Check out our New Publishers' Select for Free Articles

Analytical and Bioanalytical Chemistry (v.401, #1)

Bernd W. Wenclawiak, Michael Koch, Evsevios Hadjicostas (Eds): Quality assurance in analytical chemistry. Training and teaching, 2nd ed. by Jürgen W. Einax (pp. 1-2).

Quantitative single-cell gene expression measurements of multiple genes in response to hypoxia treatment by Jia Zeng; Jiangxin Wang; Weimin Gao; Aida Mohammadreza; Laimonas Kelbauskas; Weiwen Zhang; Roger H. Johnson; Deirdre R. Meldrum (pp. 3-13).

Cell-to-cell heterogeneity in gene transcription plays a central role in a variety of vital cell processes. To quantify gene expression heterogeneity patterns among cells and to determine their biological significance, methods to measure gene expression levels at the single-cell level are highly needed. We report an experimental technique based on the DNA-intercalating fluorescent dye SYBR green for quantitative expression level analysis of up to ten selected genes in single mammalian cells. The method features a two-step procedure consisting of a step to isolate RNA from a single mammalian cell, synthesize cDNA from it, and a qPCR step. We applied the method to cell populations exposed to hypoxia, quantifying expression levels of seven different genes spanning a wide dynamic range of expression in randomly picked single cells. In the experiment, 72 single Barrett’s esophageal epithelial (CP-A) cells, 36 grown under normal physiological conditions (controls) and 36 exposed to hypoxia for 30 min, were randomly collected and used for measuring the expression levels of 28S rRNA, PRKAA1, GAPDH, Angptl4, MT3, PTGES, and VEGFA genes. The results demonstrate that the method is sensitive enough to measure alterations in gene expression at the single-cell level, clearly showing heterogeneity within a cell population. We present technical details of the method development and implementation, and experimental results obtained by use of the procedure. We expect the advantages of this technique will facilitate further developments and advances in the field of single-cell gene expression profiling on a nanotechnological scale, and eventually as a tool for future point-of-care medical applications.

Keywords: Single-cell; Gene expression; Hypoxia treatment

Focus on MALDI imaging by Olivier Laprévote (pp. 15-16).

is currently Professor of Toxicology at the University Paris Descartes (Faculty of Pharmacy) and at Lariboisière Hospital in Paris. He was Group Leader of the Mass Spectrometry Laboratory of the Institute for Natural Compounds Chemistry at Gif-sur-Yvette (France) until 2008. His scientific interests lie in the development of original mass spectrometric methods for characterization of natural molecules in complex biological matrices. Among his past research activities, mass spectrometry imaging took a prominent part, with a particular interest being in biomedical applications. O. Laprévote is a member of the Advisory Board of Analytical and Bioanalytical Chemistry.

MALDI tissue imaging: from biomarker discovery to clinical applications by Lisa H. Cazares; Dean A. Troyer; Binghe Wang; Richard R. Drake; O. John Semmes (pp. 17-27).

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for the generation of multidimensional spatial expression maps of biomolecules directly from a tissue section. From a clinical proteomics perspective, this method correlates molecular detail to histopathological changes found in patient-derived tissues, enhancing the ability to identify candidates for disease biomarkers. The unbiased analysis and spatial mapping of a variety of molecules directly from clinical tissue sections can be achieved through this method. Conversely, targeted IMS, by the incorporation of laser-reactive molecular tags onto antibodies, aptamers, and other affinity molecules, enables analysis of specific molecules or a class of molecules. In addition to exploring tissue during biomarker discovery, the integration of MALDI-IMS methods into existing clinical pathology laboratory practices could prove beneficial to diagnostics. Querying tissue for the expression of specific biomarkers in a biopsy is a critical component in clinical decision-making and such markers are a major goal of translational research. An important challenge in cancer diagnostics will be to assay multiple parameters in a single slide when tissue quantities are limited. The development of multiplexed assays that maximize the yield of information from a small biopsy will help meet a critical challenge to current biomarker research. This review focuses on the use of MALDI-IMS in biomarker discovery and its potential as a clinical diagnostic tool with specific reference to our application of this technology to prostate cancer.

Keywords: Matrix-assisted laser desorption ionization tissue imaging; Biomarker discovery; Cancer

Matrix-assisted laser desorption ionization imaging mass spectrometry in lipidomics by J. A. Fernández; B. Ochoa; O. Fresnedo; M. T. Giralt; R. Rodríguez-Puertas (pp. 29-51).

The relevant structural, energetics, and regulatory roles of lipids are universally acknowledged. However, the high variability of lipid species and the large differences in concentrations make unraveling the role played by the different species in metabolism a titanic task. A recently developed technique, known as imaging mass spectrometry, may shed some light on the field, as it enables precise information to be obtained on the location of lipids in tissues. A review of the state of the art of the technique is presented in this manuscript, including detailed analysis of sample-preparation steps, data handling, and the identification of the species mapped so far.

Keywords: Lipidomics; Lipid molecular species; Mass spectrometry; Imaging mass spectrometry; Time of flight

Imaging of complex sulfatides SM3 and SB1a in mouse kidney using MALDI-TOF/TOF mass spectrometry by Christian Marsching; Matthias Eckhardt; Hermann-Josef Gröne; Roger Sandhoff; Carsten Hopf (pp. 53-64).

Sulfatides, a class of acidic glycosphingolipids, are highly expressed in mammalian myelin and in kidney, where they are thought to stabilize neuronal structures and signaling and to influence osmotic stability of renal cells, respectively. Recently, 9-aminoacridine (9-AA) has been introduced as a negative ion matrix that displays high selectivity for low complexity galactosylceramid-I³-sulfate sulfatides and that is suitable for quantitative analysis by matrix-assisted desorption/ionization (MALDI) mass spectrometry (MS). Analyzing acidic fractions of lipid extracts and cryosections from kidneys of wild type and arylsulfatase A-deficient (ASA −/−) mice, we demonstrate that 9-AA also enables sensitive on-target analysis as well as imaging of complex lactosylceramide-II³-sulfate and gangliotetraosylceramide-II³, IV³ bis-sulfate sulfatides by MALDI-TOF/TOF MS. Utilizing the MALDI imaging MS technique, we show differential localization in mouse kidney of (1) sulfatides with identical ceramide anchors, but different glycan-sulfate head groups but also of (2) sulfatides with identical head groups but with different acyl- or sphingoid base moieties. A comparison of MALDI images of renal sulfatides from control and sulfatide storing arylsulfatase A-deficient (ASA −/−) mice revealed relative expression differences, very likely reflecting differences in sulfatide turnover of the various renal cell types. These results establish MALDI imaging MS with 9-AA matrix as a label-free method for spatially resolved ex vivo investigation of the relative turnover of sulfatides in animal models of human glycosphingolipid storage disease. Figure (left) MALDI IMS using 9-Amino acridine (9-AA) matrix reveals differential localization of kidney sulfatides containing different glycan headgroups. (right) Furthermore, in mice lacking aryl sulfatase A (ASA -/-) differential distribution of sulfatides with variations in their sphingoid base are observed

Keywords: MALDI-MS Imaging; Sulfatide; Kidney; Arylsulfatase A; 9-Amino acridine matrix

Mass spectrometry imaging with high resolution in mass and space (HR² MSI) for reliable investigation of drug compound distributions on the cellular level by Andreas Römpp; Sabine Guenther; Zoltan Takats; Bernhard Spengler (pp. 65-73).

Mass spectrometry (MS) imaging is a versatile method to analyze the spatial distribution of analytes in tissue sections. It provides unique features for the analysis of drug compounds in pharmacokinetic studies such as label-free detection and differentiation of compounds and metabolites. We have recently introduced a MS imaging method that combines high mass resolution and high spatial resolution in a single experiment, hence termed HR² MS imaging. In the present study, we applied this method to analyze the spatial distribution of the anti-cancer drugs imatinib and ifosfamide in individual mouse organs. The whole kidney of an animal dosed with imatinib was measured at 35 μm spatial resolution. Imatinib showed a well-defined distribution in the outer stripe of the outer medulla. This area was analyzed in more detail at 10 μm step size, which constitutes a tenfold increase in effective spatial resolution compared to previous studies of drug compounds. In parallel, ion images of phospholipids and heme were used to characterize the histological features of the tissue section and showed excellent agreement with histological staining of the kidney after MS imaging. Ifosfamide was analyzed in mouse kidney at 20 μm step size and was found to be accumulated in the inner medulla region. The identity of imatinib and ifosfamide was confirmed by on-tissue MS/MS measurements. All measurements including mass spectra from 10 μm pixels featured accurate mass (≤2 ppm root mean square) and mass resolving power of R = 30,000. Selected ion images were generated with a bin size of ∆m/z = 0.01 ensuring highly specific information. The ability of the method to cover larger areas was demonstrated by imaging a compound in the intestinal tract of a rat whole-body tissue section at 200 μm step size. The described method represents a major improvement in terms of spatial resolution and specificity for the analysis of drug compounds in tissue sections. Figure Mass spectrometry imaging of drug compounds in biological tissue acquired with high resolution in space and mass reveals deep information on biochemical and biomedical mechanisms

Keywords: Mass spectrometry imaging; Drug compounds; Accurate mass; High-resolution mass spectrometry

MALDI imaging mass spectrometry of lipids by adding lithium salts to the matrix solution by Christopher D. Cerruti; David Touboul; Vincent Guérineau; Vanessa W. Petit; Olivier Laprévote; Alain Brunelle (pp. 75-87).

Mass spectrometry imaging of lipids using MALDI–TOF/TOF mass spectrometers is of growing interest for chemical mapping of organic compounds at the surface of tissue sections. Many efforts have been devoted to the best matrix choice and deposition technique. Nevertheless, the identification of lipid species desorbed from tissue sections remains problematic. It is now well-known that protonated, sodium- and potassium-cationized lipids are detected from biological samples, thus complicating the data analysis. A new sample preparation method is proposed, involving the use of lithium salts in the matrix solution in order to simplify the mass spectra with only lithium-cationized molecules instead of a mixture of various cationized species. Five different lithium salts were tested. Among them, lithium trifluoroacetate and lithium iodide merged the different lipid adducts into one single lithium-cationized species. An optimized sample preparation protocol demonstrated that the lithium trifluoroacetate salt slightly increased desorption of phosphatidylcholines. Mass spectrometry images acquired on rat brain tissue sections by adding lithium trifluoroacetate showed the best results in terms of image contrast. Moreover, more structurally relevant fragments were generated by tandem mass spectrometry when analyzing lithium-cationized species.

Keywords: Mass spectrometry imaging; MALDI; Lithium salt; Lipid; Rat brain

Distribution of lipids in human brain by Antonio Veloso; Roberto Fernández; Egoitz Astigarraga; Gabriel Barreda-Gómez; Iván Manuel; M. Teresa Giralt; Isidro Ferrer; Begoña Ochoa; Rafael Rodríguez-Puertas; José A. Fernández (pp. 89-101).

The enormous abundance of lipid molecules in the central nervous system (CNS) suggests that their role is not limited to be structural and energetic components of cells. Over the last decades, some lipids in the CNS have been identified as intracellular signalers, while others are known to act as neuromodulators of neurotransmission through binding to specific receptors. Neurotransmitters of lipidic nature, currently known as neurolipids, are synthesized during the metabolism of phospholipid precursors present in cell membranes. Therefore, the anatomical identification of each of the different lipid species in human CNS by imaging mass spectrometry (IMS), in association with other biochemical techniques with spatial resolution, can increase our knowledge on the precise metabolic routes that synthesize these neurolipids and their localization. The present study shows the lipid distribution obtained by MALDI-TOF IMS in human frontal cortex, hippocampus, and striatal area, together with functional autoradiography of cannabinoid and LPA receptors. The combined application of these methods to postmortem human brain samples may be envisioned as critical to further understand neurological diseases, in general, and particularly, the neurodegeneration that accompanies Alzheimer’s disease.

Keywords: Cannabinoid; Lysophosphatidic acid; MALDI-TOF; Mercaptobenzothiazole; Lipidomics; Imaging mass spectrometry

Lipid analysis of flat-mounted eye tissue by imaging mass spectrometry with identification of contaminants in preservation by Timothy J. Garrett; Robert F. Menger; William W. Dawson; Richard A. Yost (pp. 103-113).

Matrix-assisted laser desorption/ionization imaging mass spectrometry was used to analyze donor eye tissue specimens for phospholipid content to evaluate lipid distribution. Phosphatidylcholines and sphingomyelins were detected in the positive ion mode using 2,5-dihydroxybenzoic acid as the matrix. During this study, unknown ion signals in the lower m/z region (less than m/z 400) were detected, mainly in the far periphery of human flat-mounted tissue but not in age-matched rhesus monkey tissue prepared in a similar manner. The unknown ion signals occurred at m/z 304, 332, 360, and 388. These ions were subjected to tandem mass spectrometry directly from the tissue sample, and exact mass measurements of extracts were prepared for further identification. These ions were identified as alkyl dimethylbenzylammonium surfactants (benzalkonium chlorides (BACs)). The classification of these species was verified by comparing an eye tissue extract to an over-the-counter eye-care product containing BACs.

Keywords: Imaging mass spectrometry; MALDI; Benzalkonium chloride; Phospholipids; Macula

MALDI-MS imaging of lipids in ex vivo human skin by Philippa J. Hart; Simona Francese; Emmanuelle Claude; M. Nicola Woodroofe; Malcolm R Clench (pp. 115-125).

Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 μm, using a MALDI quadrupole time-of-flight Q-Star Pulsar-i ^TM (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMS^TM system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations.

Keywords: Matrix-assisted laser desorption ionisation mass spectrometry imaging; Matrix-assisted laser desorption ionisation; Mass spectrometry imaging; Lipids; Skin

MALDI-TOF imaging mass spectrometry of artifacts in “dried droplet” polymer samples by Steffen Weidner; Patrick Knappe; Ulrich Panne (pp. 127-134).

Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) imaging of polystyrenes with various molecular masses was applied to study spatial molecular mass distribution of polymers in sample spots prepared by the “dried droplet” method. When different solvents and target surfaces were examined, a segregation of single homologous polymers was observed depending upon the evaporation rate of the solvent. For the observed patterns left by the evaporating droplet, a hypothesis is offered taking into account different hydrodynamic interactions and diffusion. The results illustrate that spot preparation using the conventionally “dried droplet” method is prone to artifacts and should be avoided for reliable and reproducible MALDI mass spectrometry experiments with regards to the determination of molecular masses and mass distributions. Figure The homogeneity of MALDI spots strongly depends on several parameters. MALDI-TOF imaging was applied to investigate the local distribution of polymers in sample spots prepared by the ‘dried droplet’ method. A solvent depending segregation of single polymer homologs was found and a possible explanation based on diffusion effects while solvent evaporation was presented and discussed. The results clearly suggest avoiding spot preparation using the conventionally ‘dried droplet’ method for MS experiments with regard to the determination of molecular masses and mass distributions. Optical image (left) and ion intensity distribution of PS 45 (middle) and PS 55 silver adduct ions prepared using the ‘dried droplet’ sample preparation (DMAc, retinoic acid)

Keywords: MALDI; Mass spectrometry imaging; Polymer; Droplet

MALDI mass spectrometry based molecular phenotyping of CNS glial cells for prediction in mammalian brain tissue by Jörg Hanrieder; Grzegorz Wicher; Jonas Bergquist; Malin Andersson; Åsa Fex-Svenningsen (pp. 135-147).

The development of powerful analytical techniques for specific molecular characterization of neural cell types is of central relevance in neuroscience research for elucidating cellular functions in the central nervous system (CNS). This study examines the use of differential protein expression profiling of mammalian neural cells using direct analysis by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-MS analysis is rapid, sensitive, robust, and specific for large biomolecules in complex matrices. Here, we describe a newly developed and straightforward methodology for direct characterization of rodent CNS glial cells using MALDI-MS-based intact cell mass spectrometry (ICMS). This molecular phenotyping approach enables monitoring of cell growth stages, (stem) cell differentiation, as well as probing cellular responses towards different stimulations. Glial cells were separated into pure astroglial, microglial, and oligodendroglial cell cultures. The intact cell suspensions were then analyzed directly by MALDI-TOF-MS, resulting in characteristic mass spectra profiles that discriminated glial cell types using principal component analysis. Complementary proteomic experiments revealed the identity of these signature proteins that were predominantly expressed in the different glial cell types, including histone H4 for oligodendrocytes and S100-A10 for astrocytes. MALDI imaging MS was performed, and signature masses were employed as molecular tracers for prediction of oligodendroglial and astroglial localization in brain tissue. The different cell type specific protein distributions in tissue were validated using immunohistochemistry. ICMS of intact neuroglia is a simple and straightforward approach for characterization and discrimination of different cell types with molecular specificity.

Keywords: Intact cell mass spectrometry (ICMS); MALDI-TOF-MS; Imaging mass spectrometry (IMS); Glial cells

Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer by David Bonnel; Rémi Longuespee; Julien Franck; Morad Roudbaraki; Pierre Gosset; Robert Day; Michel Salzet; Isabelle Fournier (pp. 149-165).

The large amount of data generated using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) poses a challenge for data analysis. In fact, generally about 1.10⁸–1.10⁹ values (m/z, I) are stored after a single MALDI-MSI experiment. This imposes processing techniques using dedicated informatics tools to be used since manual data interpretation is excluded. This work proposes and summarizes an approach that utilizes a multivariable analysis of MSI data. The multivariate analysis, such as principal component analysis–symbolic discriminant analysis, can remove and highlight specific m/z from the spectra in a specific region of interest. This approach facilitates data processing and provides better reproducibility, and thus, broadband acquisition for MALDI-MSI should be considered an effective tool to highlight biomarkers of interest. Additionally, we demonstrate the importance of the hierarchical classification of biomarkers by analyzing studies of clusters obtained either from digested or undigested tissues and using bottom-up and in-source decay strategies for in-tissue protein identification. This provides the possibility for the rapid identification of specific markers from different histological samples and their direct localization in tissues. We present an example from a prostate cancer study using formalin-fixed paraffin-embedded tissue.

Keywords: MALDI mass spectrometry imaging; Principal component analysis; Symbolic discriminant analysis; Hierarchical clustering; Bottom-up; In-source decay; Biomarkers; Prostate cancer

Normalization in MALDI-TOF imaging datasets of proteins: practical considerations by Sören-Oliver Deininger; Dale S. Cornett; Rainer Paape; Michael Becker; Charles Pineau; Sandra Rauser; Axel Walch; Eryk Wolski (pp. 167-181).

Normalization is critically important for the proper interpretation of matrix-assisted laser desorption/ionization (MALDI) imaging datasets. The effects of the commonly used normalization techniques based on total ion count (TIC) or vector norm normalization are significant, and they are frequently beneficial. In certain cases, however, these normalization algorithms may produce misleading results and possibly lead to wrong conclusions, e.g. regarding to potential biomarker distributions. This is typical for tissues in which signals of prominent abundance are present in confined areas, such as insulin in the pancreas or β-amyloid peptides in the brain. In this work, we investigated whether normalization can be improved if dominant signals are excluded from the calculation. Because manual interaction with the data (e.g., defining the abundant signals) is not desired for routine analysis, we investigated two alternatives: normalization on the spectra noise level or on the median of signal intensities in the spectrum. Normalization on the median and the noise level was found to be significantly more robust against artifact generation compared to normalization on the TIC. Therefore, we propose to include these normalization methods in the standard “toolbox” of MALDI imaging for reliable results under conditions of automation.

Keywords: Bioanalytical methods; Biological samples; Genomics/proteomics; MALDI imaging; Normalization; Data processing

Development of imaging mass spectrometry (IMS) dataset extractor software, IMS convolution by Takahiro Hayasaka; Naoko Goto-Inoue; Masaru Ushijima; Ikuko Yao; Akiko Yuba-Kubo; Masatoshi Wakui; Shigeki Kajihara; Masaaki Matsuura; Mitsutoshi Setou (pp. 183-193).

Imaging mass spectrometry (IMS) is a powerful tool for detecting and visualizing biomolecules in tissue sections. The technology has been applied to several fields, and many researchers have started to apply it to pathological samples. However, it is very difficult for inexperienced users to extract meaningful signals from enormous IMS datasets, and the procedure is time-consuming. We have developed software, called IMS Convolution with regions of interest (ROI), to automatically extract meaningful signals from IMS datasets. The processing is based on the detection of common peaks within the ordered area in the IMS dataset. In this study, the IMS dataset from a mouse eyeball section was acquired by a mass microscope that we recently developed, and the peaks extracted by manual and automatic procedures were compared. The manual procedure extracted 16 peaks with higher intensity in mass spectra averaged in whole measurement points. On the other hand, the automatic procedure using IMS Convolution easily and equally extracted peaks without any effort. Moreover, the use of ROIs with IMS Convolution enabled us to extract the peak on each ROI area, and all of the 16 ion images on mouse eyeball tissue were from phosphatidylcholine species. Therefore, we believe that IMS Convolution with ROIs could automatically extract the meaningful peaks from large-volume IMS datasets for inexperienced users as well as for researchers who have performed the analysis.

Keywords: Imaging mass spectrometry (IMS); Matrix-assisted laser desorption/ionization (MALDI); Analyzing software; Common peak detection; Mouse retina; Phosphatidylcholine (PC)

Direct quantification of deoxynivalenol glucuronide in human urine as biomarker of exposure to the Fusarium mycotoxin deoxynivalenol by Benedikt Warth; Michael Sulyok; Franz Berthiller; Rainer Schuhmacher; Philipp Fruhmann; Christian Hametner; Gerhard Adam; Johannes Fröhlich; Rudolf Krska (pp. 195-200).

The direct quantification of deoxynivalenol glucuronide (DON-GlcA) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its application as a biomarker of exposure to the Fusarium mycotoxin deoxynivalenol (DON) is reported. Usually, DON exposure is estimated from dietary average intakes or by measurement of the native toxin in urine after enzymatic hydrolysis with β-glucuronidase. These methods are time-consuming, expensive, and fail to determine the ratio of DON to DON-GlcA in a simple one-step procedure. One of the main reasons for the use of indirect methods is the unavailability of DON-GlcA standards. Consequently, DON-3-O-glucuronide (D3GlcA) was synthesized and used to develop a method allowing quantification of both DON and D3GlcA by a simple “dilute and shoot” approach without the need for any cleanup. Limit of detection and apparent recovery of D3GlcA was 3 μg l⁻¹ and 88%, respectively. The identity of D3GlcA in human urine was confirmed by comparison with LC-MS/MS measurements of the synthetically produced D3GlcA standard which was also used for external calibration. The applicability of the method was demonstrated through the analysis of urine samples obtained from a volunteer during regular and cereal-restricted diet, respectively. In regular-diet urine samples, D3GlcA was quantified in concentrations >30 μg l⁻¹ by this approach. Figure Human urine sample naturally contaminated with 31 μg l⁻¹ deoxynivalenol-3-O-glucuronide (D3GlcA)

Keywords: Deoxynivalenol glucuronide; Mycotoxin; Biomarker; Exposure assessment; Human urine; LC-MS/MS

Analytical strategies for improving the robustness and reproducibility of bioluminescent microbial bioreporters by Aldo Roda; Barbara Roda; Luca Cevenini; Elisa Michelini; Laura Mezzanotte; Pierluigi Reschiglian; Kaisa Hakkila; Marko Virta (pp. 201-211).

Whole-cell bioluminescent (BL) bioreporter technology is a useful analytical tool for developing biosensors for environmental toxicology and preclinical studies. However, when applied to real samples, several methodological problems prevent it from being widely used. Here, we propose a methodological approach for improving its analytical performance with complex matrix. We developed bioluminescent Escherichia coli and Saccharomyces cerevisiae bioreporters for copper ion detection. In the same cell, we introduced two firefly luciferases requiring the same luciferin substrate emitting at different wavelengths. The expression of one was copper ion specific. The other, constitutively expressed, was used as a cell viability internal control. Engineered BL cells were characterized using the noninvasive gravitational field-flow fractionation (GrFFF) technique. Homogeneous cell population was isolated. Cells were then immobilized in a polymeric matrix improving cell responsiveness. The bioassay was performed in 384-well black polystyrene microtiter plates directly on the sample. After 2 h of incubation at 37 °C and the addition of the luciferin, we measured the emitted light. These dual-color bioreporters showed more robustness and a wider dynamic range than bioassays based on the same strains with a single reporter gene and that uses a separate cell strain as BL control. The internal correction allowed to accurately evaluate the copper content even in simulated toxic samples, where reduced cell viability was observed. Homogenous cells isolated by GrFFF showed improvement in method reproducibility, particularly for yeast cells. The applicability of these bioreporters to real samples was demonstrated in tap water and wastewater treatment plant effluent samples spiked with copper and other metal ions. Figure Schematic representation of the combined strategies to obtain a robust and reproducible whole-cell biosensor format: introduction of an internal viability control to correct the analyte-specific response, field-flow fractionation of bioreporters to obtain homogeneous cell populations and immobilization of fractionated bioreporters to preserve their viability.

Keywords: Whole-cell bioreporters; Bioluminescence; Field-flow fractionation; Cell immobilization; Biosensor

Chemiluminescence aptasensor for cocaine based on double-functionalized gold nanoprobes and functionalized magnetic microbeads by Ying Li; Xiaoting Ji; Bangwei Liu (pp. 213-219).

A novel chemiluminescence (CL) aptasensor for highly sensitive detection of small-molecule targets using cocaine as a model analyte was developed in the present study. For the proposed aptasensor, the aptamers were immobilized on the surface of gold nanoparticles (AuNPs) functionalized magnetic microbeads (MB-AuNPs) and then hybridized with the signal DNA on the double-functional gold nanoprobes (DF-AuNPs) modified with horseradish peroxidase (HRP). When cocaine was introduced, a competition for the aptamer between cocaine and the signal DNA occurred and the gold nanoprobes were forced to dissociate from the MB-AuNPs surface based on the structure switching of the aptamer. The CL signals of Luminol-H₂O₂-HRP-PIP system were proportional to the concentration of cocaine. A linear range was obtained when the concentrations of cocaine were from 1 × 10⁻⁹ to 1 × 10⁻⁸ M with the detection limit of 0.48 nM (3σ), much lower than those achieved by other methods. This new system can be easily extended to a variety of small-molecules, protein, and tumor cell analysis.

Keywords: Cocaine; Aptamer; Chemiluminescence; Gold nanoprobes; Magnetic microbeads

Detection of small RNA molecules by a combination of branched rolling circle amplification and bioluminescent pyrophosphate assay by Yasumasa Mashimo; Masayasu Mie; Shigeya Suzuki; Eiry Kobatake (pp. 221-227).

Aberrant expression of miRNAs often correlates with various human diseases. Therefore, miRNAs have been focused as disease biomarkers. Here, a novel application of a bioluminescence (BL) assay for small RNA quantification is described. The assay is based on detecting pyrophosphate (PPi) molecules released during branched rolling circle amplification (BRCA) with a second primer in the presence of target RNA molecules. The number of released PPi molecules is correlated with the target RNA copy number. This assay was capable of detecting at least 20 amol of target RNA molecules, and the dynamic range extended over at least three orders of magnitude. Appropriate use of a second primer allowed for sensitive detection of RNA molecules with a high S/N ratio in less time. Moreover, the assay could specifically detect as low as 0.1 fmol of a target small RNA within a total RNA extract with high reproducibility. These data suggest that our assay has the potential to become a simple, rapid, and highly sensitive method to detect miRNA. Furthermore, this method combined with a BL assay, which utilizes a widely used inexpensive luminometer, could be used for a wider, versatile range of applications. Figure Basic concept of RNA-primed BRCA combined BL assay to detect short RNAs

Keywords: Bioluminometric assay; MicroRNA detection; Pyruvate orthophosphate dikinase; Firefly luciferase; Branched rolling circle amplification

Silver nanoparticle-initiated chemiluminescence reaction of luminol–AgNO₃ and its analytical application by Cui Liu; Baoxin Li (pp. 229-235).

Ag⁺ has been regarded as an inert chemiluminescent oxidant. In this work, it was found that in the presence of silver nanoparticles (AgNPs), AgNO₃ could react with luminol to produce strong chemiluminescence (CL). The AgNPs with smaller size could initiate stronger CL emission. To investigate the CL mechanism of the AgNPs–luminol–AgNO₃ system, the UV–visible spectra and the CL spectrum of the CL system were obtained. The CL reaction mechanism involving catalysis was proposed. Compared with the reported nanoparticles–luminol–H₂O₂ CL system, the AgNPs–luminol–AgNO₃ CL system has the advantages of low background and good stability. Moreover, the new CL system was used in immunoassay for IgG. Figure AgNPs can initiate the CL reaction between luminol and AgNO₃. The CL mechanism is likely due to the fact that AgNPs as nucleation centers can catalyze the reduction of AgNO₃ to Ag atoms by luminol. Meanwhile, luminol is oxidized to luminol radical, which further reacts with the dissolved oxygen, giving rise to light emission.

Keywords: Silver nanoparticles; Chemiluminescence; Luminol; AgNO₃

Hydrogen/deuterium exchange-LC-MS approach to characterize the action of heparan sulfate C5-epimerase by Ponnusamy Babu; Xylophone V. Victor; Emily Nelsen; Thao Kim Nu Nguyen; Karthik Raman; Balagurunathan Kuberan (pp. 237-244).

Heparan sulfate (HS) proteoglycans regulate a number of biological functions in many systems. Most of the functions of HS are attributed to its unique structure, consisting of sulfated and non-sulfated domains, arising from the differential presence of iduronyl and glucuronyl residues along the polysaccharide chain. A single glucuronyl C5-epimerase enzyme acts on HS precursors, converts glucuronyl residues into iduronyl residues, and modulates subsequent biosynthetic steps in vivo. Previously, the ratios of non-sulfated epimers within the polysaccharide chain have been calculated by resolving radiolabeled GlcA-_AMan_R and IdoA-_AMan_R disaccharides using a tedious paper chromatography technique. This radioactive assay, based on measuring either the release or incorporation of ³H at C5 carbon of uronyl residues of ³H-labeled HS precursor substrate, has been in use over three decades to characterize the action of HS C5-epimerase. We have developed a non-radioactive assay to estimate the epimerase activity through resolving GlcA-_AMan_R and IdoA-_AMan_R disaccharides on high-performance liquid chromatography in conjunction with hydrogen/deuterium exchange upon epimerization protocol-liquid chromatography mass spectrometry (DEEP-LC-MS). Utilizing this new, non-radioactive-based assay, DEEP-LC-MS, we were able to determine the extent of both forward and reverse reactions on the same substrate catalyzed by C5-epimerase. The results from this study also provide insights into the action of C5-epimerase and provide an opportunity to delineate snapshots of biosynthetic events that occur during the HSPG assembly in the Golgi. Figure Using hydrogen/deuterium exchange, Heparan Sulfate C5-Epimerase activity is characterized using a novel DEEP-LC-MS method

Keywords: Heparan sulfate; Heparin; C5-epimerase; LC-MS; Hydrogen/deuterium exchange; Proteoglycan biosynthesis

14-3-3 Protein beta/alpha as a urinary biomarker for renal cell carcinoma: proteomic analysis of cyst fluid by Satoru Minamida; Masatsugu Iwamura; Yoshio Kodera; Yusuke Kawashima; Kenichi Tabata; Kazumasa Matsumoto; Tetsuo Fujita; Takefumi Satoh; Tadakazu Maeda; Shiro Baba (pp. 245-252).

Although various samples, including tissue, cells, serum, and urine, from patients with renal cell carcinoma (RCC) have been analyzed, biomarkers with diagnostic value have yet to be identified. We used a proteomics approach to analyze cyst fluid in cases of cyst-associated RCC to identify accessible and abundant proteins that are overexpressed and/or secreted by RCC cells. Proteins in the cyst fluid were separated by reverse-phase high-performance liquid chromatography and agarose two-dimensional gel electrophoresis and were identified by tandem mass spectrometry. We conducted a National Center for Biotechnology Information search and a MEDLINE search to predict the function of these identified proteins and to select a tumor-marker candidate protein. Our search resulted in the identification and selection of the differentially regulated protein known as 14-3-3 protein beta/alpha, which was overexpressed in cyst fluid from cyst-associated RCC but has not been previously associated with RCC. We then measured its incidence through Western blotting of various normal and RCC samples (serum, urine, tissue, and cyst fluid). The expression levels of 14-3-3 protein beta/alpha were higher in urine samples from patients with RCC than in samples from healthy volunteers. Receiver operating characteristic (ROC) curve analyses were performed to assess this potential biomarker; these data (area under the ROC curve value was 0.8813) indicate a high degree of accuracy for this screening method. 14-3-3 Protein beta/alpha may be a diagnostically useful biomarker for early diagnosis of RCC.

Keywords: Renal cancer; Proteome; Marker; 14-3-3 Protein; Cyst fluid

A lysozyme and magnetic bead based method of separating intact bacteria by Ebru Diler; Ursula Obst; Katja Schmitz; Thomas Schwartz (pp. 253-265).

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO₃-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.

Keywords: Bacteria separation; Magnetic beads; Lysozyme mutation; Protein purification; Binding capacity

Fast chromatographic determination of the bile salt critical micellar concentration by Benedetto Natalini; Roccaldo Sardella; Antimo Gioiello; Emiliano Rosatelli; Federica Ianni; Emidio Camaioni; Roberto Pellicciari (pp. 267-274).

In a line of research focused on the design, synthesis and development of new bile acid-based compounds, the physico-chemical profile of the molecules must be thoroughly explored and analyzed. In this scenario, a fast and reliable information on the critical micellar concentration (CMC) of specific compounds through a profitable chromatographic parameter can be of aid to rationally direct the synthesis of new molecular entities, mainly during the early stages of the drug-discovery process. The derived ‘chromatographic hydrophobicity index’ (CHI), usually employed for a fast access to the log P/log D value of physico-chemically diverse compounds and obtained via RP-gradient elution, was for the first time engaged in the bile acid field. Accordingly, 14 unconjugated bile acids harboured with a different number, position and orientation of hydroxy groups, as well as other substituents onto the steroidal backbone and side chain, were selected to build up a calibration curve. Such a collection of compounds was rationally assembled in order to manage an almost continuous range of CMC values (spanning the spectrophotometrically obtained CMCs between 5 and 25 mM). A high degree of correlation between CMC and CHI values was obtained (R ² and cross-validated R _xv ² of the pCMC vs CHI plot equal to 0.975 and 0.966, respectively). A selected new subset of five confidential research bile acids with experimental CMCs in the range 6–19 mM was finally recruited to validate the proposed method. The high statistical quality of the established mathematical model turned out into a very appreciable predictive power. Figure pCMC vs CHI plot for the whole set of 14 unconjugated BSs

Keywords: Bile acids; Bile salts; Critical micellar concentration; Chromatographic hydrophobicity index; Reversed-phase high-performance liquid chromatography; Evaporative light scattering detector

The comparative pharmacokinetics of two pyrrolizidine alkaloids, senecionine and adonifoline, and their main metabolites in rats after intravenous and oral administration by UPLC/ESIMS by Changhong Wang; Yan Li; Jiangguo Gao; Yuqi He; Aizhen Xiong; Li Yang; Xuemei Cheng; Yueming Ma; Zhengtao Wang (pp. 275-287).

Pyrrolizidine alkaloids (PAs) are considered to be one of the most hepatotoxic groups of compounds of plant origin and are present in about 3% of the world’s flowering plants. Most PAs represent a considerable health hazard to both livestock and humans through the consumption of plants and PA-contaminated products such as milk, honey, herbal teas, and medicines. This study determined the differences in the in vivo pharmacokinetic behavior of senecionine (SEN), adonifoline (ADO), and their main metabolites in rats after intravenous administration and oral administration by ultraperformance liquid chromatography/electrospray ionization mass spectrometry. Upon intravenous administration and oral administration of SEN and ADO, significant differences in pharmacokinetics were observed, with the SEN and ADO being absorbed fast with lower bioavailability and being quickly metabolized to PA N-oxides and hydroxylation products of PAs or their N-oxides. It could be seen that the plasma concentration ratio of senecionine N-oxide (SEN-NO) to SEN (C _SEN-NO/C _SEN) was significantly larger than that for adonifoline N-oxide (ADO-NO) and ADO (C _ADO-NO/C _ADO) (P < 0.001) for both dosing routes in rats. The high N-oxygenation activity and extensive toxicity of SEN, compared with ADO, in rats raised the question of whether or not the higher metabolic rate of SEN in rats in vivo was related to its potent toxicity. The toxicity of SEN-NO and ADO-NO needs to be evaluated further and compared in vitro/in vivo. This study was most helpful for interpreting the metabolism of metabolic bioactivation and detoxication, and toxicity differences among SEN, ADO and other PAs. Figure The comparative of senecionine and adonifoline metabolized to N-oxide in rats after intravenous and oral administration. (SEN: senecionine, ADO: adonifoline, SEN-NO: senecionine N-oxide, ADO-NO: adonifoline N-oxide)

Keywords: Pyrrolizidine alkaloids; Senecionine; Adonifoline; Pharmacokinetics; Metabolites

A sensitive liquid chromatographic–mass spectrometric method for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma by Juanjuan Zhao; Xiao Han; Xu Zhao; Chao Wang; Qing Li; Xiaohui Chen; Kaishun Bi (pp. 289-296).

A liquid chromatographic–mass spectrometric (LC–MS) method has been developed and validated for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma. After addition of the internal standard, domperidone, plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Apollo C₁₈ column (250 mm × 4.6 mm, 5 μm), with methanol–0.01% formic acid water (60:40, v/v) as mobile phase, within a runtime of 12.0 min. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 1.0–500 ng mL⁻¹ for dehydroevodiamine and 2.0–1,000 ng mL⁻¹ for limonin, with lower limits of quantitation of 1.0 and 2.0 ng mL⁻¹, respectively. Intra-day and inter-day precision were within 6.0% and 10.9%, respectively, for both analytes, and the accuracy (relative error, RE, %) was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to a comparative pharmacokinetic study of dehydroevodiamine and limonin in rat plasma after oral administration of dehydroevodiamine, limonin, and an aqueous extract of Evodiae fructus. The results indicated there were obvious differences between the pharmacokinetic behavior after oral administration of an aqueous extract of Evodiae fructus compared with single substances. Figure The concentration–time curves of DHE (a) and LM (b) in rat plasma after oral administration of DHE, LM and Evodiae fructus extract

Keywords: LC–MS; Dehydroevodiamine; Limonin; Evodia rutaecarpa ; Pharmacokinetics

Determination of amino acids and catecholamines derivatized with 3-(4-chlorobenzoyl)-2-quinolinecarboxaldehyde in PC12 and HEK293 cells by capillary electrophoresis with laser-induced fluorescence detection by Niu Zhang; Xiao-Feng Guo; Hong Wang; Hua-Shan Zhang (pp. 297-304).

An effective micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF) method has been proposed for the separation and the determination of 16 amino acids and two catecholamines using a new fluorogenic reagent, 3-(4-chlorobenzoyl)-2-quinolinecarboxaldehyde (Cl-BQCA), as the derivatizing reagent. The highest derivatization efficiency was achieved in pH 8.0 borate buffer at 50 °C for 50 min. The optimal separation of Cl-BQCA-labeled amines was obtained with a running buffer (pH 9.15) containing 120 mM boric acid, 38.5 mM sodium dodecyl sulfate, and 17% acetonitrile. The detection limit (S/N = 3) was found to be as low as 1.4 nM. The present method has been successfully used to detect amino acids and catecholamines in HEK293 and PC12 cell samples. This study explores the potential of MEKC-LIF with Cl-BQCA labeling as a tool for monitoring amino acids and catecholamines during the complex physiological and behavioral processes in various matrices.

Keywords: Amino acids; Catecholamines; Cl-BQCA; MEKC-LIF; Cell samples

Agonistic and antagonistic estrogens in licorice root (Glycyrrhiza glabra) by Rudy Simons; Jean-Paul Vincken; Loes A. M. Mol; Susan A. M. The; Toine F. H. Bovee; Teus J. C. Luijendijk; Marian A. Verbruggen; Harry Gruppen (pp. 305-313).

The roots of licorice (Glycyrrhiza glabra) are a rich source of flavonoids, in particular, prenylated flavonoids, such as the isoflavan glabridin and the isoflavene glabrene. Fractionation of an ethyl acetate extract from licorice root by centrifugal partitioning chromatography yielded 51 fractions, which were characterized by liquid chromatography–mass spectrometry and screened for activity in yeast estrogen bioassays. One third of the fractions displayed estrogenic activity towards either one or both estrogen receptors (ERs; ERα and ERβ). Glabrene-rich fractions displayed an estrogenic response, predominantly to the ERα. Surprisingly, glabridin did not exert agonistic activity to both ER subtypes. Several fractions displayed higher responses than the maximum response obtained with the reference compound, the natural hormone 17β-estradiol (E₂). The estrogenic activities of all fractions, including this so-called superinduction, were clearly ER-mediated, as the estrogenic response was inhibited by 20–60% by known ER antagonists, and no activity was found in yeast cells that did not express the ERα or ERβ subtype. Prolonged exposure of the yeast to the estrogenic fractions that showed superinduction did, contrary to E₂, not result in a decrease of the fluorescent response. Therefore, the superinduction was most likely the result of stabilization of the ER, yeast-enhanced green fluorescent protein, or a combination of both. Most fractions displaying superinduction were rich in flavonoids with single prenylation. Glabridin displayed ERα-selective antagonism, similar to the ERα-selective antagonist RU 58668. Whereas glabridin was able to reduce the estrogenic response of E₂ by approximately 80% at 6 × 10⁻⁶ M, glabrene-rich fractions only exhibited agonistic responses, preferentially on ERα. Figure

Keywords: Prenylation; Isoflavonoids; Licorice; Estrogenicity; Antagonism; Superinduction

A new direct Fourier transform infrared analysis of free fatty acids in edible oils using spectral reconstitution by Xiuzhu Yu; F. R. van de Voort; Jacqueline Sedman; Jin-ming Gao (pp. 315-324).

A new transmission-based Fourier transform infrared (FTIR) spectroscopic method for the direct determination of free fatty acids (FFA) in edible oils has been developed using the developed spectral reconstitution (SR) technique. Conventional neat-oil and SR calibrations were devised by spiking hexanoic acid into FFA-free canola oil and measuring the response to added FFA at 1,712 cm⁻¹ referenced to a baseline at 1,600 cm⁻¹(1,712 cm⁻¹/1,600 cm⁻¹). To compensate for the known oil dependency of such calibration equations resulting from variation of the triacylglycerol ester (C═O) absorption with differences in oil saponification number (SN), a correction equation was devised by recording the spectra of blends of two FFA-free oils (canola and coconut) differing substantially in SN and correlating the intensity of the ester (C═O) absorption at the FFA measurement location with the intensity of the first overtone of this vibration, measured at 3,471 cm⁻¹/3,427 cm⁻¹. Further examination of the spectra of the oil blends by generalized 2D correlation spectroscopy revealed an additional strong correlation with an absorption in the near-infrared (NIR) combination band region, which led to the development of a second correction equation based on the absorbance at 4,258 cm⁻¹/4,235 cm⁻¹. The NIR-based correction equation yielded superior results and was shown to completely eliminate biases due to variations in oil SN, thereby making a single FFA calibration generally applicable to oils, regardless of SN. FTIR methodology incorporating this correction equation and employing the SR technique has been automated. Figure SR spectra of FFA-free canola oil and coconut oil, illustrating their relative absorption profiles in the FFA measurement region (1,712 cm⁻¹) (A) as well as the relative intensities of the first overtone of the triacylglycerol ester linkage ν(C═O) absorption (B), demonstrates that the correlating band (3,471 cm⁻¹) can overcome oil dependency in FTIR FFA analysis of edible oils

Keywords: Free fatty acids; Spectral reconstitution; Edible oils; Analysis

Fast quantitation of 5-hydroxymethylfurfural in honey using planar chromatography by Elena S. Chernetsova; Igor A. Revelsky; Gertrud E. Morlock (pp. 325-332).

An approach for rapid quantitation of 5-hydroxymethylfurfural (HMF) in honey using planar chromatography is suggested for the first time. In high-performance thin-layer chromatography (HPTLC) the migration time is approximately 5 min. Detection is performed by absorbance measurement at 290 nm. Polynomial calibration in the matrix over a range of 1:80 showed correlation coefficients, r, of ≥ 0.9997 for peak areas and ≥ 0.9996 for peak heights. Repeatability in the matrix confirmed the suitability of HPTLC–UV for quantitation of HMF in honey. The relative standard deviation (RSD, %, n = 6) of HMF at 10 ng/band was 2.9% (peak height) and 5.2% (peak area); it was 0.6% and 1.0%, respectively, at 100 ng/band. Other possible detection modes, for example fluorescence measurement after post-chromatographic derivatization and mass spectrometric detection, were also evaluated and can coupling can be used as an additional tool when it is necessary to confirm the results of prior quantitation by HPTLC–UV. The confirmation is provided by monitoring the HMF sodium adduct [M + Na]⁺ at m/z 149 followed by quantitation in TIC or SIM mode. Detection limits for HPTLC–UV, HPTLC–MS (TIC), and HPTLC–MS (SIM) were 0.8 ng/band, 4 ng/band, and 0.9 ng/band, respectively. If 12 μL honey solution was applied to an HPTLC plate, the respective detection limits for HMF in honey corresponded to 0.6 mg kg⁻¹. Thus, the developed method was highly suitable for quantitation of HMF in honey at the strictest regulated level of 15 mg kg⁻¹. Comparison of HPTLC–UV detection with HPTLC–MS showed findings were comparable, with a mean deviation of 5.1 mg kg⁻¹ for quantitation in SIM mode and 6.1 mg kg⁻¹ for quantitation in TIC mode. The mean deviation of the HPTLC method compared with the HPLC method was 0.9 mg kg^-1 HMF in honey. Re-evaluation of the same HPTLC plate after one month showed a deviation of 0.5 mg kg⁻¹ HMF in honey. It was demonstrated that the proposed HPTLC method is an effective method for HMF quantitation in honey. Figure Fast quantitation of 5-hydroxymethylfurfural in honey

Keywords: High-performance thin-layer chromatography; HPTLC; HPTLC–MS; 5-hydroxymethylfurfural; HMF; Honey

Rapid detection of melamine with 4-mercaptopyridine-modified gold nanoparticles by surface-enhanced Raman scattering by Tingting Lou; Yunqing Wang; Jinhua Li; Hailong Peng; Hua Xiong; Lingxin Chen (pp. 333-338).

A surface-enhanced Raman scattering (SERS) strategy based on 4-mercaptopyridine (MPY)-modified gold nanoparticles (AuNPs) was developed for the rapid and sensitive detection of melamine in milk powder. The SERS measurement of melamine strongly relied on the “hotspot” effect, in which AuNPs immediately aggregated upon the addition of melamine, leading to significantly enhanced Raman intensity of the reporter molecule MPY and a color change for the solution from red to blue-gray. The limit of detection based on a signal to noise of 3 (S/N = 3) was found to be as low as 0.1 ppb of melamine, with an excellent linearity of 0.5–100 ppb, demonstrating a higher sensitivity and a wider quantitation range than direct SERS sensing methods based on enhanced substrate. An impressive specificity for melamine detection over various common metal ions and excipients in dairy products, even at concentrations of 100-fold higher than melamine, was achieved. Good recoveries of 88.5% and 111.7% were obtained from milk samples spiked to 20 and 100 ppb levels, respectively. The proposed method is potentially applicable for the rapid in situ determination of melamine in complex matrices. Figure An indirect SERS strategy based on 4-mercaptopyridine (MPY) modified gold nanoparticles (AuNPs) for rapid and sensitive detection of melamine

Keywords: Melamine; Surface-enhanced Raman scattering; Gold nanoparticle; 4-Mercaptopyridine

Optimisation of stir bar sorptive extraction and in-tube derivatisation–thermal desorption–gas chromatography–mass spectrometry for the determination of several endocrine disruptor compounds in environmental water samples by Arantza Iparraguirre; Ailette Prieto; Patricia Navarro; Maitane Olivares; Luis-Ángel Fernández; Olatz Zuloaga (pp. 339-352).

The analysis of organic pollutants in environmental water samples requires a pre-concentration step. Pre-concentration techniques such as stir bar sorptive extraction (SBSE) have gained popularity since they minimise the use of toxic organic solvents and can be considered as green analytical techniques. Similar to other pre-concentration techniques, one of the problems when SBSE is used is the matrix effect, which often occurs during the analysis of environmental water samples such as estuarine or wastewater samples. The present work studied the matrix effect during SBSE coupled to in-tube derivatisation–thermal desorption (TD)–gas chromatography–mass spectrometry for the determination of several endocrine disruptor compounds, such as alkylphenols, bisphenol A, estrogens and sterols, in environmental water samples, after optimisation of the major variables affecting the determination. Variables such as the addition of methanol or an inert salt to the donor phase, the extraction temperature, the volume of the donor phase, the stirring rate and the extraction time were studied during the SBSE optimisation. In the case of the in-tube derivatisation and TD step, the volume of the derivatisation reagent (N,O-bis(trimethylsilyl)triufloroacetamide with 1% of trimethylchlorosilane (BSTFA + 1% TMCS)) and the cryo-focusing temperature were fixed (2 μL and −50 °C, respectively) according to a consensus between maximum signal and optimal operation conditions. Good apparent recovery values (78–124%) were obtained for most of the analytes in Milli-Q water, except for 4-tert-octylphenol (4-tOP), which showed apparent recovery values exceeding 100%. Precision (n = 4) was in the 2–27%, and method detection limits were in the low nanogrammes per litre level for most of the analytes studied. The matrix effect was studied using two different approaches. On the one hand, Milli-Q water samples were spiked with humic acids, and apparent recovery values were studied with and without correction with the corresponding deuterated analogue. On the other hand, estuarine water and wastewater samples were spiked with known concentrations of target analytes, and apparent recoveries were studied as explained above. In general, the matrix effect could be corrected with the use of deuterated analogues, except for 4-tOP and nonylphenols for which [²H₄]-n-nonylphenol did not provide good corrections.

Keywords: Endocrine disruptor compounds; Environmental water samples; Stir bar sorptive extraction; Optimisation; Matrix effect

Identification of biotransformation products of macrolide and fluoroquinolone antimicrobials in membrane bioreactor treatment by ultrahigh-performance liquid chromatography/quadrupole time-of-flight mass spectrometry by Senka Terzic; Ivan Senta; Marin Matosic; Marijan Ahel (pp. 353-363).

Ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was applied for the identification of transformation products (TPs) of fluoroquinolone (norfloxacin and ciprofloxacin) and macrolide (azithromycin, erythromycin, and roxitromycin) antimicrobials in wastewater effluents from a Zenon hollow-fiber membrane bioreactor (MBR). The detected TPs were thoroughly characterized using the accurate mass feature for the determination of the tentative molecular formulae and MS-MS experiments for the structural elucidation of unknowns. Several novel TPs, which have not been previously reported in the literature, were identified. The TPs of azithromycin and roxithromycin, identified in MBR effluent, were conjugate compounds, which were formed by phosphorylation of desosamine moiety. Transformation of fluoroquinolones yielded two types of products: conjugates, formed by succinylation of the piperazine ring, and smaller metabolites, formed by an oxidative break-up of piperazine moiety to form the 7-[(2-carboxymethyl)amino] group. A semi-quantitative assessment of these TPs suggested that they might have contributed significantly to the overall balance of antimicrobial residues in MBR effluents and thus to the overall removal efficiency. Determination of TPs during a period of 2 months indicated a conspicuous dynamics, which warrants further research to identify microorganisms involved and treatment conditions leading to their formation. Figure Proposed structures of the novel transformation products of antimicrobials, formed during MBR wastewater treatment. AZI TP – phosphorylated azithromycin; ROX TP -phosphorylated roxithromycin; NOR TP1 – succinyl norfloxacin; CIP TP1 – succinyl ciprofloxacin; NOR TP2 - 7-[(carboxymethyl)amino]-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid; CIP TP2 - 7-[(carboxymethyl)amino]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid.

Keywords: Antimicrobials; Fluoroquinolone; Macrolide; Transformation products; Membrane bioreactor; Liquid chromatography–mass spectrometry

Sequential spectrofluorimetric determination of free and total glycerol in biodiesel in a multicommuted flow system by Sidnei G. Silva; Ángel Morales-Rubio; Miguel de La Guardia; Fábio R. P. Rocha (pp. 365-371).

A new procedure for spectrofluorimetric determination of free and total glycerol in biodiesel samples is presented. It is based on the oxidation of glycerol by periodate, forming formaldehyde, which reacts with acetylacetone, producing the luminescent 3,5-diacetyl-1,4-dihydrolutidine. A flow system with solenoid micro-pumps is proposed for solution handling. Free glycerol was extracted off-line from biodiesel samples with water, and total glycerol was converted to free glycerol by saponification with sodium ethylate under sonication. For free glycerol, a linear response was observed from 5 to 70 mg L⁻¹ with a detection limit of 0.5 mg L⁻¹, which corresponds to 2 mg kg⁻¹ in biodiesel. The coefficient of variation was 0.9% (20 mg L⁻¹, n = 10). For total glycerol, samples were diluted on-line, and the linear response range was 25 to 300 mg L⁻¹. The detection limit was 1.4 mg L⁻¹ (2.8 mg kg⁻¹ in biodiesel) with a coefficient of variation of 1.4% (200 mg L⁻¹, n = 10). The sampling rate was ca. 35 samples h⁻¹ and the procedure was applied to determination of free and total glycerol in biodiesel samples from soybean, cottonseed, and castor beans.

Keywords: Flow analysis; Multicommutation; Spectrofluorimetry; Biodiesel; Glycerol

Effect of sample preparation methods on the d,l-enantiomer ratio of extracted selenomethionine by Orsolya Egressy-Molnár; Andrea Vass; Anikó Németh; Juan F. García-Reyes; Mihály Dernovics (pp. 373-380).

Effects of the two most widespread sample preparation techniques on the d,l-enantiomer ratio of extracted selenomethionine were monitored through the analysis of the certified reference material selenium-enriched yeast and the isolated protein fraction of high selenium monkeypot nut. The extracted selenomethionine (SeMet) fractions were orthogonally cleaned up with anion exchange chromatography before carrying out the enantiomer-specific detection to increase the robustness and the efficiency of the subsequent o-phthal-aldehyde and n-isobutyril-cysteine-based derivatisation process and reversed phase-high-performance liquid chromatography-inductively coupled plasma mass spectroscopy (ICP-MS) detection. The two techniques, namely methanesulphonic acid (MSA) based digestion and proteolytic digestion with protease XIV, resulted in significantly different ratio of d,l-selenomethionine with the final results of 2.2–2.7% and 0.5–0.6% of d-SeMet, respectively. The study revealed significant differences in the ICP-MS-related sensitivity of the derivatised selenomethionine enantiomers, which calls attention to the quantification of this selenoamino acid after MSA hydrolysis. Figure Sample preparation methods influence the amount of D-selenomethionine formed during protein hydrolysis.

Keywords: Selenium; Selenomethionine; Enantiomer; Selenised yeast; Monkeypot nut; Methanesulphonic acid; Enzymatic digestion; SELM-1

Determination of superoxide dismutase and SOD-mimetic activities by a chemical system: Co₂/H₂O₂/lucigenin by T. V. Zhidkova; E. V. Proskurnina; E. A. Parfenov; Yu. A. Vladimirov (pp. 381-386).

The bright chemiluminescence has been observed in the system: Co²⁺/hydrogen peroxide/lucigenin. The chemiluminescence intensity was directly proportional to either cobalt, hydrogen peroxide, or lucigenin concentrations. A procedure of determination of superoxide dismutase (SOD) activity by the chemiluminescence method in the cobalt–hydrogen peroxide–lucigenin system at pH 8.5 is suggested. A linear dependence was established between a relative chemiluminescence intensity and SOD concentration in the range of SOD concentrations between 0 and 4.5 nM, c _1/2 = 0.8 nM. The determination of SOD activity was performed in several tissue samples (rat plasma, erythrocyte hemolysate, and liver mitochondria). A technique of tissue sample preparation with the use of thermal inactivation of interfering proteins at 60 °C was used. The method was successfully applied for comparison of the efficiency of SOD mimetics.

Keywords: Superoxide dismutase/*analysis; Chemiluminescent measurements; Lucigenin; SOD mimetics; Animals

Sections

Personal tools

Analytical and Bioanalytical Chemistry (v.401, #1)

Sections

Personal tools

Local resources

Analytical and Bioanalytical Chemistry (v.401, #1)