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Analytical and Bioanalytical Chemistry (v.396, #5)

ABC presents Nobel Prize Winners in Chemistry by Aldo Roda (pp. 1615-1617).

is Full Professor of Analytical Chemistry at Bologna University. His main research interests center on the area of analytical and bioanalytical chemistry applied to clinical chemistry, pharmaco-toxicology, medicinal chemistry, and environmental and food analysis. He is the owner of more than 20 international patents on new bile acids and antioxidant drugs, new lanthanide chelates as luminescent labels and new luciferases.

ABC presents Nobel Prize Winners in Chemistry by Aldo Roda (pp. 1615-1617).

Discovery and development of the green fluorescent protein, GFP: the 2008 Nobel Prize by Aldo Roda (pp. 1619-1622).

is Full Professor of Analytical Chemistry at Bologna University. His main research interests center on the area of analytical and bioanalytical chemistry applied to clinical chemistry, pharmaco-toxicology, medicinal chemistry, environmental and food analysis. Roda is the owner of more than 20 international patents on new bile acids and antioxidant drugs, new lanthanide chelates as luminescent labels and new luciferases.

Discovery and development of the green fluorescent protein, GFP: the 2008 Nobel Prize by Aldo Roda (pp. 1619-1622).

is Full Professor of Analytical Chemistry at Bologna University. His main research interests center on the area of analytical and bioanalytical chemistry applied to clinical chemistry, pharmaco-toxicology, medicinal chemistry, environmental and food analysis. Roda is the owner of more than 20 international patents on new bile acids and antioxidant drugs, new lanthanide chelates as luminescent labels and new luciferases.

Enabling technologies in discovery: the 2009 Nobel Prize and its implications in antibiotic design by Yinan Wei; Sylvia Daunert (pp. 1623-1626).

joined the faculty of the Department of Chemistry, University of Kentucky as an Assistant Professor in 2008. Her research interests include the study of proteins involved in the multidrug resistance, investigation of the membrane protein folding pathway, and development of biosensors for the detection of pathogenic microbes. is Gill Eminent Professor of Analytical and Biological Chemistry, Professor of Chemistry, and Professor of Pharmaceutical Sciences, Biological and Bioanalytical Chemistry at the University of Kentucky, Lexington. Dr Daunert’s research group uses recombinant DNA technology to design new molecular diagnostic tools and biosensors based on genetically engineered proteins and cells for applications in biomedical and environmental fields. Additionally, her research focuses on the design of sensing arrays for the detection of molecules in small volumes and microfluidic platforms, and in the development of smart biomaterials for responsive drug-delivery systems. Among other awards and distinctions, she received a Fulbright Scholarship, and is the recipient of the 2001 A. F. Findeis Award from the American Chemical Society, and the 2002 Special Creativity Award of the National Science Foundation.

Enabling technologies in discovery: the 2009 Nobel Prize and its implications in antibiotic design by Yinan Wei; Sylvia Daunert (pp. 1623-1626).

Quality assurance challenge 9 by Manfred Reichenbächer; Jürgen W. Einax (pp. 1627-1629).

Solution to spectroscopy challenge 14 by Reinhard Meusinger (pp. 1631-1632).

The 1000 bar and 24 hour limits of one-dimensional HPLC – graphical representations by Veronika R. Meyer (pp. 1633-1640).

is a chemist and works at EMPA, the Swiss Federal Laboratories for Materials Testing and Research in St Gallen. She is a book author in the field of HPLC and a lecturer at the University of Bern, Switzerland. Her scientific interests are analytical chemistry, especially liquid chromatography, quality assurance, and measurement uncertainty.

The 1000 bar and 24 hour limits of one-dimensional HPLC – graphical representations by Veronika R. Meyer (pp. 1633-1640).

Uranium speciation in biofilms studied by laser fluorescence techniques by Thuro Arnold; Kay Großmann; Nils Baumann (pp. 1641-1653).

Biofilms may immobilize toxic heavy metals in the environment and thereby influence their migration behaviour. The mechanisms of these processes are currently not understood, because the complexity of such biofilms creates many discrete geochemical microenvironments which may differ from the surrounding bulk solution in their bacterial diversity, their prevailing geochemical properties, e.g. pH and dissolved oxygen concentration, the presence of organic molecules, e.g. metabolites, and many more, all of which may affect metal speciation. To obtain such information, which is necessary for performance assessment studies or the development of new cost-effective strategies for cleaning waste waters, it is very important to develop new non-invasive methods applicable to study the interactions of metals within biofilm systems. Laser fluorescence techniques have some superior features, above all very high sensitivity for fluorescent heavy metals. An approach combining confocal laser scanning microscopy and laser-induced fluorescence spectroscopy for study of the interactions of biofilms with uranium is presented. It was found that coupling these techniques furnishes a promising tool for in-situ non-invasive study of fluorescent heavy metals within biofilm systems. Information on uranium speciation and uranium redox states can be obtained. Figure Spectroscopic information, e.g. different oxidation states, can be visualized and spectroscopically identified within a confocal volume by a combination of confocal laser scanning microscopy (CLSM) and laser-induced fluorescence spectroscopy (LIFS)

Keywords: Biofilm; Uranium; Confocal laser scanning microscopy (CLSM); Laser-induced fluorescence spectroscopy (LIFS)

Uranium speciation in biofilms studied by laser fluorescence techniques by Thuro Arnold; Kay Großmann; Nils Baumann (pp. 1641-1653).

Keywords: Biofilm; Uranium; Confocal laser scanning microscopy (CLSM); Laser-induced fluorescence spectroscopy (LIFS)

Multitarget quantitative metabolic profiling of hydrophilic metabolites in fermentation broths of β-lactam antibiotics production by HILIC–ESI–MS/MS by Simone Schiesel; Michael Lämmerhofer; Wolfgang Lindner (pp. 1655-1679).

The presented work deals with the development and comprehensive validation of a quantitative LC–electrospray ionization (ESI)–tandem mass spectrometry (MS/MS) method using a triple quadrupole instrument in the MRM mode for the metabolic profiling of amino acids, organic acids, vitamins, some biogenic amines, secondary metabolites of β-lactam antibiotics biosynthesis as well as their intermediates, and degradation products in fermentation broths of β-lactam antibiotics production (in total 57 hydrophilic compounds). A great number of chromatographic systems (22 different stationary phase/mobile phase conditions) were screened for their adequate chromatographic selectivity to cope with isobaric compounds and other critical analyte pairs. Finally, a hydrophilic interaction liquid chromatography (HILIC) method employing a zwitterionic ZIC-HILIC column was selected as best compromise. Particular focus was given on the elucidation of absolute and relative matrix effects via comparison of slopes of calibration functions of spiked matrix and standard solutions. These data as well as precision and accuracy data confirm suitability of the HILIC–ESI–MS/MS assay for metabolic profiling studies in fermentation samples. Detailed comprehensive data sets are presented which should illustrate critical issues, problems, and challenges of multitarget quantitative metabolic profiling and should outline possible strategies to circumvent pitfalls and overcome common problems.

Keywords: Microbial metabolomics; Process analysis technology (PAT); Process control; HILIC; HPLC–MS/MS; Matrix effects

Time-resolved biomarker discovery in ¹H-NMR data using generalized fuzzy Hough transform alignment and parallel factor analysis by Erik Alm; Ralf J. O. Torgrip; K. Magnus Åberg; Ina Schuppe-Koistinen; Johan Lindberg (pp. 1681-1689).

This work addresses the subject of time-series analysis of comprehensive ¹H-NMR data of biological origin. One of the problems with toxicological and efficacy studies is the confounding of correlation between the administered drug, its metabolites and the systemic changes in molecular dynamics, i.e., the flux of drug-related molecules correlates with the molecules of system regulation. This correlation poses a problem for biomarker mining since this confounding must be untangled in order to separate true biomarker molecules from dose-related molecules. One way of achieving this goal is to perform pharmacokinetic analysis. The difference in pharmacokinetic time profiles of different molecules can aid in the elucidation of the origin of the dynamics, this can even be achieved regardless of whether the identity of the molecule is known or not. This mode of analysis is the basis for metabonomic studies of toxicology and efficacy. One major problem concerning the analysis of ¹H-NMR data generated from metabonomic studies is that of the peak positional variation and of peak overlap. These phenomena induce variance in the data, obscuring the true information content and are hence unwanted but hard to avoid. Here, we show that by using the generalized fuzzy Hough transform spectral alignment, variable selection, and parallel factor analysis, we can solve both the alignment and the confounding problem stated above. Using the outlined method, several different temporal concentration profiles can be resolved and the majority of the studied molecules and their respective fluxes can be attributed to these resolved kinetic profiles. The resolved time profiles hereby simplifies finding true biomarkers and bio-patterns for early detection of biological conditions as well as providing more detailed information about the studied biological system. The presented method represents a significant step forward in time-series analysis of biological ¹H-NMR data as it provides almost full automation of the whole data analysis process and is able to analyze over 800 unique features per sample. The method is demonstrated using a ¹H-NMR rat urine dataset from a toxicology study and is compared with a classical approach: COW alignment followed by bucketing.

Keywords: Urine; ¹H-NMR; Alignment; Multivariate; Metabolic profiling; PARAFAC; Drug metabolism; Toxicology

Characterisation of B(a)P metabolites formed in an ex vivo pig skin model using three complementary analytical methods by Carine Jacques; Emilien L. Jamin; Elisabeth Perdu; Hélène Duplan; Alain Mavon; Daniel Zalko; Laurent Debrauwer (pp. 1691-1701).

An original method was developed to separate, identify and quantify the different benzo(a)pyrene (B(a)P) metabolites formed through oxidative and conjugative pathways. All B(a)P metabolites were separated by an improved high-performance liquid chromatography method, then detected and quantified relatively by online radioactivity detection. At the same time, metabolite structures were characterised by tandem mass spectrometry using two complementary ionisation modes: electrospray ionisation in the negative mode and atmospheric pressure chemical ionisation in the positive mode. This method was successfully applied to the analysis of B(a)P metabolites, produced by incubation of B(a)P with the ex vivo pig ear skin model. These include glucuronic acid and sulphate conjugates of B(a)P-OHs and B(a)P-diols, as well as direct phase I metabolites: B(a)P-tetrol, B(a)P-diones, B(a)P-catechols, B(a)P-diols and B(a)P-OHs.

Keywords: HPLC; Radioactivity; Mass spectrometry; Skin; Benzo(a)pyrene; Metabolites

Hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) determination of cocaine and its metabolites benzoylecgonine, ecgonine methyl ester, and cocaethylene in hair samples by Oscar Quintela; Elena Lendoiro; Angelines Cruz; Ana de Castro; Alfredo Quevedo; Carmen Jurado; Manuel López-Rivadulla (pp. 1703-1712).

This study reports the development and validation of a method using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) for the analysis of cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME), and cocaethylene (CE) in hair samples. Decontamination was performed as follows: Firstly, the aliquot of hair was briefly rinsed with 2 mL dichloromethane, then was washed three times with 10 mL 0.01 M phosphate buffer, pH 6, for 15 min, followed by 2 mL 2-propanol for less than 2 min, and, finally, a last rinse with 2 mL dichloromethane was again done. Cocaine compounds were extracted from 10 mg of hair by incubation with 2 mL 0.1 M HCl at 50 °C for 12 h and purified by solid phase extraction with Oasis MCX cartridges. Analysis was performed by LC-MS/MS using an Atlantis HILIC silica chromatographic column. The method was fully validated. Linearity was established over the concentration range 0.020–10.0 ng/mg for cocaine (COC), 0.010–10.0 ng/mg for BE and CE, and 0.005–2.0 ng/mg for EME, and the correlation coefficients were all >0.99. Extraction efficiency was >70% for all analytes. Limits of detection were 0.0005 ng/mg for CE and 0.001 ng/mg for the other analytes (COC, BE, and EME). Lower limits of quantification were the lowest points of the calibration curves with acceptable accuracy and precision (coefficient of variation ≤20%). Intra- and inter-day imprecision ranged between 1.5% and 9.5% and 0.7% and 12.6%, respectively. Intra- and inter-day inaccuracy ranged from 0.5% to 12.3% and from 0.7% to 7.1%, respectively. With regard to matrix effects, suppression was <27.5% in all cases. The method was applied to the analysis of several samples derived from forensic cases.

Keywords: Hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS); Cocaine and metabolites; Ecgonine methyl ester; Hair analysis

Measurement of progesterone in human serum by isotope dilution liquid chromatography–tandem mass spectrometry and comparison with the commercial chemiluminescence immunoassay by Hwashim Lee; Chang Joon Park; Gaeho Lee (pp. 1713-1719).

Progesterone is one of the steroid hormones. The hormone is especially important in preparing the uterus for the implantation of the blastocyst and in maintaining pregnancy. Its concentration in serum is measured to determine ovarian function and to predict early pregnancy. The progesterone concentration is also important for in-vitro fertilization and embryo-transfer outcomes. We have established isotope dilution liquid chromatography–tandem mass spectrometry as a primary method for the measurement of progesterone in human serum. Progesterone and its isotopic analogue, progesterone-¹³C₂, in serum were monitored at mass transitions of m/z 315.2/109.2 and 317.2/111.2 respectively in multiple-reaction monitoring (MRM) mode with electrospray positive ionization. For validation of the method, progesterone in a National Institute of Standards and Technology standard reference material (NIST SRM) was measured, and the measured results were in good agreement with the reference values within the uncertainty. On the basis of the established method, progesterone certified reference material (CRM) was developed in this work. The certified value was (1.41 ± 0.036) μg kg⁻¹. The repeatability of 1.1% and reproducibility of 0.14% showed that ID LC–MS–MS is a reliable and reproducible method. The expanded uncertainty for the measurement of progesterone in the CRM was approximately 2.6% within 95% confidence limits. The detection limit of progesterone was approximately 0.6 μg kg⁻¹. The progesterone CRMs were distributed to representative clinical laboratories in the Republic of Korea for comparison with the chemiluminescence immunoassay (CLIA), which is the most sensitive immunoassay method. The results from the comparison showed quite a large bias among the participating laboratories. This implies that the CRM is a very important material for establishment of traceability to its practical use. Figure

Keywords: Progesterone; ID LC–MS–MS; Traceability; Primary method

Impacts of different promoters on the mammalian one-hybrid assay for detecting nuclear receptor agonists by Zhi-Hui Zheng; Xin-Hua Lu; Hua Zhang; Guo-Ping Lv; Jian-Gong He; Bao-Hua Zhao; Shu-Yi Si (pp. 1721-1730).

Nuclear receptors are a superfamily of ligand-activated transcription factors that play key roles in many biological processes, and have become one class of the most important targets in drug discovery. Mammalian one-hybrid system has been used to develop a cell-based functional transactivation high-throughput screening (HTS) assay for detecting nuclear receptors ligands. In the present study, we proved that different promoters used in the reporter vector had significant different impacts on the performance of HTS assays. The assay using the SV40 promoter in the reporter vector showed the characteristics of much higher signal/noise ratios, acceptable Z′ factors (>0.6), low coefficient variation (<12.5%) and higher hits rate, which could be more robust, reproducible, and sensitive. In contrast, utilizing a TATA box promoter in the assay resulted in higher variance and low sensitivity. In addition, it was found that the assay using SV40 had longer signal decay time and was easier to be miniaturized in 384-well format. It has been confirmed that the choice of a promoter is a critical factor in developing a reporter gene HTS assay. However, the SV40 promoter used in the present study has been shown to be more adaptable than the minimal promoter TATA box in the Mammalian one-hybrid HTS assays for detecting nuclear receptor agonists. Figure During construction of a cell-based functional transactivation high-throughput screening (HTS) assay for detecting nuclear receptors ligands, different promoters used in the reporter vector had significant different impacts on the performance of HTS assays. The assay using the SV40 promoter in reporter vector showed the characteristics of much higher signal/noise ratios, acceptable Z′ factors (>0.6%), low coefficient variation (<12.5%) and higher hits rate, which could be more robust, reproducible and sensitive. In contrast, utilizing TATA box promoter in the assay resulted in higher variance and low sensitivity

Keywords: Nuclear receptor agonists; High-throughput screening; Mammalian one-hybrid; GAL4 chimeric receptor assay; TATA box promoter; SV40 promoter

Screening of antinociceptive components in Corydalis yanhusuo W.T. Wang by comprehensive two-dimensional liquid chromatography/tandem mass spectrometry by Chen Wang; Shuowen Wang; Guorong Fan; Hanfa Zou (pp. 1731-1740).

Formalin-induced pain models were used in rats to evaluate the antinociceptive effect of the total alkaloids of Corydalis yanhusuo (TAC). The results indicated that formalin-evoked spontaneous nociceptive responses (licking behavior) could be inhibited significantly by giving (intragingival) TAC at a single dose of 150 mg/kg. Subsequently, an online comprehensive two-dimensional biochromatography method with a silica-bonded human serum albumin (HSA) column in the first dimension and a monolithic ODS column in the second was developed. The absorbed bioactive components were screened by comparing and contrasting the components detected in the plasma and striatum with those in TAC. More than 100 compounds were separated and detected in the TAC, among which 13 compounds were identified. About 40 compounds (seven compounds identified) were absorbed into the plasma with appropriate concentrations and about 20 compounds (four compounds identified) passed through the blood-brain barrier into the striatum. Of interest, four compounds (protopine, glaucine, tetrahydropalmatine, and corydaline) which were reported to possess profound antinociceptive effects exhibited high concentrations in the striatum. This may result from their synergistic effects in regulating the formalin-induced nociception. The results indicated that the comprehensive two-dimensional biochromatography method developed is capable of screening the bioactive components in Corydalis yanhusuo and providing valuable information for understanding the mechanisms by which Corydalis yanhusuo alleviates nociception.

Keywords: Comprehensive two-dimensional biochromatography; Corydalis yanhusuo W.T. Wang; Human serum albumin column; Traditional Chinese medicine; Formalin test

Development and validation of a rapid method for the detection of latrunculol A in plasma by Jiajiu Shaw; Frederick A. Valeriote; Joseph Media; Tyler A. Johnson; Taro Amagata; Karen Tenney; Phillip Crews (pp. 1741-1744).

Latrunculol A is a recently discovered 6,7-dihydroxy analog of the potent actin inhibitor latrunculin A. Latrunculol A has exhibited greater cytotoxicity than latrunculin A against both murine and human colon tumor cell lines in vitro. Currently, there are no reports regarding the bioavailability of latrunculol A in vivo. This study was undertaken as a prelude to pharmacokinetic assessments and it is the first work where bioavailability of latrunculol A was studied. In the present work, a simple plasma preparation and a rapid HPLC method have been developed. Mouse plasma containing latrunculol A was first treated by acetonitrile and then centrifuged at 14,000 rpm at 4 °C for 25 min. The supernatant was injected in an HPLC system comprising a Waters Symmetry NH₂ column, a mobile phase of acetonitrile/water (95/5, v/v), a flow rate of 1.0 mL/min, at 220 nm. The method was validated by parameters including a good linear correlation, a limit of quantification of 9 ng/mL, and a good precision with a coefficient variation of 1.65, 1.86, and 1.26% for 20, 400, and 800 ng/mL, respectively. With this simple method, excellent separation and sensitivity of latrunculol A are achieved, thus allowing a rapid analysis of the plasma samples for absorption, distribution, and metabolism studies.

Keywords: Latrunculins; Latrunculol A; Anti-actin activity; HPLC analysis; Bioavailability

An enzyme-linked immunosorbent assay to compare the affinity of chemical compounds for β-amyloid peptide as a monomer by Chunyi Jiang; Yu Feng; Xiaotong Huang; Yechun Xu; Yaping Zhang; Naiming Zhou; Xu Shen; Kaixian Chen; Hualiang Jiang; Dongxiang Liu (pp. 1745-1754).

Aβ_1–42 is the proteolytic cleavage product of cleavage of the amyloid precursor protein by β- and γ-secretases. The aggregation of Aβ_1–42 plays a causative role in the development of Alzheimer’s disease. To lock Aβ_1–42 in a homogenous state, we embedded the Aβ_1–42 sequence in an unstructured region of Bcl-x_L. Both the N-terminus and the C-terminus of Aβ_1–42 were constrained in the disordered region, whereas the conjunction did not introduce any folding to Aβ_1–42 but maintained the sequence as a monomer in solution. With Bcl-x_L-Aβ₄₂, we developed an enzyme-linked immunosorbent assay to compare the affinity of compounds for monomeric Aβ_1–42. Bcl-x_L-Aβ₄₂ was coated on a microplate and this was followed by incubation with different concentrations of compounds. Compounds binding to Leu17-Val24 of Aβ_1–42 inhibited the interaction between Bcl-x_L-Aβ₄₂ and antibody 4G8. The method can not only reproduce the activities of the reported Aβ_1–42 inhibitors such as dopamine, tannin, and morin but can also differentiate decoy compounds that do not bind to Aβ_1–42. Remarkably, using this method, we discovered a new inhibitor that binds to monomeric Aβ_1–42 and inhibits Aβ_1–42 fibril formation. As the structure of Bcl-x_L-Aβ₄₂ monomer is stable in solution, the assay could be adapted for high-throughput screening with a series of antibodies that bind the different epitopes of Aβ_1–42. In addition, the monomeric form of the Aβ_1–42 sequence in Bcl-x_L-Aβ₄₂ would also facilitate the identification of Aβ_1–42 binding partners by coimmunoprecipitation, cocrystallization, surface plasmon resonance technology, or the assay as described here. Figure Schematic diagram of an enzyme-linked immunosorbent assay to compare the affinity of chemical compounds for β-amyloid peptide in monomer

Keywords: Enzyme-linked immunosorbent assay; β-Amyloid peptide; Binding affinity; Inhibitor; Bcl-x_L

A highly selective and sensitive dopamine and uric acid biosensor fabricated with functionalized ordered mesoporous carbon and hydrophobic ionic liquid by Junping Dong; Yanyan Hu; Shenmin Zhu; Jiaqiang Xu; Yinjuan Xu (pp. 1755-1762).

An ordered mesoporous carbon material functionalized with carboxylic acid groups was synthesized. It was characterized by powder X-ray diffraction, transmission electron microscopy, Fourier transform IR spectroscopy and N₂ adsorption/desorption. Furthermore, this material was used to modify an electrode surface combined with a hydrophobic ionic liquid. The functionalized ordered mesoporous carbon/ionic liquid gel modified electrode shows excellent electrocatalytic performances for the oxidation of dopamine, uric acid and ascorbic acid. The presence of the ionic liquid promotes the electron transfer. Linear responses for dopamine and uric acid were obtained in the ranges of 0.1 to 500 μM and from 0.1 to 100 μM with detection limits of 4.1 and 2.5 nM (signal-to-noise ratio of 3), respectively, under optimum conditions. A quick and sensitive biosensor based on functionalized ordered mesoporous carbon and an ionic liquid has been developed for the first time for the detection of dopamine and uric acid in the presence of a large amount of ascorbic acid. Figure Fig. DPV curves recorded at f-OMC/IL-gel modified electrode in pH 7.0 PBS buffer solution containing 2 mΜ AA, 20 μM UA and different concentrations of DA (from a to h): 0.5, 1, 2.5, 5, 10, 25, 50 and 100 μM.

Keywords: Ordered mesoporous carbon; Ionic liquid; Dopamine; Uric acid; Ascorbic acid

HPLC-UV measurements of metabolites in the supernatant of endothelial cells exposed to oxidative stress by Mehjabin Kathiwala; Andrews Obeng Affum; Anna Brajter-Toth (pp. 1763-1771).

2,8-Dihydroxyadenine (2,8-DHA) was identified by high-performance liquid chromatography with ultraviolet detection as a major metabolite in the supernatant of endothelial cells of the pulmonary artery (PAECs) and aorta (AECs), in addition to hypoxanthine, xanthine, uric acid, and uracil. Under normoxic, hypoxic, and hyperoxic conditions, the concentrations of all the identified metabolites change with time, marking the response of endothelial cells to stress, as a result of changes in cellular metabolism. Thus, the metabolites can serve as stress markers, and their concentrations can indicate the type and the level of cell stress. The results verify that PAECs adapt to survive oxidative stress of hyperoxia. However, AECs can adapt to hypoxia only for a short time and do not survive prolonged hypoxia. The role of the polyamine synthesis pathway in the formation of the unsalvaged adenine, as a possible source of 2,8-DHA, is discussed.

Keywords: HPLC-UV; Endothelial cells; Oxidative stress; 2, 8-Dihydroxyadenine; Uric acid; Xanthine; Hypoxanthine; Uracil

HPLC-UV measurements of metabolites in the supernatant of endothelial cells exposed to oxidative stress by Mehjabin Kathiwala; Andrews Obeng Affum; Anna Brajter-Toth (pp. 1763-1771).

Keywords: HPLC-UV; Endothelial cells; Oxidative stress; 2, 8-Dihydroxyadenine; Uric acid; Xanthine; Hypoxanthine; Uracil

Determination of diketopiperazines of Burkholderia cepacia CF-66 by gas chromatography–mass spectrometry by Jian-Hua Wang; Chun-Shan Quan; Xiao-Hui Qi; Xin Li; Sheng-Di Fan (pp. 1773-1779).

Bacteria communicate with each other by a process termed “quorum sensing” (QS), and diffusible, low-molecular-weight chemicals, called signal molecules, are used as the communication languages. In cell-free Burkholderia cepacia CF-66 culture supernatants, five compounds suspected of being signal molecules were identified. The gene (cepI) related with AHLs synthesis were not detected by polymerase chain reaction (PCR) using specific primers. Gas chromatography–mass spectrometry (GC–MS) revealed that these compounds were not AHLs but the diketopiperazines (DKPs) cyclo(Pro–Phe), cyclo(Pro–Tyr), cyclo(Ala–Val), cyclo(Pro–Leu), and cyclo(Pro–Val), all of which were both d and l-type. Four kinds of DKPs had been isolated from other Gram-negative bacteria, but the other was a novel kind discovered in CF-66, and l-cyclo (Pro–Phe) was quantified by GC–MS. It was found that exogenous DKPs had a negative effect on the candidacidal activity of the culture supernatant extracts.

Keywords: Quorum sensing; Signal molecules; Burkholderia cepacia CF-66; Gas chromatography–mass spectrometry; Polymerase chain reaction; Diketopiperazines

Determination of diketopiperazines of Burkholderia cepacia CF-66 by gas chromatography–mass spectrometry by Jian-Hua Wang; Chun-Shan Quan; Xiao-Hui Qi; Xin Li; Sheng-Di Fan (pp. 1773-1779).

Keywords: Quorum sensing; Signal molecules; Burkholderia cepacia CF-66; Gas chromatography–mass spectrometry; Polymerase chain reaction; Diketopiperazines

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy by Aidan D. Meade; Colin Clarke; Florence Draux; Ganesh D. Sockalingum; Michel Manfait; Fiona M. Lyng; Hugh J. Byrne (pp. 1781-1791).

The in vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) employing fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy’s fixative and methanol–acetic acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level. Figure Chemical preservation of cells for Raman microspectroscopy is shown to be strongly dependent on the cell type and the fixative used, in a variety of cell lines, with formalin fixation show to result in spectral content most comparable to that in the live cell

Keywords: Micro-Raman spectroscopy; Chemical fixation mechanisms; Cell culture; Formalin; Carnoy’s fixative; Methanol–acetic acid; PCA; HCA; k-means clustering

Evaluation of the derivates of phosphorescent Pt-coproporphyrin as intracellular oxygen-sensitive probes by Andreas Fercher; Gelii V. Ponomarev; Dmitri Yashunski; Dmitri Papkovsky (pp. 1793-1803).

Several new derivatives of the phosphorescent Pt(II)-coproporphyrin (PtCP) were evaluated with respect to the sensing of intracellular oxygen by phosphorescence quenching. Despite the more favorable molecular charge compared to PtCP, self-loading into mammalian cells was rather inefficient for all the dyes, while cell loading by facilitated transport using transfection reagents produced promising results. The PtCP-NH₂ derivative, which gave best loading efficiency and S/N ratio, was investigated in detail including the optimisation of loading conditions, studies of sub-cellular localization, cytotoxicity, oxygen sensitivity and long-term signal stability. Being spectrally similar to the macromolecular MitoXpress™ probe currently used in this application, the PtCP-NH₂ demonstrated higher loading efficiency and phosphorescent signals, suitability for several problematic cell lines and a slightly increased lifetime scale for the physiological range (0–200 μM O₂). In physiological experiments with different cell types, mitochondrial uncouplers and inhibitors performed on a time-resolved fluorescence plate reader, this probe produced the anticipated profiles of intracellular oxygen concentration and responses to cell stimulation. Therefore, PtCP-NH₂ represents a convenient probe for the experiments and applications in which monitoring of cellular oxygen levels is required.

Keywords: Cellular oxygen; Phosphorescent oxygen-sensitive probes; Pt-porphyrins; Intracellular probe; Cell metabolism and mitochondrial function

Evaluation of the derivates of phosphorescent Pt-coproporphyrin as intracellular oxygen-sensitive probes by Andreas Fercher; Gelii V. Ponomarev; Dmitri Yashunski; Dmitri Papkovsky (pp. 1793-1803).

Keywords: Cellular oxygen; Phosphorescent oxygen-sensitive probes; Pt-porphyrins; Intracellular probe; Cell metabolism and mitochondrial function

Simultaneous concentration and separation of microorganisms: insulator-based dielectrophoretic approach by Héctor Moncada-Hernández; Blanca H. Lapizco-Encinas (pp. 1805-1816).

Microanalytical methods offer attractive characteristics for rapid microbial detection and concentration. There is a growing interest in the development of microscale separation techniques. Dielectrophoresis (DEP), a nondestructive electrokinetic transport mechanism, is a technique with great potential for microbe manipulation, since it can achieve concentration and separation in a single step. DEP is the movement of particles due to polarization effects in nonuniform electric fields. The majority of the work on dielectrophoretic manipulation of microbes has employed alternating current fields in arrays of microelectrodes, an approach with some disadvantages. An alternative is to employ insulator-based DEP (iDEP), a dielectrophoretic mode where nonuniform fields are produced by employing arrays of insulating structures. This study presents the concentration and fractionation of a mixture of bacteria and yeast cells employing direct current-iDEP in a microchannel containing an array of cylindrical insulating structures. Negative dielectrophoretic trapping of both types of microorganisms was demonstrated, where yeast cells exhibited a stronger response, opening the possibility for dielectrophoretic differentiation. Simultaneous concentration and fractionation of a mixture of both types of cells was carried out analogous to a chromatographic separation, where a dielectropherogram was obtained in less than 2 min by applying an electric field gradient and achieving concentration factors in the order of 50 and 37 times the inlet concentration for Escherichia coli and Saccharomyces cerevisiae cells, respectively. Encouraging results were also obtained employing a sample of water taken from a pond. The findings demonstrated the great potential of iDEP as a rapid and effective technique for intact microorganism concentration and separation. Figure Simultaneous concentration and separation of a mixture of bacteria and yeast in a single step, employing an electric field gradient with insulator-based dielectrophoresis

Keywords: Dielectrophoresis; Electrokinetic; Electroosmotic flow; Microfluidics; Microorganisms

Simultaneous concentration and separation of microorganisms: insulator-based dielectrophoretic approach by Héctor Moncada-Hernández; Blanca H. Lapizco-Encinas (pp. 1805-1816).

Keywords: Dielectrophoresis; Electrokinetic; Electroosmotic flow; Microfluidics; Microorganisms

New insights into the formation of volatile compounds in mainstream cigarette smoke by C. Liu; S. Feng; J. van Heemst; K. G. McAdam (pp. 1817-1830).

A sampling system has been set up to monitor a group of volatile smoke analytes (nitric oxide, acetaldehyde, acetone, benzene, toluene, 1,3 butadiene, isoprene and carbon dioxide) from mainstream cigarette smoke on a puff-resolved basis. The system was able to record gas evolution profiles during puffing and interpuff periods without interruption (e.g. taking clearing puffs). Gas phase smoke analytes were sampled as close to the mouth end of the cigarette filter as possible in order to minimise any dead volume effect. The results revealed that, for some volatile species, a significant fraction (e.g. up to 30% for benzene) in the cigarette mainstream smoke had been generated during the preceding smoulder period. These species were trapped or absorbed within the cigarette rod and then subsequently eluted during the puff. The identification of the two sources of the mainstream smoke, a smouldering source and a puffing source, has not been reported before. The observation contributes to the fundamental knowledge of the cigarette smoke formation and may have implications on wider smoke chemistry and associated effects. Figure A cross-sectional schematic of a burning cigarette, illustrating the main chemical and physical processes involved in the smoke formation

Keywords: Real-time sampling; Cigarette; Smoke chemistry; Volatile species

New insights into the formation of volatile compounds in mainstream cigarette smoke by C. Liu; S. Feng; J. van Heemst; K. G. McAdam (pp. 1817-1830).

Keywords: Real-time sampling; Cigarette; Smoke chemistry; Volatile species

Multiplex real-time PCR using SYBR^® GreenER™ for the detection of DNA allergens in food by Simona Pafundo; Mariolina Gullì; Nelson Marmiroli (pp. 1831-1839).

We describe the development of a six-target real-time multiplex PCR assay with the SYBR® GreenER™ fluorescent dye, targeted to genes encoding for allergenic proteins commonly present in many processed food products (patent application pending). The assay was successfully trialled on reconstructed food matrices and on a range of commercial foodstuffs, and is proposed as a ready-to-use analytical tool for food manufacturers to detect the presence or confirm the absence of sequences encoding for important allergenic proteins of plant origin. Figure Multiplex real-time PCR for the detection of DNA allergens in food using ABI Prism® 7000 Sequence detection system

Keywords: Allergen; Multiplex real-time PCR; Traceability; Food; Melting curve

Multiplex real-time PCR using SYBR^® GreenER™ for the detection of DNA allergens in food by Simona Pafundo; Mariolina Gullì; Nelson Marmiroli (pp. 1831-1839).

Keywords: Allergen; Multiplex real-time PCR; Traceability; Food; Melting curve

Development and validation of an analytical method by LC-MS/MS for the quantification of estrogens in sewage sludge by Virginie Gabet-Giraud; Cécile Miege; Bernard Herbreteau; Guillermina Hernandez-Raquet; Marina Coquery (pp. 1841-1851).

A sensitive method for the simultaneous analysis of five estrogens in sewage sludge was developed. The extraction and purification steps were optimized and the matrix effects were evaluated. The chromatographic gradient was optimized to limit matrix effects and the analysis step was performed by LC-MS/MS. The method consists of an ASE® extraction with a solvent mixture water/methanol 80/20 v/v at 100 °C followed by two consecutive purifications on Oasis HLB® and florisil cartridges. A thorough validation of the developed method was performed. Recoveries determined at two different spiking levels ranged between 86% and 126% depending on the molecule. Repeatability was evaluated on five replicates of the same sludge sample spiked at two different levels and measuring native estrogens in triplicates of 12 sludge samples. Relative standard deviations obtained a range of between 2% and 27%. Reproducibility was also studied by analyzing the same sludge on four different days: the relative standard deviation ranged between 14% and 20% for E1, βE2 and E3. For αE2, poor reproducibility (68%) was observed but it was linked to the very low quantity of αE2 present in the sludge sample and not to the method performance. The specificity of the method was evaluated on various sludge samples spiked at different spiking levels showing that performances of the proposed method were not modified by matrix effects. Finally, sensitivity of the method was evaluated taking into account both instrumental sensitivity and matrices; the estimated limits of quantification were around 1 ng/g for E1, between 2 and 4 ng/g for αE2, βE2, and E3 and around 5 ng/g for EE2.

Keywords: Estrogens; LC-MSMS analysis; Sewage sludge; Matrix effects; Wastewater treatment plant; Method validation

Stir-bar-sorptive extraction and liquid desorption combined with large-volume injection gas chromatography–mass spectrometry for ultra-trace analysis of musk compounds in environmental water matrices by Ana Rita M. Silva; J. M. F. Nogueira (pp. 1853-1862).

Stir-bar-sorptive extraction with liquid desorption followed by large-volume injection and capillary gas chromatography coupled to mass spectrometry in selected ion monitoring acquisition mode (SBSE–LD/LVI-GC–MS(SIM)) has been developed to monitor ultra-traces of four musks (celestolide (ADBI), galaxolide (HHCB), tonalide (AHTN) and musk ketone (MK)) in environmental water matrices. Instrumental calibration (LVI-GC–MS(SIM)) and experimental conditions that could affect the SBSE-LD efficiency are discussed. Assays performed on 30-mL water samples spiked at 200 ng L⁻¹ under optimized experimental conditions yielded recoveries ranging from 83.7 ± 8.1% (MK) to 107.6 ± 10.8% (HHCB). Furthermore, the experimental data were in very good agreement with predicted theoretical equilibria described by octanol–water partition coefficients (K _PDMS/W ≈ K _O/W). The methodology also showed excellent linear dynamic ranges for the four musks studied, with correlation coefficients higher than 0.9961, limits of detection and quantification between 12 and 19 ng L⁻¹ and between 41 and 62 ng L⁻¹, respectively, and suitable precision (< 20%). Application of this method for analysis of the musks in real water matrices such as tap, river, sea, and urban wastewater samples resulted in convenient selectivity, high sensitivity and accuracy using the standard addition methodology. The proposed method (SBSE–LD/LVI-GC–MS(SIM)) was shown to be feasible and sensitive, with a low-sample volume requirement, for determination of musk compounds in environmental water matrices at the ultra-trace level, overcoming several disadvantages presented by other sample-preparation techniques.

Keywords: Stir-bar-sorptive extraction; Large-volume injection; GC–MS(SIM); Musk compounds; Environmental water matrices; Trace analysis

A fast, simple, and reliable hydrophilic interaction liquid chromatography method for the determination of ascorbic and isoascorbic acids by Ana I. R. N. A. Barros; Ana P. Silva; Berta Gonçalves; Fernando M. Nunes (pp. 1863-1875).

A reliable method for the determination of total vitamin C must be able to resolve ascorbic acid (AA) and the epimeric isoascorbic acid (IAA) and determine the sum of AA and its oxidized form dehydroascorbic acid. AA and IAA are polar molecules with a low retention time in conventional reversed phase systems, and hence of difficult resolution. Hydrophilic interaction chromatography using a TSKgel Amide-80 stationary phase with isocratic elution was successful in resolving the two epimers. The column was compatible with injections of high concentrations of metaphosphoric acid, tris(2-carboxyethyl)-phosphine, and EDTA without drift of baseline and retention time. Total AA and IAA were extracted, stabilized, and reduced in one step at 40 °C, using 5% m-phosphoric acid, 2 mM of EDTA, and 2 mM of tris(2-carboxyethyl)-phosphine as reducing agent. This simple, fast, and robust hydrophilic interaction chromatography-DAD method was applied for the analysis of food products namely fruit juices, chestnut, and ham and also in pharmaceutical and multivitamin tablets. Method validation was performed on the food products, including parameters of precision, accuracy, linearity, limit of detection, and quantification (LOQ). The absence of matrix interferences was assessed by the standard addition method and Youden calibration. The method was fast, accurate, and precise with a LOQ_AA of 1.5 mg/L and LOQ_IAA of 3.7 mg/L. The simple experimental procedure, completed in 1 h, the possibility of using IAA as an internal standard, and low probability of artifacts are the major advantages of the proposed method for the routine determination of these compounds in a large number of samples. Figure Hydrophilic interaction liquid chromatography separation of isoascorbic and ascorbic acids.

Keywords: Hydrophilic interaction chromatography; Vitamin C; Ascorbic acid; Isoascorbic acid

Isotope dilution gas chromatography/mass spectrometry method for determination of pyrethroids in apple juice by Siu-kay Wong; Kwok-chiu Yu; Chi-ho Lam (pp. 1877-1884).

This paper presents the development of a highly precise and accurate analytical method for the determination of three matrix-bound pyrethroids, namely, cypermethrin, permethrin, and bifenthrin, using an isotope dilution gas chromatography/mass spectrometry technique. Identification of the analytes was confirmed under selective ion monitoring mode by the presence of two dominant ion fragments within specific time windows and matching of relative ion intensities of the ions concerned in samples and calibration standards. Quantitation was based on the measurement of concentration ratios of the natural and isotope analogues in the sample and calibration blends. Intraday and interday repeatabilities of replicate analyses of the pyethroids in an apple juice sample were below 0.5%. The expanded relative uncertainty ranged from 3 to 6%, which was significantly lower than the range obtained using internal or external calibration methods. As a labeled analogue is not available for bifenthrin, bifenthrin was determined using labeled cis-permethrin as the internal standard. The results were counterchecked by a gas chromatography-electron capture detection technique using PCB 209 as the internal standard. The method developed was applied to a recent pilot study organized by CCQM and the results were consistent with those of other participants.

Keywords: Pyrethroids; Isotope dilution mass spectrometry; Cypermethrin; cis-Permethrin; Bifenthrin

Isotope dilution gas chromatography/mass spectrometry method for determination of pyrethroids in apple juice by Siu-kay Wong; Kwok-chiu Yu; Chi-ho Lam (pp. 1877-1884).

Keywords: Pyrethroids; Isotope dilution mass spectrometry; Cypermethrin; cis-Permethrin; Bifenthrin

Non-destructive mapping of dampness and salts in degraded wall paintings in hypogeous buildings: the case of St. Clement at mass fresco in St. Clement Basilica, Rome by Valeria Di Tullio; Noemi Proietti; Marco Gobbino; Donatella Capitani; Roberto Olmi; Saverio Priori; Cristiano Riminesi; Elisabetta Giani (pp. 1885-1896).

As is well known, the deterioration of wall paintings due to the capillary rise of water through the walls is a very widespread problem. In this paper, a study of microclimate monitoring, unilateral nuclear magnetic resonance (NMR), and evanescent-field dielectrometry (EFD) was applied to map non-destructively, in situ, and in a quantitative way the distribution of the moisture in an ancient deteriorated wall painting of the eleventh century. Both unilateral NMR and EFD are quite new, fully portable, and non-destructive techniques, and their combination is absolutely new. The approach reported here is proposed as a new analytical protocol to afford the problem of mapping, non-destructively, the moisture in a deteriorated wall painting in a hypogeous building such as that of the second level of St. Clement Basilica, Rome (Italy), where the use of IR thermography is impaired due to the environmental conditions, and the gravimetric tests are forbidden due to the preciousness of the artifact. The moisture distribution was mapped at different depths, from the very first layers of the painted film to a depth of 2 cm. It has also been shown how the map obtained in the first layers of the artwork is affected by the environmental conditions typical of a hypogeous building, whereas the maps obtained at higher depths are representative of the moisture due to the capillary rise of water from the ground. The quantitative analysis of the moisture was performed by calibrating NMR and EFD signals with purposely prepared specimens. This study may be applied before and after performing any intervention aimed at restoring and improving the state of conservation of this type of artwork and reducing the dampness or extracting salts (driven by the variation of moisture content) and monitoring the effectiveness of the performed interventions during the time. This protocol is applicable to any type of porous material. Figure Map of the distribution of moisture content obtained by unilateral NMR at about 0.5 cm of depth (left) and by EFD at about 2 cm of depth (right)

Keywords: Unilateral NMR; Evanescent-field dielectrometry; Rising damp; Wall paintings; Porous materials

Development of a sequential injection–square wave voltammetry method for determination of paraquat in water samples employing the hanging mercury drop electrode by Luciana B. O. dos Santos; Carlos M. C. Infante; Jorge C. Masini (pp. 1897-1903).

This work describes the development and optimization of a sequential injection method to automate the determination of paraquat by square-wave voltammetry employing a hanging mercury drop electrode. Automation by sequential injection enhanced the sampling throughput, improving the sensitivity and precision of the measurements as a consequence of the highly reproducible and efficient conditions of mass transport of the analyte toward the electrode surface. For instance, 212 analyses can be made per hour if the sample/standard solution is prepared off-line and the sequential injection system is used just to inject the solution towards the flow cell. In-line sample conditioning reduces the sampling frequency to 44 h⁻¹. Experiments were performed in 0.10 M NaCl, which was the carrier solution, using a frequency of 200 Hz, a pulse height of 25 mV, a potential step of 2 mV, and a flow rate of 100 µL s⁻¹. For a concentration range between 0.010 and 0.25 mg L⁻¹, the current (i _p, µA) read at the potential corresponding to the peak maximum fitted the following linear equation with the paraquat concentration (mg L⁻¹): i _p = (−20.5 ± 0.3)C _paraquat − (0.02 ± 0.03). The limits of detection and quantification were 2.0 and 7.0 µg L⁻¹, respectively. The accuracy of the method was evaluated by recovery studies using spiked water samples that were also analyzed by molecular absorption spectrophotometry after reduction of paraquat with sodium dithionite in an alkaline medium. No evidence of statistically significant differences between the two methods was observed at the 95% confidence level. Figure Sequential injection manifold to perform sequential injection square wave voltammetry

Keywords: Paraquat; Sequential injection analysis; Environmental analysis; Water

High-resolution investigations of ripple structures formed by femtosecond laser irradiation of silicon by M. Schade; O. Varlamova; J. Reif; H. Blumtritt; W. Erfurth; H. S. Leipner (pp. 1905-1911).

We report on the structural investigation of self-organized periodic microstructures (ripples) generated in Si(100) targets after multishot irradiation by approximately 100-fs to 800-nm laser pulses at intensities near the single shot ablation threshold. Inspection by surface sensitive microscopy, e.g., atomic force microscopy (AFM) or scanning electron microscopy (SEM), and conventional and high-resolution transmission electron microscopy reveal complex structural modifications upon interaction with the laser: even well outside the ablated area, the target surface exhibits fine ripple-like undulations, consisting of alternating crystalline and amorphous silicon. Inside the heavily modified area, amorphous silicon is found only in the valleys but not on the crests which, instead, consist of highly distorted crystalline phases, rich in defects.

Keywords: Laser ablation; Ripple structure; Electron microscopy; Amorphous silicon

High-resolution investigations of ripple structures formed by femtosecond laser irradiation of silicon by M. Schade; O. Varlamova; J. Reif; H. Blumtritt; W. Erfurth; H. S. Leipner (pp. 1905-1911).

Keywords: Laser ablation; Ripple structure; Electron microscopy; Amorphous silicon

Atomic layer deposition (ALD) as a coating tool for reinforcing fibers by A. K. Roy; W. Baumann; I. König; G. Baumann; S. Schulze; M. Hietschold; T. Mäder; D. J. Nestler; B. Wielage; W. A. Goedel (pp. 1913-1919).

Layers of alumina were deposited on to bundled carbon fibers in an atomic layer deposition (ALD) process via sequential exposure to vapors of aluminium chloride and water, respectively. Scanning electron microscopic (SEM) images of the coated fibers revealed that each individual fiber within a bundle was coated evenly and separately, fibers are not bridged by the coating. SEM and transmission electron microscopic (TEM) images indicate that the coating was uniform and conformal with good adhesion to the fiber surface. Average deposition rate, measured from SEM images, was 0.06 nm per cycle at 500 °C. SEM also revealed that at deposition temperatures of 500 °C few of the fibers were damaged. At temperatures of 300 °C, no damaged fibers were observed, the average deposition rate decreased down to 0.033 nm per cycle. Oxidation resistance of the alumina-coated fibers was characterized by thermogravimetric analysis (TGA). The alumina coating improved oxidation resistance of the carbon fiber significantly. Oxidation onset temperature was 600 °C for fibers coated with a 45 nm thick alumina. Uncoated fibers, on the other hand, started to oxidize at temperatures as low as 250 °C. Figure Layers of alumina were deposited onto bundled carbon fibers in an atomic layer deposition (ALD) process via repeated cycles of sequential exposure to vapors of aluminum chloride and water. A 45 nm thick coating was sufficient to improve the oxidation resitance of the fibres significantly.

Keywords: Atomic layer deposition; Carbon fiber; Oxidation resistance; Reinforcing fiber; Alumina coating; Aluminium chloride precursor

Coupled stochastic resonance to improve chromatography determinations by Shaofei Xie; Bingren Xiang; Haishan Deng; Jingfang Wu (pp. 1921-1927).

Traditional bistable stochastic resonance has been demonstrated as an effective tool to detect the weak signal in a strong noise background. To achieve a better signal-to-noise ratio for the output signal, a coupled stochastic resonance was developed by nonlinearly coupling two double-well potential systems. The response characteristics of coupled stochastic resonance subjected to analytical signals have been investigated and compared with those of bistable stochastic resonance. The improvement of chromatographic determination with the proposed coupled stochastic resonance was validated by both simulated signals and chromatographic signals. The weak signals from both simulated data and plasma samples with different concentrations were all amplified significantly and the quantitative relationship between different concentrations and responses was kept well. It is reasonable to believe that coupled stochastic resonance could play an important role in applications where quantitative determination of low-concentration samples is crucial. Figure The coupled stochastic resonance procedure

Keywords: Coupled stochastic resonance; Bistable stochastic resonance; Weak signal; Signal-to-noise ratio; Chromatographic signal

Coupled stochastic resonance to improve chromatography determinations by Shaofei Xie; Bingren Xiang; Haishan Deng; Jingfang Wu (pp. 1921-1927).

Keywords: Coupled stochastic resonance; Bistable stochastic resonance; Weak signal; Signal-to-noise ratio; Chromatographic signal

Continuous solid-phase extraction method for the determination of amines in human urine following on-line microwave-assisted acid hydrolysis by Beatriz Jurado-Sánchez; Evaristo Ballesteros; Mercedes Gallego (pp. 1929-1937).

Humans are exposed to endogenous or exogenous formation of aromatic amines (AAs) and N-nitrosamines (NAms), which are considered to be potent carcinogens. The objective of this study was to monitor AAs and NAms in human urine to obtain a way to assess exposure. However, while NAms can be directly detected in urine samples, AAs require hydrolysis to convert their conjugates into free amines. A semiautomatic flow-base method is proposed for the simultaneous determination of aliphatic and aromatic NAms, anilines and chloroanilines in human urine in one analytical run. Conjugated AAs are released from urine by on-line microwave-assisted acid hydrolysis without degradation of NAms; all amines were then preconcentrated using solid-phase extraction. Separation/determinations are carried out by gas chromatography and mass spectrometry operating in selected ion monitoring mode. The method is fast (∼15 min for 25 mL of sample) and provides low limits of detection (from 2 to 26 ng/L) with good precision (relative standard deviation within and between days less than 7%). Finally, the proposed method was successfully applied to check AAs and NAms in the human urine of exposed and unexposed researchers. The kinetics of amine excretion in the urine of the researchers exposed is calculated after termination of the exposure and shows half-life times between 1.3 and 2.1 h, and that the dosage absorbed was eliminated within 6 h after exposure. Figure On-line microwave-assisted acid hydrolysis–solid-phase extraction and gas chromatography–mass spectrometry were combined to monitor aromatic amines and N-nitrosamines in the urine of exposed researchers

Keywords: Solid-phase extraction; Microwave-assisted hydrolysis; Mass spectrometry; N-Nitrosamines; Amines; Urine

Detection of hazelnuts and almonds using commercial ELISA test kits by Eric A. E. Garber; Jesse Perry (pp. 1939-1945).

Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 μg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits. Figure Almonds and hazelnuts

Keywords: Almond; Hazelnut; ELISA; Food allergen; Foods/beverages; Immunoassays/ELISA; Bioanalytical methods

Detection of hazelnuts and almonds using commercial ELISA test kits by Eric A. E. Garber; Jesse Perry (pp. 1939-1945).

Keywords: Almond; Hazelnut; ELISA; Food allergen; Foods/beverages; Immunoassays/ELISA; Bioanalytical methods

Letter to the Editor: ESI-MS–MS library of 1,253 compounds for application in forensic and clinical toxicology by François-Ludovic Sauvage; Pierre Marquet (pp. 1947-1947).

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Analytical and Bioanalytical Chemistry (v.396, #5)