Analytical and Bioanalytical Chemistry (v.399, #4)

Separation science of macromolecules by André M. Striegel (1399-1400).
is Assistant Professor in the Department of Chemistry and Biochemistry at Florida State University (FSU), where his group’s research focuses on the separation science of macromolecules. Dr Striegel is the author of nearly 50 peer-reviewed papers, lead co-author of the second edition of the book “Modern Size-Exclusion Liquid Chromatography” (2009), and editor of the book “Multiple Detection in Size-Exclusion Chromatography” (2005). He has served as inaugural Professor in Residence for Preservation Research and Testing at the US Library of Congress. Dr Striegel was the inaugural recipient of the ACS-DAC Award for Young Investigators in Separation Science, and has also received the Eli Lilly Analytical Chemistry Grantee Award, an FSU First Year Assistant Professor Award, and a Solutia Technical Achievement Award for his research in private industry before coming to academia. He has served on the editorial board of Analytical and Bioanalytical Chemistry since 2008 and, since 2009, as associate editor of the Encyclopedia of Analytical Chemistry.

An overview is given of the recent literature on (bio) analytical applications of flow field-flow fractionation (FlFFF). FlFFF is a liquid-phase separation technique that can separate macromolecules and particles according to size. The technique is increasingly used on a routine basis in a variety of application fields. In food analysis, FlFFF is applied to determine the molecular size distribution of starches and modified celluloses, or to study protein aggregation during food processing. In industrial analysis, it is applied for the characterization of polysaccharides that are used as thickeners and dispersing agents. In pharmaceutical and biomedical laboratories, FlFFF is used to monitor the refolding of recombinant proteins, to detect aggregates of antibodies, or to determine the size distribution of drug carrier particles. In environmental studies, FlFFF is used to characterize natural colloids in water streams, and especially to study trace metal distributions over colloidal particles. In this review, first a short discussion of the state of the art in instrumentation is given. Developments in the coupling of FlFFF to various detection modes are then highlighted. Finally, application studies are discussed and ordered according to the type of (bio) macromolecules or bioparticles that are fractionated.
Keywords: Flow field-flow fractionation; Biomacromolecules; Bioparticles; Molecular weight distribution; Particle size distribution

Size-exclusion chromatography (SEC) of branched polymers and polysaccharides by Marianne Gaborieau; Patrice Castignolles (1413-1423).
Branched polymers are among the most important polymers, ranging from polyolefins to polysaccharides. Branching plays a key role in the chain dynamics. It is thus very important for application properties such as mechanical and adhesive properties and digestibility. It also plays a key role in viscous properties, and thus in the mechanism of the separation of these polymers in size-exclusion chromatography (SEC). Critically reviewing the literature, particularly on SEC of polyolefins, polyacrylates and starch, we discuss common pitfalls but also highlight some unexplored possibilities to characterize branched polymers. The presence of a few long-chain branches has been shown to lead to a poor separation in SEC, as evidenced by multiple-detection SEC or multidimensional liquid chromatography. The local dispersity can be large in that case, and the accuracy of molecular weight determination achieved by current methods is poor, although hydrodynamic volume distributions offer alternatives. In contrast, highly branched polymers do not suffer from this extensive incomplete separation in terms of molecular weight. Figure Representation of (a) a linear polymer chain and various branched polymer structures with (b) longchain branches (amylose-like), (c) short-chain branches (amylopectin-like), (d) both short-chain and long-chain branches (polyacrylate- or polyethylene-like).
Keywords: Size-exclusion chromatography; Gel permeation chromatography; Branching; Polyolefins; Polyacrylates; Starch

Starch and glycogen are highly branched polymers of glucose of great importance to humans in managing and mitigating nutrition-related diseases, especially diabetes and obesity, and in industrial uses, for example in food and paper-making. Size-separation characterization using multiple-detection size-exclusion chromatography (SEC, also known as gel-permeation chromatography, GPC) is able to furnish substantial amounts of information on the relationships between the biosynthesis, processing, structure, and properties of these biopolymers, and achieves superior characterization for use in industrial product and process improvements. Multi-detector SEC is able to give much more information about structure than simple averages such as total molecular weight or size; the detailed information yielded by this technique has already given new information on important biosynthesis–structure–property reactions, and has considerable potential in this field in the future. However, it must be used with care to avoid artifacts arising from incomplete dissolution of the substrate and shear scission during separation. It is also essential in interpreting data to appreciate that this size-separation technique can only ever give size distributions, never true molecular weight distributions. Other size-separation techniques, particularly field-flow fractionation, require substantial technical development to be used on undegraded native starches. Figure The three structural levels of glycogen
Keywords: Biomaterials; Separations/theory; Field-flow fractionation; Foods/beverages; Polymers; Separations/instrumentation

Flow field-flow fractionation: recent trends in protein analysis by Diana Cristina Rambaldi; Pierluigi Reschiglian; Andrea Zattoni (1439-1447).
Flow field-flow fractionation (F4) is the gentlest flow-assisted separation technique for analysis of macromolecules. The use of an empty channel as separation device and of a second mobile phase flow as perpendicular field enable F4 to separate analytes under native conditions without any modification of their original structure. Because of this unique peculiarity, F4 has been shown to be ideal for “gentle” separation of biological samples, for example intact proteins and protein complexes, since its early development. Today’s F4 is an appealing technique which complements most established separation techniques, for example liquid chromatography and electrophoresis. The number of applications that show the unique advantages of F4 for analysis of protein samples is constantly increasing. In particular, F4 is finding increasing application on very high-molecular-weight species such as protein oligomers, aggregates, and complexes. This review critically discusses recent literature on the application of F4 to proteins. Either stand-alone or coupled with other characterization techniques, F4 is particularly promising for quality control of protein therapeutics, characterization of amyloid proteins, lipoprotein profiling, and as a pre-MS separation step in proteomics.
Keywords: Flow field-flow fractionation; Protein aggregation; Protein therapeutics; Amyloid proteins; Proteomics; Lipoproteins

Size-exclusion chromatography (gel filtration chromatography or gel permeation chromatography) in conjunction with online synchrotron radiation solution small-angle X-ray scattering optics, absorbance, and/or refractive index detectors was further assessed by application of biological macromolecules, such as the hollow sphere protein complex, apoferritin, and a linear polysaccharide, pullulan. The net X-ray scattering patterns of the eluted 24-mer molecule of apoferritin showed the specific character for the hollow spherical shape. The chromatographic (time-resolved) X-ray scattering data of the linear polysaccharide pullulan revealed the flexible chain structure during the chromatographic separation in an aqueous solution. These further applications demonstrated that the present measurement technique will be useful for not only the determination of the radius of gyration value of less than about 10 nm and molecular weight below several hundred thousand but also for the structural characterization of the various macromolecules during the chromatography. Figure Typical time-resolved scattering patterns and chromatograms in the SEC-SAXS measurement system
Keywords: Small-angle X-ray scattering; SEC-SAXS; GPC-SAXS; Radius of gyration; Molecular weight; Conformation

Asymmetrical-flow field-flow fractionation combined with multiangle light scattering and refractive index detection has been revealed to be a powerful tool for starch characterization. It is based on size separation according to the hydrodynamic diameter of the starch components. Starch from a wide range of different botanical sources were studied, including normal starch and high-amylose and high-amylopectin starch. The starch was dissolved by heat treatment at elevated pressure in a laboratory autoclave. This gave clear solutions with no granular residues. Amylose retrogradation was prevented by using freshly dissolved samples. Programmed cross flow starting at 1.0 mL min−1 and decreasing exponentially with a half-life of 4 min was utilised. The starches showed two size populations representing mainly amylose and mainly amylopectin with an overlapping region where amylose and amylopectin were possibly co-eluted. Most of the first population had molar masses below 106 g mol−1, and most of the second size population had molar masses above 107 g mol−1. Large differences were found in the relative amounts of the two populations, the molar mass, and hydrodynamic diameters, depending on the plant source and its varieties.
Keywords: Molar mass; Starch; Size separation; Hydrodynamic diameter; Field-flow fractionation; Multiangle light scattering

Behavior of polysaccharide assemblies in field-flow fractionation and size-exclusion chromatography by Leena Pitkänen; Maija Tenkanen; Päivi Tuomainen (1467-1472).
Asymmetric flow field-flow fractionation (AsFlFFF) and high-performance size-exclusion chromatography (HPSEC) are techniques for separating and characterizing macromolecules; until now the latter is more utilized for analyzing polysaccharides. The demand for characterizing complex, high-molar-mass polysaccharides has raised interest in the use of AsFlFFF in analyzing polymeric carbohydrates in addition to HPSEC. In this paper, we compare the behavior of arabinoxylan aggregates present in aqueous solution in AsFlFFF and HPSEC and their effect on the obtained molecular characteristics (molar mass averages and size). Although the amount of aggregates in aqueous arabinoxylan solutions may be low, their role needs to be understood to avoid erroneous interpretations of AsFlFFF and HPSEC data. When these two separation systems were compared, AsFlFFF seemed to possess more separation power for the differentiation of aggregates from individual chains than HPSEC. To our knowledge, this is the first report on the characterization of xylans with AsFlFFF.
Keywords: Size-exclusion chromatography; Asymmetric flow field-flow fractionation; Polysaccharides; Arabinoxylans; Aggregates

Comparison of separation performance of laser-ablated and wet-etched microfluidic devices by Christopher A. Baker; Rayford Bulloch; Michael G. Roper (1473-1479).
Laser ablation of glass allows for production of microfluidic devices without the need for hydrofluoric acid and photolithography. The goal of this study was to compare the separation performance of microfluidic devices produced using a low-cost laser ablation system and conventional wet etching. During laser ablation, cracking of the glass substrate was prevented by heating the glass to 300 °C. A range of laser energy densities was found to produce channel depths ranging from 4 to 35 μm and channel widths from 118 to 162 μm. The electroosmotic flow velocity was lower in laser-ablated devices, 0.110 ± 0.005 cm   s−1, as compared to wet-etched microfluidic chips, 0.126 ± 0.003 cm   s−1. Separations of both small and large molecules performed on both wet- and laser-ablated devices were compared by examining limits of detection, theoretical plate count, and peak asymmetry. Laser-induced fluorescence detection limits were 10 pM fluorescein for both types of devices. Laser-ablated and wet-etched microfluidic chips had reproducible migration times with ≤   2.8% relative standard deviation and peak asymmetries ranged from 1.0 to 1.8. Numbers of theoretical plates were between 2.8- and 6.2-fold higher on the wet-etched devices compared to laser-ablated devices. Nevertheless, resolution between small and large analytes was accomplished, which indicates that laser ablation may find an application in pedagogical studies of electrophoresis or microfluidic devices, or in settings where hydrofluoric acid cannot be used.
Keywords: Laser ablation; Electrophoresis; Microfluidic; Separation

Relaxation of microparticles exposed to hydrodynamic forces in microfluidic conduits by Josef Janča; Věra Halabalová; Vladimír Polášek; Martin Vašina; Anastasia Yu. Menshikova (1481-1491).
The behavior of microparticles exposed to gravitational and lift forces and to the velocity gradient in flow velocity profile formed in microfluidic conduits is studied from the viewpoint of the transient period (the relaxation) between the moment at which a particle starts to be transported by the hydrodynamic flow and the time at which it reaches an equilibrium position, characterized by a balance of all active forces. The theoretical model allowing the calculation of the relaxation time is proposed. The numerical calculus based on the proposed model is compared with the experimental data obtained under different experimental conditions, namely, for different lengths of microfluidic channels, different average linear velocities of the carrier liquid, and different sizes and densities of the particles used in the study. The results are important for the optimization of microfluidic separation units such as microthermal field-flow fractionation channels in which the separation or manipulation of the microparticles of various origin, synthetic, natural, biological, etc., is performed under similar experimental conditions but by applying an additional thermodynamic force. Figure Trajectory of a particle transported by fluid flow in microfluidic channel and undergoing the effect of external filed force and lift force.
Keywords: Microthermal field-flow fractionation; Focusing mechanism; Relaxation; Microfluidic separations; Micrometer-sized particles; Theoretical model and experiments at low Reynolds numbers

Starch consists of a mixture of two α-glucans built mainly upon α-(1,4) linkages: amylose, an essentially linear polymer, and amylopectin, a branched polymer containing 5–6% of α-(1,6) linkages. The aim of the present work was to analyze the structural properties of native starches displaying different amylose-to-amylopectin ratios and arising from different botanical sources, using asymmetrical flow field flow fractionation (A4F) and a combination of hydrodynamic and size-exclusion chromatography (HDC-SEC) coupled with multiangle laser light scattering, online quasi-elastic light scattering, and differential refractive index techniques. The procedure, based upon dimethyl sulfoxide pretreatment and then solubilization in water, generates a representative injected sample without altering the initial degree of polymerization. The amylopectin weight-average molar masses and radii of gyration were around 1.0 × 108–4.8 × 108 g mol-1 and 110–267 nm, respectively. For each starch sample, the hydrodynamic radius (R H) distributions and the molar mass distributions obtained from the two fractionation systems coupled with light scattering techniques were analyzed. The size determination scales were extended by means of R H calibration curves. HDC-SEC and A4F data could be matched. However, A4F enabled a better separation of amylopectins and therefore an enhanced structural characterization of the starches. The two advantages of this experimental approach are (1) it can directly obtain distributions as a function of both molar mass and size, while taking account of sample heterogeneity, and (2) it is possible to compare the results obtained using the different techniques through the direct application of R H distributions. Figure Compared macromolecular size distributions of Wild Type Rice Starch obtained using A4F and HDC-SEC
Keywords: Asymmetrical flow field flow fractionation; Hydrodynamic chromatography; Hydrodynamic radius calibration curves; Macromolecular size distribution; Starch

The biological application of nanoparticles has resulted in an increased need for the development of robust, accurate, and precise methods for quality control analysis and characterization. Parameters such as particle size, particle shape, and their distributions affect end-use properties such as chemical reactivity, diffusivity, permeability, and transport. Introduced here is a hydrodynamic chromatography (HDC) method utilizing multi-angle static light scattering, quasi-elastic light scattering, differential viscometry, and differential refractometry detection for characterizing nanoscale vesicles. Quadruple-detector HDC was used to determine multiple sizing parameters and their statistical moments and distributions. Molar mass and molar mass averages were determined in a calibrant-independent fashion. Both the sizing parameters and the molar mass were measured across the HDC elution profile. The shape and structure of the nanoparticle were monitored as a function of HDC elution volume through the dimensionless ratio ρ ≡ R G,z /R H,z . The HDC results were comparable to those obtained by transmission electron microscopy, but more extensive characterization was possible by HDC, which provided distributions of both particle size and particle shape. Table of contents graphic
Keywords: Polymers; Hydrodynamic chromatography; Particle sizing; Shape analysis; Structural analysis; Drug delivery

The effect of different types of short- and long-range intrachain interactions along the polymeric backbone on the persistence length of a polymer, as well as on other properties such as solvation (characterized by the second virial coefficient), dilute solution conformation, specific refractive index increment, and intrinsic viscosity, were studied using multi-detector size-exclusion chromatography and off-line techniques. The polymers in this study, namely, polystyrene (PS), poly(vinyl chloride) (PVC), and poly(p-vinylbenzyl chloride) (PpVBC), were chosen based on intrachain interactions specific to each, intrachain repulsion in PVC, attraction in PS, and hindered attraction in PpVBC, and also based on a coincidence in molar mass averages and distributions between the polymers. The latter allowed polymeric properties of the three polymers to be compared to each other at the same molar mass and/or degree of polymerization. From the comparisons emerged the effects of intrachain repulsion between consecutive monomers and of the second virial coefficient on chain stiffness and solvation. The increase in the second virial coefficient corresponded to an increase in both polymer solvation and rigidity, while increased intrachain repulsion between consecutive monomers increased polymer solvation while decreasing chain rigidity. Figure Factors affecting persistence length
Keywords: Polymers; Multi-detector size-exclusion chromatography; Persistence length; Second virial coefficient; Intrachain interactions; Dilute solution properties

It is shown that the molar mass distribution of polyethylene glycols (PEGs) and their monomethyl ethers can be determined by liquid adsorption chromatography (LAC) on reversed phases using isocratic or gradient elution. In gradient LAC, the evaporative light scattering detector (ELSD) has to be used, which is, however, problematic with respect to quantification. The response factors of the individual oligomers depend strongly on the operating conditions, molar mass, and sample size. These problems do not arise with density and refractive index detection, which can, however, only be applied with isocratic elution. A comparison of the results obtained with these three detectors showed that calibration of the ELSD has to be performed very carefully. Figure Molar mass distribution of PEG 600, as obtained by LAC with different detectors
Keywords: Polyethylene glycol; Liquid adsorption chromatography; Density detector; RI detector; ELSD; Molar mass distributions

Different polymer homologous series having the same repeat unit, but different end groups, can be separated by one-dimensional LAC according to the number of repeat units and functionality, if a favorable combination of the interaction parameters of the repeat unit and the end groups can be found. As an example, polyethylene glycol (PEG) can be determined in PEG monomethyl ethers. The molar mass distribution of the minor component in such samples can be determined even at concentrations of a few percent. LAC of PEG-MME 350 containing different amounts of PEG 600 and the pure samples on the Synergi MAX RP column in 12.15 wt-% acetone at 25°C. Detection: RI
Keywords: Polyethylene glycol; Monomethyl ether; Functionaliy; Liquid adsorption chromatography; Interaction parameter

Separation of short-chain branched polyolefins by high-temperature gradient adsorption liquid chromatography by Tibor Macko; Robert Brüll; Rufina G. Alamo; Florian J. Stadler; Simone Losio (1547-1556).
A new separation principle was recently introduced into the analytical characterization of polyolefins by researchers from the German Institute for Polymers in Darmstadt. It was demonstrated that polyolefins can be selectively separated via high-performance liquid chromatography on the basis of their adsorption/desorption behaviours at temperatures as high as 160 °C. A Hypercarb® column packed with porous graphite gave the best results. The mobile phase consisted of a mixture of 1-decanol and 1,2,4-trichlorobenzene. In this work, the same chromatographic system is applied to the separation of ethylene/alkene and ethylene/norbornene copolymers. It was found that the elution volumes of the samples correlate linearly with the average chemical composition of samples. The elution volume is indirectly proportional to the concentration of branches in the ethylene/alkene copolymer. Branching shortens the length of continuous methylene sequences of the polymer backbone, thus decreasing the probability of orientation of a methylene sequence in a flat conformation on the graphite surface, which enables the most intensive van der Waals interactions between the methylene backbone and the carbon surface. An opposite trend in the elution order has been found for ethylene/norbornene copolymers. The elution volume of the ethylene/norbornene copolymers increased with the concentration of norbornene. It indicates pronounced attractive interactions between graphite and the cyclic comonomer. Figure Overlay of chromatograms and dependence of the elution volume on the average chemical composition of ethylene/1-butene copolymers.
Keywords: Polyolefins; Liquid chromatography; Adsorption; Graphite

The introduction of single-site catalysts in the polyolefins industry opens new routes to design resins with improved performance through multicatalyst-multireactor processes. Physical combination of various polyolefin types in a secondary extrusion process is also a common practice to achieve new products with improved properties. The new resins have complex structures, especially in terms of composition distribution, and their characterization is not always an easy task. Techniques like temperature rising elution fractionation (TREF) or crystallization analysis fractionation (CRYSTAF) are currently used to characterize the composition distribution of these resins. It has been shown that certain combinations of polyolefins may result in equivocal results if only TREF or CRYSTAF is used separately for their characterization.
Keywords: CRYSTAF; TREF; Polypropylene; Polyethylene; Fractionation of polymers

A high-throughput three-capillary noise-cancelling viscometer for chromatographic systems by Wallace W. Yau; Rongjuan Cong; David Gillespie (1563-1570).
This paper describes an analytical three-capillary viscometer detector that eliminates the traditional viscometer “hold-up” volume (commonly found in four-capillary designs) while maintaining cancellation of short-term pump noise and long-term baseline drift of temperature and solvent flow rate that are inherent in chromatography systems. This improvement allows a staggered sample injection approach in chromatography, yielding a significant increase in sample throughput by cutting down the chromatographic run time. It also provides a more robust design as it does not require capillary rebalancing, complex purging, flushing or changing the hold-up volume to accommodate long-term chromatographic use. Example of a four-capillary viscometer in a quad-detector GPC system.
Keywords: Polymers; Online viscometer; HPLC SEC–GPC

Zhiquo Wang and Baofeng Yang: MicroRNA expression detection methods by David Broyles; Sapna K. Deo (1575-1576).

Nanoparticles for the development of improved (bio)sensing systems by Briza Pérez-López; Arben Merkoçi (1577-1590).
Nanoparticles serve as fundamental building blocks for nanobiotechnology, especially in several applications in the development of novel (bio)sensing systems. Nanoparticles can be used for modification of the surfaces of (bio)sensing transducers or as optical or electroactive labels to improve different aspects of performance, for example sensitivity, detection limit, multidetection capability, and response stability. Nanoparticles can be integrated into the transducer materials on an individual basis or inside other matrices to ensure the immobilization of recognition biomolecules and/or receptors which are the principal components of the (bio)sensing systems. Incorporation of nanoparticles into optical and electrochemical (bio)sensing systems, including their use in microfluidic based systems has the advantages of enabling the design of robust, easy to use, portable, and cost-effective devices. Figure The various detection platforms opportunities ranging from electrochemical, optical including the integrated microfluidics offered by nanoparticles so as to improve (bio)sensing systems
Keywords: Nanoparticles; Electrochemical methods; Optical methods; (Bio)sensing systems; Analytical applications

Nucleic acid aptamers have recently attracted significant attention in the field of clinical diagnosis because they have numerous merits, such as high affinity, high specificity, small size, little immunogenicity, stable structures, and ease of synthesis. This review focuses on discussing the potential applications of aptamers in cell detection and molecular imaging. For the ex vivo cell detection, this review discusses the status of five strategies: endogenous nucleic acid analysis, flow cytometry analysis, nanoparticle-based cell sensing, microfluidic cell separation, and histological examination. This review also discusses in vivo molecular and cell imaging by introducing aptamer-based molecular imaging, cell imaging, and integrated imaging and therapy. On the basis of the status of these promising studies, this review summarizes several challenging issues and unmet needs that may require more effort or attention in the future.
Keywords: Nucleic acid aptamer; Antibody mimic; Antibody; Dendrimer; Nanomaterial; Nanoparticle; Molecular recognition; Ultrasensitive detection; Molecular imaging; Cell imaging

Recombinant therapeutic antibodies have shown a great potential in the treatment of several severe medical conditions such as cancer and autoimmune diseases. Glycosylation plays a critical role in biological activity and immunogenic properties of these compounds. The analysis of glycan profiles is therefore necessary in many steps of the development and manufacturing process from early development to quality control of the final product. In this paper, a fast, parallel, and robust sample preparation platform for glycosylation profiling using a microfluidic compact disc (CD) is presented. A sequential process including selective capture of antibody from a crude cell supernatant using protein A beads, enzymatic release of glycans, purification with a graphitized carbon black column, and crystallisation for MALDI-TOF analysis were performed on the CD. Glycosylation profiles of an antibody intended for therapeutic use produced in two different cell lines were compared. Figure Automated sample preparation for MALDI-MS profiling of therapeutic antibody glycosylation is performed using a microfluidic compact disc.
Keywords: Microfluidics/Microfabrication; Microfluidic CD; Glycosylation profiling; Recombinant therapeutic antibodies; MALDI-MS; Biotechnological products

Interpretation of 41Ca data using compartmental modeling in post-menopausal women by Wang-Hee Lee; Meryl E. Wastney; George S. Jackson; Berdine R. Martin; Connie M. Weaver (1613-1622).
Calcium-41 (t 1/2 = 105 years) can be used after a single dose to follow calcium metabolism over a subject's lifetime. The aims of this study were to expand a 41Ca kinetic model and estimate bone resorption in women with stable bone loss, compare the rates with those calculated with classical isotope studies, and to use the model to simulate dynamic changes in urinary 41Ca:Ca ratios and bone balance for the design and interpretation of 41Ca studies. Forty-two women >5 years post-menopause were given 41Ca intravenously. Bone mineral content and bone mineral density of total body were measured by dual-energy X-ray absorptiometry at the beginning of the study. Urine collections were made periodically for up to ∼5 years while subjects were free living. Urinary 41Ca:Ca ratios were measured using accelerator mass spectrometry. The isotope data were analyzed by compartmental modeling. Four compartments were necessary to fit the urinary tracer data and total bone calcium. The final model included pathways for absorption, distribution, urinary excretion, and endogenous excretion and was used to calculate rates of bone turnover. Estimates of bone resorption in a subset of the women (n = 13), studied previously in a 3-week balance and full kinetic study with 45Ca, agreed with those using 41Ca methodology. Thus, rates of bone resorption can be estimated from 41Ca urinary data in stable post-menopausal women. The model was used to simulate dynamic changes in urinary 41Ca:Ca ratios and bone balance, as a result of interventions that perturb calcium metabolism to aid in study design and interpretation.
Keywords: 41Ca; Compartmental modeling; Bone resorption; Bone balance; Dynamic simulation

A fast gas chromatography (GC)-MS method has been developed and validated for the simultaneous screening of different classes of drugs of abuse in urine. Tetrahydrocannabinol metabolite, cocaine, opiates such as morphine, O-6-monoacetylmorphine (O-6-MAM), codeine, opioids such as buprenorphine, methadone, pentazocine, fentanyl and analogues and their main metabolites can be detected and quantified after a simple liquid–liquid extraction in alkaline conditions and derivatisation to obtain the corresponding trimethylsilyl derivatives. The chromatographic separation is performed in a total time of 6 min, using a short GC column (5% phenyl methyl silicone, 10-m length × 0.18-mm internal diameter). The Limits of Detection are satisfactory for forensic purposes for all the substances; the repeatability of concentrations (percent coefficients of variation) are always lower than 15% at high and low concentration levels, and accuracy, intended as % error on the true value, is always lower than 15% for all the analytes. The method can successfully be applied for screening analyses in many fields of forensic toxicology.
Keywords: Drugs of abuse; Fast GC; Forensic toxicology; Drugs screening; GC-MS

Evaluation of extraction methods for quantification of aqueous fullerenes in urine by Troy M. Benn; Benny F. G. Pycke; Pierre Herckes; Paul Westerhoff; Rolf U. Halden (1631-1639).
There is a growing concern about the human and environmental health effects of fullerenes (e.g., C60) due to their increasing application in research, medicine, and industry. Toxicological and pharmacokinetic research requires standard methods for extraction and detection of fullerenes from biological matrices such as urine. The present study validates the use of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) methods in conjunction with liquid chromatography–mass spectrometry (LC–MS) for the quantitative determination of C60 in human and synthetic urine as compared with ultrapure water. Glacial acetic acid, which is necessary to prevent emulsions during LLE, inhibited C60 detection by LC–MS, but this could be mitigated with evaporation. Aqueous C60 aggregates (nC60) were spiked at 180 μg/L into the components of a synthetic urine recipe to determine their individual impacts on extraction and detection. Urea, creatinine, and a complex protein (i.e., gelatin) were found to impair SPE, leading to a low recovery rate of 43 ± 4% for C60 spiked into human urine. In contrast, C60 was consistently recovered from synthetic matrices using LLE, and recovery in human urine was 80 ± 6%. These results suggest that LLE combined with LC–MS is suitable for studying the clearance of fullerenes from the body. LLE is a robust technique that holds promise for extracting C60 from other complex biological matrices (e.g., blood, sweat, amniotic fluid) in toxicological studies, enabling a better understanding of the behavior of fullerenes in human and animal systems and facilitating a more comprehensive risk evaluation of fullerenes.
Keywords: Solid-phase extraction; Nano; C60 ; Environment; Fate; Chromatography; Mass spectrometry

This paper analyzes the equilibria between immunoglobulins (R 2), homo-bifunctional ligands (L 2), monovalent ligands (I), and their complexes. We present a mathematical model that can be used to estimate the concentration of each species present in a mixture of R 2, L 2, and I, given the initial conditions defining the total concentration of R 2, L 2, I, and four dissociation constants ( $$ K_{ m{d}}^{ m{inter}} $$ , $$ K_{ m{d}}^{ m{intra}} $$ , $$ K_{ m{d}}^{ m{mono}} $$ , and α). This model is based on fewer assumptions than previous models and can be used to describe exactly a broad range of experimental conditions. A series of curves illustrates the dependence of the equilibria upon the total concentrations of receptors and ligands, and the dissociation constants. We provide a set of guidelines for the design and analysis of experiments with a focus on estimating the binding constants from experimental binding isotherms. Two analytical equations relate the conditions for maximum aggregation in this system to the binding constants. This model is a tool to quantify the binding of immunoglobulins to antigens and a guide to understanding and predicting the experimental data of assays and techniques that employ immunoglobulins. Figure The fraction of immunoglobulin-bivalent ligand complexes that contain two or more immunoglobulins ( $$ {F_{{ ilde{1}}}} $$ ) depends on the total concentration of immunoglobulins. A series of curves illustrates the dependence of the equilibria upon the total concentrations of immunoglobulins, bivalent ligands, monovalent ligands, and the dissociation constants that describe the system
Keywords: Affinity; Aggregation; Antibody; Bivalent ligand; Binding isotherm

Labeling of fibronectin by fluorescent and paramagnetic nanoprobes for exploring the extracellular matrix: bioconjugate synthesis optimization and biochemical characterization by Céline Hoffmann; Anne-Charlotte Faure; Cedric Vancaeyzeele; Stéphane Roux; Olivier Tillement; Emmanuel Pauthe; Fabrice Goubard (1653-1663).
In this study, fibronectin–nanoparticles bioconjugates are developed and characterized. Multilabeled nanoparticles are composed of a core of the rare-earth oxide Gd2O3:Tb3+, capped with a set of Rhodamine B isothiocyanate encapsulated in a silica matrix and functionalized by a carboxylated polyethylene glycol shell. These nanoparticles are stabilized in aqueous solution and are found to contain about 400 carboxyl groups on their surface. Nanoparticle bioconjugation with highly purified human plasma fibronectin (Fn) is mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, resulting in an amide linkage between the carboxylic acid-terminated surface of the nanoparticle and the primary amine of Fn. The bioconjugation temperature and pH are optimized. The Local structure and global conformation of fibronectin–nanoparticle bioconjugates (FnNP*) are studied by fluorescence spectroscopy and enzymatic sites accessibility. Protein biochemical functionalities are globally conserved, and the protein is actually labeled. Elaboration of such complexes provides a promising bimodal contrasting agent for in vivo imaging. Figure Fluorescence microscopy image of double labeled fibronectin; Left : immunostaining signal; Right: core-shell nanoparticle signal
Keywords: Nanoparticles; Fibronectin; Bioconjugation; EDC/NHS

Selective determination of cysteines through precolumn double-labeling and liquid chromatography followed by detection of intramolecular FRET by Makoto Yoshitake; Hitoshi Nohta; Naoko Sejima; Kenichiro Todoroki; Hideyuki Yoshida; Masatoshi Yamaguchi (1665-1675).
In this paper we introduce a novel approach for highly selective and sensitive analysis of cysteines (glutathione, cysteine, and homocysteine). This method is based on the detection of intramolecular fluorescence resonance energy transfer (FRET) in a liquid chromatography (LC) system after double-labeling of the amino and sulfhydryl groups of the cysteines. In this detection process, we monitored the FRET between the amine-derivatized and thiol-derivatized fluorophores. We screened 16 combinations of fluorescent reagents. As a result, FRET occurred most effectively when the sulfhydryl and amino groups of the cysteines were derivatized with 7-diethylamino-3-[{4′-(iodoacetyl)amino}phenyl]-4-methylcoumarin (DCIA, Ex/Em 390/480 nm) and 4-fluoro-7-nitrobenz-2-oxo-1,3-diazole (NBD-F, Ex/Em 480/540 nm), respectively, in this order. The double-labeled cysteines emitted NBD-F fluorescence (540 nm) through an intramolecular FRET process when they were excited at the wavelength of maximum excitation of DCIA (390 nm). The generation of FRET was confirmed by comparison with analysis of n-amylamine or tryptophan (amines without a sulfhydryl group) and 6-mercaptohexanol (thiol without an amino group) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the double-labeled cysteines (DCIA and NBD-F) when performing LC on an ODS column with isocratic elution. The limits of quantification (signal-to-noise ratio = 10) and detection (signal-to-noise ratio = 3) for the cysteines, for a 20-μL injection volume, were in the range 150–670 fmol and 46–200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of a system which takes advantage of conventional detection of the derivatives. Furthermore, this method provides sufficient selectivity and sensitivity to determine the total cysteines present in the plasma of healthy humans. Figure Fret- inducing derivatization of homocysteine
Keywords: FRET; Double-labeling; Liquid chromatography; Glutathione; Cysteine; Homocysteine

A dissociative fluorescence enhancement technique for one-step time-resolved immunoassays by Kaj R. Blomberg; Veli-Matti Mukkala; Harri H. O. Hakala; Pauliina H. Mäkinen; Mikko U. Suonpää; Ilkka A. Hemmilä (1677-1682).
The limitation of current dissociative fluorescence enhancement techniques is that the lanthanide chelate structures used as molecular probes are not stable enough in one-step assays with high concentrations of complexones or metal ions in the reaction mixture since these substances interfere with lanthanide chelate conjugated to the detector molecule. Lanthanide chelates of diethylenetriaminepentaacetic acid (DTPA) are extremely stable, and we used EuDTPA derivatives conjugated to antibodies as tracers in one-step immunoassays containing high concentrations of complexones or metal ions. Enhancement solutions based on different β-diketones were developed and tested for their fluorescence-enhancing capability in immunoassays with EuDTPA-labelled antibodies. Characteristics tested were fluorescence intensity, analytical sensitivity, kinetics of complex formation and signal stability. Formation of fluorescent complexes is fast (5 min) in the presented enhancement solution with EuDTPA probes withstanding strong complexones (ethylenediaminetetra acetate (EDTA) up to 100 mM) or metal ions (up to 200 μM) in the reaction mixture, the signal is intensive, stable for 4 h and the analytical sensitivity with Eu is 40 fmol/L, Tb 130 fmol/L, Sm 2.1 pmol/L and Dy 8.5 pmol/L. With the improved fluorescence enhancement technique, EDTA and citrate plasma samples as well as samples containing relatively high concentrations of metal ions can be analysed using a one-step immunoassay format also at elevated temperatures. It facilitates four-plexing, is based on one chelate structure for detector molecule labelling and is suitable for immunoassays due to the wide dynamic range and the analytical sensitivity. Figure
Keywords: Time-resolved fluorometry; Dissociative fluorescence enhancement; Immunoassay; Multiplexing; Eu; Sm; Tb; Dy; Antibody

Dye-doped silica nanoparticles (C dots) were synthesized in reverse microemulsions and used to quantitatively examine DNA cleavage in the presence of transition metal ions. The cores were synthesized as fluorescein isothiocyanate (FITC)-doped silica nanoparticles and the shells’ surfaces were modified with single-stranded DNA oligomers tagged with Cy5 fluorophores. DNA cleavage induced by heavy metal ions was estimated by comparing the fluorescence of Cy5 before and after reaction with metal ions. For this, a lab-built laser-induced fluorescence microscope equipped with a charge coupled device (CCD) camera, for imaging, and photomultiplier tube, for photon counting, was used. FITC fluorescence from the core was measured as an internal standard to compensate for possible loss of the beads during the treatment. The cleavage of DNA in air in the presence of Pb2+, Cd2+, and Hg2+ at 1 ng/mL was found to be 14%, 6%, and 20%, respectively, and was significantly reduced to below 9% under N2 gas, indicating that the main cleavage source was oxygen in air. The most significant DNA cleavage was observed with the addition of hydrogen peroxide. This analytical method using dye-doped C dots provided convenient handling and quantification of the estimation of metal–DNA interaction with a detection limit of 34.9 pmol/mL. Figure Core-shell nanoparticles for quantitative measurement by LIFM
Keywords: Core-shell nanoparticle; DNA cleavage; Laser-induced fluorescence microscopy; Heavy metal ions; Reactive oxygen species (ROS)

Heat sterilization of peritoneal dialysis (PD) fluids leads to the formation of glucose degradation products (GDPs), which considerably impair long-term application of PD. Knowledge of the exact composition of GDPs present in a PD fluid is important to improve the biocompatibility of dialysis solutions. The present study conducted a targeted screening for novel GDPs with α-dicarbonyl structure in PD fluids. Thus, 3-deoxygalactosone (3-DGal) was identified for the first time in PD fluids. Quantification of 3-DGal was achieved by high-performance liquid chromatography (HPLC)/DAD/MSMS after derivatization with o-phenylendiamine to yield the quinoxaline derivative. Baseline separation of all α-dicarbonyl GDPs, particularly of the diastereomers 3-deoxyglucosone (3-DG) and 3-DGal, required the application of a polar, phenyl-based RP column for HPLC and additional pH-gradient elution. Concentrations of 3-DGal ranged between 55.8 and 136.9 μM in single-chamber PD fluids, and between 2.5 and 12.4 μM in double-chamber PD fluids. In solutions containing glucose, 3-DGal is formed from 3-DG via the intermediate 3,4-dideoxyglucosone-3-ene (3,4-DGE). Further studies are now required to determine the (patho-)physiological properties of 3-DGal. Figure Unexpected formation of 3-deoxygalactosone from glucose in peritoneal dialysis fluids
Keywords: [alpha]-Dicarbonyl compounds; Glucose degradation products (GDPs); 3-Deoxygalactosone (3-DGal); HPLC; Peritoneal dialysis fluid; Quinoxaline derivatives

A crystallinity study of dental tissues and tartar by infrared spectroscopy by J. A. Abraham; H. J. Sánchez; C. A. Marcelli; M. Grenón; M. C. Guidi; M. Piccinini (1699-1704).
In this paper we report a study of an important property of biomineralized phases, crystallinity, on the basis of previous results for synthetic apatite. Crystallinity is not only important for understanding biomineralization, it is also related to the maturation and mechanisms of growth of calcium phosphates in biological surroundings. We studied two kinds of sample, teeth as an example of biomineralized tissues and dental calculi (adhering) as an example of mineralization without participation of biological agents, except possibly bacteria. The investigation focused on study of ν 1ν 3 infrared absorption bands of PO 4 3– phosphates. We used ATR (attenuated total reflection) analysis to examine human dental tissues and tartar on several samples. The results confirm for the first time previous assumptions about the growth and maturation of dental calculi, i.e., crystallinity progresses from regions of high crystallinity to regions of lower crystallinity, and, in addition, its quantification with spatial resolution in the sample. A gradual pattern was observed in dental calculus. Another result from this study was that cementum and dentine had similar crystallinity, despite their different biological and mechanical functions.
Keywords: Crystallinity; Dental calculi; Tooth; Infrared spectroscopy; Biomineralization; Attenuated total reflection

The environmental interest of sulfonylurea herbicides was derived from the possibility of diffusion and penetration of these herbicides in the deepest layers of the ground, in particular in sandy or clay-poor soils, up to the ground waters; another interest of the study is their natural degradation pathway which leads to the formation of new species that are potentially more toxic and stable than the precursor herbicides. In this case, a lower persistence in the environment unfortunately does not correspond to a lower toxicity: hence, the importance of the identification of the species can be potentially formed. Here, nicosulfuron, a typical sulfonylurea herbicide, is considered in order to outline the environmental fate of the molecules generating from the simulation of one of the natural processes that can occur, i.e. photoinduced degradation. Aqueous nicosolfuron solutions underwent a simulated sun irradiation: the new species formed during the degradation process were identified by HPLC-DAD-MS/MS and a degradation pathway was proposed. The effect of temperature and the contribution of the hydrolysis were also evaluated. The use of ESI in both positive ion (PI) and negative ion (NI) mode and APCI in PI mode permits to obtain integrated information about the transformation products that can form; moreover, a study of the total ion chromatogram followed by the extraction of the SIM chromatograms of the most intense m/z signals made possible the identification of five possible photodegradation transformation products.
Keywords: Photoinduced degradation; Sulfonylurea herbicides; Degradation pathway; ESI-MS/MS; APCI-MS/MS

Analysis of the constituents and quality control of Viola odorata aqueous preparations by HPLC-DAD and HPLC-ESI-MS by Anastasia Karioti; Claudia Furlan; Franco Francesco Vincieri; Anna Rita Bilia (1715-1723).
In the present study, a method based on liquid chromatography with diode array detection (HPLC-DAD) coupled to an electrospray ionisation (ESI) interface was developed for the determination of the constituents in the aqueous preparations of Viola odorata L. flowering tops. The developed assay was fast, simple and effective and permitted the quality control of the preparations. The aim of this work was to assess the qualitative and quantitative profile of the investigated preparations, which find until today wide applications in food and cosmetic industry, and to propose a validated method for their quality control. Characteristic constituents of V. odorata flowers are considered to be the anthocyanins; however, a detailed literature research showed that data concerning their chemical content are scarce. HPLC-DAD-ESI-MS analyses supported by extensive preparative chromatographic investigations and 2D NMR analyses revealed the predominance of complex flavonol glycosides and permitted the complete characterisation of the content of V. odorata preparations. This is the first report of detailed analysis of the chemical composition of V. odorata flowers.
Keywords: Viola odorata ; Sweet violet; Flavonol glycosides; HPLC-DAD-ESI-MS

Optimization of extraction procedure and chromatographic separation of glyphosate, glufosinate and aminomethylphosphonic acid in soil by Coline Druart; Olivier Delhomme; Annette de Vaufleury; Evodie Ntcho; Maurice Millet (1725-1732).
Analysing herbicides in soil is a complex issue that needs validation and optimization of existing methods. An extraction and analysis method was developed to assess concentrations of glyphosate, glufosinate and aminomethylphophonic acid (AMPA) in field soil samples. After testing extractions by accelerated solvent extraction and ultrasonic extraction, agitation was selected with the best recoveries. Water was preferred as solvent extraction because it resulted in a cleaner chromatogram with fewer impurities than was the case with alkaline solvents. Analysis was performed by FMOC pre-column derivatization followed by high-performance liquid chromatography (HPLC) on a 300 mm C18 column which permitted enhanced separation and sensitivity than a 250 mm C18 column and increased resistance than the NH2 column for soil samples. This extraction and analysis method allowing a minimum of steps before the injection in the HPLC with fluorescence detection is efficient and sensitive for a clay-loamy soil with detection limits of 103 μg kg−1 for glyphosate, 15 μg kg−1 for glufosinate and 16 μg kg−1 for AMPA in soil samples.
Keywords: Glyphosate and glufosinate herbicides; Extraction; Soil; FMOC derivatization