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Analytical and Bioanalytical Chemistry (v.392, #3)
Jacquie T. Keer and Lyndsey Birch (Eds.): Essentials of nucleic acid analysis. A robust approach
by Sapna K. Deo (pp. 321-322).
Jacquie T. Keer and Lyndsey Birch (Eds.): Essentials of nucleic acid analysis. A robust approach
by Sapna K. Deo (pp. 321-322).
Kourosh Kalantar-zadeh and Benjamin Fry: Nanotechnology-enabled sensors
by Peter A. Lieberzeit (pp. 325-326).
Kourosh Kalantar-zadeh and Benjamin Fry: Nanotechnology-enabled sensors
by Peter A. Lieberzeit (pp. 325-326).
The genetically modified foods debate: demystifying the controversy through analytical chemistry by Sylvia Daunert; Sapna Deo; Xenia Morin; Aldo Roda (pp. 327-331).
is the Gill Eminent Professor of Analytical and Biological Chemistry, the 2004–2005 Distinguished Professor from the College of Arts and Sciences at the University of Kentucky, and a 2005–2006 University of Kentucky Research Professor. Her research interests are in bioanalytical chemistry, at the interface between analytical chemistry, molecular biology, and bioengineering. More specifically, her group uses recombinant DNA technology to design new molecular diagnostic tools and biosensors based on genetically engineered proteins and cells for biomedical and environmental applications. Additionally, the research of her group 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. Dr Daunert has served as Editor of Analytical and Bioanalytical Chemistry since its inception in 2001. has been an Assistant Professor of Bioanalytical Chemistry in the Department of Chemistry and Chemical Biology at the Indiana University–Purdue University Indianapolis since 2005. She is author and co-author of over 55 scientific publications and several patents. Dr Deo is an editor of the book Photoproteins in Bioanalysis. Her research interest is in the development of novel bioanalytical techniques for detection of microRNAs and RNA molecules based on luminescence detection for application in diagnosis and pathogen detection. Other areas of research interest in Dr Deo’s laboratory include the development of molecular probes for biosensing and bioimaging applications. Dr Deo’s research is funded by the National Science Foundation and the National institutes of Health. is a plant biochemist by training and currently holds the position of lecturer in the Princeton Writing Program and the Princeton Environmental Institute at Princeton University. She is interested in the interplay between science, technology, and society, particularly in terms of agriculture and food production. She is concerned with a range of issues, from the acceptance of genetically modified foods to the emergence of the organic, local, and slow food movements. She also explores issues such as how to feed the world, food security, hunger and malnutrition, environmentalism and sustainability in agriculture, and the production of biofuels. is Professor of Analytical Chemistry at Bologna University. His main research interests include the development of ultrasensitive bio-chemiluminescence imaging techniques for the localization of target molecules (proteins, nucleic acids) in biological samples, such as cells and tissues.
The genetically modified foods debate: demystifying the controversy through analytical chemistry by Sylvia Daunert; Sapna Deo; Xenia Morin; Aldo Roda (pp. 327-331).
is the Gill Eminent Professor of Analytical and Biological Chemistry, the 2004–2005 Distinguished Professor from the College of Arts and Sciences at the University of Kentucky, and a 2005–2006 University of Kentucky Research Professor. Her research interests are in bioanalytical chemistry, at the interface between analytical chemistry, molecular biology, and bioengineering. More specifically, her group uses recombinant DNA technology to design new molecular diagnostic tools and biosensors based on genetically engineered proteins and cells for biomedical and environmental applications. Additionally, the research of her group 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. Dr Daunert has served as Editor of Analytical and Bioanalytical Chemistry since its inception in 2001. has been an Assistant Professor of Bioanalytical Chemistry in the Department of Chemistry and Chemical Biology at the Indiana University–Purdue University Indianapolis since 2005. She is author and co-author of over 55 scientific publications and several patents. Dr Deo is an editor of the book Photoproteins in Bioanalysis. Her research interest is in the development of novel bioanalytical techniques for detection of microRNAs and RNA molecules based on luminescence detection for application in diagnosis and pathogen detection. Other areas of research interest in Dr Deo’s laboratory include the development of molecular probes for biosensing and bioimaging applications. Dr Deo’s research is funded by the National Science Foundation and the National institutes of Health. is a plant biochemist by training and currently holds the position of lecturer in the Princeton Writing Program and the Princeton Environmental Institute at Princeton University. She is interested in the interplay between science, technology, and society, particularly in terms of agriculture and food production. She is concerned with a range of issues, from the acceptance of genetically modified foods to the emergence of the organic, local, and slow food movements. She also explores issues such as how to feed the world, food security, hunger and malnutrition, environmentalism and sustainability in agriculture, and the production of biofuels. is Professor of Analytical Chemistry at Bologna University. His main research interests include the development of ultrasensitive bio-chemiluminescence imaging techniques for the localization of target molecules (proteins, nucleic acids) in biological samples, such as cells and tissues.
Genetically modified food from crops: progress, pawns, and possibilities by Xenia K. Morin (pp. 333-340).
is a plant biochemist by training and currently holds the position of lecturer in the Princeton Writing Program and the Princeton Environmental Institute at Princeton University. She is interested in the interplay between science, technology, and society, particularly in terms of agriculture and food production. She is concerned with a range of issues, from the acceptance of genetically modified foods to the emergence of the organic, local, and slow food movements. She also explores issues such as how to feed the world, food security, hunger, and malnutrition, environmentalism and sustainability in agriculture, and the production of biofuels.
Genetically modified food from crops: progress, pawns, and possibilities by Xenia K. Morin (pp. 333-340).
is a plant biochemist by training and currently holds the position of lecturer in the Princeton Writing Program and the Princeton Environmental Institute at Princeton University. She is interested in the interplay between science, technology, and society, particularly in terms of agriculture and food production. She is concerned with a range of issues, from the acceptance of genetically modified foods to the emergence of the organic, local, and slow food movements. She also explores issues such as how to feed the world, food security, hunger, and malnutrition, environmentalism and sustainability in agriculture, and the production of biofuels.
Genetically modified food from crops: progress, pawns, and possibilities by Xenia K. Morin (pp. 333-340).
is a plant biochemist by training and currently holds the position of lecturer in the Princeton Writing Program and the Princeton Environmental Institute at Princeton University. She is interested in the interplay between science, technology, and society, particularly in terms of agriculture and food production. She is concerned with a range of issues, from the acceptance of genetically modified foods to the emergence of the organic, local, and slow food movements. She also explores issues such as how to feed the world, food security, hunger, and malnutrition, environmentalism and sustainability in agriculture, and the production of biofuels.
Should genetically modified foods be abandoned on the basis of allergenicity? by Stephanie Bachas-Daunert; Sapna K. Deo (pp. 341-346).
is a second-year undergraduate student in Civil and Environmental Engineering at Princeton University. Prior to enrolling at Princeton, she performed scientific research with Dr. Deo on several projects, mostly of a biochemical or chemical engineering nature. Her work has been featured in the literature, and she contributed to a book co-authored by Dr. Deo, Photoproteins in Bioanalysis. Stephanie is now focusing on environmental engineering, and has particular interests in sustainability, the effects of the environment on health, and environmental problem-solving. is an Assistant Professor of Bioanalytical Chemistry in the Department of Chemistry & Chemical Biology at the Indiana University-Purdue University Indianapolis since 2005. She is author and co-author of over 55 scientific publications and several patents. Dr. Deo is an editor of the book Photoproteins in Bioanalysis. Dr. Deo’s research interest is in the development of novel bioanalytical techniques for the detection of microRNAs and RNA molecules based on luminescence detection for application in diagnosis and pathogen detection. Other areas of research interest in Dr. Deo’ laboratory include development of molecular probes for biosensing and bioimaging applications. Dr. Deo’s research is funded by the National Science Foundation and the National institutes of Health.
Should genetically modified foods be abandoned on the basis of allergenicity? by Stephanie Bachas-Daunert; Sapna K. Deo (pp. 341-346).
is a second-year undergraduate student in Civil and Environmental Engineering at Princeton University. Prior to enrolling at Princeton, she performed scientific research with Dr. Deo on several projects, mostly of a biochemical or chemical engineering nature. Her work has been featured in the literature, and she contributed to a book co-authored by Dr. Deo, Photoproteins in Bioanalysis. Stephanie is now focusing on environmental engineering, and has particular interests in sustainability, the effects of the environment on health, and environmental problem-solving. is an Assistant Professor of Bioanalytical Chemistry in the Department of Chemistry & Chemical Biology at the Indiana University-Purdue University Indianapolis since 2005. She is author and co-author of over 55 scientific publications and several patents. Dr. Deo is an editor of the book Photoproteins in Bioanalysis. Dr. Deo’s research interest is in the development of novel bioanalytical techniques for the detection of microRNAs and RNA molecules based on luminescence detection for application in diagnosis and pathogen detection. Other areas of research interest in Dr. Deo’ laboratory include development of molecular probes for biosensing and bioimaging applications. Dr. Deo’s research is funded by the National Science Foundation and the National institutes of Health.
Advances in molecular techniques for the detection and quantification of genetically modified organisms by Dimitrios S. Elenis; Despina P. Kalogianni; Kyriaki Glynou; Penelope C. Ioannou; Theodore K. Christopoulos (pp. 347-354).
Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.
Keywords: Genetically modified organisms; GMO; Molecular techniques; DNA hybridization; Biosensors; Microarrays; Capillary electrophoresis; Real-time PCR; Competitive PCR
Advances in molecular techniques for the detection and quantification of genetically modified organisms by Dimitrios S. Elenis; Despina P. Kalogianni; Kyriaki Glynou; Penelope C. Ioannou; Theodore K. Christopoulos (pp. 347-354).
Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.
Keywords: Genetically modified organisms; GMO; Molecular techniques; DNA hybridization; Biosensors; Microarrays; Capillary electrophoresis; Real-time PCR; Competitive PCR
New trends in bioanalytical tools for the detection of genetically modified organisms: an update by Elisa Michelini; Patrizia Simoni; Luca Cevenini; Laura Mezzanotte; Aldo Roda (pp. 355-367).
Despite the controversies surrounding genetically modified organisms (GMOs), the production of GM crops is increasing, especially in developing countries. Thanks to new technologies involving genetic engineering and unprecedented access to genomic resources, the next decade will certainly see exponential growth in GMO production. Indeed, EU regulations based on the precautionary principle require any food containing more than 0.9% GM content to be labeled as such. The implementation of these regulations necessitates sampling protocols, the availability of certified reference materials and analytical methodologies that allow the accurate determination of the content of GMOs. In order to qualify for the validation process, a method should fulfil some criteria, defined as “acceptance criteria” by the European Network of GMO Laboratories (ENGL). Several methods have recently been developed for GMO detection and quantitation, mostly based on polymerase chain reaction (PCR) technology. PCR (including its different formats, e.g., double competitive PCR and real-time PCR) remains the technique of choice, thanks to its ability to detect even small amounts of transgenes in raw materials and processed foods. Other approaches relying on DNA detection are based on quartz crystal microbalance piezoelectric biosensors, dry reagent dipstick-type sensors and surface plasmon resonance sensors. The application of visible/near-infrared (vis/NIR) spectroscopy or mass spectrometry combined with chemometrics techniques has also been envisaged as a powerful GMO detection tool. Furthermore, in order to cope with the multiplicity of GMOs released onto the market, the new challenge is the development of routine detection systems for the simultaneous detection of numerous GMOs, including unknown GMOs.
Keywords: Genetically modified organism detection; PCR technology; Biosensors; Multiplexing; Sampling; Reference materials
New trends in bioanalytical tools for the detection of genetically modified organisms: an update by Elisa Michelini; Patrizia Simoni; Luca Cevenini; Laura Mezzanotte; Aldo Roda (pp. 355-367).
Despite the controversies surrounding genetically modified organisms (GMOs), the production of GM crops is increasing, especially in developing countries. Thanks to new technologies involving genetic engineering and unprecedented access to genomic resources, the next decade will certainly see exponential growth in GMO production. Indeed, EU regulations based on the precautionary principle require any food containing more than 0.9% GM content to be labeled as such. The implementation of these regulations necessitates sampling protocols, the availability of certified reference materials and analytical methodologies that allow the accurate determination of the content of GMOs. In order to qualify for the validation process, a method should fulfil some criteria, defined as “acceptance criteria” by the European Network of GMO Laboratories (ENGL). Several methods have recently been developed for GMO detection and quantitation, mostly based on polymerase chain reaction (PCR) technology. PCR (including its different formats, e.g., double competitive PCR and real-time PCR) remains the technique of choice, thanks to its ability to detect even small amounts of transgenes in raw materials and processed foods. Other approaches relying on DNA detection are based on quartz crystal microbalance piezoelectric biosensors, dry reagent dipstick-type sensors and surface plasmon resonance sensors. The application of visible/near-infrared (vis/NIR) spectroscopy or mass spectrometry combined with chemometrics techniques has also been envisaged as a powerful GMO detection tool. Furthermore, in order to cope with the multiplicity of GMOs released onto the market, the new challenge is the development of routine detection systems for the simultaneous detection of numerous GMOs, including unknown GMOs.
Keywords: Genetically modified organism detection; PCR technology; Biosensors; Multiplexing; Sampling; Reference materials
Methods for detection of GMOs in food and feed by Nelson Marmiroli; Elena Maestri; Mariolina Gullì; Alessio Malcevschi; Clelia Peano; Roberta Bordoni; Gianluca De Bellis (pp. 369-384).
This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.
Keywords: GMO traceability; Regulation and legislation; PCR methods; Sensor and microsensor; Validation of methods
Methods for detection of GMOs in food and feed by Nelson Marmiroli; Elena Maestri; Mariolina Gullì; Alessio Malcevschi; Clelia Peano; Roberta Bordoni; Gianluca De Bellis (pp. 369-384).
This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.
Keywords: GMO traceability; Regulation and legislation; PCR methods; Sensor and microsensor; Validation of methods
Binding properties of a monoclonal antibody against the Cry1Ab from Bacillus Thuringensis for the development of a capillary electrophoresis competitive immunoassay by Cristina Giovannoli; Laura Anfossi; Claudio Baggiani; Gianfranco Giraudi (pp. 385-393).
Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation conditions. The two binding constants appear comparable—(5.0 ± 1.2) × 106 M−1 and (9.06 ± 5.7) × 106 M−1—reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 μg L−1). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matrices.
Keywords: Capillary electrophoresis immunoassay; Binding constant determination; Cry1Ab; Genetically modified organism; Bacillus thuringensis
Binding properties of a monoclonal antibody against the Cry1Ab from Bacillus Thuringensis for the development of a capillary electrophoresis competitive immunoassay by Cristina Giovannoli; Laura Anfossi; Claudio Baggiani; Gianfranco Giraudi (pp. 385-393).
Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation conditions. The two binding constants appear comparable—(5.0 ± 1.2) × 106 M−1 and (9.06 ± 5.7) × 106 M−1—reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 μg L−1). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matrices.
Keywords: Capillary electrophoresis immunoassay; Binding constant determination; Cry1Ab; Genetically modified organism; Bacillus thuringensis
Real-time and conventional PCR detection of Liberty Link® rice varieties and transgenic soy in rice sampled in the Mexican and American retail markets by Maricarmen Quirasco; Bernd Schoel; Pradheep Chhalliyil; John Fagan; Amanda Gálvez (pp. 395-404).
Samples of rice from Mexican and USA retail stores were analyzed for the presence of transgenic (GM) events using real-time PCR. In screening for the CaMV35S promoter sequence (35SP), positive results were found in 49 and 35% of the Mexican and American samples, respectively. In further investigations in Mexican samples, 43% were positive for P35S::bar, with two above the quantifiable limit; these were 0.07% and 0.05% GMO. Fourteen out of the sixteen positive samples were labeled as imported from the USA. In testing samples bought in American retail shops, 24% showed positive results, all below the quantifiable range. It could be deduced that P35S::bar positive samples were Liberty Link® (LL) rice. In distinguishing between LL601 and LL62, end-point PCR was used, corroborating the P35S::bar amplicon length difference of these events. LL62 was found in one rice sample purchased in Mexico and two in the USA samples. Its presence was verified with the 35S terminator sequence. All other LL positive samples contained LL601. None of the samples analyzed showed the presence of Bt63 rice. The LL rice varieties found have been identified as not being commercially cultivated, and so their presence requires further investigation. 35SP was also present in samples which did not have any LL rice. Maize sequences could not be detected in any of the samples; however, soybean DNA was found in Mexican and USA rice samples. The Roundup Ready® trait was detected in trace amounts in 16 and 6% of the rice samples bought in Mexico and the USA, respectively. Real-time PCR was shown to be the method of choice for the sensitive and rapid screening of commodities and retail samples for the detection of GM and other contamination.
Keywords: GM rice; Quantitative PCR; LLRICE601; LLRICE62; P35S::bar ; Adventitious presence
Real-time and conventional PCR detection of Liberty Link® rice varieties and transgenic soy in rice sampled in the Mexican and American retail markets by Maricarmen Quirasco; Bernd Schoel; Pradheep Chhalliyil; John Fagan; Amanda Gálvez (pp. 395-404).
Samples of rice from Mexican and USA retail stores were analyzed for the presence of transgenic (GM) events using real-time PCR. In screening for the CaMV35S promoter sequence (35SP), positive results were found in 49 and 35% of the Mexican and American samples, respectively. In further investigations in Mexican samples, 43% were positive for P35S::bar, with two above the quantifiable limit; these were 0.07% and 0.05% GMO. Fourteen out of the sixteen positive samples were labeled as imported from the USA. In testing samples bought in American retail shops, 24% showed positive results, all below the quantifiable range. It could be deduced that P35S::bar positive samples were Liberty Link® (LL) rice. In distinguishing between LL601 and LL62, end-point PCR was used, corroborating the P35S::bar amplicon length difference of these events. LL62 was found in one rice sample purchased in Mexico and two in the USA samples. Its presence was verified with the 35S terminator sequence. All other LL positive samples contained LL601. None of the samples analyzed showed the presence of Bt63 rice. The LL rice varieties found have been identified as not being commercially cultivated, and so their presence requires further investigation. 35SP was also present in samples which did not have any LL rice. Maize sequences could not be detected in any of the samples; however, soybean DNA was found in Mexican and USA rice samples. The Roundup Ready® trait was detected in trace amounts in 16 and 6% of the rice samples bought in Mexico and the USA, respectively. Real-time PCR was shown to be the method of choice for the sensitive and rapid screening of commodities and retail samples for the detection of GM and other contamination.
Keywords: GM rice; Quantitative PCR; LLRICE601; LLRICE62; P35S::bar ; Adventitious presence
Gene delivery to cells on a miniaturized multiwell plate for high-throughput gene function analysis by Ellyana Njatawidjaja; Hiroo Iwata (pp. 405-408).
The transfectional microarray is a promising tool to conduct high-throughput analysis of gene function. In this study, a miniaturized multiwell plate was prepared by applying a silicone rubber sheet with holes on a gold electrode. The combination of electroporation and a miniaturized multiwell plate successfully achieved spatially controlled gene delivery with absence of cross-contamination between genes. Furthermore, we found that gene delivery efficiency was not dependent on conditions such as plasmid loading, electric field strength, and pulse duration.
Keywords: Miniaturized multiwell plate; Transfectional microarray; Electroporation; Plasmid DNA; Gene delivery
Gene delivery to cells on a miniaturized multiwell plate for high-throughput gene function analysis by Ellyana Njatawidjaja; Hiroo Iwata (pp. 405-408).
The transfectional microarray is a promising tool to conduct high-throughput analysis of gene function. In this study, a miniaturized multiwell plate was prepared by applying a silicone rubber sheet with holes on a gold electrode. The combination of electroporation and a miniaturized multiwell plate successfully achieved spatially controlled gene delivery with absence of cross-contamination between genes. Furthermore, we found that gene delivery efficiency was not dependent on conditions such as plasmid loading, electric field strength, and pulse duration.
Keywords: Miniaturized multiwell plate; Transfectional microarray; Electroporation; Plasmid DNA; Gene delivery
A high-efficiency sample introduction system for capillary electrophoresis analysis of amino acids from dynamic samples and static dialyzed human vitreous samples by Eric E. Patterson II; Sujeewa C. Piyankarage; Kyaw ThetMaw Myasein; Jose S. Pulido; Robert F. Dundervill III; R. Mark Hatfield; Scott A. Shippy (pp. 409-416).
A low-volume automated injection system for the analysis of chemically complex, amino acid samples is presented. This system utilizes submicroliter sample volumes stored on a 75-μm inner diameter capillary. A pulse of positive pressure (82 kPa) is used to load nanoliter sample volumes into an in-house fabricated interface and onto a separation capillary. Residual sample solution in the interface is immediately washed away by a continuous transverse flow through the injection interface, yielding a sharp and reproducible sample plug. By performing multiple injections of a static sample, one may average the signals to yield a signal-to-noise ratio improvement of up to 4.07-fold for 20 injections compared with a theoretical maximum of a 4.47-fold improvement. Without interruption of the applied voltage, injections performed every 150 s were used to monitor the progress of the reaction of multiple amino acids with the fluorogenic dye 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde. Analysis of dialyzed clinical vitreous samples demonstrates the resolution and quantitation of arginine, lysine, leucine, glutamine, and glutamate. Observed levels are comparable with those of nonautomated injection methods and reports by others. Figure Multiple injections of fluorescently labeled human vitreous with a detailed view of a single injection (above) and with all injections segmented and averaged for signal-to-noise ratio improvement (right)
Keywords: Signal averaging; Capillary electrophoresis; Injection; Sample introduction; Dialysis; Vitreous
A high-efficiency sample introduction system for capillary electrophoresis analysis of amino acids from dynamic samples and static dialyzed human vitreous samples by Eric E. Patterson II; Sujeewa C. Piyankarage; Kyaw ThetMaw Myasein; Jose S. Pulido; Robert F. Dundervill III; R. Mark Hatfield; Scott A. Shippy (pp. 409-416).
A low-volume automated injection system for the analysis of chemically complex, amino acid samples is presented. This system utilizes submicroliter sample volumes stored on a 75-μm inner diameter capillary. A pulse of positive pressure (82 kPa) is used to load nanoliter sample volumes into an in-house fabricated interface and onto a separation capillary. Residual sample solution in the interface is immediately washed away by a continuous transverse flow through the injection interface, yielding a sharp and reproducible sample plug. By performing multiple injections of a static sample, one may average the signals to yield a signal-to-noise ratio improvement of up to 4.07-fold for 20 injections compared with a theoretical maximum of a 4.47-fold improvement. Without interruption of the applied voltage, injections performed every 150 s were used to monitor the progress of the reaction of multiple amino acids with the fluorogenic dye 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde. Analysis of dialyzed clinical vitreous samples demonstrates the resolution and quantitation of arginine, lysine, leucine, glutamine, and glutamate. Observed levels are comparable with those of nonautomated injection methods and reports by others. Figure Multiple injections of fluorescently labeled human vitreous with a detailed view of a single injection (above) and with all injections segmented and averaged for signal-to-noise ratio improvement (right)
Keywords: Signal averaging; Capillary electrophoresis; Injection; Sample introduction; Dialysis; Vitreous
Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay by J. C. W. Rijk; T. F. H. Bovee; M. J. Groot; A. A. C. M. Peijnenburg; M. W. F. Nielen (pp. 417-425).
Prohormones such as dehydroepiandrosterone (DHEA) are steroid precursors that do not show hormonal activity by themselves. Abuse of these prohormones in cattle fattening is hard to prove because of strong in vivo metabolism and the difficulty to detect metabolites which are not significantly above endogenous levels. The aim of the present work was to develop an in vitro assay capable of detecting the indirect hormonal activity of prohormones that might be present in feed supplements and injection preparations. Sample extracts were incubated with a bovine liver S9 fraction in order to mimic the in vivo metabolic activation. Subsequently incubated extracts were exposed to a highly androgen-specific yeast bioassay to detect hormonal activity. Metabolic activation of DHEA, 4-androstene-3,17-dione (4-adione) and 5-androstene-3,17-diol (5-adiol) resulted in an increased androgenic activity caused by the formation of the active androgen 17β-testosterone (17β-T), as shown by ultra-performance liquid chromatography and time-of-flight mass spectrometry with accurate mass measurement. The developed in vitro system successfully mimics the hydroxysteroid dehydrogenase (HSD)- and cytochrome P450-mediated in vivo metabolic transitions, thus allowing assessment of both bioactivity and chemical identification without the use of animal experiments. Screening of unknown supplement samples claimed to contain DHEA resulted in successful bioactivation and positive screening results according to the androgen yeast biosensor.
Keywords: Bioactivation; Prohormone; Steroid; Bovine; Liquid chromatography; Mass spectrometry
Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay by J. C. W. Rijk; T. F. H. Bovee; M. J. Groot; A. A. C. M. Peijnenburg; M. W. F. Nielen (pp. 417-425).
Prohormones such as dehydroepiandrosterone (DHEA) are steroid precursors that do not show hormonal activity by themselves. Abuse of these prohormones in cattle fattening is hard to prove because of strong in vivo metabolism and the difficulty to detect metabolites which are not significantly above endogenous levels. The aim of the present work was to develop an in vitro assay capable of detecting the indirect hormonal activity of prohormones that might be present in feed supplements and injection preparations. Sample extracts were incubated with a bovine liver S9 fraction in order to mimic the in vivo metabolic activation. Subsequently incubated extracts were exposed to a highly androgen-specific yeast bioassay to detect hormonal activity. Metabolic activation of DHEA, 4-androstene-3,17-dione (4-adione) and 5-androstene-3,17-diol (5-adiol) resulted in an increased androgenic activity caused by the formation of the active androgen 17β-testosterone (17β-T), as shown by ultra-performance liquid chromatography and time-of-flight mass spectrometry with accurate mass measurement. The developed in vitro system successfully mimics the hydroxysteroid dehydrogenase (HSD)- and cytochrome P450-mediated in vivo metabolic transitions, thus allowing assessment of both bioactivity and chemical identification without the use of animal experiments. Screening of unknown supplement samples claimed to contain DHEA resulted in successful bioactivation and positive screening results according to the androgen yeast biosensor.
Keywords: Bioactivation; Prohormone; Steroid; Bovine; Liquid chromatography; Mass spectrometry
Development of saw palmetto (Serenoa repens) fruit and extract standard reference materials by Michele M. Schantz; Mary Bedner; Stephen E. Long; John L. Molloy; Karen E. Murphy; Barbara J. Porter; Karsten Putzbach; Catherine A. Rimmer; Lane C. Sander; Katherine E. Sharpless; Jeanice B. Thomas; Stephen A. Wise; Laura J. Wood; James H. Yen; Takashi Yarita; Agnes NguyenPho; Wendy R. Sorenson; Joseph M. Betz (pp. 427-438).
As part of a collaboration with the National Institutes of Health’s Office of Dietary Supplements and the Food and Drug Administration’s Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed two standard reference materials (SRMs) representing different forms of saw palmetto (Serenoa repens), SRM 3250 Serenoa repens fruit and SRM 3251 Serenoa repens extract. Both of these SRMs have been characterized for their fatty acid and phytosterol content. The fatty acid concentration values are based on results from gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS) analysis while the sterol concentration values are based on results from GC-FID and liquid chromatography with mass spectrometry analysis. In addition, SRM 3250 has been characterized for lead content, and SRM 3251 has been characterized for the content of β-carotene and tocopherols. SRM 3250 (fruit) has certified concentration values for three phytosterols, 14 fatty acids as triglycerides, and lead along with reference concentration values for four fatty acids as triglycerides and 16 free fatty acids. SRM 3251 (extract) has certified concentration values for three phytosterols, 17 fatty acids as triglycerides, β-carotene, and γ-tocopherol along with reference concentration values for three fatty acids as triglycerides, 17 fatty acids as free fatty acids, β-carotene isomers, and δ-tocopherol and information values for two phytosterols. These SRMs will complement other reference materials currently available with concentrations for similar analytes and are part of a series of SRMs being developed for dietary supplements.
Keywords: Saw palmetto; Serenoa repens ; Fatty acids; Phytosterols; Certified reference material; Standard reference material
Development of saw palmetto (Serenoa repens) fruit and extract standard reference materials by Michele M. Schantz; Mary Bedner; Stephen E. Long; John L. Molloy; Karen E. Murphy; Barbara J. Porter; Karsten Putzbach; Catherine A. Rimmer; Lane C. Sander; Katherine E. Sharpless; Jeanice B. Thomas; Stephen A. Wise; Laura J. Wood; James H. Yen; Takashi Yarita; Agnes NguyenPho; Wendy R. Sorenson; Joseph M. Betz (pp. 427-438).
As part of a collaboration with the National Institutes of Health’s Office of Dietary Supplements and the Food and Drug Administration’s Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed two standard reference materials (SRMs) representing different forms of saw palmetto (Serenoa repens), SRM 3250 Serenoa repens fruit and SRM 3251 Serenoa repens extract. Both of these SRMs have been characterized for their fatty acid and phytosterol content. The fatty acid concentration values are based on results from gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS) analysis while the sterol concentration values are based on results from GC-FID and liquid chromatography with mass spectrometry analysis. In addition, SRM 3250 has been characterized for lead content, and SRM 3251 has been characterized for the content of β-carotene and tocopherols. SRM 3250 (fruit) has certified concentration values for three phytosterols, 14 fatty acids as triglycerides, and lead along with reference concentration values for four fatty acids as triglycerides and 16 free fatty acids. SRM 3251 (extract) has certified concentration values for three phytosterols, 17 fatty acids as triglycerides, β-carotene, and γ-tocopherol along with reference concentration values for three fatty acids as triglycerides, 17 fatty acids as free fatty acids, β-carotene isomers, and δ-tocopherol and information values for two phytosterols. These SRMs will complement other reference materials currently available with concentrations for similar analytes and are part of a series of SRMs being developed for dietary supplements.
Keywords: Saw palmetto; Serenoa repens ; Fatty acids; Phytosterols; Certified reference material; Standard reference material
MALDI-TOF mass signatures for differentiation of yeast species, strain grouping and monitoring of morphogenesis markers by Jiang Qian; Jim E. Cutler; Richard B. Cole; Yang Cai (pp. 439-449).
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is demonstrated to be a potentially useful tool for the rapid identification of yeasts, the grouping of Candida albicans strains, and the monitoring of germ tube-specific markers. Co-crystallized with sinapinic acid as the MALDI matrix, intact yeast cells yielded a sufficient number of medium-sized ions (4–15 kDa) in MALDI mass spectra to provide “mass signatures” that were diagnostic of strain type. For most isolates, the mass signatures were affected by the growth medium, length of incubation and the cell preparation method. While the overall past success of this methodology for fungal cells has been relatively low compared to its application to bacteria, fixing the yeast cells in 50% methanol inactivated the cells, reduced cell aggregation in aqueous suspension solution, and more importantly, it significantly improved the mass signature quality. This simple but critical advance in sample treatment improved mass spectrometric signal-to-noise ratios and allowed the identification of yeasts by a mass signature approach. Under optimized conditions, Candida species (C. albicans, C. glabrata, C. krusei, C. kefyr), Aspergillus species (A. terreus, A. fumigatus, A. syndowii) and other yeast genera (Cryptococcus neoformans, Saccharomyces cerevisiae and a Rhodotorula sp.) could be distinguished. Within the C. albicans species, several common ions in the m/z 5,000–10,000 range were apparent in the mass spectra of all tested strains. In addition to shared ions, the mass spectra of individual C. albicans strains permitted grouping of the strains. Principal component analysis (PCA) was employed to confirm spectral reproducibility and C. albicans strain grouping by mass signatures. Finally, C. albicans germ tubes produced MALDI-TOF mass signatures that differed from yeast forms of this species. This is a rapid, sensitive and simple method for identifying yeasts, grouping strains and following the morphogenesis of C. albicans. Figure
Keywords: Mass signature; MALDI-TOF mass spectrometry; Alcohol fixation; Yeast differentiation; Principal component analysis
MALDI-TOF mass signatures for differentiation of yeast species, strain grouping and monitoring of morphogenesis markers by Jiang Qian; Jim E. Cutler; Richard B. Cole; Yang Cai (pp. 439-449).
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is demonstrated to be a potentially useful tool for the rapid identification of yeasts, the grouping of Candida albicans strains, and the monitoring of germ tube-specific markers. Co-crystallized with sinapinic acid as the MALDI matrix, intact yeast cells yielded a sufficient number of medium-sized ions (4–15 kDa) in MALDI mass spectra to provide “mass signatures” that were diagnostic of strain type. For most isolates, the mass signatures were affected by the growth medium, length of incubation and the cell preparation method. While the overall past success of this methodology for fungal cells has been relatively low compared to its application to bacteria, fixing the yeast cells in 50% methanol inactivated the cells, reduced cell aggregation in aqueous suspension solution, and more importantly, it significantly improved the mass signature quality. This simple but critical advance in sample treatment improved mass spectrometric signal-to-noise ratios and allowed the identification of yeasts by a mass signature approach. Under optimized conditions, Candida species (C. albicans, C. glabrata, C. krusei, C. kefyr), Aspergillus species (A. terreus, A. fumigatus, A. syndowii) and other yeast genera (Cryptococcus neoformans, Saccharomyces cerevisiae and a Rhodotorula sp.) could be distinguished. Within the C. albicans species, several common ions in the m/z 5,000–10,000 range were apparent in the mass spectra of all tested strains. In addition to shared ions, the mass spectra of individual C. albicans strains permitted grouping of the strains. Principal component analysis (PCA) was employed to confirm spectral reproducibility and C. albicans strain grouping by mass signatures. Finally, C. albicans germ tubes produced MALDI-TOF mass signatures that differed from yeast forms of this species. This is a rapid, sensitive and simple method for identifying yeasts, grouping strains and following the morphogenesis of C. albicans. Figure
Keywords: Mass signature; MALDI-TOF mass spectrometry; Alcohol fixation; Yeast differentiation; Principal component analysis
Cholesteric bonded stationary phases for high-performance liquid chromatography: a comparative study of the chromatographic behavior by Cédric Courtois; Guilhem Pagès; Stefano Caldarelli; Corinne Delaurent (pp. 451-461).
The properties of four cholesteric bonded stationary phases differing in the nature of the spacer and the end-capping were assessed using simple chromatographic tests based on the retention of nonpolar compounds and of planar or nonplanar probe solutes. All cholesteric columns showed a hydrophobicity close to that of conventional octadecyldimethylsilyl (ODS) materials. Non-end-capped cholesteric bonded phases showed greater selectivity than ODS ones and both end-capped cholesteric bonded phases exhibit behavior intermediate between that of the non-end-capped original material and that of the ODS bonded phase.
Keywords: High-performance liquid chromatography; Column characterization; Cholesteric bonded stationary phase
Cholesteric bonded stationary phases for high-performance liquid chromatography: a comparative study of the chromatographic behavior by Cédric Courtois; Guilhem Pagès; Stefano Caldarelli; Corinne Delaurent (pp. 451-461).
The properties of four cholesteric bonded stationary phases differing in the nature of the spacer and the end-capping were assessed using simple chromatographic tests based on the retention of nonpolar compounds and of planar or nonplanar probe solutes. All cholesteric columns showed a hydrophobicity close to that of conventional octadecyldimethylsilyl (ODS) materials. Non-end-capped cholesteric bonded phases showed greater selectivity than ODS ones and both end-capped cholesteric bonded phases exhibit behavior intermediate between that of the non-end-capped original material and that of the ODS bonded phase.
Keywords: High-performance liquid chromatography; Column characterization; Cholesteric bonded stationary phase
Thermoresponsive polymeric gel as a medium for examining interactions between dsDNA and an anticancer drug by Agata Kowalczyk; Anna M. Nowicka; Marcin Karbarz; Zbigniew Stojek (pp. 463-469).
A piece of dry N-isopropylacrylamide polymer was soaked in phosphate buffer to obtain a hydrogel which was then employed in the examination of interactions between an anticancer drug C-1311 (5-diethylaminoethyl-amino-8-hydroxyimidazoacridinone) and dsDNA. dsDNA was introduced into the polymer at the polymerization stage. The drug was added to the buffer. Using the volume phase transition of the gel at 40 °C, the unbound drug could be determined in the solution released during the transition, which made the calculations more reliable. The interaction parameters were calculated using the McGhee and von Hippel model. It appeared that in the gel medium, the interaction between the drug and dsDNA is spatially limited, since the number of binding units of the polymer chain occupied by one drug molecule was found to be one, while it was two in the regular buffer solution. Figure
Keywords: Drug–dsDNA interaction; Volume phase transition gel; Voltammetry
Thermoresponsive polymeric gel as a medium for examining interactions between dsDNA and an anticancer drug by Agata Kowalczyk; Anna M. Nowicka; Marcin Karbarz; Zbigniew Stojek (pp. 463-469).
A piece of dry N-isopropylacrylamide polymer was soaked in phosphate buffer to obtain a hydrogel which was then employed in the examination of interactions between an anticancer drug C-1311 (5-diethylaminoethyl-amino-8-hydroxyimidazoacridinone) and dsDNA. dsDNA was introduced into the polymer at the polymerization stage. The drug was added to the buffer. Using the volume phase transition of the gel at 40 °C, the unbound drug could be determined in the solution released during the transition, which made the calculations more reliable. The interaction parameters were calculated using the McGhee and von Hippel model. It appeared that in the gel medium, the interaction between the drug and dsDNA is spatially limited, since the number of binding units of the polymer chain occupied by one drug molecule was found to be one, while it was two in the regular buffer solution. Figure
Keywords: Drug–dsDNA interaction; Volume phase transition gel; Voltammetry
Controlled diffusion for laboratory solution preparation by Makoto Yoshida; Koji Tohda; Miklόs Gratzl (pp. 471-477).
We report here on a method for preparation of precise laboratory solutions using controlled diffusion for dosing. The desired chemical is delivered into the target solution from a stock solution through a mass-transport-limiting delivery port that blocks convection but allows reproducible diffusive transport. Solution making with this approach involves a single step irrespective of how low the desired concentration is. Diffusional delivery of chemicals involves no appreciable movement of water and, thus, no addition of volume. The approach is therefore particularly suitable for standard addition. Precise solutions of usual laboratory volumes can be made within a short time period with proper design of the delivery port or ports. Comparison with the performance of conventional methods of routine solution preparation shows that better precision can be achieved with less labor using this approach.
Keywords: Laboratory solution; Diffusional buret; Standard addition; Diffusional delivery; Precise reagent
Controlled diffusion for laboratory solution preparation by Makoto Yoshida; Koji Tohda; Miklόs Gratzl (pp. 471-477).
We report here on a method for preparation of precise laboratory solutions using controlled diffusion for dosing. The desired chemical is delivered into the target solution from a stock solution through a mass-transport-limiting delivery port that blocks convection but allows reproducible diffusive transport. Solution making with this approach involves a single step irrespective of how low the desired concentration is. Diffusional delivery of chemicals involves no appreciable movement of water and, thus, no addition of volume. The approach is therefore particularly suitable for standard addition. Precise solutions of usual laboratory volumes can be made within a short time period with proper design of the delivery port or ports. Comparison with the performance of conventional methods of routine solution preparation shows that better precision can be achieved with less labor using this approach.
Keywords: Laboratory solution; Diffusional buret; Standard addition; Diffusional delivery; Precise reagent
Development and validation of an efficient HPLC method for quantification of voriconazole in plasma and microdialysate reflecting an important target site by Franziska Simmel; Jens Soukup; Alexander Zoerner; Joachim Radke; Charlotte Kloft (pp. 479-488).
Voriconazole is a very potent antifungal agent used to treat serious fungal infections (candidiasis); it is also the therapy of choice for aspergillosis. After standard dosing, several factors affect exposure of voriconazole, resulting in large variability and demanding further elucidation of drug distribution. For measurements at the site of action, microdialysis is considered to be an outstanding minimally invasive method. For determination of voriconazole in microdialysate and human plasma a new, efficient, reliable, and robust HPLC assay using UV detection at 254 nm has been developed and validated. After simple sample preparation using acetonitrile for plasma and for microdialysate, 20 μL were injected and separated on an RP-18 column. The chromatographic run time was less than 4 min. Overall, the assay showed high precision (CV 93.9 to 99.5%) and accuracy (RE −96.7 to +107%) for both matrices. Of the 36 drug products typically co-administered with voriconazole, none except ambroxol interfered with its peak signal, and this interference was successfully managed. In summary, the method is highly suitable for application in (pre)clinical microdialysis studies, e.g., of critically ill patients with invasive mycoses. Figure Eye-catching image_abstract (PPT 369 KB)
Keywords: Voriconazole; Microdialysate; Bioanalytical methods; HPLC; Target site; Microlitre
Development and validation of an efficient HPLC method for quantification of voriconazole in plasma and microdialysate reflecting an important target site by Franziska Simmel; Jens Soukup; Alexander Zoerner; Joachim Radke; Charlotte Kloft (pp. 479-488).
Voriconazole is a very potent antifungal agent used to treat serious fungal infections (candidiasis); it is also the therapy of choice for aspergillosis. After standard dosing, several factors affect exposure of voriconazole, resulting in large variability and demanding further elucidation of drug distribution. For measurements at the site of action, microdialysis is considered to be an outstanding minimally invasive method. For determination of voriconazole in microdialysate and human plasma a new, efficient, reliable, and robust HPLC assay using UV detection at 254 nm has been developed and validated. After simple sample preparation using acetonitrile for plasma and for microdialysate, 20 μL were injected and separated on an RP-18 column. The chromatographic run time was less than 4 min. Overall, the assay showed high precision (CV 93.9 to 99.5%) and accuracy (RE −96.7 to +107%) for both matrices. Of the 36 drug products typically co-administered with voriconazole, none except ambroxol interfered with its peak signal, and this interference was successfully managed. In summary, the method is highly suitable for application in (pre)clinical microdialysis studies, e.g., of critically ill patients with invasive mycoses. Figure Microdialysis probe situated in the interstitial space fluid containing voriconazole drug molecules (magenta coloured) extracting an important target site representative matrix (microdialysate) [Courtesy of CMA]
Keywords: Voriconazole; Microdialysate; Bioanalytical methods; HPLC; Target site; Microlitre
Gas chromatography retention data of environmentally relevant polybrominated compounds by Walter Vetter; Natalie Rosenfelder (pp. 489-504).
Polybrominated organic compounds are ubiquitous throughout the environment. This generic term comprises several classes of brominated flame retardants (e.g., polybrominated diphenyl ethers, polybrominated biphenyls, hexabromocyclododecane, dibromopropyltribromophenyl ether, 1,2-bis(2,4,6-tribromophenoxy)ethane) as well as a range of marine halogenated natural products (HNPs). Here we present gas chromatography retention times and elution orders (on DB-5) of 122 polybrominated compounds that may be found in food and environmental samples. Organobromine compounds in fish samples determined with gas chromatography interfaced to electron-capture negative ion mass spectrometry (GC/ECNI-MS) are discussed. The environmental relevance and important mass spectrometric features of the compounds are described as well. Our database aims to support the closer inspection and identification of peaks in gas chromatograms and to initiate dedicated screening for less frequently studied organobromines in samples.
Keywords: Retention times; GC elution order; Polybrominated compounds; Polybrominated flame retardants; Naturally produced organobromines
Gas chromatography retention data of environmentally relevant polybrominated compounds by Walter Vetter; Natalie Rosenfelder (pp. 489-504).
Polybrominated organic compounds are ubiquitous throughout the environment. This generic term comprises several classes of brominated flame retardants (e.g., polybrominated diphenyl ethers, polybrominated biphenyls, hexabromocyclododecane, dibromopropyltribromophenyl ether, 1,2-bis(2,4,6-tribromophenoxy)ethane) as well as a range of marine halogenated natural products (HNPs). Here we present gas chromatography retention times and elution orders (on DB-5) of 122 polybrominated compounds that may be found in food and environmental samples. Organobromine compounds in fish samples determined with gas chromatography interfaced to electron-capture negative ion mass spectrometry (GC/ECNI-MS) are discussed. The environmental relevance and important mass spectrometric features of the compounds are described as well. Our database aims to support the closer inspection and identification of peaks in gas chromatograms and to initiate dedicated screening for less frequently studied organobromines in samples.
Keywords: Retention times; GC elution order; Polybrominated compounds; Polybrominated flame retardants; Naturally produced organobromines
Comparison of extraction and derivatization methods for fatty acid analysis in solid environmental matrixes by María Gómez-Brandón; Marta Lores; Jorge Domínguez (pp. 505-514).
This study involved comparison of different extraction and derivatization methods for determining FAs in soil and in four highly organic matrixes (cattle manure, pig slurry, compost, and vermicompost), by application of a multifactor categorical design. Although some studies have been carried out regarding the application of FA analysis to highly organic matrixes, comparison and verification are still required to test which methods of extraction and derivatization of FAs function best for these matrixes. We compared three extraction methods (one in which the same extraction mixture as used in the Folch method was employed, a modification of the Bligh and Dyer method, and a microwave-assisted extraction) and two derivatization procedures (alkaline methanolysis and derivatization with trimethylsulfonium hydroxide (TMSH)). The highest yields of FAs belonging to different structural classes, and of individual FAs used as microbial biomarkers were obtained by application of the same extraction mixture as in the Folch method and use of TMSH as derivatization agent. These methods also involved a significant reduction in the complexity and time involved in sample preparation.
Keywords: Fatty acid extraction; Fatty acid derivatization; Soil; Compost; Vermicompost; Microbial biomarkers
Comparison of extraction and derivatization methods for fatty acid analysis in solid environmental matrixes by María Gómez-Brandón; Marta Lores; Jorge Domínguez (pp. 505-514).
This study involved comparison of different extraction and derivatization methods for determining FAs in soil and in four highly organic matrixes (cattle manure, pig slurry, compost, and vermicompost), by application of a multifactor categorical design. Although some studies have been carried out regarding the application of FA analysis to highly organic matrixes, comparison and verification are still required to test which methods of extraction and derivatization of FAs function best for these matrixes. We compared three extraction methods (one in which the same extraction mixture as used in the Folch method was employed, a modification of the Bligh and Dyer method, and a microwave-assisted extraction) and two derivatization procedures (alkaline methanolysis and derivatization with trimethylsulfonium hydroxide (TMSH)). The highest yields of FAs belonging to different structural classes, and of individual FAs used as microbial biomarkers were obtained by application of the same extraction mixture as in the Folch method and use of TMSH as derivatization agent. These methods also involved a significant reduction in the complexity and time involved in sample preparation.
Keywords: Fatty acid extraction; Fatty acid derivatization; Soil; Compost; Vermicompost; Microbial biomarkers
An ensemble method based on a self-organizing map for near-infrared spectral calibration of complex beverage samples by Chao Tan; Xin Qin; Menglong Li (pp. 515-521).
Based on a so-called ensemble strategy, an algorithm is proposed for near-infrared (NIR) spectral calibration of complex beverage samples. This algorithm is a combination of a novel training set/test set sample-selection procedure based on a Kohonen self-organizing map (SOM) with a simple procedure to calculate an average partial least-squares (PLS) calibration model, which is therefore named SOMEPLS. In order to verify the proposed SOMEPLS, two NIR beverage datasets involving the determination of sugar content are considered, and three kinds of reference algorithm, i.e., conventional PLS (CPLS), the Kennard-Stone (KS) algorithm in combination with PLS (KSPLS), and sample set partitioning based on the joint x-y distance (SPXY) algorithm in combination with PLS (SPXYPLS), are used. Of these, both KS and SPXY are well-known representative sample-selection algorithms. By comparison, it was found that when there is a training set of appropriate size, SOMEPLS can achieve better prediction accuracy than the three reference algorithms, but without increasing the complexity of the corresponding calibration model for the future application, indicating that SOMEPLS can serve as a promising tool for NIR spectral calibration.
Keywords: Near-infrared; Calibration; Accuracy; Ensemble; Self-organizing map; Sugar concentration
An ensemble method based on a self-organizing map for near-infrared spectral calibration of complex beverage samples by Chao Tan; Xin Qin; Menglong Li (pp. 515-521).
Based on a so-called ensemble strategy, an algorithm is proposed for near-infrared (NIR) spectral calibration of complex beverage samples. This algorithm is a combination of a novel training set/test set sample-selection procedure based on a Kohonen self-organizing map (SOM) with a simple procedure to calculate an average partial least-squares (PLS) calibration model, which is therefore named SOMEPLS. In order to verify the proposed SOMEPLS, two NIR beverage datasets involving the determination of sugar content are considered, and three kinds of reference algorithm, i.e., conventional PLS (CPLS), the Kennard-Stone (KS) algorithm in combination with PLS (KSPLS), and sample set partitioning based on the joint x-y distance (SPXY) algorithm in combination with PLS (SPXYPLS), are used. Of these, both KS and SPXY are well-known representative sample-selection algorithms. By comparison, it was found that when there is a training set of appropriate size, SOMEPLS can achieve better prediction accuracy than the three reference algorithms, but without increasing the complexity of the corresponding calibration model for the future application, indicating that SOMEPLS can serve as a promising tool for NIR spectral calibration.
Keywords: Near-infrared; Calibration; Accuracy; Ensemble; Self-organizing map; Sugar concentration
Method development and validation for melamine and its derivatives in rice concentrates by liquid chromatography. Application to animal feed samples by Roberto Muñiz-Valencia; Silvia G. Ceballos-Magaña; Daniel Rosales-Martinez; Raquel Gonzalo-Lumbreras; Ana Santos-Montes; Angel Cubedo-Fernandez-Trapiella; Roberto C. Izquierdo-Hornillos (pp. 523-531).
An isocratic LC method for the determination of melamine and its degradation products (ammelide, ammeline, and cyanuric acid), used to increase the apparent protein content of rice protein concentrate, has been developed. Method development involved optimization of different RP columns, aqueous mobile phases, pH, phosphate concentration, and temperature. The optimum separation of these compounds was achieved using a Luna CN column (30 °C), 5 mmol L−1 sodium phosphate (pH 5.0) as mobile phase, 1 mL min−1 flow-rate, UV absorbance-DAD detection at 220 nm, and resorcine as internal standard; this enabled separation of these compounds with baseline resolution (values in the 2.1–10.1 range) in about 8 min. Prior to HPLC, the developed sample preparation procedure consisted in a leaching process using the above mentioned mobile phase. Method validation was carried out in rice protein concentrates in accordance with the European Commission decision 2002/657/EC criteria. For this purpose, eight mandatory performance characteristics for the conventional validation approach were determined: calibration graphs, extraction efficiencies, decision limits, detection capabilities, precision (repeatability and within-laboratory reproducibility), accuracy, selectivity, and robustness. The extraction efficiencies for these compounds were in the range 99–100% and the within-laboratory reproducibility at 1.0, 1.5, and 2.0 detection capabilities concentration levels were smaller than 5, 4, and 3%, respectively. Finally, the proposed method was successfully applied to the analysis of other rice protein concentrates and several animal feed samples.
Keywords: Rice protein concentrate; Animal feed; Melamine; Cyanuric acid; Ammelide; Ammeline; Liquid chromatography
Method development and validation for melamine and its derivatives in rice concentrates by liquid chromatography. Application to animal feed samples by Roberto Muñiz-Valencia; Silvia G. Ceballos-Magaña; Daniel Rosales-Martinez; Raquel Gonzalo-Lumbreras; Ana Santos-Montes; Angel Cubedo-Fernandez-Trapiella; Roberto C. Izquierdo-Hornillos (pp. 523-531).
An isocratic LC method for the determination of melamine and its degradation products (ammelide, ammeline, and cyanuric acid), used to increase the apparent protein content of rice protein concentrate, has been developed. Method development involved optimization of different RP columns, aqueous mobile phases, pH, phosphate concentration, and temperature. The optimum separation of these compounds was achieved using a Luna CN column (30 °C), 5 mmol L−1 sodium phosphate (pH 5.0) as mobile phase, 1 mL min−1 flow-rate, UV absorbance-DAD detection at 220 nm, and resorcine as internal standard; this enabled separation of these compounds with baseline resolution (values in the 2.1–10.1 range) in about 8 min. Prior to HPLC, the developed sample preparation procedure consisted in a leaching process using the above mentioned mobile phase. Method validation was carried out in rice protein concentrates in accordance with the European Commission decision 2002/657/EC criteria. For this purpose, eight mandatory performance characteristics for the conventional validation approach were determined: calibration graphs, extraction efficiencies, decision limits, detection capabilities, precision (repeatability and within-laboratory reproducibility), accuracy, selectivity, and robustness. The extraction efficiencies for these compounds were in the range 99–100% and the within-laboratory reproducibility at 1.0, 1.5, and 2.0 detection capabilities concentration levels were smaller than 5, 4, and 3%, respectively. Finally, the proposed method was successfully applied to the analysis of other rice protein concentrates and several animal feed samples.
Keywords: Rice protein concentrate; Animal feed; Melamine; Cyanuric acid; Ammelide; Ammeline; Liquid chromatography
Electroanalytical study of proflavine intercalation in 5-methyl or inosine-containing amplicons by Despina K. Alexiadou; Andrea K. Ioannou; Sofia A. Kouidou-Andreou; Anastasios N. Voulgaropoulos; Stella Th. Girousi (pp. 533-539).
Amplicons corresponding to the GC-rich p53 exon 5 and its analogues, synthesized by substituting 60% of cytosine by 5-methyl-cytosine, or 60% of guanosine by inosine and GC-poor p53 exon 6 were synthesized and investigated electrochemically, in the presence and absence of proflavine, by differential pulse voltammetry (DPV). Incorporation of base analogues and the thermal stability of the resulting amplicons were tested in the presence of a fluorescent probe (Sybr–Green). Peak current at 1.0 V was lower for methylated than for unmethylated PCR amplicons and was similarly affected by proflavine intercalation. In contrast, considerable peak current differences were observed in the presence of proflavine for unmodified exon 5 v.s. exon 6 or inosine-containing amplicons. Thermal analysis verified the expected shifts in melting temperature (T m) due to the base analogue incorporation and GC-content variations. In conclusion, methylated and unmethylated PCR amplicons could be distinguished in model DNA systems using differential pulse voltammetry (DPV) and use of proflavine could serve as an electrochemical probe for identifying different DNA conformations.
Keywords: Amplicons; 5-Methylcytosine; Inosine; Melting temperature; Differential pulse voltammetry; Proflavine
Electroanalytical study of proflavine intercalation in 5-methyl or inosine-containing amplicons by Despina K. Alexiadou; Andrea K. Ioannou; Sofia A. Kouidou-Andreou; Anastasios N. Voulgaropoulos; Stella Th. Girousi (pp. 533-539).
Amplicons corresponding to the GC-rich p53 exon 5 and its analogues, synthesized by substituting 60% of cytosine by 5-methyl-cytosine, or 60% of guanosine by inosine and GC-poor p53 exon 6 were synthesized and investigated electrochemically, in the presence and absence of proflavine, by differential pulse voltammetry (DPV). Incorporation of base analogues and the thermal stability of the resulting amplicons were tested in the presence of a fluorescent probe (Sybr–Green). Peak current at 1.0 V was lower for methylated than for unmethylated PCR amplicons and was similarly affected by proflavine intercalation. In contrast, considerable peak current differences were observed in the presence of proflavine for unmodified exon 5 v.s. exon 6 or inosine-containing amplicons. Thermal analysis verified the expected shifts in melting temperature (T m) due to the base analogue incorporation and GC-content variations. In conclusion, methylated and unmethylated PCR amplicons could be distinguished in model DNA systems using differential pulse voltammetry (DPV) and use of proflavine could serve as an electrochemical probe for identifying different DNA conformations.
Keywords: Amplicons; 5-Methylcytosine; Inosine; Melting temperature; Differential pulse voltammetry; Proflavine
Screening of humic and fulvic acids in estuarine sediments by near-infrared spectrometry by Javier Moros; Paloma Herbello-Hermelo; Antonio Moreda-Piñeiro; Pilar Bermejo-Barrera; Salvador Garrigues; Miguel de la Guardia (pp. 541-549).
Diffuse reflectance near-infrared spectroscopy (NIR) combined with partial least squares (PLS) data treatment has been employed for the rapid and nondestructive determination of sedimentary humic substances. Forty one samples of surface estuarine sediments, taken during distinct seasonal periods from different locations across Ria de Arousa (northwest of Spain), were scanned at wavelengths from 833 to 2,976 nm (12,000 to 3,360 cm−1). Twenty four samples were randomly selected, from previous hierarchical cluster analysis of their NIR spectra, for the calibration set, and the 17 remaining samples were assigned to the validation set. NIR spectra of calibration samples were correlated to measured values of humic acids (HAs) and fulvic acids (FAs), which ranged from 1.53 to 28.17 mg/g and from 0.37 to 2.45 mg/g, respectively, using PLS regression and multiplicative scattering correction on the raw and first-derivative NIR spectra, respectively. Low root mean square error of prediction values of 4.3 mg HA/g sediment and 0.25 mg FA/g sediment were obtained. Good residual prediction deviation values of 1.16 and 1.2 were obtained for HA and FA, respectively, allowing the PLS models built to be considered as appropriate tools for screening purposes.
Keywords: Hierarchical cluster analysis; Partial least squares; Diffuse reflectance Fourier transform near-infrared; Estuarine sediments; Humic and fulvic acids
Screening of humic and fulvic acids in estuarine sediments by near-infrared spectrometry by Javier Moros; Paloma Herbello-Hermelo; Antonio Moreda-Piñeiro; Pilar Bermejo-Barrera; Salvador Garrigues; Miguel de la Guardia (pp. 541-549).
Diffuse reflectance near-infrared spectroscopy (NIR) combined with partial least squares (PLS) data treatment has been employed for the rapid and nondestructive determination of sedimentary humic substances. Forty one samples of surface estuarine sediments, taken during distinct seasonal periods from different locations across Ria de Arousa (northwest of Spain), were scanned at wavelengths from 833 to 2,976 nm (12,000 to 3,360 cm−1). Twenty four samples were randomly selected, from previous hierarchical cluster analysis of their NIR spectra, for the calibration set, and the 17 remaining samples were assigned to the validation set. NIR spectra of calibration samples were correlated to measured values of humic acids (HAs) and fulvic acids (FAs), which ranged from 1.53 to 28.17 mg/g and from 0.37 to 2.45 mg/g, respectively, using PLS regression and multiplicative scattering correction on the raw and first-derivative NIR spectra, respectively. Low root mean square error of prediction values of 4.3 mg HA/g sediment and 0.25 mg FA/g sediment were obtained. Good residual prediction deviation values of 1.16 and 1.2 were obtained for HA and FA, respectively, allowing the PLS models built to be considered as appropriate tools for screening purposes.
Keywords: Hierarchical cluster analysis; Partial least squares; Diffuse reflectance Fourier transform near-infrared; Estuarine sediments; Humic and fulvic acids
Understanding otolith biomineralization processes: new insights into microscale spatial distribution of organic and mineral fractions from Raman microspectrometry by Aurélie Jolivet; Jean-François Bardeau; Ronan Fablet; Yves-Marie Paulet; Hélène de Pontual (pp. 551-560).
It is generally accepted that the formation of otolith microstructures (L- and D-zones) and in particular the organic and mineral fractions vary on a daily basis. Raman microspectrometry provides a nondestructive technique that can be used to provide structural information on organic and mineral compounds. We applied it to thin otolith sections of hake in order to address the following issues: (1) the simultaneous characterization of variations in the organic and mineral fractions both in the core area and along successive otolith microstructures; (2) elucidation of significant differences between these fractions; (3) quantification of the effects of etching and staining protocols on otolith structures. The primordium appeared as a punctual area depicting higher luminescence and greater concentrations in organic compounds containing CH groups. Sulcus side showed similar composition suggesting that the contact of the otolith with the macula and its orientation in otosac occur rapidly (about 10 days). The characterization of L- and D-zones in the opaque zones indicated that both structures contained organic and aragonitic fractions with cyclic and synchronous variations. Contrary to the results obtained after EDTA etching, L-zones depicted greater concentrations in organic compounds containing CH groups, whereas D-zones appear richer in aragonite. This organic fraction seemed to be revealed by Mutvei’s staining and was affected by EDTA etching which suggests that it corresponds to the soluble fraction of organic matrix. Such results indicate that L- and D-zones differ in their respective organic constituents. Raman microspectrometry thus appears as a powerful technique to acquire quantitative information that is required for a better understanding of otolith biomineralization. Figure Raman microspectrometry is a powerful technique for studying otolith biomineralization
Keywords: Core; L- and D-zones; Aragonite; Staining; Acid etching
Understanding otolith biomineralization processes: new insights into microscale spatial distribution of organic and mineral fractions from Raman microspectrometry by Aurélie Jolivet; Jean-François Bardeau; Ronan Fablet; Yves-Marie Paulet; Hélène de Pontual (pp. 551-560).
It is generally accepted that the formation of otolith microstructures (L- and D-zones) and in particular the organic and mineral fractions vary on a daily basis. Raman microspectrometry provides a nondestructive technique that can be used to provide structural information on organic and mineral compounds. We applied it to thin otolith sections of hake in order to address the following issues: (1) the simultaneous characterization of variations in the organic and mineral fractions both in the core area and along successive otolith microstructures; (2) elucidation of significant differences between these fractions; (3) quantification of the effects of etching and staining protocols on otolith structures. The primordium appeared as a punctual area depicting higher luminescence and greater concentrations in organic compounds containing CH groups. Sulcus side showed similar composition suggesting that the contact of the otolith with the macula and its orientation in otosac occur rapidly (about 10 days). The characterization of L- and D-zones in the opaque zones indicated that both structures contained organic and aragonitic fractions with cyclic and synchronous variations. Contrary to the results obtained after EDTA etching, L-zones depicted greater concentrations in organic compounds containing CH groups, whereas D-zones appear richer in aragonite. This organic fraction seemed to be revealed by Mutvei’s staining and was affected by EDTA etching which suggests that it corresponds to the soluble fraction of organic matrix. Such results indicate that L- and D-zones differ in their respective organic constituents. Raman microspectrometry thus appears as a powerful technique to acquire quantitative information that is required for a better understanding of otolith biomineralization. Figure Raman microspectrometry is a powerful technique for studying otolith biomineralization
Keywords: Core; L- and D-zones; Aragonite; Staining; Acid etching