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Analytical and Bioanalytical Chemistry (v.389, #7-8)

B. Stuart: Analytical techniques in materials conservation by Peter Vandenabeele (pp. 2035-2036).

Marvin McMaster: HPLC: a practical user’s guide, 2nd edition by Catherine Rimmer (pp. 2037-2038).

Christoph A. Schalley (Ed.): Analytical methods in supramolecular chemistry by Franz L. Dickert (pp. 2039-2040).

Thanks to reviewers in 2007 (pp. 2041-2049).

Contributions of Professor Pier Giorgio Zambonin to analytical chemistry by Elio Desimoni; Francesco Palmisano; Luigia Sabbatini (pp. 2051-2053).

Analytical investigations of poly(acrylic acid) coatings electrodeposited on titanium-based implants: a versatile approach to biocompatibility enhancement by E. De Giglio; S. Cometa; N. Cioffi; L. Torsi; L. Sabbatini (pp. 2055-2063).

A polyacrylic acid film was synthesized on titanium substrates from aqueous solutions via an electroreductive process for the first time. This work was done in order to develop a versatile coating for titanium-based orthopaedic implants that acts as both an effective bioactive surface and an effective anti-corrosion barrier. The chemical structure of the PAA coating was investigated by X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy (SEM) was employed to evaluate the effect of annealing treatment on the morphology of the coatings in terms of their uniformity and porosity. Inductively coupled plasma mass spectrometry was used to measure ion concentrations in ion release tests performed on Ti-6Al-4V sheets modified with PAA coatings (annealed and unannealed). Results indicate that the annealing process produces coatings that possess considerable anti-corrosion performance. Moreover, the availability and the reactivity of the surface carboxylic groups were exploited in order to graft biological molecules onto the PAA-modified titanium implants. The feasibility of the grafting reaction was tested using a single aminoacid residue. A fluorinated aminoacid was selected, and the grafting reaction was monitored both by XPS, using fluorine as a marker element, and via quartz crystal microbalance (QCM) measurements. The success of the grafting reaction opens the door to the synthesis of a wide variety of PAA-based coatings that are functionalized with selected bioactive molecules and promote positive reactions with the biological system interfacing the implant while considerably reducing ion release into surrounding tissues. Figure Vanadium release from bare Ti-6Al-4V sheets compared with the release from sheets coated with annealed and unannealed electrosynthesised PAA

Keywords: Titanium; PAA; Electrosynthesis; Corrosion protection; Biomaterial; Surface modification

Evaluation of the thermal history of bovine milk from the lactosylation of whey proteins: an investigation by liquid chromatography–electrospray ionization mass spectrometry by Ilario Losito; Teresa Carbonara; Linda Monaci; Francesco Palmisano (pp. 2065-2074).

Reversed-phase high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (RP-HPLC–ESI-MS) has been used for analysis of the native and lactosylated forms of the main whey proteins, α-lactalbumin and β-lactoglobulins A and B, in commercial bovine milk samples after different thermal treatment (pasteurisation and ultra high-temperature, UHT, treatment), of different lipid content, and of different brands, to find markers of the thermal history of the milk. A new quantification strategy was developed, based on peak-area integration after multiple ion current extraction and considering all the ions detectable in the multi-charge ESI mass spectrum for each type of protein. Validation of the procedure for native forms was first accomplished by calibration with model solutions. Linearity was always good. Sensitivity was different for α-lactalbumin and β-lactoglobulins; the signal was stronger for the latter with only a slight difference between variants A and B of β-lactoglobulins. Application of the quantification approach to pasteurised and UHT milk samples showed that the distributions of the three proteins and of their three main forms (native, and mono and bi-lactosylated) in whey extracts can be used as statistically robust discriminatory properties for recognition of commercial thermal treatment of milk.

Keywords: Bovine milk; Whey proteins; Thermal denaturation; Lactosylation; Liquid chromatography–electrospray ionization mass spectrometry

A laser desorption ionization time-of-flight mass spectrometry investigation into triacylglycerols oxidation during thermal stressing of edible oils by Cosima Damiana Calvano; Antonella Aresta; Francesco Palmisano; Carlo G. Zambonin (pp. 2075-2084).

Laser desorption ionization time-of-flight mass spectrometry (LDI–TOF MS) was used to characterize olive and sunflower oils before and after thermally assisted oxidation in order to develop a rapid fingerprinting method for oil that contains unchanged and oxidized components. No matrix was used to assist laser desorption, and simplified mass spectra were obtained in the mass range of interest (m/z 500–1000), where triacyl- and diacylglycerol ions were observed. Sample preparation was reduced to dissolving oil in chloroform saturated with NaCl. Sodiated triacylglycerols (TAGs), their epoxy/hydroxy and hydroperoxy derivatives, as well as TAGs with shortened chain fatty acids (β-scission products) were clearly observed in the spectra. LDI–TOF MS rapidly provides semiquantitative information about the oxidation level of edible oil, and thus represents a very useful quality control tool.

Keywords: LDI; Triacylglycerols; Olive oil; Sunflower oil; Oxidation

Progress in direct solid sampling analysis using line source and high-resolution continuum source electrothermal atomic absorption spectrometry by Bernhard Welz; Maria Goreti R. Vale; Daniel L. G. Borges; Uwe Heitmann (pp. 2085-2095).

The literature about direct solid sample analysis of the past 10–15 years using electrothermal atomic absorption spectrometry has been reviewed. It was found that in the vast majority of publications aqueous standards were reported as having been used for calibration after careful program optimization. This means the frequently expressed claim that certified reference materials with a matrix composition and analyte content close to that of the sample have to be used for calibration in solid sample analysis is not confirmed in the more recent literature. There are obviously limitations, and there are examples in the literature where even calibration with certified reference materials did not lead to accurate results. In these cases the problem is typically associated with spectral interferences that cannot be corrected properly by the systems available for conventional line source atomic absorption spectrometry, including Zeeman-effect background correction. Using high-resolution continuum source atomic absorption spectrometry, spectral interferences become visible owing to the display of the spectral environment at both sides of the analytical line at high resolution, which makes program optimization straightforward. Any spectrally continuous background absorption is eliminated automatically, and even rapidly changing background absorption does not cause any artifacts, as measurement and correction of background absorption are truly simultaneous. Any kind of fine-structured background can be eliminated by “subtracting” reference spectra using a least-squares algorithm. Aqueous standards are used for calibration in all published applications of high-resolution continuum source atomic absorption spectrometry to direct solid sample analysis.

Keywords: Direct solid sample analysis; Electrothermal atomic absorption spectrometry; High-resolution continuum source atomic absorption spectrometry; Background correction; Calibration

Boronic acid–lectin affinity chromatography. 1. Simultaneous glycoprotein binding with selective or combined elution by Alex Monzo; Guenther K. Bonn; András Guttman (pp. 2097-2102).

We introduce a novel combination of boronic acid affinity chromatography with lectin affinity chromatography, dubbed as boronic acid–lectin affinity chromatography (BLAC). Concanavalin A and wheat germ agglutinin lectins were mixed with the pesudo-lectin boronic acid to form the BLAC affinity column and their performance was evaluated with standard glycoproteins. Optimization of the binding and elution buffers for the BLAC system is described. The BLAC columns were employed to isolate glycoproteins of interest using both selective and/or combined elution.

Keywords: Lectin; Pseudo-lectin; Affinity chromatography; Electrophoresis

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of zearalenone and deoxynivalenol by Anna Yu Kolosova; Sarah De Saeger; Liberty Sibanda; Ron Verheijen; Carlos Van Peteghem (pp. 2103-2107).

A multianalyte lateral-flow technique using colloidal gold-labeled monoclonal antibodies was developed for the rapid simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEA). The results of this qualitative one-step test were interpreted visually. A very simple and fast sample preparation was used, and the assay procedure could be accomplished within 10 min. When applied to spiked wheat samples, the technique gave accurate and reproducible results. Cut-off levels of 1500 and 100 μg kg⁻¹ for DON and ZEA, respectively, were observed. The described multianalyte format can be used as a reliable, rapid and cost-effective on-site screening technique for the simultaneous determination of mycotoxins in grain samples.

Keywords: Lateral-flow immunoassay; Colloidal gold; Deoxynivalenol; Zearalenone; Multianalyte

A receptor-binding-based bioassay to determine the potency of a plasmid biopharmaceutical encoding VEGF-C by Thomas Waerner; Thomas Girsch; Sandra Varga; Lichun Huang; Alexander Gornikiewicz; Gerhard Loeber (pp. 2109-2113).

Over the last decade biological assays (bioassays) have gained much importance for quality control in biopharmaceutical development and manufacturing. Here we describe the development and validation of a bioassay to determine the biological activity (potency) of the plasmid biopharmaceutical pVGI.1 which encodes the VEGF-C (VEGF-2) protein. This assay was developed to test drug substance and drug product for release and stability testing for phase I and II clinical trials. The main focus was on fast assay development and easy handling of the assay, combined with valid results representing the specific therapeutic mechanism. The method includes the expression of the VEGF-C protein in mammalian cells and its binding to the cell surface receptor VEGFR-3. The binding activity of VEGF-C to its immobilized receptor is quantified in a colorimetric assay. IC₅₀ values of VEGF-C expressed after transfection with sample plasmid and an in-house standard plasmid are determined. The ratio of the IC₅₀ value of the test article to that of the reference standard reflects the potency of the sample. The potency assay meets the criteria generally requested by authorities for precision, linearity, accuracy, and range. Therefore the assay can be used in pharmaceutical quality control and is a suitable basis for development of related bioassays.

Keywords: Bioassays; Biotechnological products; Bioanalytical methods; Quality assurance/control; Pharmaceuticals; Immunoassays; ELISA

Photonic crystal sensor for organophosphate nerve agents utilizing the organophosphorus hydrolase enzyme by Jeremy P. Walker; Kyle W. Kimble; Sanford A. Asher (pp. 2115-2124).

We developed an intelligent polymerized crystalline colloidal array (IPCCA) photonic crystal sensing material which reversibly senses the organophosphate compound methyl paraoxon at micromolar concentrations in aqueous solutions. A periodic array of colloidal particles is embedded in a poly-2-hydroxyethylacrylate hydrogel. The particle lattice spacing is such that the array Bragg-diffracts visible light. We utilize a bimodular sensing approach in which the enzyme organophosphorus hydrolase (OPH) catalyzes the hydrolysis of methyl paraoxon at basic pH, producing p-nitrophenolate, dimethylphosphate, and two protons. The protons lower the pH and create a steady-state pH gradient. Protonation of the phenolates attached to the hydrogel makes the free energy of mixing of the hydrogel less favorable, which causes the hydrogel to shrink. The IPCCA’s lattice constant decreases, which blueshifts the diffracted light. The magnitude of the steady-state diffraction blueshift is proportional to the concentration of methyl paraoxon. The current detection limit is 0.2 μmol methyl paraoxon per liter.

Keywords: Sensor; Pesticide; Organophosphate; Organophosphorus hydrolase; Enzyme; Polymerized Crystalline Colloidal Array (PCCA)

Bootstrap method for the estimation of measurement uncertainty in spotted dual-color DNA microarrays by Tobias K. Karakach; Robert M. Flight; Peter D. Wentzell (pp. 2125-2141).

DNA microarrays permit the measurement of gene expression across the entire genome of an organism, but the quality of the thousands of measurements is highly variable. For spotted dual-color microarrays the situation is complicated by the use of ratio measurements. Studies have shown that measurement errors can be described by multiplicative and additive terms, with the latter dominating for low-intensity measurements. In this work, a measurement-error model is presented that partitions the variance into general experimental sources and sources associated with the calculation of the ratio from noisy pixel data. The former is described by a proportional (multiplicative) structure, while the latter is estimated using a statistical bootstrap method. The model is validated using simulations and three experimental data sets. Monte-Carlo fits of the model to data from duplicate experiments are excellent, but suggest that the bootstrap estimates, while proportionately correct, may be underestimated. The bootstrap standard error estimates are particularly useful in determining the reliability of individual microarray spots without the need for replicate spotting. This information can be used in screening or weighting the measurements.

Keywords: DNA microarrays; Measurement errors; Bootstrap; Gene expression; Transcriptomics; Measurement quality; Uncertainty estimation

Raman spectroscopic study of bacterial endospores by Joke De Gelder; Patsy Scheldeman; Karen Leus; Marc Heyndrickx; Peter Vandenabeele; Luc Moens; Paul De Vos (pp. 2143-2151).

Endospores and endospore-forming bacteria were studied by Raman spectroscopy. Raman spectra were recorded from Bacillus licheniformis LMG 7634 at different steps during growth and spore formation, and from spore suspensions obtained from diverse Bacillus and Paenibacillus strains cultured in different conditions (growth media, temperature, peroxide treatment). Raman bands of calcium dipicolinate and amino acids such as phenylalanine and tyrosine are more intense in the spectra of sporulating bacteria compared with those of bacteria from earlier phases of growth. Raman spectroscopy can thus be used to detect sporulation of cells by a characteristic band at 1,018 cm^–1 from calcium dipicolinate. The increase in amino acids could possibly be explained by the formation of small acid-soluble proteins that saturate the endospore DNA. Large variations in Raman spectra of endospore suspensions of different strains or different culturing conditions were observed. Next to calcium dipicolinate, tyrosine and phenylalanine, band differences at 527 and 638 cm^–1 were observed in the spectra of some of the B. sporothermodurans spore suspensions. These bands were assigned to the incorporation of cysteine residues in spore coat proteins. In conclusion, Raman spectroscopy is a fast technique to provide useful information about several spore components. Figure A difference spectrum between Raman spectra of B. licheniformis LMG 7634 cultured for 6 days and 1 day, together with the reference Raman spectrum of calcium dipicolinate

Keywords: Raman spectroscopy; Bacteria; Endospore; Dipicolinic acid; Bacillus

Ex-vivo NMR of unprocessed tissue in water: a simplified procedure for studying intracranial neoplasms by Maosheng Xu; Jieru Ye; Daiwen Yang; Xiaoli Xu; Tseng Tsai Yeo; Wai Hoe Ng; C. C. Lim (pp. 2153-2159).

Ex-vivo and in-vitro nuclear magnetic resonance (NMR) spectroscopy techniques have been used for studying chemical metabolites in surgically resected specimens of human neoplasms, and may provide complementary information to in-vivo whole-body magnetic-resonance spectroscopy (MRS). We describe an ex-vivo NMR in water method for measurement of water-soluble metabolites in unprocessed normal rat brain tissue and human intracranial neoplasms. The NMR spectra obtained using the method described here were comparable to those obtained using high-resolution magic-angle spinning (HRMAS) NMR methods, with good correlation in metabolite concentrations relative to creatine (r ² = 0.7635). Improved spectral resolution and baseline were noted compared to HRMAS, but macromolecule resonances were not detected. Ex-vivo NMR of unprocessed tissue in water is rapid and technically simple to perform, and has the potential to be used for direct assessment of intracranial neoplasms.

Keywords: Nuclear magnetic resonance; Brain neoplasm; Rat brain; High-resolution magic-angle spinning; Choline

A 2D NMR method to study peptide phosphorylation by Christian Raeck; Stefan Berger (pp. 2161-2165).

We demonstrate a 2D NMR method which distinguishes between phosphorylated and non-phosphorylated amino acids. The method is capable of monitoring the amino acid and site-specific enzymatic phosphorylation and dephosphorylation of peptides. The method was developed using O-phosphorylated amino acids and its potential is shown with a peptide fragment of the myelin basic protein (MBP).

Keywords: Phosphorylation; NMR; Peptides; Kinase; Phosphatase

Selenium methylselenocysteine protects human hepatoma HepG2 cells against oxidative stress induced by tert-butyl hydroperoxide by Susana Cuello; Sonia Ramos; Raquel Mateos; M. Angeles Martín; Yolanda Madrid; Carmen Cámara; Laura Bravo; Luis Goya (pp. 2167-2178).

Selenium methylselenocysteine (Se-MeSeCys) is a common selenocompound in the diet with a tested chemopreventive effect. This study investigated the potential protective effect of Se-MeSeCys against a chemical oxidative stress induced by tert-butyl hydroperoxide (t-BOOH) on human hepatoma HepG2 cells. Speciation of selenium derivatives by liquid chromatography–inductively coupled plasma mass spectrometry depicts Se-MeSeCys as the only selenocompound in the cell culture. Cell viability (lactate dehydrogenase) and markers of oxidative status—concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR)—were evaluated. Pretreatment of cells with Se-MeSeCys for 20 h completely prevented the enhanced cell damage, MDA concentration and GR and GPx activity and the decreased GSH induced by t-BOOH but did not prevent increased ROS generation. The results show that treatment of HepG2 cells with concentrations of Se-MeSeCys in the nanomolar to micromolar range confers a significant protection against an oxidative insult.

Keywords: Antioxidant defences; Biomarkers for oxidative stress; Dietary antioxidants; Selenium compounds

Application of cadmium sulfide nanoparticles as oligonucleotide labels for the electrochemical detection of NOS terminator gene sequences by Wei Sun; Jianghua Zhong; Bo Zhang; Kui Jiao (pp. 2179-2184).

A mercaptoacetic acid (MAA)-modified cadmium sulfide (CdS) nanoparticle was synthesized in aqueous solution and used as an oligonucleotide label for the electrochemical detection of nopaline synthase (NOS) terminator gene sequence. The carboxyl groups on the surface of the CdS nanoparticle can be easily covalently linked with NH₂-modified NOS oligonucleotide probe sequences. The target ssDNA sequence was fixed onto the electrode surface by covalently linking to a mercaptoethanol self-assembled gold electrode, and the DNA hybridization of target ssDNA with probe ssDNA was accomplished on the electrode surface. The CdS nanoparticles anchored on the hybrids were dissolved in the solution by the oxidation with HNO₃ and further detected by a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used for monitoring the hybridization, and the NOS target sequence was satisfactorily detected in the approximate range from 8.0 × 10⁻¹² to 4.0 × 10⁻⁹ mol L⁻¹ with a detection limit of 2.75 × 10⁻¹² mol L⁻¹ (3σ). The established method extended the nanoparticle-labeled electrochemical DNA analysis to genetically modified organisms (GMOs) specific sequence samples with higher sensitivity and selectivity.

Keywords: DNA hybridization; CdS nanoparticle; NOS sequence; Anodic stripping voltammetry; Genetically modified organisms

Functional toxicity and tolerance patterns of bioavailable Pd(II), Pt(II), and Rh(III) on suspended Saccharomyces cerevisiae cells assayed in tandem by a respirometric biosensor by Chiara Frazzoli; Roberto Dragone; Alberto Mantovani; Cristiana Massimi; Luigi Campanella (pp. 2185-2194).

Toxicological implications of exposure to bioavailable platinum group metals, here Pd, Pt, and Rh, are still to be clarified. This study obtained by a biosensor-based method preliminary information on potential effects on cellular metabolism as well as on possible tolerance mechanisms. Aerobic respiration was taken as the toxicological end point to perform tandem tests, namely functional toxicity test and tolerance test. Cells were suspended in the absence of essential constituents for growth. The dose–response curves obtained by exposure (2 h) to the metals (nanogram per gram range) suggested the same mechanisms of action, with Rh showing the greatest curve steepness and the lowest EC₅₀ value. Conservative (95% lower confidence interval) EC₁₀ values were 187, 85 and 51 ng g⁻¹ for Pt, Pd, and Rh respectively. Tolerance patterns were tested during the same runs. The full tolerance obtained after 12 h of exposure to each metal suggested mitochondrial inhibition of aerobic respiration as a target effect. The hazard rating of the metals in the tolerance test changed in the Rh EC₅₀ range, where Rh showed the lowest toxicity. The observed tolerance might suggest a protective mechanism such as metallothionein induction at concentrations around the EC₅₀ values. The performance of the bioassay was satisfactory, in terms of the limit of detection, repeatability, reproducibility, roboustness, sensibility, and stability; the method’s critical uncertainty sources were identified for improvements. Figure Respirometric curved

Keywords: Palladium; Platinum; Rhodium; Mitochondrial toxicity; Metallothionein

Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR by Natalia Ferraz; Daniel Carnevia; Gonzalo Nande; Martin Rossotti; María N. Miguez; Jerold A. Last; Gualberto Gonzalez-Sapienza (pp. 2195-2202).

Vitellogenin (VTG) and choriogenin (CHO) are valuable biomarkers of endocrine-disrupting compound (EDC) exposure in fish. Existing immunoassays are limited to a few species, which restricts their use for the analysis of local wildlife sentinels. Using C. facetum as a relevant South American model fish, this work presents a new strategy for the preparation of antibodies to VTG and CHO, with zero cross-reactivity with fish serum components. Recombinant fragments of Cichlasoma facetum VTG (280-mer) and CHO (223-mer) were prepared by degenerate primer RT-PCR and expression in E. coli. Polyclonal and monoclonal antibodies prepared with these antigens were used to develop rapid dotblot assays for VTG and CHO. Both the polyclonal and monoclonal antibodies prepared with the recombinant antigens reacted against the native proteins adsorbed on to nitrocellulose allowing the set up of sensitive dotblot assays. The VTG assay was further validated with spiked samples and purified native VTG. Exposure experiments with several estrogenic compounds revealed the potential of C. facetum as a sensitive biomonitor that produced measurable responses at concentrations of 100 ng L⁻¹ of 17-beta-estradiol, 100 ng L⁻¹ of ethynylestradiol, and 6.6 μg L⁻¹ of nonylphenol. The approach described here may be applied to other native species to produce highly specific and sensitive rapid tests. It may be particularly advantageous for species that cannot be kept in captivity or when homogeneous purification of the immunizing proteins is particularly challenging. In conclusion, we present a novel approach to develop a strategy for the generation of immunoassay reagents for vitellogenin (VTG) and choriogenin (CHO), which will facilitate regional studies on the impact of endocrine-disrupting chemicals on local wildlife.

Keywords: Endocrine disruptors; Recombinant antigens; Rapid assay; Estradiol; Nonylphenol

Analytical discrimination between sources of ginseng using Raman spectroscopy by H. G. M. Edwards; T. Munshi; K. Page (pp. 2203-2215).

Ginseng is a widely used medicinal product that grows mainly in Korea, China and America. American ginseng is classified as an endangered species, and so the import and export of this type of ginseng is illegal in certain countries. Due to this restriction it is becoming increasingly important to be able to distinguish between different types of ginseng. FT-Raman spectroscopy has the ability to discriminate between ginseng specimens according to the country of origin and the effects of processing on the ginseng material. The ginsenoside content of ginseng differs in both conformation and concentration depending on the source of the ginseng, which means that ginseng grown in different countries should express unique spectral features. The presence or absence of these features, therefore, could indicate the geographical origin of the sample. Several spectral features were identified for a range of ginsengs, such as a peak at 980 cm⁻¹ that was only found in Chinese ginseng, and the different wavenumber positions of characteristic ginseng bands near 1600 cm⁻¹. This indicates that Raman spectroscopy can be used to pinpoint the origin of an unknown ginseng sample and that it would provide a rapid nondestructive analytical technique for formally discriminating between restricted and permitted imports.

Keywords: Ginseng; Forensic; Raman spectroscopy; Origin; Real or counterfeit

Determination of loratadine and pseudoephedrine sulfate in pharmaceuticals based on non-linear second-order spectrophotometric data generated by a pH-gradient flow injection technique and artificial neural networks by María J. Culzoni; Héctor C. Goicoechea (pp. 2217-2225).

Loratadine (LOR) and pseudoephedrine sulfate (PES) were determined in pharmaceutical samples by using non-linear second-order data generated by a pH-gradient flow injection analysis (FIA) system with diode-array detection. Determination of both analytes was performed on the basis of differences between the acid–base and spectral features of each drug species. Non-linearities were detected by using both qualitative and quantitative tools. As a consequence of the non-linearity, a recently reported algorithm, artificial neural networks followed by residual bilinearization (ANN/RBL), was shown to furnish more satisfactory results. Recoveries of 99.7% (LOR) and 95.6% (PES) were obtained when analyzing a validation set containing unexpected components (the usual excipients found in pharmaceutical preparations). The average value obtained by implementation of the method on four replicates was compared with that obtained by a reference method based on HPLC (difference not significant; p > 0.05).

Keywords: Loratadine; Pseudoephedrine; Pharmaceutical; Artificial neural networks; Second-order advantage

Immobilised tyrosinase-based biosensor for the detection of tea polyphenols by K. S. Abhijith; P. V. Sujith Kumar; M. A. Kumar; M. S. Thakur (pp. 2227-2234).

An amperometric principle-based biosensor containing immobilized enzyme tyrosinase has been used for detection of polyphenols in tea. The immobilized tyrosinase-based biosensor could detect tea polyphenols in the concentration range 10–80 mmol L⁻¹. Immobilization of the enzyme by the crosslinking method gave good stable response to tea polyphenols. The biosensor response reached the steady state within 5 min. The voltage response was found to have a direct linear relationship with the concentration of polyphenols in black tea samples. Enzyme membrane fouling was observed with number of analyses with a single immobilised enzyme membrane. The tyrosinase-based biosensor gave maximum response to tea polyphenols at 30°C. The optimum pH was 7.0. This biosensor system can be applied for analysis of tea polyphenols. Variation in the biosensor response to black tea infusions gave an indication of the different amounts of theaflavins in the samples, which is an important parameter in evaluating tea quality. A comparative study of the quality attributes of a variety of commercially available brands of tea were performed using the biosensor and conventional analytical techniques such as spectrophotometry.

Keywords: Biosensor; Tyrosinase; Catechin; Amperometric; Crosslinking; Black tea

Biosensor for total cholesterol estimation using N-(2-aminoethyl)-3-aminopropyltrimethoxysilane self-assembled monolayer by Sunil K. Arya; Monika Datta; S. P. Singh; Bansi D. Malhotra (pp. 2235-2242).

Cholesterol oxidase (ChOx), cholesterol esterase (ChEt), and horseradish peroxidase (HRP) have been co-immobilized covalently on a self-assembled monolayer (SAM) of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTS) deposited on an indium–tin–oxide (ITO) glass surface. These enzyme-modified (ChOx-ChEt-HRP/AEAPTS/ITO) biosensing electrodes have been used to estimate cholesteryl oleate from 10 to 500 mg dL⁻¹. The sensitivity, K _m value, and shelf-life of these ChEt-ChOx-HRP/AEAPTS/ITO biosensing electrodes have been found to be 124 nA mg⁻¹ dL, 95.098 mg dL⁻¹ (1.46 mmol L⁻¹), and ten weeks, respectively. The ChEt-ChOx-HRP/AEAPTS/ITO bio-electrodes have been used to estimate total cholesterol in serum samples. Figure Covalent immobilization of enzymes onto AEAPTS/ITO surface using EDC/NHS chemistry

Keywords: Cholesterol oleate; N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTS); Self-assembled monolayer (SAM); Cyclic voltammetry; Biosensor

Characterization and application of quantum dot nanocrystal–monoclonal antibody conjugates for the determination of sulfamethazine in milk by fluoroimmunoassay by Jianzhong Shen; Fei Xu; Haiyang Jiang; Zhanhui Wang; Jing Tong; Pengju Guo; Shuangyang Ding (pp. 2243-2250).

Quantum dot (Qdot) nanocrystals have been increasingly used as fluorescence labels in fluoroimmunoassays recently because of their excellent optical characteristics. In this paper, a new monoclonal antibody (MAb) against sulfamethazine (SMZ) was successfully produced and linked to Qdot nanocrystals by covalent coupling. The Qdot–MAb conjugates were characterized by SDS-PAGE and high-performance capillary electrophoresis (HPCE). An enzyme-linked immunosorbent assay (ELISA) method was utilized to evaluate the antigen–antibody binding affinity and then a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of SMZ in milk by using Qdots as fluorescent labels was evaluated. The results showed that the 50% inhibition values (IC₅₀) of the cFLISA were 4.3 ng/mL in milk and 5.2 ng/mL in PBS, and the limits of detection (LODs) were 0.6 ng/mL in milk and 0.4 ng/mL in PBS, respectively. The recoveries of SMZ from spiked milk samples at levels of 10–100 ng/mL ranged from 94 to 106%, with coefficients of variation (CVs) of 2.1–9.2%. Figure Shematic diagram of the direct cFLISA procedure

Keywords: Quantum dots; Monoclonal antibody; Sulfamethazine; Milk residue

Characteristics of a liposome immunoassay on a poly(methyl methacrylate) surface by Sang Youn Hwang; Yoichi Kumada; Gi Hoon Seong; Jaebum Choo; Shigeo Katoh; Eun Kyu Lee (pp. 2251-2257).

Liposome immunoassay (LIA) is based on enzyme immunoassay (EIA) but the detection sensitivity could be significantly enhanced by using antibody-coupled immunoliposomes encapsulating HRP (horse radish peroxidase). Here, we applied LIA to non-porous poly(methyl methacrylate) (PMMA) and polystyrene (PS) surfaces to compare its detection sensitivity with that of EIA, using rabbit IgG (a ligand molecule) and anti-rabbit IgG antibody (a capture molecule) as the model system. LIA developed much stronger color signals than EIA, especially at a lower concentration range (< ca. 1 μg mL⁻¹). PMMA showed higher affinity toward rabbit IgG than the PS surface, and the anti-rabbit IgG antibody adsorbed on PMMA was more stable than that on PS. Furthermore, the effects of spot volume and antibody concentration on the signal density were analyzed. The signal density increased as the antibody concentration increased, but it was not significantly affected by the spot volume (2.5–20 μL). In conclusion, LIA on PMMA as a solid support is a very useful, highly sensitive microarray detection system.

Keywords: Liposome immunoassay; Immunoliposome; PMMA; Microarray detection; Signal enhancement

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by high-performance liquid chromatography: application to a pharmacokinetic study by Peifan Li; Yunhui Zhang; Li Xiao; Xinghua Jin; Kun Yang (pp. 2259-2264).

Radix Scrophulariae (Xuanshen) is one of the famous Chinese herbal medicines widely used to treat rheumatism, tussis, pharyngalgia, arthritis, constipation, and conjunctival congestion. Harpagoside and cinnamic acid are the main bioactive components of Xuanshen. The purpose of this study was to develop an HPLC–UV method for simultaneous determination of harpagoside and cinnamic acid in rat plasma and investigate pharmacokinetic parameters of harpagoside and cinnamic acid after oral administration of Xuanshen extract (760 mg kg⁻¹). After addition of syringin as internal standard, the analytes were isolated from plasma by liquid–liquid extraction. Separation was achieved on a Kromasil C₁₈ column, and detection was by UV absorption at 272 nm. The described assay was validated in terms of linearity, accuracy, precision, recovery, and limit of quantification according to the FDA validation guidelines. Calibration curves for both analytes were linear with the coefficient of variation (r) for both was greater than 0.999. Accuracy for harpagoside and cinnamic acid ranged from 100.7–103.5% and 96.9–102.9%, respectively, and precision for both analytes were less than 8.5%. The main pharmacokinetic parameters found for harpagoside and cinnamic acid after oral infusion of Xuanshen extract were as follows: C _max 1488.7 ± 205.9 and 556.8 ± 94.2 ng mL⁻¹, T _max 2.09 ± 0.31 and (1.48 ± 0.14 h, AUC_0–24 10336.4 ± 1426.8 and 3653.1 ± 456.4 ng h mL⁻¹, $$ AUC_{{0 - infty }} $$ 11276.8 ± 1321.4 and 3704.5 ± 398.8 ng h mL⁻¹, and t _1/2 4.9 ± 1.3 and 2.5 ± 0.9 h, respectively. These results indicated that the proposed method is simple, selective, and feasible for pharmacokinetic study of Radix Scrophulariae extract in rats. Figure Radix Scrophulariae

Keywords: Radix Scrophulariae (Xuanshen) extract; Harpagoside; Cinnamic acid; HPLC–UV; Pharmacokinetics

PEG-linked geminal dicationic ionic liquids as selective, high-stability gas chromatographic stationary phases by Ke Huang; Xinxin Han; Xiaotong Zhang; Daniel W. Armstrong (pp. 2265-2275).

It is known that room-temperature ionic liquids (RTILs) have wide applicability in many scientific and technological fields. In this work, a series of three new dicationic room-temperature ionic liquids functionalized with poly(ethylene glycol) (PEG) linkages were synthesized and characterized via a linear solvation model. The application of these ILs as new GC stationary phases was studied. The efficient separation of several mixtures containing compounds of different polarities and 24 components of a flavor and fragrance mixture indicated comparable or higher resolving power for the new IL stationary phases compared to the commercial polysiloxane and poly(ethylene glycol)-based stationary phases. In addition, the selectivities of the IL stationary phases could be quite unique. The separation of a homologous alkane and alcohol mixture displayed the “dual nature” of these ionic liquids as GC stationary phases. The thermal stability study showed the column robustness up to 350 °C. The high separation power, unique selectivity, high efficiency and high thermal stability of the new dicationic ionic liquids indicate that they may be applicable as a new type of robust GC stationary phase.

Keywords: PEG; Geminal dicationic RTIL; Gas chromatography; Orthogonal stationary phase; Thermal stability

Rapid-resolution HPLC with spectrometric detection for the determination and identification of isoflavones in soy preparations and plant extracts by B. Klejdus; J. Vacek; L. Benešová; J. Kopecký; O. Lapčík; V. Kubáň (pp. 2277-2285).

A rapid-resolution HPLC/UV-VIS DAD separation method (which takes <1 min) for the determination and identification of genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin and biochanin A in fmol quantities in submicroliter sample volumes was optimized. A linear gradient elution (0 min 22% B, 1.0 min 80% B, 1.4 min 100% B, 1.8 min 22% B) using a mobile phase containing 0.2 % (v/v) acetic acid (solvent A) and methanol (solvent B) was applied on a Zorbax SB C₁₈ column (1.8 μm particle size) at 80 °C. The method was verified using samples of bits of soy and methanolic extracts from Trifolium pratense, Iresine herbstii and Ononis spinosa plants. Pseudobaptigenin glucoside, irilone, prunetin, texasin, tlatlancuayin and other isoflavones, in addition to aglycones of isoflavones and their β-glucosides and malonyl and acetyl derivatives, were identified by UV-VIS DAD and electrospray mass spectrometric (ESI-MS) detection in the extracts. Figure Rapid resolution HPLC for determination and identification of isoflavones in soy preparations and plant extracts

Keywords: Rapid resolution; Fast chromatography; Reversed phase; HPLC; Isoflavone identification; Aglycones; β-glucosides; Soy food; Red clover

Enhancement of intensities in glow discharge mass spectrometry by using mixtures of argon and helium as plasma gases by Britta Lange; Ralf Matschat; Heinrich Kipphardt (pp. 2287-2296).

Glow discharge mass spectrometry (GD-MS) is an excellent technique for fast multi-element analysis of pure metals. In addition to metallic impurities, non-metals also can be determined. However, the sensitivity for these elements can be limited due to their high first ionization potentials. Elements with a first ionization potential close to or higher than that of argon, which is commonly used as discharge gas in GD-MS analysis, are ionized with small efficiency only. To improve the sensitivity of GD-MS for such elements, the influence of different glow-discharge parameters on the peak intensity of carbon, chlorine, fluorine, nitrogen, phosphorus, oxygen, and sulfur in pure copper samples was investigated with an Element GD (Thermo Fisher Scientific) GD-MS. Discharge current, discharge gas flow, and discharge gas composition, the last of which turned out to have the greatest effect on the measured intensities, were varied. Argon–helium mixtures were used because of the very high potential of He to ionize other elements, especially in terms of the high energy level of its metastable states. The effect of different Ar–He compositions on the peak intensity of various impurities in pure copper was studied. With Ar–He mixtures, excellent signal enhancements were achieved in comparison with use of pure Ar as discharge gas. In this way, traceable linear calibration curves for phosphorus and sulfur down to the μg kg⁻¹ range could be established with high sensitivity and very good linearity using pressed powder samples for calibration. This was not possible when pure argon alone was used as discharge gas.

Keywords: Glow discharge mass spectrometry; Discharge gas; Argon–helium mixtures; Copper matrix; Non-metals

Surfactant to dye binding degree based approach for the selective determination of l-glutamate in foodstuffs by Ana Pedraza; María Dolores Sicilia; Soledad Rubio; Dolores Pérez-Bendito (pp. 2297-2302).

A selective method for the determination of l-glutamate in foodstuffs has been developed. It was based on the competition established between the analyte and the dye Coomassie brilliant blue G (CBBG) to interact with the surfactant didodecyldimethylammonium bromide (DDABr). The measurement parameter was the amount of DDABr required to reach a given dye-to-surfactant binding degree. It was obtained by photometric titration on the basis of the changes observed in the spectral characteristics of the dye when CBBG–DDABr aggregates were formed. The calibration graph obtained was linear in the l-glutamate concentration interval 0.2–5 mM (detection limit 0.05 mM). The high selectivity of the proposed method (other amino acids and food additives did not interfere at the concentrations present in foodstuffs) permitted the direct analysis of food samples after dissolution of the analyte in hot water. The accuracy of the surfactant to the dye binding degree method was demonstrated by determining l-glutamate in different kinds of foodstuffs (liquid and dried soups, seasonings, pasta sauces and dried mushroom creams) and comparing the results obtained with those provided by the commercial Boehringer Mannheim essay.

Keywords: Surfactant to dye binding degree method; l-Glutamate; Food analysis; Routine quality control

Use of the diffusive gradients in thin films technique (DGT) with various diffusive gels for characterization of sewage sludge-contaminated soils by Vladěna Kovaříková; Hana Dočekalová; Bohumil Dočekal; Martina Podborská (pp. 2303-2311).

The diffusive gradients in thin film technique (DGT) was used for characterization of South Moravian arable soils (sampling sites Zlín, Tuřany, and Chrlice) amended by sewage sludge in the 1980s. Two types of polyacrylamide diffusive gel with different pore size (APA gels—cross-linked with agarose and RG gels—cross-linked with bis-acrylamide) were employed. The (bio)available parts of Cd, Cu, and Ni and the proportions of inorganically and organically complexed species of these metals were assessed. The degree of metal resupply from the soil solid phase to the soil solution was also determined. Metal concentrations obtained by the DGT technique were lower by almost 4 to 5 orders of magnitude in comparison with those obtained by extraction with aqua regia. DGT concentrations of metals were also lower by approximately 1 to 2 orders of magnitude in comparison with those obtained by extraction with sodium nitrate (commonly used for assessment of the (bio)available part of metals). Results obtained by DGT measurement were expected to be closer to the actual content of available metal species than results obtained by extraction with sodium nitrate. Using RG gels together with APA gels provided resolution of inorganically and organically complexed metal species and their proportional representation. Inorganic metal species (particles smaller than 1 nm) formed a predominant part of assessed metal content in all studied soil samples and horizons. However, there was the exception of the cadmium content in the middle profile of Chrlice sandy soil sample. Ratio R values indicated that resupply of Cd, Cu, and Ni from the solid phase to the soil solution varied for individual soil samples and individual depth profiles. Mobile and labile species of Cd, Cu, and Ni were much more closely related to upper rather than deeper horizons. This observation correlates very well with the mechanical treatment and amendment of the studied soils.

Keywords: DGT; Diffusive gels; Soils; Metals; Metal species; Extraction procedures

Quantitative determination of soybean meal content in compound feeds: comparison of near-infrared spectroscopy and real-time PCR by Hui Li; Xiaowen Lv; Jing Wang; Junguo Li; Haifeng Yang; Yuchang Qin (pp. 2313-2322).

Standard methods for determining the raw material content of compound feed are little exploited, except for the identification of meat and bone meal in feeds. In this work, near-infrared (NIR) spectroscopy and real-time polymerase chain reaction (PCR) were applied in order to establish new and fast methods for quantification of soybean meal content in compound feeds. The best prediction quality was achieved by using a model based on NIR spectroscopy (R ² = 0.9857, standard error of cross-validation 1.1065). Furthermore, a sensitive qualitative detection method by using the real-time PCR was developed (R ² = 0.976, slope −3.7599). Finally, the differences between the real-time PCR result and the NIR spectroscopy result for a given sample were also treated, and we found that the NIR spectroscopy method provided quite accurate results which approach closely those of the real-time PCR method.

Keywords: Near-infrared spectroscopy; Real-time PCR; Soybean meal; Determination

A thermo extraction–UV/Vis spectrophotometric method for total iodine quantification in soils and sediments by B. S. Gilfedder; F. Althoff; M. Petri; H. Biester (pp. 2323-2329).

Iodine in soils and sediments is a difficult element to analyze due to its volatility in acidic conditions. Traditionally it has been quantified using neutron activation analysis techniques, which, unfortunately, requires access to a nuclear reactor. We present here a simple method for solid-phase iodine analysis by thermo extraction at 1000°C and quantification by UV/Vis photometry. Samples are combusted in an oxygen stream and trapped in Milli-Q water. The extracts are then quantified by an As³⁺–Ce⁴⁺ spectrometric method whereby iodide catalyzes the oxidation of As³⁺ to As⁵⁺ and reduction of Ce⁴⁺ to Ce³⁺. Three standard reference materials were analyzed with excellent recoveries (97–113%) and RSDs (<5%). Moreover, the detection limit was less than 50 ng absolute iodine with a confidence limit of 95%. When applied to carbonate-rich samples from sediment traps deployed in Lake Constance we found very low iodine levels (0.8–2 mg kg⁻¹). Despite the low concentrations, the precision of the method was consistently better than 5% RSD. However, the method needed to be slightly modified for organic and iodine-rich sediments (20–30% organic carbon) from a lake in the Black Forest by increasing the oxygen flow rate and decreasing the combustion time. Using the modified method we were able to achieve RSDs lower than 5%.

Keywords: Iodine analysis; Iodine quantification; Iodine in sediments; Iodine in soils; Iodine extraction

Chemically driven variable selection by focused multimodal genetic algorithms in mid-IR spectra by M. P. Gómez-Carracedo; M. Gestal; J. Dorado; J. M. Andrade (pp. 2331-2342).

Four genetic-algorithm-based approaches to variable selection in spectral data sets are presented. They range from a pure black-box approach to a chemically driven one. The latter uses a fitness function that takes into account not only typical parameters like the number of errors when classifying a training set but also the chemical interpretability of the selected variables. In order to cope with the fact that multiple solutions may be acceptable, a multimodal genetic algorithm (GA) is employed and the most satisfactory solution selected. The multimodal GA uses two populations (denominated “hybrid two populations” GA or HTP-GA): a classical population, from which potential solutions emerge, and a new population, which maintains diversity in the search space (as required by multimodal problems). Results show that the HTP-GA approach improves the chemical understanding of the selected solution (compared to other GA approaches) and that the classification capabilities of the approach are still good. All of the GA strategies for variable selection were compared with a classical parametric technique, Procrustes rotation, which does not consider interpretability.

Keywords: Variable selection; Genetic algorithms; Procrustes rotation; Neural networks

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Analytical and Bioanalytical Chemistry (v.389, #7-8)