples were successfully differentiated. Therefore, the present study successfully extends the applicability of EESI-MS to the direct analysis of complex liquid samples with high gas content. Figure By gently bubbling nitrogen gas through beer, both volatile and non-volatile compounds can be simultaneously sampled in the form of aerosol for further analysis, allowing fast chemically fingerprinting using extractive electrospray ionization mass spectrometry (EESI-MS).

Keywords: Extractive electrospray ionization; Beer analysis; EESI mechanism

LA-ICP-MS analysis of waste polymer materials by T. Stehrer; J. Heitz; J. D. Pedarnig; N. Huber; B. Aeschlimann; D. Günther; H. Scherndl; T. Linsmeyer; H. Wolfmeir; E. Arenholz (pp. 415-424).

Waste polymer materials were analyzed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The concentrations of 35 elements were determined by using different types of external standards, namely glass and polyethylene (PE) based. Prior to the LA-ICP-MS analysis of determined elements, Na and/or Zn were used as internal standards. The investigations concentrated mainly on the detection of Cr, As, Cd, Sn, Sb, Hg, and Pb. Using PE-based calibration standards, the measured concentrations in the waste polymers were within 49% of the wet chemical data. The determined deviation was up to 102% when using the glass standards. Trace concentration of As and Hg (and also of S) could be determined with a concentration below 1 mg/kg. However, Hg provided very low intensity with a high relative standard deviation (RSD) and was therefore not further evaluated. Cryomilling of polymers was applied to reduce the particle size of the material and improved the precision and accuracy of LA-ICP-MS analysis. On average, the LA-ICP-MS results showed a deviation from the wet chemical reference analysis of 38% and an RSD of 56% for pressed polymer powder samples prepared by cryomilling. In general, waste pellets without sample preparation (i.e., use of pellets as delivered) are too heterogeneous, not suitable for micro-beam techniques, and showed a strong matrix dependence. With homogeneous pellets that appear similar to each other agreement in the determined concentrations was found for some elements.

Keywords: Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS); Polymer waste pellets; Certified reference materials; Cryogenic impact mill; Trace element detection

Determination of organic acids in Vaccinium berry standard reference materials by Melissa M. Phillips; Ryan J. Case; Catherine A. Rimmer; Lane C. Sander; Katherine E. Sharpless; Stephen A. Wise; James H. Yen (pp. 425-434).

Nine organic acids (citric acid, galacturonic acid, glycolic acid, isocitric acid, malic acid, oxalic acid, quinic acid, shikimic acid, and tartaric acid) and two anions (phosphate and sulfate) were determined in a suite of Vaccinium berry-containing dietary supplement standard reference materials (SRMs). Following solvent extraction, three independent methods were utilized in the quantification of these compounds. The first method involved reversed-phase liquid chromatography with ultraviolet absorbance detection at 210 nm and isotope dilution mass spectrometry. The second method utilized ion chromatography with conductivity detection. Finally, gas chromatography with isotope dilution mass spectrometry detection was used following derivatization with N-methyl-N-trifluoroacetamide (MSTFA). The combined data from these methods was used for the assignment of organic acid levels in the seven candidate SRMs.

Keywords: Organic acids; Berry; Liquid chromatography; Gas chromatography; Ion chromatography; Mass spectrometry; Isotope dilution

Characterization of metal glycinate complexes by electrospray Q-TOF-MS/MS and their determination by capillary electrophoresis–ICP-MS: application to premix samples by Véronique Vacchina; Sébastien Oguey; Catherine Ionescu; David Bravo; Ryszard Lobinski (pp. 435-449).

A method was developed for the determination of metal complexes with glycine (glycinates, [M(Gly)_x(H₂O)_y(SO₄)_z]_n, where M denotes Zn, Cu, Mn and Fe) in premix samples used for the preparation of animal feeds enriched in essential trace elements. The method was based on the extraction of the glycinates with 10 mM ammonium acetate (pH 7.4) followed by their determination using capillary electrophoresis with ICP MS detection. The stability of the glycinates in solution was verified by electrospray TOF-MS. Each supplement was shown to be a mixture of complexes, with polymerization degrees ranging from n = 1 to n = 4 (depending on the metal), that were fully or partially dehydrated. The metal glycine complex moiety was found to be preserved during capillary electrophoresis. The detection limits, calculated as three times the standard deviation of the blank plus the blank, were between 0.05 and 0.2 µg mL⁻¹ (as the metal), and the calibration curves were linear, allowing the analysis of premix samples. Repeatability for glycinate standards was below 12%, and analytical precision was typically within 15%.

Keywords: Cu, Zn, Mn and Fe glycinates; Capillary electrophoresis; Speciation; ICP-MS; Electrospray MS

Selective extraction of sulfonamides from food by use of silica-coated molecularly imprinted polymer nanospheres by Ruixia Gao; Junjie Zhang; Xiwen He; Langxing Chen; Yukui Zhang (pp. 451-461).

We report the use of nanospheres prepared by coating silica with molecularly imprinted polymer (MIP) for sulfamethoxazole (SMO). The resulting SiO₂–SMO–MIP nanoparticles have highly improved imprinting and adsorption capacity, and can be used for separation and determination of sulfonamides in eggs and milk. In the synthesis, monodispersed SiO₂ nanoparticles (Si–NP) of diameter 80 nm were amino-modified by reaction with 3-aminopropyltriethoxylsilane. The acryloyl monolayer was then grafted onto the amine-modified Si–NP. Finally, the MIP films were coated on to the surface of Si–NP by the copolymerization of the vinyl end groups with functional monomer (acrylamide), cross-linking agent (ethylene glycol dimethacrylate), initiator (azo-bis-isobutyronitrile), and template molecule (sulfamethoxazole). The resulting SiO₂–SMO–MIP–NP were characterized by transmission electron microscopy, scanning electron microscopy, and Fourier transform infrared spectrometry. The adsorption properties were demonstrated by equilibrium rebinding experiments and Scatchard analysis. The results showed that the binding sites of the SiO₂–SMO–MIP were highly accessible, and the maximum adsorption capacity of the SiO₂–SMO–MIP for SMO was 20.21 mg g⁻¹. The selectivity of the SiO₂–SMO–MIP–NP obtained was elucidated by using SMO and structurally related sulfonamides. The results indicated that the SiO₂–SMO–MIP had significant selectivity for SMO. The feasibility of removing SMO and sulfadiazine (SDZ) from food samples was proved by use of spiked milk and eggs. A method for the separation and determination of trace SMO and SDZ in milk and egg samples was developed, with recoveries ranging from 69.8% to 89.1%. Figure The nanosized and uniform silica-molecularly imprinted polymers (MIPs) for sulfamethoxazole (SMO) was described. The resulting SiO₂/SMO-MIPs nanoparticles possess a highly improved imprinting effect and adsorption capacity, and can be applied in separation and determination of sulfonamides in the eggs and milk.

Keywords: Molecularly imprinted polymers; Nanospheres; Sulfonamide antibiotics; Adsorbents; High-performance liquid chromatography

Exploratory analysis of human urine by LC–ESI-TOF MS after high intake of olive oil: understanding the metabolism of polyphenols by Rocío García-Villalba; Alegría Carrasco-Pancorbo; Ekaterina Nevedomskaya; Oleg A. Mayboroda; André M. Deelder; Antonio Segura-Carretero; Alberto Fernández-Gutiérrez (pp. 463-475).

Olive oil polyphenols have important biological properties which closely depend on their bioavailability; it is, therefore, essential to understand how polyphenols are absorbed, metabolized, and eliminated from the body. An analytical method based on rapid-resolution liquid chromatography (RRLC) coupled with mass spectrometric detection with a time-of-flight analyzer (RRLC–ESI-TOF MS) has been developed for analysis of the main olive oil phenolic compounds and their metabolites in human urine. Urine samples from ten healthy volunteers were collected before and 2, 4, and 6 h after intake of 50 mL extra-virgin olive oil. The proposed method includes liquid–liquid extraction with ethyl acetate, which provides extraction recoveries of the phenolic compounds studied between 35 and 75% from spiked urine samples. Good repeatability was obtained—the relative standard deviations (RSDs) of peak areas in intra-day and inter-day studies were 4.3 and 6.5%, respectively. Statistical studies enabled us to discriminate between urine samples before and after intake, and facilitated the search for m/z values enabling this discrimination. Based on the very accurate mass information and the isotopic pattern provided by the TOF MS analyzer, together with other available information, ten of these biomarkers and more than 50 metabolites, obtained through phase I and phase II biotransformation reactions, were tentatively identified. Additionally, kinetic studies were conducted on the metabolites identified as possible biomarkers; for most of the compounds concentrations were maximum in the first two hours.

Keywords: RRLC–ESI-TOF MS; Olive oil; Phenolic compounds; Urine sample; Metabolites

Ex vivo and in vivo diagnosis of C6 glioblastoma development by Raman spectroscopy coupled to a microprobe by Abdelilah Beljebbar; Sylvain Dukic; Nadia Amharref; Michel Manfait (pp. 477-487).

The potential of Raman spectroscopy for ex vivo and in vivo classification of normal and glioblastoma brain tumor development was investigated. High-quality spectra of normal and tumor tissues were obtained using a portable Raman spectrometer coupled to a microprobe with a signal integration time of 5 s. Ex vivo results demonstrated that by using the biochemical information contained in the spectra, we were able to distinguish between normal brain features (white and gray matter), invasion, and tumor tissues with a classification accuracy of 100%. Differences between these features resulted from variations in their lipid signal contributions, which probably reflect differences in the level of myelinization. This finding supports the ability of in vivo Raman spectroscopy to delineate tumor margins during surgery. After implanting C6 cells in rat brain, we monitored, in vivo, the development of glioblastoma tumor from days 0 to 20 post-implantation (PI). The classification exhibited a clear separation of the data into two clusters: one cluster was associated with normal brain tissues (cortex), and the second was related to data measured from tumor evolution. The second cluster could be divided into two subclusters, one associated with tumor tissue from 4 to 13 days PI and the second related to tumor tissue from 15 to 20 days PI. Histological analysis reveals that the differences between these two subclusters are: the presence of a massive infiltration zone in the brain tissue from 4 to 13 days PI, and; a maturation of the tumor characterized by the appearance of edematous and necrotic zones, as well as a diminution in the proliferative and invasive area, from 15 days. This work demonstrates the potential of Raman spectroscopy to provide diagnostic information for the early detection of tumors in vivo.

Keywords: In vivo Raman spectroscopy; Glioblastoma development; Raman microprobe; Multivariate statistical analysis; Classification

Ultra-fast two-dimensional microchip electrophoresis using SDS μ-CGE and microemulsion electrokinetic chromatography for protein separations by John K. Osiri; Hamed Shadpour; Steven A. Soper (pp. 489-498).

A poly(methyl methacrylate) microfluidic chip was used to perform a two-dimensional (2-D) separation of a complex protein mixture in short development times. The separation was performed by combining sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS μ-CGE) with microemulsion electrokinetic chromatography (μ-MEEKC), which were used for the first and second dimensions, respectively. Fluorescently labeled Escherichia coli cytosolic proteins were profiled by this 2-D approach with the results compared to a similar 2-D separation using SDS μ-CGE × μ-MEKC (micelle electrokinetic chromatography). The relatively short column lengths (effective length = 10 mm) for both dimensions were used to achieve separations requiring only 220 s of development time. High spot production rates (131 ± 11 spots min⁻¹) and reasonable peak capacities (481 ± 18) were generated despite the fact that short columns were used. In addition, the use of μ-MEEKC in the second dimension was found to produce higher peak capacities compared to μ-MEKC (481 ± 18 for μ-MEEKC and 332 ± 17 for μ-MEKC) due to the higher plate numbers associated with μ-MEEKC.

Keywords: Two-dimensional electrophoresis; Protein profiling; E. coli proteins; SDS micro-CGE; micro-MEEKC; PMMA microchip

1H-Pteridine-2,4-dione (lumazine): a new MALDI matrix for complex (phospho)lipid mixtures analysis by Cosima D. Calvano; Saverio Carulli; Francesco Palmisano (pp. 499-507).

Nowadays, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry represents an emerging and versatile tool for analysis of lipids. However, direct (i.e., with no previous separation of lipid classes) analysis of crude extracts containing a complex mixture of lipids (a problem typically encountered in shotgun lipidomics) is still a quite challenging task using a conventional MALDI matrix such as 2,5-dihydroxybenzoic acid (DHB). Indeed, in the presence of phospholipids containing quaternary ammonium groups, such as phosphatidylcholines and sphingomyelins, strong ionization-suppression effects are experienced especially in positive ion mode. To overcome this limitation, lumazine (1H-pteridine-2,4-dione) was evaluated as an alternative matrix. Lumazine in the solid state showed an absorption maximum at 350 nm, ionizes/desorbs without appreciable decomposition and extensive cluster formation, and can be used in both ion modes. In positive ion mode, the main species were M •⁺ and 2M •⁺ radical cations and cationized species ([M+H]⁺, [M+Na]⁺, [M+2Na+2Li-3H]⁺). In negative ion mode, the main signals observed were the deprotonated molecular ion and the radical anion. The signal-to-noise ratio for phosphatidylglycerols and phosphatidylethanolamines using lumazine was almost 1 order of magnitude higher than that observed for DHB. Lumazine was successfully used for MALDI analysis (positive and negative ion modes) of crude lipid extracts of milk, soymilk, and hen egg, where phosphatidylethanolamines, phosphatidylserines, and phosphatidylinositols could additionally be detected.

Keywords: Lumazine; Matrix-assisted laser desorption/ionization; Mass spectrometry; Phospholipids; Food

Spatially resolved determination of the structure and composition of diatom cell walls by Raman and FTIR imaging by Martin Kammer; René Hedrich; Hermann Ehrlich; Jürgen Popp; Eike Brunner; Christoph Krafft (pp. 509-517).

Vibrational spectroscopic imaging has developed into a versatile tool to study the local composition of various materials. Here, we present for the first time that Raman mapping and Fourier transform infrared imaging are useful tools to study diatom cell walls as is demonstrated for the species Stephanopyxis turris. The unicellular diatoms exhibit intricately micro- and nano-patterned cell walls, which consist of amorphous silica as well as various organic and inorganic constituents, thus making up an extremely interesting inorganic/organic hybrid material. The structure and composition of this material as well as the biochemical and biophysical processes leading to its formation remain to be challenges for ongoing research. Whereas the lateral resolution of Fourier transform infrared imaging is limited to 5 μm by diffraction, Raman maps are shown to be capable of detecting the spatial distribution of the silica as well as an additional inorganic component and the organic material down to 330-nm resolution. Due to the spherical shape of the sample with a radius of 40 μm and the requirement to accurately focus the laser before each Raman measurement within the micrometer range, Raman maps of whole diatom cell walls were registered after an adjustment of the axial position. The results reveal local differences in the cell wall composition of the honeycomb-like structures and the bottom layer.

Keywords: Biomineralization; Diatoms; Organic cell wall material; Raman mapping; FTIR imaging

Determination of total and non-water soluble iodine in atmospheric aerosols by thermal extraction and spectrometric detection (TESI) by B. S. Gilfedder; R. Chance; U. Dettmann; S. C. Lai; A. R. Baker (pp. 519-526).

Iodine has recently been of interest in atmospheric chemistry due to its role in tropospheric ozone depletion, modification of the HO/HO₂ ratio and aerosol nucleation. Gas-phase iodine chemistry is tightly coupled to the aerosol phase through heterogeneous reactions, which are dependent on iodine concentrations and speciation in the aerosol. To date, the only method available for total iodine determination in aerosols is collection on filters by impaction and quantification by neutron activation analysis (NAA). NAA is not widely available to all working groups and is costly to commission. Here, we present a method to determine total iodine concentrations in aerosol impact filter samples by combustion of filter sub-samples (∼5 cm²) at 1,000 °C, trapping in deionised water and quantification by UV/Vis spectroscopy. Both quartz and cellulose filters were analysed from four separate sampling campaigns. The method proved to be sensitive (3σ = 6 ng absolute iodine ≈ 3 pmol m⁻³) precise (RSD ∼ 5%) and accurate, as determined by external and standard addition calibrations. Total iodine concentrations ranged from 10 pmol m⁻³ over the Southern Ocean to 100 pmol m⁻³ over the tropical Atlantic, in agreement with previous estimates. The soluble iodine concentration (extracted with water and measured by ICP-MS) was then subtracted from the total iodine to yield non-water-soluble iodine (NSI). The NSI fraction ranged from 20% to 53% of total iodine, and thus can be significant in some cases.

Keywords: Total iodine measurement; Iodine and aerosols; Non-soluble iodine; Iodine speciation

Quantitation of plasmid DNA deposited on gold particles for particle-mediated epidermal delivery using ICP-MS by Colin D. Medley; Bilikallahalli K. Muralidhara; Steven Chico; Stephen Durban; Paul Mehelic; Charles Demarest (pp. 527-535).

DNA-plasmid-based vaccines are a promising class of next generation therapeutics. Particle-mediated epidermal delivery is an attractive method for the administration of DNA plasmid vaccines. This technology utilizes minute quantities of DNA plasmid which have been deposited onto the surface of 2–3-μm gold particles, and so the development of this technology requires the use of analytical methods that can accurately quantitate the amount of the DNA on the particle. Spectroscopic methods are generally insufficient for this task due to interference from the gold particle. ICP-MS circumvents this issue while allowing for the sensitive, reproducible, and accurate determination of the quantity of DNA on the particle surface. This report will detail the development and application of such a method. Figure SEM image of DNA plasmid vaccine on gold particles

Keywords: ICP-MS; Nucleic acids

Spectroscopic and analytical characteristics of an inductively coupled argon plasma combined with hydride generation with or without simultaneous introduction of the sample aerosol for optical emission spectrometry by Pawel Pohl; Jose A. C. Broekaert (pp. 537-545).

A radially viewed inductively coupled argon plasma was used for optical emission spectrometry of volatile species formed by reaction with NaBH₄ (hydride generation). The volatile hydrides were either introduced into the plasma alone or at the same time as a sample aerosol generated by pneumatic nebulization with a commercially available Concomitant Metals Analyzer. The effects of the forward power, the presence of pre-reducing agents [(NH₂)₂SC, KI, KBr and hot HCl], the occurrence of easily ionized elements (Ca, K, Mg and Na) in the analyte solutions on the excitation temperature (as measured via Ar atomic lines) and the electron number density were investigated for both of the sample introduction modes applied. The detection limits and the signal-to-background intensity ratios for As, Bi, Sb, Se and Sn lines were also evalutated and were observed to deteriorate with increasing power. When simultaneous hydride generation and pneumatic nebulization was employed under optimized experimental conditions, detection limits of 3.5, 2.9, 4.3, 1.5 and 2.1 μg L⁻¹ for As, Bi, Sb, Se and Sn, respectively, were obtained, and the intensities of the analytical lines for elements that do not form volatile hydrides were found to be 40% (Cd), 30% (Ni), 20% (Co, Cr, Fe, Mn and Zn) and 10% (Cu, Mg, V) greater than those obtained when only pneumatic nebulization was used.

Keywords: Inductively coupled plasma; Optical emission spectrometry; Hydride generation; Excitation temperatures; Electron number densities; Detection limits

A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates by Kathryn L. Gilroy; Sarah A. Cumming; Andrew R. Pitt (pp. 547-554).

Quantum dots (Qdots) are fluorescent nanoparticles that have great potential as detection agents in biological applications. Their optical properties, including photostability and narrow, symmetrical emission bands with large Stokes shifts, and the potential for multiplexing of many different colours, give them significant advantages over traditionally used fluorescent dyes. Here, we report the straightforward generation of stable, covalent quantum dot–protein A/G bioconjugates that will be able to bind to almost any IgG antibody, and therefore can be used in many applications. An additional advantage is that the requirement for a secondary antibody is removed, simplifying experimental design. To demonstrate their use, we show their application in multiplexed western blotting. The sensitivity of Qdot conjugates is found to be superior to fluorescent dyes, and comparable to, or potentially better than, enhanced chemiluminescence. We show a true biological validation using a four-colour multiplexed western blot against a complex cell lysate background, and have significantly improved previously reported non-specific binding of the Qdots to cellular proteins. Figure Stable covalent conjugates of Qdots with a range of emission frequencies and protein A/G have been generated. These can be bound to appropriate primary antibodies from many species and used for the selective detection of target proteins within a sample. We have demonstrated this using four-colour detection in a western blot format from a cell lysate.

Keywords: Quantum dot; Protein A/G; Multiplexing; Western blot

Comparison of CIM discs and CPG glass as solid supports for bioanalytical columns used in allergen detection by Ana Čevdek; Mladen Franko (pp. 555-562).

This work presents a comparison of convective interaction media (CIM) and controlled pore glass (CPG) as solid supports for immunoglobulin antibodies used in bioanalytical detection of allergens in foodstuffs. A flow-injection manifold with highly sensitive thermal lens spectrometric detection was used for this purpose. Using beta-lactoglobulin, a milk allergen, as a model analyte, CIM disc supports had a higher linear range (0.2–3.5 μg L⁻¹), better reproducibility (intra-day RSD = 1%, inter-day RSD = 10%), lower consumption of reagents, and better immunocolumn stability (1 month, over 240 injections of substrate), while providing comparable LODs (0.1 μg L⁻¹). Application of CIM discs as solid supports in immunocolumns for allergen detection enables fast and sensitive screening of allergens in foodstuffs with sample throughput of up to eight samples per hour.

Keywords: Bioanalytical columns; Controlled-pore glass (CPG); Convective interaction media (CIM); Allergen detection; Thermal lens spectrometry

Aptamer-based electrochemical approach to the detection of thrombin by modification of gold nanoparticles by Lidong Li; Hongtao Zhao; Zhengbo Chen; Xiaojiao Mu; Lin Guo (pp. 563-570).

This paper presents a simple electrochemical approach for the detection of thrombin, using aptamer-modified electrodes. The use of gold nanoparticles results in significant signal enhancement for subsequent detection. 1,6-Hexanedithiol was used as the medium to link Au nanoparticles to a bare gold electrode. Anti-thrombin aptamers were immobilized on the gold nanoparticles’ surfaces by self-assembly. The packing density of aptamers was determined by cyclic voltammetric (CV) studies of redox cations (e.g., [Ru(NH₃)₆]³⁺) which were electrostatically bound to the DNA phosphate backbones. The results indicate that the total amount of aptamer probes immobilized on the gold nanoparticle surface is sixfold higher than that on the bare electrode, leading to increased sensitivity of the aptasensor and a detection limit of 1 pmol L⁻¹. Based on the Langmuir model, the sensor signal displayed an almost perfect linear relationship over the range of 1 pmol L⁻¹ to 30 nmol L⁻¹. Moreover, the proposed aptasensor is highly selective and stable. In summary, this biosensor is simple, highly sensitive, and selective, which is beneficial to the ever-growing interest in fabricating portable bio-analytical devices with simple electrical readout procedures. Figure Schematic diagram showing biosensor fabrication

Keywords: Aptamers; Gold nanoparticles; Thrombin; Cyclic voltammetry (CV); Electrochemical impedance spectroscopy (EIS)

Transfer of bisphenol A from thermal printer paper to the skin by Sandra Biedermann; Patrik Tschudin; Koni Grob (pp. 571-576).

Of 13 thermal printing papers analyzed, 11 contained 8–17 g/kg bisphenol A (BPA). When taking hold of a receipt consisting of thermal printing paper for 5 s, roughly 1 μg BPA (0.2–6 μg) was transferred to the forefinger and the middle finger if the skin was rather dry and about ten times more if these fingers were wet or very greasy. This amount transferred to dry skin was neither significantly increased when taking hold of the paper at up to 10 sites, nor reduced when BPA-free paper was contacted afterwards. After 60–90 min, BPA applied to the skin as a solution in ethanol was only partially or no longer at all extractable with ethanol, whereas BPA transferred to the skin by holding thermal printer paper remained largely extractable after 2 h. This suggests that penetration of the skin depends on the conditions. Extractability experiments did not enable us to conclude whether BPA passes through the skin, but indicated that it can enter the skin to such a depth that it can no longer be washed off. If this BPA ends up in the human metabolism, exposure of a person repeatedly touching thermal printer paper for 10 h/day, such as at a cash register, could reach 71 μg/day, which is 42 times less than the present tolerable daily intake (TDI). However, if more than just the finger pads contact the BPA-containing paper or a hand cream enhances permeability of the skin, this margin might be smaller. Figure Thermal printer paper as used in numerous every-day-applications

Keywords: Thermal printer receipt; Skin contact; Exposure to bisphenol A; Skin permeability

IUPAC International Congress for Analytical Sciences 2011 (ICAS2011) (pp. 577-577).

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Analytical and Bioanalytical Chemistry (v.398, #1)

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Analytical and Bioanalytical Chemistry (v.398, #1)