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Analytical and Bioanalytical Chemistry (v.397, #5)

“Measurement Science in Chemistry” consortium – a new force in analytical chemistry higher education in Europe by Ivo Leito; Anneli Kruve (pp. 1635-1637).

is professor of analytical chemistry at the University of Tartu. He teaches analytical chemistry and metrology in chemistry at UT. He is the initiator and director of the international master’s program “Applied Measurement Science” ( http://www.ut.ee/ams ) that has been awarded the label of “cool curriculum” by the Estonian Ministry of education and science. This program is part of the Euromaster labeled consortium “Measurement Science in Chemistry”. His research interests include metrological foundations of analytical chemistry, chromatographic and mass spectrometric methods, and chemistry of acids and bases. ( http://tera.chem.ut.ee/~ivo/ ) is a PhD student at the University of Tartu. She teaches analytical chemistry at UT. She is working as an assistant program director for the “Applied Measurement Science” masters program. Her research interests are related to liquid chromatography and mass spectrometry, in particular to liquid chromatography columns, electrospray ionization mechanisms, ionization efficiency in the ESI source, and matrix effects.

Whipped egg-white challenge by Hervé This (pp. 1639-1640).

Solution to quality assurance challenge 9 by Manfred Reichenbächer; Jürgen W. Einax (pp. 1641-1643).

The schizophrenia of the administrative path by John Fetzer (pp. 1645-1646).

is the author or coauthor of over 130 research articles, reviews, and book chapters. He is a member of the International Advisory Board of Analytical and Bioanalytical Chemistry. He worked for over 20 years as an analytical chemist for Chevron Corporation and now runs his own consulting company, Fetzpahs Consulting, in Pinole, CA, USA. His book Career management for chemists—a guide to success in a chemistry career was published by Springer.

European analytical column no. 38 (January 2010) by Bo Karlberg; Paul Worsfold; Jens E. T. Andersen (pp. 1647-1651).

Advances in the detection of phycotoxins and cyanotoxins by J. F. Humbert (pp. 1653-1654).

is currently a senior researcher (directeur de recherche) at the Institut National de la Recherche Agronomique (INRA, France), and he works at UMR Bioemco in Paris. He is involved in both basic and applied research work on the microbial communities of freshwater ecosystems, and has a special interest in toxic cyanobacteria. In recent years he has been involved in several programs investigating the genetic and genomic diversity of these microorganisms, and also in developing new tools for monitoring cyanobacteria and their toxins in freshwater ecosystems.

Comparison of analytical tools and biological assays for detection of paralytic shellfish poisoning toxins by A. R. Humpage; V. F. Magalhaes; S. M. Froscio (pp. 1655-1671).

The paralytic shellfish poisoning toxins (PSTs) were, as their name suggests, discovered as a result of human poisoning after consumption of contaminated shellfish. More recently, however, the same toxins have been found to be produced by freshwater cyanobacteria. These organisms have worldwide distribution and are common in our sources of drinking water, thus presenting another route of potential human exposure. However, the regulatory limits for PSTs in drinking water are considerably lower than in shellfish. This has increased the need to find alternatives to the mouse bioassay, which, apart from being ethically questionable, does not have a limit of detection capable of detecting the PSTs in water at the regulated concentrations. Additionally, the number of naturally occurring PSTs has grown substantially since saxitoxin was first characterised, markedly increasing the analytical challenge of this group of compounds. This paper summarises the development of chromatographic, toxicity, and molecular sensor binding methodologies for detection of the PSTs in shellfish, cyanobacteria, and water contaminated by these toxins. It then summarises the advantages and disadvantages of their use for particular applications. Finally it recommends some future requirements that will contribute to their improvement for these applications.

Keywords: Saxitoxin; Gonyautoxin; C-toxin; Dinoflagellate; Cyanobacteria; Drinking water; Bioassay; ELISA; HPLC; LC–MS; PSTs

Biological methods for marine toxin detection by Natalia Vilariño; M. Carmen Louzao; Mercedes R. Vieytes; Luis M. Botana (pp. 1673-1681).

The presence of marine toxins in seafood poses a health risk to human consumers which has prompted the regulation of the maximum content of marine toxins in seafood in the legislations of many countries. Most marine toxin groups are detected by animal bioassays worldwide. Although this method has well known ethical and technical drawbacks, it is the official detection method for all regulated phycotoxins except domoic acid. Much effort by the scientific and regulatory communities has been focused on the development of alternative techniques that enable the substitution or reduction of bioassays; some of these have recently been included in the official detection method list. During the last two decades several biological methods including use of biosensors have been adapted for detection of marine toxins. The main advances in marine toxin detection using this kind of technique are reviewed. Biological methods offer interesting possibilities for reduction of the number of biosassays and a very promising future of new developments.

Keywords: Phycotoxin; Seafood; Shellfish poisoning; Biosensor; SPR

Requirements for screening and confirmatory methods for the detection and quantification of marine biotoxins in end-product and official control by Philipp Hess (pp. 1683-1694).

An overview is given of the biological origin of phycotoxins, as well as their chemical characteristics. Major poisoning types are described and examples of poisoning events are given to illustrate the importance of the phenomenon to both shellfish consumers and the shellfish producing industry. The characteristics of phycotoxins as natural products, the lack of predictability of their occurrence, economic drivers and the freshness of shellfish consumed in many countries result in a number of requirements for methods to be used in the efficient detection of these compounds. Subsequently, the performance of mouse bioassays and mass spectrometry as detection tools are compared for examples from Irish and French monitoring programmes to assess the usefulness of qualitative and quantitative tools in official control, and their fitness for purpose compared with the requirements. The final part of the paper critically reviews methods available for the end-product and official control of shellfish toxins and their use in screening and confirmatory approaches in monitoring. Recent expert consultations on the methodology for phycotoxins at European and global level are summarised and recommendations are made for future progress in this area.

Keywords: Bioanalytical methods; Bioassays; Mass spectrometry; Liquid chromatography; Biological samples

Ligand-binding assays for cyanobacterial neurotoxins targeting cholinergic receptors by Rómulo Aráoz; Natalia Vilariño; Luis M. Botana; Jordi Molgó (pp. 1695-1704).

Toxic cyanobacterial blooms are a threat to public health because of the capacity of some cyanobacterial species to produce potent hepatotoxins and neurotoxins. Cyanobacterial neurotoxins are involved in the rapid death of wild and domestic animals by targeting voltage gated sodium channels and cholinergic synapses, including the neuromuscular junction. Anatoxin-a and its methylene homologue homoanatoxin-a are potent agonists of nicotinic acetylcholine receptors. Since the structural determination of anatoxin-a, several mass spectrometry-based methods have been developed for detection of anatoxin-a and, later, homoanatoxin-a. Mass spectrometry-based techniques provide accuracy, precision, selectivity, sensitivity, reproducibility, adequate limit of detection, and structural and quantitative information for analyses of cyanobacterial anatoxins from cultured and environmental cyanobacterial samples. However, these physicochemical techniques will only detect known toxins for which toxin standards are commercially available, and they require highly specialized laboratory personnel and expensive equipment. Receptor-based assays are functional methods that are based on the mechanism of action of a class of toxins and are thus, suitable tools for survey of freshwater reservoirs for cyanobacterial anatoxins. The competition between cyanobacterial anatoxins and a labelled ligand for binding to nicotinic acetylcholine receptors is measured radioactively or non-radioactively providing high-throughput screening formats for routine detection of this class of neurotoxins. The mouse bioassay is the method of choice for marine toxin monitoring, but has to be replaced by fully validated functional methods. In this paper we review the ligand-binding assays developed for detection of cyanobacterial and algal neurotoxins targeting the nicotinic acetylcholine receptors and for high-throughput screening of novel nicotinic agents. Figure Non-radioactive ligand-binding assay for cyanobacterial anatoxins. The detection method (**) is among the highly sensitive ones

Keywords: Receptor binding assay; Cyanobacterial neurotoxins; Anatoxin-a; Nicotinic acetylcholine receptors

A review of cyanobacteria and cyanotoxins removal/inactivation in drinking water treatment by Judy A. Westrick; David C. Szlag; Benjamin J. Southwell; James Sinclair (pp. 1705-1714).

This review focuses on the efficiency of different water treatment processes for the removal of cyanotoxins from potable water. Although several investigators have studied full-scale drinking water processes to determine the efficiency of cyanotoxin inactivation, many of the studies were based on ancillary practice. In this context, “ancillary practice” refers to the removal or inactivation of cyanotoxins by standard daily operational procedures and without a contingency operational plan utilizing specific treatment barriers. In this review, “auxiliary practice” refers to the implementation of inactivation/removal treatment barriers or operational changes explicitly designed to minimize risk from toxin-forming algae and their toxins to make potable water. Furthermore, the best drinking water treatment practices are based on extension of the multibarrier approach to remove cyanotoxins from water. Cyanotoxins are considered natural contaminants that occur worldwide and specific classes of cyanotoxins have shown regional prevalence. For example, freshwaters in the Americas often show high concentrations of microcystin, anatoxin-a, and cylindrospermopsin, whereas Australian water sources often show high concentrations of microcystin, cylindrospermopsin, and saxitoxins. Other less frequently reported cyanotoxins include lyngbyatoxin A, debromoaplysiatoxin, and β-N-methylamino-l-alanine. This review focuses on the commonly used unit processes and treatment trains to reduce the toxicity of four classes of cyanotoxins: the microcystins, cylindrospermopsin, anatoxin-a, and saxitoxins. The goal of this review is to inform the reader of how each unit process participates in a treatment train and how an auxiliary multibarrier approach to water treatment can provide safer water for the consumer.

Keywords: Drinking water treatment; Cyanotoxins; Cyanobacteria

A strategy to study genotoxicity: application to aquatic toxins, limits and solutions by Valérie Fessard; Ludovic Le Hégarat (pp. 1715-1722).

Humans can be exposed to aquatic toxins mainly through contamination of food and water (drinking and recreational). Among these toxins, contamination by both phycotoxins occurring in shellfish and cyanotoxins mostly involved in freshwater bodies are of concern for public health. Whereas regulations exist to evaluate the genotoxicity of most compounds to which humans are exposed, including drugs and chemicals, no regulations have been established for these compounds. In this paper, we show that the same strategy including both in vitro and in vivo tests can be followed to evaluate the genotoxicity of aquatic toxins (phycotoxins and cyanotoxins). However, this strategy encountered different limits which arise when completing an overview of the genotoxic potential of toxins. The most restrictive one is undoubtedly the low amount (even the lack sometimes) of purified toxins available. Solutions and recommendations for testing the genotoxicity of aquatic toxins are suggested to overcome the specific problems encountered with these compounds. It must be kept in mind that recent developments in drug toxicology should be considered and that experiments must be conducted in respect of the 3Rs principle of refinement, reduction and replacement for animal experimentation.

Keywords: Cyanotoxin; Phycotoxin; Genotoxicity; Strategy

Molecular approaches for monitoring potentially toxic marine and freshwater phytoplankton species by J. F. Humbert; C. Quiblier; M. Gugger (pp. 1723-1732).

Harmful phytoplankton species are a growing problem in freshwater and marine ecosystems, because of their ability to synthesize toxins that threaten both animal and human health. The monitoring of these microorganisms has so far been based on conventional methods, mainly involving the microscopic counting and identification of cells, and using analytical and bioanalytical methods to identify and quantify the toxins. However, the increasing number of microbial sequences in the GeneBank database and the development of new tools in the last 15 years nowadays enables the use of molecular methods for detection and quantification of harmful phytoplankton species and their toxins. These methods provide species-level identification of the microorganisms of interest, and their early detection in the environment by PCR techniques. Moreover, real time PCR can be used to quantify the cells of interest, and in some cases to evaluate the proportion of toxin-producing and non-toxin-producing genotypes in a population. Recently, microarray technologies have also been used to achieve simultaneous detection and semi-quantification of harmful species in environmental samples. These methods look very promising, but so far their use remains limited to research. The need for validation for routine use and the cost of these methods still hamper their use in monitoring programs.

Keywords: Aquatic ecosystems; Phytoplankton; Phycotoxins; Cyanotoxins; Detection; Molecular approaches

A fluorescent immunochromatographic test using immunoliposomes for detecting microcystins and nodularins by Nathalie Khreich; Patricia Lamourette; Bernard Lagoutte; Cyril Ronco; Xavier Franck; Christophe Créminon; Hervé Volland (pp. 1733-1742).

Microcystins (MCs), a group of cyclic heptapeptides produced by common cyanobacteria (blue green algae), cause both acute and chronic toxicity. Due to their toxicity, constant monitoring in drinking water, recreational waters as well as other potential exposure through ingestion of contaminated sea food, is very important. In this context, an immunochromatographic test (ICT) using a monoclonal antibody labeled with fluorescent liposomes (immunoliposomes) as tracer was developed, allowing a rapid and simple detection of a large number of MC and nodularin variants in field samples. The present ICT using immunoliposomes proved to be ten times more sensitive than the ICT using colloidal gold for labeling. To achieve quantitative measurement, this ICT was improved by including a stable signal on the control band allowing the expression of the results as a ratio of the fluorescence signals of the specific band versus the control band (SB/CB). Very low concentrations of MC-LR were detected in the analysis buffer (0.06 ng/ml), well below the guideline value of 1 ng/ml proposed by the World Health Organization (WHO), with a dynamic range from 0.06 to 1.5 ng/ml of MC-LR. This method was also validated using a hand-held commercial fluorometer (from ESE®), providing the same performances obtained via the analysis station (from Kodak®) used in our laboratory. Repeatability tests performed with both devices showed good accuracy (CV < 13%). Furthermore, quantification of MCs in natural samples (water bloom and Microcystis culture) was achieved using ICT, leading to similar results obtained via an EIA previously described. All these results demonstrate that this new fluorescent ICT could be used not only as a sensitive detection tool but also to quantify MCs in field samples.

Keywords: Microcystins; Nodularins; Monoclonal antibody; Immunoliposomes; Immunochromatographic test; Enzyme immunoassay

Celiac disease diagnosis and gluten-free food analytical control by Marta Maria Pereira da Silva Neves; Maria Begoña González-Garcia; Hendrikus Petrus Antonius Nouws; Cristina Delerue-Matos; Alice Santos-Silva; Agustín Costa-García (pp. 1743-1753).

Celiac disease (CD) is an autoimmune enteropathy, characterized by an inappropriate T-cell-mediated immune response to the ingestion of certain dietary cereal proteins in genetically susceptible individuals. This disorder presents environmental, genetic, and immunological components. CD presents a prevalence of up to 1% in populations of European ancestry, yet a high percentage of cases remain underdiagnosed. The diagnosis and treatment should be made early since untreated disease causes growth retardation and atypical symptoms, like infertility or neurological disorders. The diagnostic criteria for CD, which requires endoscopy with small bowel biopsy, have been changing over the last few decades, especially due to the advent of serological tests with higher sensitivity and specificity. The use of serological markers can be very useful to rule out clinical suspicious cases and also to help monitor the patients, after adherence to a gluten-free diet. Since the current treatment consists of a life-long gluten-free diet, which leads to significant clinical and histological improvement, the standardization of an assay to assess in an unequivocal way gluten in gluten-free foodstuff is of major importance.

Keywords: Celiac disease; Autoimmune; Transglutaminase; Gliadin

Simultaneous speciation of arsenic (As(III), MMA, DMA, and As(V)) and selenium (Se(IV), Se(VI), and SeCN⁻) in petroleum refinery aqueous streams by Gisele B. Tonietto; José M. Godoy; Ana Cristina Oliveira; Marcia V. de Souza (pp. 1755-1761).

High-performance liquid chromatography (HPLC) coupled to an ICP-MS with an octapole reaction system (ORS) has been used to carry out quantitative speciation of selenium (Se) and arsenic (As) in the stream waters of a refining process. The argon dimers interfering with the ⁷⁸Se and ⁸⁰Se isotopes were suppressed by pressurizing the octapole chamber with 3.1 mL min⁻¹ H₂ and 0.5 mL min⁻¹ He. Four arsenic species arsenite—As(III), arsenate (As(V)), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA)—and three inorganic Se species—selenite Se(IV), selenate Se(VI), and selenocyanate (SeCN⁻)—were separated in a single run by ion chromatography (IC) using gradient elution with 100 mmol L⁻¹ NH₄NO₃, pH 8.5, adjusted by addition of NH₃, as eluent. Repeatabilities of peak position and of peak area evaluation were better than 1% and about 3%, respectively. Detection limits (as 3σ of the baseline noise) were 81, 56, and 75 ng L⁻¹ for Se(IV), Se(VI), and SeCN⁻, respectively, and 22, 19, 25, and 16 ng L⁻¹ for As(III), As(V), MMA, and DMA, respectively. Calibration curve R ² values ranged between 0.996 and 0.999 for the arsenic and selenium species. Column recovery for ion chromatography was calculated to be 97 ± 6% for combined arsenic species and 98 ± 3% for combined selenium species. Because certified reference materials for As and Se speciation studies are still not commercially available, in order to check accuracy and precision the method was applied to certified reference materials, BCR 714, BCR 1714, and BCR 715 and to two different refinery samples—inlet and outlet wastewater. The method was successfully used to study the quantitative speciation of selenium and arsenic in petroleum refinery wastewaters.

Keywords: Speciation; Selenium; Arsenic; Wastewaters; Stream waters; HPLC-ICP-MS

Biased spectroscopic protein quantification in the presence of ionic liquids by Patrick Mester; Martin Wagner; Peter Rossmanith (pp. 1763-1766).

Owing to their unique physicochemical properties, ionic liquids have gained much recognition as solvents or co-solvents in a wide variety of biochemical applications. In the context of protein analytics, four similar 1-alkyl-3-methylimidazolium-based ionic liquids have been analysed for their applicability as co-solvents. Spectroscopic bovine serum albumin (BSA) quantification experiments in the presence of ionic liquids were performed and for two ionic liquids a concentration-dependent effect has been found that can lead to biased protein quantification. It could be shown that the biased spectroscopic analysis of the tested ionic liquids is dependent on the length of the alkyl side chain (>C₄) of the 1-alkyl-3-methylimidazolium-based cation, and the chaotropicity of the anion. Once accounted for and properly calibrated when using spectroscopic methods, these effects can be avoided thus facilitating correct protein quantification in the presence of ionic liquids. Figure Concentration dependent but non-linear shift of UV/Vis spectra of ionic liquids

Keywords: Ionic liquids; Protein quantification; UV/Vis; BSA; Aggregation

Characterization of dielectric barrier electrospray ionization for mass spectrometric detection by Ann Kathrin Stark; Michael Schilling; Dirk Janasek; Joachim Franzke (pp. 1767-1772).

A novel electrospray interface is presented which induces an electric field by dielectric polarization through a non-conductive barrier. Therefore, a square-wave high-voltage signal is applied. This technique allows mass spectrometric measurements in the positive as well as in the negative mass spectrometry mode without changing the polarity of the potential applied, and it decreases the risk of undesired discharges, induced by high electric currents. The applicability of this technique is demonstrated by mass spectrometric determination of reserpine.

Keywords: ESI; MS; Dielectric barrier

An LC-ESI/MS method for determining theanine in green tea dietary supplements by Mary Bedner; Lane C. Sander; Katherine E. Sharpless (pp. 1773-1777).

Theanine is the major amino acid present in Camellia sinensis or green tea. A method for determining theanine in its native state using liquid chromatography with positive-mode electrospray ionization mass spectrometric detection was developed. Quantitation of theanine was achieved using theanine-[²H₅] as an internal standard. This approach was utilized on different green tea matrix materials that are commonly used as dietary supplements including powdered plant leaves, a powdered plant leaf extract, and an oral dosage form that contains green tea. The theanine response was linear over several orders of magnitude, and excellent measurement precision was obtained for all three materials using the developed method.

Keywords: Theanine; Green tea; Camellia sinensis ; Liquid chromatography; Mass spectrometry; Standard Reference Material

Modifications to the NIST reference measurement procedure (RMP) for the determination of serum glucose by isotope dilution gas chromatography/mass spectrometry by Jocelyn L. Prendergast; Lorna T. Sniegoski; Michael J. Welch; Karen W. Phinney (pp. 1779-1785).

The definitive method (DM), now known as the reference measurement procedure (RMP), for the analysis of glucose in serum was originally published in 1982 by the National Institute of Standards and Technology (NIST). Over the years the method has been subject to a number of modifications to adapt to newer technologies and simplify sample preparation. We discuss here an adaptation of the method associated with serum glucose measurements using a modified isotope dilution gas chromatography/mass spectrometry (ID-GC/MS) method. NIST has used this modified method to certify the concentrations of glucose in SRM 965b, Glucose in Frozen Human Serum, and SRM 1950, Metabolites in Human Plasma. Comparison of results from the revised method with certified values for existing Standard Reference Materials (SRMs) demonstrated that these modifications have not affected the quality of the measurements, giving both good precision and accuracy, while reducing the sample preparation time by a day and a half.

Keywords: Glucose; Serum; Isotope dilution; Gas chromatography/mass spectrometry; GC/MS

Multiplexed identification of different fish species by detection of parvalbumin, a common fish allergen gene: a DNA application of multi-analyte profiling (xMAP™) technology by Sabine Hildebrandt (pp. 1787-1796).

Fish are a common cause of allergic reactions associated with food consumption, with parvalbumin being the major allergenic protein. Some fish-hypersensitive patients tolerate some fish species while being allergic to others. Reliable detection methods for allergenic fish species in foods are necessary to ensure compliance with food allergen labeling guidelines to protect fish-allergic consumers. The objective of this project was to develop a multi-analyte detection method for the presence of fish in food. Therefore, conserved parvalbumin exon sequences were utilized for the design of universal PCR primers amplifying intron DNA and small regions of exons flanking the enclosed intron from even very distantly related fish species. An assay for the identification of eight fish species was developed using xMAP™ technology with probes targeting species-specific parvalbumin intron regions. Additionally, a universal fish probe was designed targeting a highly conserved exon region located between the intron and the reverse primer region. The universal fish assay showed no cross-reactivity with other species, such as beef, pork, lamb, chicken, turkey, and shrimp. Importantly, with the exception of one notable case with fish in the same subfamily, species-specific detection showed no cross-reactivity with other fish species. Limits of detection for these eight species were experimentally estimated to range from 0.01% to 0.04%, with potential to increase the detection sensitivity. This report introduces a newly developed method for the multiplex identification of at least eight allergenic fish species in food, which could conceivably be extended to detect up to 100 species simultaneously in one sample. Figure Schematic overview of the xMAP™ assay. Amine-modified capture oligonucleotides were covalently coupled to color-coded MagPlex™-C magnetic carboxylated microspheres (Luminex Corporation, TX, USA) using a carbodiimide coupling method described by Fulton et al. [25]. Parvalbuminencoding DNA was amplified with real-time PCR, whereas one of the primers was labeled with biotin. Biotin-labeled DNA strands were hybridized to their corresponding capture oligonucleotides. The fluorophor streptavidin–phycoerythrin (SAPE) was added and bound to the biotin molecules. Reaction tubes containing the magnetic microspheres were put on a magnet, unbound oligonucleotides and SAPE was removed by washing. The reaction mixture was analyzed on the BioPlex 200 analyzer (BioRad, CA, USA). Microspheres were interrogated individually in a rapidly flowing fluid stream as they passed by two separate lasers. A red diode laser excited two fluorochromes contained within the colorcoded microspheres and a green laser excited the reporter fluorochrome (SAPE) bound to the microsphere surface. High-speed digital signal processing classified the microsphere based on its spectral address and quantified the reaction on the surface

Keywords: Fish allergy; Parvalbumin; PCR; Universal primer; xMAP

Validation and application of an LC-MS/MS method for the simultaneous quantification of 13 pharmaceuticals in seawater by Klaas Wille; Herlinde Noppe; Karolien Verheyden; Julie Vanden Bussche; Eric De Wulf; Peter Van Caeter; Colin R. Janssen; Hubert F. De Brabander; Lynn Vanhaecke (pp. 1797-1808).

Knowledge of the presence of micropollutants such as pharmaceuticals, in coastal areas, is very limited; therefore, the main objective of this study was to optimize and validate a new analytical method for the quantitative analysis of 13 multiclass pharmaceuticals in seawater. Target compounds included antibiotics, non-steroidal anti-inflammatory drugs, β-blockers, lipid regulators and one psychiatric drug. A combination of solid-phase extraction and liquid chromatography coupled with multiple mass spectrometry enabled their detection at the low nanogram per litre level. The limits of quantification varied between 1 and 50 ng L^-1, for most components the linearities were more than 0.99 and the recoveries obtained in seawater (95–108%) were satisfactory. This method was applied to seawater and estuarine water samples collected in the Belgian coastal zone, to assess the prevalence of common pharmaceuticals in this marine environment. Seven pharmaceuticals, including compounds of which the presence in marine environments had not been reported earlier, were detected, with salicylic acid and carbamazepine being the most abundant, in concentrations up to 855 ng L^-1.

Keywords: Pharmaceuticals; Liquid chromatography–tandem mass spectrometry; Validation; Marine environment; Persistence

Proteomic biomarkers in plasma that differentiate rapid and slow decline in lung function in adult cigarette smokers with chronic obstructive pulmonary disease (COPD) by Gaurav S. J. B. Rana; Timothy P. York; Jeffery S. Edmiston; Barbara K. Zedler; Joel G. Pounds; Joshua N. Adkins; Richard D. Smith; Zaigang Liu; Guoya Li; Bradley T. Webb; Edward L. Murrelle; Jason W. Flora (pp. 1809-1819).

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of morbidity and mortality in the United States and cigarette smoking is a primary determinant of the disease. COPD is characterized by chronic airflow limitation as measured by the forced expiratory volume in one second (FEV₁). In this study, the plasma proteomes of 38 middle-aged or older adult smokers with mild to moderate COPD, with FEV₁ decline characterized as either rapid (RPD, n = 20) or slow or absent (SLW, n = 18), were interrogated using a comprehensive high-throughput proteomic approach, the accurate mass and time (AMT) tag technology. This technology is based upon a putative mass and time tag database (PMT), high-resolution LC separations and high mass accuracy measurements using FT-ICR MS with a 9.4-T magnetic field. The peptide and protein data were analyzed using three statistical approaches to address ambiguities related to the high proportion of missing data inherent to proteomic analysis. The RPD and SLW groups were differentiated by 55 peptides which mapped to 33 unique proteins. Twelve of the proteins have known roles in the complement or coagulation cascade and, despite an inability to adjust for some factors known to affect lung function decline, suggest potential mechanistic biomarkers associated with the rate of lung function decline in COPD. Whether these proteins are the cause or result of accelerated decline will require further research.

Keywords: Amino acids/peptides; Bioanalytical methods; Biological samples; Clinical/biomedical analysis; Genomics/proteomics

Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva by Scott A. Klasner; Alexander K. Price; Kurt W. Hoeman; Rashaun S. Wilson; Kayla J. Bell; Christopher T. Culbertson (pp. 1821-1829).

We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 μm wide with barriers as narrow as 250 μm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated (Martinez et al., Angew Chem Int Ed 8:1318–1320, 1; Martinez et al., Anal Chem 10:3699–3707, 2; Martinez et al., Proc Nat Acad Sci USA 50:19606–19611, 3; Lu et al., Electrophoresis 9:1497–1500, 4; Abe et al., Anal Chem 18:6928–6934, 5). Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 μM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 μM) were generated. The time of device fabrication to the time of test results was about 25 min. Paper-based microfluidic chip illustrating the colorimetric detection of salivary nitrite. Color intensities were quantified using a flatbed scanner and image manipulation software and plotted against concentration to produce calibration curves for the assay

Keywords: Paper-based devices; Paper microfluidics; Clinical assays; Colorimetric detection; Photolithography; Photoactive polymers

Analysis of thyroid hormones in serum by liquid chromatography-tandem mass spectrometry by Dongli Wang; Heather M. Stapleton (pp. 1831-1839).

Thyroid hormones are essential hormones for regulating growth and development in humans and wildlife. Methods to monitor precise and low levels of these hormones in serum and tissues are needed to assess overall health, whether from disease considerations or possibly from environmental contaminant exposures. Common and routine methods typically rely upon radioimmunoassays, which can be expensive, and typically only measure thyroxine and 3,3′,5-triidothyronine, which can be a limitation in fully evaluating impacts on thyroid regulation. In this study we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of five thyroid hormones—thyroxine, 3,3′,5-triidothyronine, 3,3′,5′-triiodothyronine, 3,3′-diiodothyronine, and 3,5-diiodothyronine—in serum samples. The LC-MS/MS parameters were optimized and calibrated over a wide concentration range (1.0–500 ng/mL) with on-column detection limits of 1.5–7.0 pg. With use of spiked bovine serum samples, the mean method recoveries were calculated to be 81.3–111.9% with relative standard deviations of 1.2–9.6% at spiking levels ranging from 10 to 100 ng/mL. This method was compared with measurements made by standard radioimmunoassays and with measurements made in a serum Standard Reference Material (SRM 1951b). Development of this method expands the capacity to measure thyroid hormones by including a larger suite of thyroid hormones, and has promising applications for measuring catabolism of thyroid hormones in vitro.

Keywords: Thyroid hormones; Tandem mass spectrometry; Serum; Solid-phase extraction; Thyroxine

Bioanalytical method development for a generation 5 polyamidoamine folic acid methotrexate conjugated nanoparticle by Leon van Haandel; John F. Stobaugh (pp. 1841-1852).

Generation 5 poly(amidoamine) dendrimer nanoparticles conjugated with folic acid and methotrexate (G5-MTX-FA) for targeted treatment of cancer are of recent interest. The increased efficacy of these nanodevices over the free methotrexate has been shown in vitro and in vivo. The heterogeneous nature of this nanoparticle together with possible release of active compounds complicated the method development. This work presents a bioanalytical assay for the detection of nanoparticle-conjugated methotrexate, released methotrexate, and its main plasma metabolite 7-hydroxymethotrexate in rat plasma. Determination of G5-MTX-FA-associated methotrexate occurred by a reductive cleavage of the C9–N10 bond in methotrexate, resulting in a highly fluorescent 2,4-diamino-6-methylpteridine reporter molecule that could be measured by reversed-phase chromatography and fluorescence detection. It was found that reduction should occur directly in the plasma matrix to avoid irreversible adsorption of the nanodevice during sample preparation. The method was linear over a range from 50 to 10,000 nM G5-MTX-FA utilizing 100 μL of plasma. Nanoparticle-released methotrexate and its metabolite 7-hydroxymethotrexate were determined by reversed-phase chromatography followed by online post-column photochemical derivatization and fluorescence detection. The method was specific for these analytes irrespective of nanoparticle concentration. Sample preparation consisted of perchloric acid protein precipitation followed by a strong anion exchange solid-phase extraction. Limits of quantification were about 50 nM for methotrexate and 10 nM for 7-hydroxymethotrexate. Preliminary pharmacokinetic profiles of intravenous and subcutaneous administered G5-MTX-FA in rats were obtained. These data indicated that less than 0.1% of the methotrexate mass is released from the nanoparticle in plasma.

Keywords: Poly(amidoamine); Dendrimer; Methotrexate conjugate

An improved one-tube RT-PCR protocol for analyzing single-cell gene expression in individual mammalian cells by Yongzhong Li; Hansa Thompson; Courtney Hemphill; Fan Hong; Jessica Forrester; Roger H. Johnson; Weiwen Zhang; Deirdre R. Meldrum (pp. 1853-1859).

It is well known that gene expression is regulated at the level of individual cells, and more evidence is now emerging that heterogeneity of cell regulation is orders of magnitude greater than previously thought. In order to detect meaningful variations in transcription levels, it is necessary to measure gene expression at single cell levels rather than in bulk cells, where individual differences or heterogeneity could be lost. In this work, we report an improved reverse-transcriptase polymerase chain reaction (RT-PCR) protocol which allows the direct measurement of gene expression in one tube (5–25 μl of total PCR mixture) at the single mammalian cell level. The protocol employs a new cell lysis buffer, and involves no RNA isolation or nested PCR steps, significantly reducing the possibility of contamination and errors. We successfully applied this protocol in qRT-PCR and linear-after-the-exponential (LATE)-PCR to analyze selected genes of various expression levels from three cell lines. Although further characterization of RNA stability is important, the preliminary results showed that gene expression heterogeneity could be common among members of genetically identical cell populations. The protocol illustrated can be utilized for a wide array of applications without much modification, such as cancer cell analysis and preimplantation genetic diagnostics. In addition, the protocol is based on intercalator-based (SYBR Green PCR) chemistry, which is less expensive and suitable for high-throughput platforms. Figure Gene expression heterogeneity in genetically identical cell population. qRT-PCR analysis of p53 transcript in HeLa cells with two negative controls in red. (A) qRT-PCR analysis of p53 transcript with 10 cells in each PCR. 10 PCR were analyzed; (B) qRT-PCR analysis of p53 transcript with 5 cells in each reaction. 8 PCR were analyzed; (C) qRT-PCR analysis of p53 transcript with single cell in each reaction tube. 9 reactions were analyzed; and (D) Statistical analysis on Ct values from panel A, B, and C above. The final total volume is 25 μl for all PCR analysis.

Keywords: Single-cell; Gene expression; One-tube; RT-PCR

Heterobifunctional modification of DNA for conjugation to solid surfaces by Hana I. Lim; Piercen M. Oliver; Jutta Marzillier; Dmitri V. Vezenov (pp. 1861-1872).

Many biosensors, DNA arrays, and next-generation DNA sequencing technologies need common methods for end modification of random DNA sequences generated from a sample of DNA. Surface immobilization of chemically modified DNA is often the first step in creating appropriate sensing platforms. We describe a simple technique for efficient heterobifunctional modification of arbitrary double-stranded DNA fragments with chosen chemical groups. The modification requires the use of short (10–20 base pairs) synthetic adaptors having desired terminal functional groups and installs known sequences, which can be used for hybridization of primers in the sequencing-by-synthesis approaches. The method, based on ligation under optimized conditions, is selective and provides high yields of the target heterobifunctional DNA product. An additional two-step procedure can be applied to select further for the desired bifunctionalized product using PCR amplification with a chemically modified primer. Both functional groups in the modified DNA are chemically active and can be used in surface immobilization of the DNA strands to create the surface of a biosensor or sequencing chip. Figure We describe a simple adapter ligation method for efficient heterobifunctional modification of arbitrary dsDNA fragments with chosen chemical groups. The modification requires the use of short (10–20 base pairs) synthetic adaptors having desired terminal functional groups and installs known sequences. Both functional groups in the modified DNA are chemically active and can be used in surface immobilization of the DNA strands to create a biosensor or the sequencing chip.

Keywords: Biosensors; DNA modification; Next-generation sequencing; DNA attachment; Bioconjugation; Genomic DNA

Surface plasmon resonance aided electrochemical immunosensor for CK-MB determination in undiluted serum samples by Fernando Garay; Greggory Kisiel; Aiping Fang; Ernő Lindner (pp. 1873-1881).

This article presents a simple chronoamperometric immunosensor for the quantitative assessment of creatine kinase MB (CK-MB) in 50 µL undiluted serum samples. The immunosensor consists of gold working and counter electrodes patterned onto a glass chip by thin-film photolithography and an external Ag|AgCl reference electrode. The detection limit (DL) of the chronoamperometric method is 13 ng mL⁻¹ (DL = 2×RMSD/S, where RMSD is the residual mean standard deviation of the measured points around a calibration curve with a slope of S). In spiked serum samples, the response was linear up to 300 ng mL⁻¹ of CK-MB. A surface plasmon resonance (SPR) system with simultaneous electrochemical detection (EC-SPR) aided the development of the sandwich immunoassay. Real-time monitoring of the SPR signal was used to optimize the capture antibody immobilization, CK-MB and detection antibody binding, as well as to minimize the nonspecific adsorption of serum proteins to the sensor surface. The detection antibody has been labeled with alkaline phosphatase (ALP) enzyme for sensitive electrochemical detection. ALP catalyzes the hydrolysis of ascorbic acid phosphate and generates ascorbic acid, which is measured chronoamperometrically. The electrochemical immunoassay for CK-MB was less sensitive to nonspecific adsorption related interferences, had a better detection limit, and required a lower volume of sample than the SPR method. Figure SPR (A) and electrochemical (B) cells for CK-MB measurement in serum. (C) counter electrode, (R) reference electrode, (W) working electrode, (r) reference chamber, and (s) signal chamber

Keywords: Creatine kinase MB; Immunosensor; SPR; Electrochemistry; Serum

Optimization and single-laboratory validation study of a high-performance liquid chromatography (HPLC) method for the determination of phenolic Echinacea constituents by Paula N. Brown; Michael Chan; Joseph M. Betz (pp. 1883-1892).

Three species of Echinacea (Echinacea purpurea, Echinacea angustifolia, and Echinacea pallida) are commonly used for medicinal purposes. The phenolic compounds caftaric acid, cichoric acid, echinacoside, cynarin, and chlorogenic acid are among the phytochemical constituents that may be responsible for the purported beneficial effects of the herb. Although methods for the analysis for these compounds have been published, documentation of their validity was inadequate as the accuracy and precision for the detection and quantification of these phenolics was not systematically determined and/or reported. To address this issue, the high-performance liquid chromatography method, originally developed by the Institute for Nutraceutical Advancement (INA), was reviewed, optimized, and validated for the detection and quantification of these phenolic compounds in Echinacea roots and aerial parts.

Keywords: Echinacea ; Phenolic; Chromatography; Method validation; Quality control

Multiobjective evolutionary optimisation for surface-enhanced Raman scattering by Roger M. Jarvis; William Rowe; Nicola R. Yaffe; Richard O’Connor; Joshua D. Knowles; Ewan W. Blanch; Royston Goodacre (pp. 1893-1901).

In most optimisation experiments, a single parameter is first optimised before a second and then third one are subsequently modified to give the best result. By contrast, we believe that simultaneous multiobjective optimisation is more powerful; therefore, an optimisation of the experimental conditions for the colloidal SERS detection of l-cysteine was carried out. Six aggregating agents and three different colloids (citrate, borohydride and hydroxylamine reduced silver) were tested over a wide range of concentrations for the enhancement and the reproducibility of the spectra produced. The optimisation was carried out using two methods, a full factorial design (FF, a standard method from the experimental design literature) and, for the first time, a multiobjective evolutionary algorithm (MOEA), a method more usually applied to optimisation problems in computer science. Simulation results suggest that the evolutionary approach significantly out-performs random sampling. Real experiments applying the evolutionary method to the SERS optimisation problem led to a 32% improvement in enhancement and reproducibility compared with the FF method, using far fewer evaluations.

Keywords: SERS; SERRS; PESA-II; Evolutionary; Optimisation; l-cysteine

Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry by Gianluca Maddalo; Mohammadreza Shariatgorji; Christopher M. Adams; Eva Fung; Ulrika Nilsson; Roman A. Zubarev; Jan Sedzik; Leopold L. Ilag (pp. 1903-1910).

Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain–Barré syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-Å crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination. Figure FT-ICR MS/MS spectrum (left) of porcine myelin P2 protein (green) and GC profile (right) of associated lipids extracted/identified from protein crystals by GC-MS. (Note: Ribbon diagram was generated by Rasmol based on PDB file 1YIV. Crystals depicted are not of the sample used.)

Keywords: CID; ECD; De novo sequencing; GC/MS; Lipid identification; Myelin proteins

Visual screening for JAK2V617F mutation by a disposable dipstick by Jessica K. Konstantou; Alexandra C. Iliadi; Penelope C. Ioannou; Theodore K. Christopoulos; Nikolaos I. Anagnostopoulos; Emmanuel Kanavakis; Jan Traeger-Synodinos (pp. 1911-1916).

During the last 5 years, it was discovered that the JAK2V617F somatic mutation is present in virtually all patients with polycythemia vera and a large proportion of patients with essential thrombocythemia, primary myelofibrosis, and refractory anemia with ring sideroblasts and thrombocytosis. As a result, JAK2V617F was incorporated as a new clonal marker in the 2008 revision of the WHO diagnostic criteria. Current methods for JAK2 genotyping include direct sequencing, pyrosequencing, allele-specific PCR with electrophoresis, restriction fragment length polymorphism, real-time PCR, DNA-melting curve analysis, and denaturing HPLC. Some of these methods are labor intensive and time consuming, while the others require specialized costly equipment and reagents. We report a method for direct detection of the JAK2V617F allele by the naked eye using a dipstick test in a dry-reagent format. The method comprises a triprimer PCR combined with visual detection of the products within minutes by the dipstick test. Specialized instrumentation is not involved. The requirements for highly qualified technical personnel are minimized. Because the detection reagents exist in dry form on the dipstick, there is no need for multiple pipetting and incubation steps.

Keywords: JAK2V617F ; Mutation detection; Triprimer PCR; Dipstick test

A validated method for the quantification of curcumin in plasma and brain tissue by fast narrow-bore high-performance liquid chromatography with fluorescence detection by Christina Schiborr; Gunter P. Eckert; Gerald Rimbach; Jan Frank (pp. 1917-1925).

Curcumin, a lipophilic polyphenol derived from the rhizome of the plant turmeric (Curcuma longa), might be useful in the prevention and treatment of a number of degenerative brain disorders, including glioma multiforma and Alzheimer’s disease. Thus, there is growing interest in measuring curcumin concentrations in the brain and other target tissues in relevant animal models. We therefore developed and validated (according to the Food and Drug Administration guidelines for bioanalytical method validation), a simple, fast and reliable method for the quantification of curcumin in biological matrices by fast high-performance liquid chromatography with fluorescence detection. This method involves a simple extraction with 95% ethyl acetate and 5% methanol, rapid separation (<2 min if external standards and <4 min if the internal standard β-estradiol 17-acetate is used) on a Jasco Reprosil-Pur Basic C₁₈ column (75 × 2 mm, 1.8 μm) with an eluent of acetonitrile, methanol, de-ionised water and acetic acid (49:20:30:1, v/v; flow rate, 0.4 mL/min) and fluorescence detection (excitation wavelength, 420 nm; emission wavelength, 470 nm). The method is selective, precise (<15% RSD at the lower limit of quantification), accurate (<15% of the coefficient of variation at the lower limit of quantification) and sensitive over a linear range of 0.05–10 μg/mL for curcumin. The developed method was used for the quantification of curcumin in the brains of mice force-fed (50 mg/kg bw) or i.p. injected (100 mg/kg bw) with curcumin. Brain curcumin concentrations of the mice were below the limit of detection at 30, 60 and 120 min after oral gavage and reached 4–5 μg/g brain 20–40 min after i.p. injection. In conclusion, the developed and validated method should be useful for the accurate and precise quantification of curcumin in target organs from relevant animal models of human diseases. Figure Representative chromatograms of curcumin extracted from brains of mice that were intraperitoneally injected with curcumin (100 mg/kg bw). Brains were removed 20, 30 or 40 min (red, blue, and green trace, respectively) after injection.

Keywords: Curcumin; Fast HPLC; Small particle size; Validation; Murine brain; Plasma

Noninvasive detection of counterfeited ampoules of dexamethasone using NIR with confirmation by HPLC-DAD-MS and CE-UV methods by Oxana Rodionova; Alexey Pomerantsev; Lars Houmøller; Alexey Shpak; Oleg Shpigun (pp. 1927-1935).

Application of near-infrared (NIR) measurements together with chemometric data processing is widely used for counterfeit drug detection. The most difficult counterfeits to detect are the “high quality fakes”, which have the proper composition but are produced in violation of technological regulations by underground manufacturers. This study uses such forgeries and addresses important issues. The first is the possibility of applying the NIR/chemometric approach to the detection of injectable formulations of drugs (in this case dexamethasone), which are aqueous solutions with low concentration of active ingredients, directly in the closed ampoules. The second issue is the comparison of NIR/chemometric conclusions with detailed chemical analysis.

Keywords: High quality forgeries; PCA; NIR; HPLC-DAD-MS; CE-UV

PCR-free MDR1 polymorphism identification by gold nanoparticle probes by Bing Yang; Guohua Zhou; Lequn Lee Huang (pp. 1937-1945).

Single nucleotide polymorphisms (SNPs) represent the most abundant source of genetic variation in the human genome, and they can be linked to genetic susceptibilities or varied pharmaceutical responses. Established SNP detection techniques are mainly PCR-based, which means that they involve complex, labor-intensive procedures, are easy contaminated, and can give false-positive results. Therefore, we have developed a simple and rapid MS-based disulfide barcode methodology that relies on magnifying the signal from a dual-modified gold nanoparticle. This approach permits direct SNP genotyping of total human genomic DNA without the need for primer-mediated enzymatic amplification. Disulfides that are attached to the gold nanoparticle serve as a “barcode” that allows different sequences to be discerned using MS detection. Specificity is based on two sequential oligonucleotide hybridizations, which include two steps: the first is the capture of the target by gene-specific probes immobilized onto magnetic beads; the second is the recognition of gold nanoparticles functionalized with allele-specific oligonucleotides. The sensitivity of this new method reaches down to the 0.1 fM range, thus approaching that of PCR. The feasability of this SNP identification methodology based on an MS-based disulfide barcode assay was demonstrated by applying it to genomic DNA samples representing all possible genotypes of the SNPs G2677T and C3435T in the human MDR1 gene. Due to its great advantage—the ability to perform SNP typing without the use of PCR—the assay was found to be simple, rapid and robust, and so may be highly suited to routine clinical detection as well as basic medical research. Figure SNP typing procedures of the MS-based disulfide-barcode assay

Keywords: Gold nanoparticle probe; Single nucleotide polymorphism (SNP); PCR-free; Disulfide barcode; MS detection

Investigation of calcium antagonist–L-type calcium channel interactions by a vascular smooth muscle cell membrane chromatography method by Hui Du; Jianyu He; Sicen Wang; Langchong He (pp. 1947-1953).

The dissociation equilibrium constant (K _D) is an important affinity parameter for studying drug–receptor interactions. A vascular smooth muscle (VSM) cell membrane chromatography (CMC) method was developed for determination of the K _D values for calcium antagonist–L-type calcium channel (L-CC) interactions. VSM cells, by means of primary culture with rat thoracic aortas, were used for preparation of the cell membrane stationary phase in the VSM/CMC model. All measurements were performed with spectrophotometric detection (237 nm) at 37 °C. The K _D values obtained using frontal analysis were 3.36 × 10⁻⁶ M for nifedipine, 1.34 × 10⁻⁶ M for nimodipine, 6.83 × 10⁻⁷ M for nitrendipine, 1.23 × 10⁻⁷ M for nicardipine, 1.09 × 10⁻⁷ M for amlodipine, and 8.51 × 10⁻⁸ M for verapamil. This affinity rank order obtained from the VSM/CMC method had a strong positive correlation with that obtained from radioligand binding assay. The location of the binding region was examined by displacement experiments using nitrendipine as a mobile-phase additive. It was found that verapamil occupied a class of binding sites on L-CCs different from those occupied by nitrendipine. In addition, nicardipine, amlodipine, and nitrendipine had direct competition at a single common binding site. The studies showed that CMC can be applied to the investigation of drug–receptor interactions. Figure The analytical process of calcium antagonist—L-type calcium channel (L-CC) interactions using a vascular smooth muscle (VSM) cell membrane chromatography (CMC) method (a) and measurement of As (b).

Keywords: Vascular smooth muscle; Cell membrane chromatography; Frontal analysis; Dissociation equilibrium constant; Binding site

Evidence of natural occurrence of the banned antibiotic chloramphenicol in herbs and grass by Bjorn Berendsen; Linda Stolker; Jacob de Jong; Michel Nielen; Enkhtuya Tserendorj; Ruuragchas Sodnomdarjaa; Andrew Cannavan; Christopher Elliott (pp. 1955-1963).

Chloramphenicol (CAP), a broad-spectrum antibiotic, was detected in several herb and grass samples from different geographic origins. Due to its suspected carcinogenicity and linkages with the development of aplastic anemia in humans, CAP is banned for use in food-producing animals in the European Union (EU) and many other countries. However, products of animal origin originating from Asian countries entering the European market are still found noncompliant (containing CAP) on a regular basis, even when there is no history of chloramphenicol use in these countries. A possible explanation for the continued detection of these residues is the natural occurrence of CAP in plant material which is used as animal feed, with the consequent transfer of the substance to the animal tissues. Approximately 110 samples were analyzed using liquid chromatography coupled with mass spectrometric detection. In 26 samples, the presence of CAP was confirmed using the criteria for banned substances defined by the EU. Among other plant materials, samples of the Artemisia family retrieved from Mongolia and from Utah, USA, and a therapeutic herb mixture obtained from local stores in the Netherlands proved to contain CAP at levels ranging from 0.1 to 450 µg/kg. These findings may have a major impact in relation to international trade and safety to the consumer. The results of this study demonstrate that noncompliant findings in animal-derived food products may in part be due to the natural occurrence of chloramphenicol in plant material. This has implications for the application of current EU, USA, and other legislation and the interpretation of analytical results with respect to the consideration of CAP as a xenobiotic veterinary drug residue and the regulatory actions taken upon its detection in food.

Keywords: Plant material; 2002/657/EC; Confirmation; Analysis; LC–MS/MS; Chloramphenicol

Key parameters for the development of a NIR microscopic method for the quantification of processed by-products of animal origin in compound feedingstuffs by O. Abbas; J. A. Fernández Pierna; A. Boix; C. von Holst; P. Dardenne; V. Baeten (pp. 1965-1973).

The aim of this work is to show new advances in the analytical methods developed in the frame of the ban of processed animal by-products in compound feed that is currently applied within the European Union. With this aim, studies to develop a quantitative near infrared microscopy (NIRM) approach have been undertaken in order to fulfil future requirements of European legislation like the introduction of tolerance levels that would require for official control purposes the availability of specific quantitative methods. The capabilities of the NIRM method have been improved; no sample preparation is required and the acquisition parameters are optimised. Both the gross and the fine fractions of the samples are considered; the reflexion mode was used to analyse the gross raw fraction and the transmission mode was chosen to analyse the fine raw fraction. Parameters for reflexion analyses were already fixed in our previous studies while those of transmission mode have been determined in the present study. Because particles are too small, it is difficult to mark them; spectra were collected using the mapping technique. Quantitative analyses have been carried out for different percentages of adulteration (0.5, 1, 2 and 5%). Results were depending on the particle size distribution of the feed and of the fish meal which led to experimental values of adulteration varying between 0.13–0.92%, 0.93–3.7%, 2.42–5.83% and 1.95–9.39% for theoretical percentages of adulteration equal to 0.5, 1, 2 and 5%, respectively. The established protocol with the key parameters proposed has to be considered for the development of an accurate method of quantification.

Keywords: NIR microscopy; Transmission; Mapping; Animal feed; Contamination; Quantification

Characterization of bioactive thiophenes from the dichloromethane extract of Echinops grijisii as Michael addition acceptors by Xiaoyu Zhang; Zhongjun Ma (pp. 1975-1984).

Michael addition acceptors are considered as biologically active molecules, which regulate many signal pathways in cells. In the present study, it was demonstrated that the dichloromethane extract of Echinops grijisii had phase II detoxifying enzyme-inducing and nuclear factor-κB (NF-κB)-inhibiting activities, which might be attributed to the modification of key cysteine residues in Keap1 and NF-κB by Michael addition acceptors in it. To screen these Michael addition acceptors, glutathione (GSH) was employed, and a simple liquid chromatography–tandem mass spectrometry screening method was established to investigate the formation of GSH conjugates. Three thiophenes, 5-(penta-1,3-diynyl)-2-(3,4-diacetoxybut-1-ynyl)-thiophene (6), 2-(penta-1,3-diynyl)-5-(4-hydroxybut-1-ynyl)-thiophene (7), and 2-(penta-1,3-diynyl)-5-(3,4-dihydroxybut-1-ynyl)-thiophene (8) were demonstrated to react with GSH. Then NAD(P)H/quinone oxidoreductase1(NQO1) induction assay and an ultrafiltration mass spectrometric screening method were performed to investigate whether the above three compounds had NQO1-inducing and NF-κB (p65) alkylating activities. The result indicated that compounds 6–8, which had a common structural moiety, a penta-1,3-diynyl group, had strong NQO1-inducing activities, and compounds 7 and 8 could effectively alkylate the cysteine residues in NF-κB (p65). Figure A simple LC-MS screening method was established to discover the Michael addition acceptors in the dichloromethane extract of Echinops grijisii.

Keywords: Michael addition acceptors; LC-MS screening; Glutathione; Thiophenes; Dichloromethane extract of Echinops grijisii

Oxidative modifications in glycated insulin by Sofia Guedes; Rui Vitorino; Maria R. M. Domingues; Francisco Amado; Pedro Domingues (pp. 1985-1995).

At the present, the term “glycoxidation” is recognized as the synergistic interaction between glycation and oxidative processes which, with the help of redox-active metals, consequently leads to the production of deleterious tissue modifications. The association between glycation and oxidation events is considered one of the major factors in the accumulation of non-functional damaged proteins, enhancing the oxidative damage at the cellular level. Because of the central role of insulin in the biology of diabetes, we investigated the site-specific oxidation of native and glycated insulin (mono, di, and tri-glycated forms), through metal-catalyzed oxidation, with a combination of liquid chromatography and mass spectrometry. With this approach we were able to identify the residues that were mainly oxidized, and peptide sequences resulting from oxidative cleavage of insulin. Tyrosine, phenylalanine, and cysteine were the main affected residues. Time-course analysis (0–48 h) of the oxidative damage enabled to detect more pronounced and earlier oxidative modifications in the case of glycated insulin. We also observed more severe oxidative damage as the number of glycation sites increased in insulin. These oxidative modifications included other oxidized residues, namely proline, histidine, valine, leucine, and glycine, which were shown to be carbonylated. In addition, we identified new sites of peptide cleavage with the formation of new fragments, derived mainly from chain B, which were both glycated and oxidatively modified. Peptide fragmentation occurred mainly between the residues phenylalanine, glycine, leucine, and tyrosine. Moreover, for diglycated and triglycated forms we observed further oxidative cleavage occurring in both chains, with oxidation and fragmentation of residues occurring near cysteine bridges, especially in chain A. Figure Schematic structure of glycated insulin showing modified residues resulting from oxidative damage marked with red circles and one possible glycated residues (bold and underlined). Two examples of tandem mass spectra that allowed the identification of the modified residues are shown.

Keywords: Insulin; Glycation; Oxidative damage; Oxidative stress; Mass spectrometry

Chiral ligand-exchange separation and resolution of extremely rigid glutamate analogs: 1-aminospiro[2.2]pentyl-1,4-dicarboxylic acids by Benedetto Natalini; Roccaldo Sardella; Nicola Giacchè; Samantha Palmiotto; Emidio Camaioni; Maura Marinozzi; Antonio Macchiarulo; Roberto Pellicciari (pp. 1997-2011).

Owing to their chelation ability, a series of fully constrained l-Glu analogs formed by the spiro-union of two cyclopropane rings (1-aminospiro[2.2]pentyl-1,4-dicarboxylic acids, ASPED A–D), was submitted to chiral ligand-exchange chromatographic (CLEC) analysis. As the initial step, two methodologically different chiral devices were evaluated. A chiral stationary phase (CSP) obtained by dynamic coating of C₁₈ chains with the S-trityl-(R)-cysteine ((R)-STC) was used first with this objective. The lack of separation of the enantiomers of ASPED C and D prompted us to utilize the chiral mobile phase (CMP) prepared from O-benzyl-(S)-serine ((S)-OBS). The latter afforded complete separation of the four pairs of enantiomers. For all the pairs, quantum mechanical investigations shed light on the main features responsible for the different enantiomer recognition mechanism with (S)-OBS. The validated analytical method was then fruitfully adopted for semi-preparative-scale isolation of the enantiomers of ASPED C.

Keywords: Glutamate constrained analogs; Coated chiral stationary phase; Chiral mobile phase; Semi-preparative scale-up; Mechanism of chiral recognition

Electrochemical anion sensing using electrodes chemically modified with Au(I)–Cu(I) heterotrimetallic alkynyl cluster complexes containing ferrocenyl groups by Antonio Doménech; Igor O. Koshevoy; Noemí Montoya; Tapani A. Pakkanen (pp. 2013-2022).

A novel family of electrochemical anion sensors operating in aqueous media, based on the heterometallic Au(I)–Cu(I) [{Au₃Cu₂(C₂R)₆}Au₃(PPh₂C₆H₄PPh₂)₃](PF₆)₂ (L1, R = Fc; L2, R = C₆H₄Fc) alkynyl cluster complexes, is presented. Upon attachment to graphite and gold electrodes, these compounds exhibit a well-defined, essentially reversible, solid-state electrochemistry in contact with aqueous media, based on ferrocenyl-centered oxidation processes involving anion insertion, leading to distinctive pH-independent electrochemical responses for fluoride, chloride, bromide, perchlorate, bicarbonate, carbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, and nitrate anions. Cluster-modified electrodes can be used as potentiometric sensors as a result of the reversible, diffusion-controlled electrochemistry obtained for the anion-assisted electrochemical oxidation of L1 and L2.

Keywords: Gold–copper alkynyl metal clusters; Ferrocenyl units; Selective anion sensing; Electrochemical anion insertion

Determination of mitragynine in plasma with solid-phase extraction and rapid HPLC–UV analysis, and its application to a pharmacokinetic study in rat by Suhanya Parthasarathy; Surash Ramanathan; Sabariah Ismail; Mohd Ilham Adenan; Sharif Mahsufi Mansor; Vikneswaran Murugaiyah (pp. 2023-2030).

A new solid phase extraction method for rapid high performance liquid chromatography–UV determination of mitragynine in plasma has been developed. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile–ammonium acetate buffer, 50 mM at pH 5.0 (50:50, v/v). The method had limits of detection and quantification of 0.025 and 0.050 μg/mL, respectively. The method was accurate and precise for the quantitative analysis of mitragynine in human and rat plasma with within-day and between-day accuracies between 84.0 and 109.6%, and their precision values were between 1.7 and 16.8%. Additional advantages over known methods are related to the solid phase extraction technique for sample preparation which yields a clean chromatogram, a short total analysis time, requires a smaller amount of plasma samples and has good assay sensitivity for bioanalytical application. The method was successfully applied in pharmacokinetic and stability studies of mitragynine. In the present study, mitragynine was found to be fairly stable during storage and sample preparation. The present study showed for the first time the detailed pharmacokinetic profiles of mitragynine. Following intravenous administration, mitragynine demonstrated a biphasic elimination from plasma. Oral absorption of the drug was slow, prolonged and was incomplete, with a calculated absolute oral bioavailability value of 3.03%. The variations observed in previous pharmacokinetic studies after oral administration of mitragynine could be attributed to its poor bioavailability rather than to the differences in assay method, metabolic saturation or mitragynine dose.

Keywords: Mitragynine; High performance liquid chromatography–UV detection; Solid phase extraction; Stability; Pharmacokinetic

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Analytical and Bioanalytical Chemistry (v.397, #5)