Analytical and Bioanalytical Chemistry (v.398, #2)

The emerging field of nanotoxicology by Christy L. Haynes (587-588).
is Associate Professor of Chemistry at the University of Minnesota. Since arriving in 2005, she has developed a diverse research program that includes single-cell assays for nanotoxicology, fabrication of drug-delivery nanoparticles, monitoring chemical messenger release from immune system cells, and development of surface-enhanced Raman sensing platforms. She is the author or coauthor of 50 scientific publications and serves on the editorial board of multiple journals. During her time as a faculty member, she has received multiple prestigious awards, including the NIH New Innovator, the Searle Scholar Award, the Sloan Fellowship, and a Dreyfus Teacher-Scholar Award. Her work in the area of nanotoxicology has recently been recognized by the Society for Electroanalytical Chemistry Young Investigator Award as well as the American Chemical Society Division of Analytical Chemistry Findeis Award.

Toxicity assessment of nanomaterials: methods and challenges by Alok Dhawan; Vyom Sharma (589-605).
The increasing use of nanomaterials in consumer and industrial products has aroused global concern regarding their fate in biological systems, resulting in a demand for parallel risk assessment. A number of studies on the effects of nanoparticles in in vitro and in vivo systems have been published. However, there is still a need for further studies that conclusively establish their safety/toxicity, due to the many experimental challenges and issues encountered when assessing the toxicity of nanomaterials. Most of the methods used for toxicity assessment were designed and standardized with chemical toxicology in mind. However, nanoparticles display several unique physicochemical properties that can interfere with or pose challenges to classical toxicity assays. Recently, some new methods and modified versions of pre-existing methods have been developed for assessing the toxicity of nanomaterials. This review is an attempt to highlight some important methods employed in nanomaterial toxicology and to provide a critical analysis of the major issues/challenges faced in this emerging field. Figure Nanospecific properties leading to interference with some commonly used in vitro assays.
Keywords: Nanomaterial toxicology; In vitro; In vivo; Methods; Interference; Challenges

This brief discussion provides an overview of current concepts and perceptions regarding the pulmonary toxicity of ultrafine or nanoparticles. These aspects include, but are not limited to comparisons of fine particle vs. ultrafine particle effects and the unique response of pulmonary effects in rats vs. other rodent species, particularly at particle overload concentrations. In the final section, two studies are described which demonstrate that particle size is not the most significant particulate factor in producing exposure-related pulmonary effects.
Keywords: Nanoparticles; Nanotechnology; Clinical; Biomedical analysis; Pulmonary hazards

Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA by Elijah J. Petersen; Bryant C. Nelson (613-650).
Many of the current investigations on the environmental and human health risks of engineered nanomaterials focus on their short-term acute toxicity. However, the long-term chronic effects of nanomaterials on living systems, and in particular, on the genetic components of living systems, also warrant attention. An increasing number of nanomaterial safety studies include an assessment of genotoxicity as part of the overall risk evaluation. The potential of nanomaterials to directly or indirectly promote the formation of reactive oxygen species is one of the primary steps in their genotoxic repertoire. The subsequent modification of genomic DNA by reactive oxygen species could lead to the development of mutagenesis, carcinogenesis, or other age-related diseases if the DNA damage is not repaired. This review focuses on the interactions of nanomaterials with DNA and specifically on the capacity of some nanomaterials to induce oxidative damage to DNA. A critical assessment of the analytical methodology and the potential biochemical mechanisms involved in nanomaterial induction of oxidative damage to DNA is presented, results obtained for the various studies with each nanomaterial are compared, and recommendations for future research are discussed. Researchers should consider, among other experimental recommendations, (1) the application of more chromatography-based and mass-spectrometry-based analytical techniques to the assessment of oxidative damage to DNA to facilitate an enhanced understanding of DNA damage mechanisms and (2) the verification of cellular viability before conducting genotoxicity assays to reduce the impact of fragmented DNA, formed as a consequence of cell death, on DNA damage measurements.
Keywords: Base lesions; Comet assay; DNA damage; Engineered nanomaterials; Genotoxicity; Toxicity

The comet assay in nanotoxicology research by Hanna L. Karlsson (651-666).
Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The article reviews studies that have used the comet assay to investigate the toxicity of manufactured nanoparticles. It is shown that at least 46 cellular in vitro studies and several in vivo studies have used the comet assay and that the majority of the nanoparticles tested cause DNA strand breaks or oxidative DNA lesions. This is not surprising considering the sensitivity of the method and the reactivity of many nanomaterials. Interactions between the particles and the assay cannot be totally excluded and need further consideration. It is concluded that studies including several particle types, to enable the assessment of their relative potency, are valuable as are studies focusing both on comet assay end points and mutagenicity. Finally, the article discusses the potential future use of the comet assay in human biomonitoring studies, which could provide valuable information for hazard identification of nanoparticles.
Keywords: Comet assay; Nanomaterials; Particles; Genotoxicity; Toxicity; Test methods

New methods for nanotoxicology: synchrotron radiation-based techniques by Bing Wang; Zhe Wang; Weiyue Feng; Meng Wang; Zhongbo Hu; Zhifang Chai; Yuliang Zhao (667-676).
Nanotoxicology, a new branch of bionanoscience, deals with the study and application of the toxic or biological effects of nanomaterials or nanostructures, and aims to fill gaps in our knowledge of interactions between nano- and biosystems. However, progress in this new discipline largely relies on developing methodology to characterize nanomaterials in biological samples, quantify nanoparticles in living systems, and study their uptake, translocation, biodistribution, location and chemical status in vitro and in vivo, etc. In this review article, we focus on the main features of synchrotron radiation-based methods and their application to the study of the toxicological activities of nanomaterials. Synchrotron radiation-based analytical techniques are shown to provide a potent means for characterizing the toxic or biological behaviors of nanoparticles in biological systems.
Keywords: Nanotoxicology; Nanomaterials; Methodology; Synchrotron radiation techniques

Using two of the most commonly synthesized noble metal nanoparticle preparations, citrate-reduced Au and Ag, the impacts of short-term accidental nanoparticle exposure are examined in primary culture murine adrenal medullary chromaffin cells. Transmission electron microscopy (TEM), inductively coupled plasma atomic emission spectroscopy (ICP-AES) and Alamar Blue viability studies revealed that nanoparticles are taken up by cells but do not decrease cell viability within 48 hours of exposure. Carbon-fiber microelectrode amperometry (CFMA) examination of exocytosis in nanoparticle-exposed cells revealed that nanoparticle exposure does lead to decreased secretion of chemical messenger molecules, of up to 32.5% at 48 hours of Au exposure. The kinetics of intravesicular species liberation also slows after nanoparticle exposure, between 30 and 50% for Au and Ag, respectively. Repeated stimulation of exocytosis demonstrated that these effects persisted during subsequent stimulations, meaning that nanoparticles do not interfere directly with the vesicle recycling machinery but also that cellular function is unable to recover following vesicle content expulsion. By comparing these trends with parallel studies done using mast cells, it is clear that similar exocytosis perturbations occur across cell types following noble metal nanoparticle exposure, supporting a generalizable effect of nanoparticle-vesicle interactions.
Keywords: Nanoparticle; Toxicity; MAMC; CFMA

Comparative toxicity study of Ag, Au, and Ag–Au bimetallic nanoparticles on Daphnia magna by Ting Li; Brian Albee; Matti Alemayehu; Rocio Diaz; Leigha Ingham; Shawn Kamal; Maritza Rodriguez; Sandra Whaley Bishnoi (689-700).
A comparative assessment of the 48-h acute toxicity of aqueous nanoparticles synthesized using the same methodology, including Au, Ag, and Ag–Au bimetallic nanoparticles, was conducted to determine their ecological effect in freshwater environments through the use of Daphnia magna, using their mortality as a toxicological endpoint. D. magna are one of the standard organisms used for ecotoxicity studies due to their sensitivity to chemical toxicants. Particle suspensions used in toxicity testing were well-characterized through a combination of absorbance measurements, atomic force or electron microscopy, flame atomic absorption spectrometry, and dynamic light scattering to determine composition, aggregation state, and particle size. The toxicity of all nanoparticles tested was found to be dose and composition dependent. The concentration of Au nanoparticles that killed 50% of the test organisms (LC50) ranged from 65–75 mg/L. In addition, three different sized Ag nanoparticles (diameters = 36, 52, and 66 nm) were studied to analyze the toxicological effects of particle size on D. magna; however, it was found that toxicity was not a function of size and ranged from 3–4 μg/L for all three sets of Ag nanoparticles tested. This was possibly due to the large degree of aggregation when these nanoparticles were suspended in standard synthetic freshwater. Moreover, the LC50 values for Ag–Au bimetallic nanoparticles were found to be between that of Ag and Au but much closer to that of Ag. The bimetallic particles containing 80% Ag and 20% Au were found to have a significantly lower toxicity to Daphnia (LC50 of 15 μg/L) compared to Ag nanoparticles, while the toxicity of the nanoparticles containing 20% Ag and 80% Au was greater than expected at 12 μg/L. The comparison results confirm that Ag nanoparticles were much more toxic than Au nanoparticles, and that the introduction of gold into silver nanoparticles may lower their environmental impact by lowering the amount of Ag which is bioavailable. Figure Citrate-capped silver, gold, and bimetallic gold nanomaterial synthesis, characterization, and ecotoxicity profiling using Daphnia magna as a model organism
Keywords: Forensics/toxicology; AAS; AFM; X-ray spectroscopy; Nanoparticles/nanotechnology; Metals/heavy metals

We propose a novel combination of high-throughput luminescent bacterial tests for the evaluation of the reactive oxygen species (ROS)-generating potential of engineered nanoparticles (eNPs) and the role of solubilised metal ions in this process. The set of tests consists of differently engineered recombinant Escherichia coli strains: (1) a new sensor strain, which bioluminescence is induced by superoxide anions; (2) six recombinant E. coli strains (superoxide dismutase (sod) single, double and triple mutants and a respective wild-type strain), transformed with luxCDABE genes responding to toxic compounds by decreasing their luminescence; and (3) three strains in which bioluminescence is specifically induced by bioavailable metals (Cu, Zn and Ag). The applicability of this battery of tests in profiling oxidative potential of eNPs was evaluated on nTiO2, nCuO, nZnO and nAg (25, 30, 70 and <100 nm, respectively) NPs and fullerenes. As controls for the size or solubility, the bulk formulations (bTiO2, bCuO and bZnO) and soluble salts (ZnSO4, CuSO4 and AgNO3) were also analysed. Bacterial toxicity tests showed that nCuO was four-fold more toxic, and nAg was 15-fold more toxic to triple sod mutant than to wild type (2-h EC50 values were 8.1 and 2.0 mg Cu l−1, respectively, and 46 and 3.1 mg Ag l−1, respectively). Formation of ROS by nCuO and nAg was proved by superoxide anion-inducible strain. The metal sensor bacteria showed that the ROS formation by CuO NPs was caused by solubilised Cu ions, but in case of nAg, particles also had an effect. nZnO was remarkably more toxic to sod triple mutant than to wild type strain (2-h EC50 were 4.5 and 54 mg Zn l−1, respectively). Fullerenes inhibited the bioluminescence of sod triple mutant at 3,882 mg l−1 but had no effect on the wild-type strain even at 20,800 mg l−1. Nano and bTiO2 showed some effect on viability of bacteria only at high concentrations (>4,000 mg l−1) although nTiO2 (but not bTiO2) induced the bioluminescence of the superoxide anion sensing bacteria starting from 100 mg l−1. Thus, our innovative combined approach is expected to provide more consistent and informative data concerning the general toxicity, ROS-production potential and also solubilisation of metals in the case of metallic NPs. Figure This study discusses the potential of nTiO2, nCuO, nZnO, nAg and fullerene to generate reactive oxygen species and release metal ions as analysed by a combined set of bacterial tests.
Keywords: Reactive oxygen species; Bioluminescence; Oxidative stress; Sensor bacteria; sodABC ; Mutant

Polycyclic aromatic hydrocarbons (PAHs) in a coal tar standard reference material—SRM 1597a updated by Stephen A. Wise; Dianne L. Poster; Stefan D. Leigh; Catherine A. Rimmer; Stephanie Mössner; Patricia Schubert; Lane C. Sander; Michele M. Schantz (717-728).
SRM 1597 Complex Mixture of Polycyclic Aromatic Hydrocarbons from Coal Tar, originally issued in 1987, was recently reanalyzed and reissued as SRM 1597a with 34 certified, 46 reference, and 12 information concentrations (as mass fractions) for polycyclic aromatic hydrocarbons (PAHs) and polycyclic aromatic sulfur heterocycles (PASHs) including methyl-substituted PAHs and PASHs. The certified and reference concentrations (as mass fractions) were based on results of analyses of the coal tar material using multiple analytical techniques including gas chromatography/mass spectrometry on four different stationary phases and reversed-phase liquid chromatography. SRM 1597a is currently the most extensively characterized SRM for PAHs and PASHs.
Keywords: Polycyclic aromatic hydrocarbons (PAHs); Polycyclic aromatic sulfur heterocycles (PASHs); Standard reference material (SRM); Coal tar; Gas chromatography/mass spectrometry (GC/MS); Liquid chromatography (LC)

This paper describes the application of plasmonics-based nanoprobes that combine the modulation of the plasmonics effect to change the surface-enhanced Raman scattering (SERS) of a Raman label and the specificity of a DNA hairpin loop sequence to recognize and discriminate a variety of molecular target sequences. Hybridization with target DNA opens the hairpin and physically separates the Raman label from the metal nanoparticle thus reducing the plasmonics effect and quenching the SERS signal of the label. We have successfully demonstrated the specificity and selectivity of the nanoprobes in the detection of a single-nucleotide polymorphism (SNP) in the breast cancer BRCA1 gene in a homogenous solution at room temperature. In addition, the potential application of plasmonics nanoprobes for quantitative DNA diagnostic testing is discussed.
Keywords: Plasmonics; Surface-enhanced Raman scattering; Single-nucleotide polymorphism; Breast cancer

Measuring the intra-individual variability of the plasma proteome in the chicken model of spontaneous ovarian adenocarcinoma by Adam M. Hawkridge; Rebecca B. Wysocky; James N. Petitte; Kenneth E. Anderson; Paul E. Mozdziak; Oscar J. Fletcher; Jonathan M. Horowitz; David C. Muddiman (737-749).
The domestic chicken (Gallus domesticus) has emerged as a powerful experimental model for studying the onset and progression of spontaneous epithelial ovarian cancer (EOC) with a disease prevalence that can exceed 35% between 2 and 7 years of age. An experimental strategy for biomarker discovery is reported herein that combines the chicken model of EOC, longitudinal plasma sample collection with matched tissues, advanced mass spectrometry-based proteomics, and concepts derived from the index of individuality (Harris, Clin Chem 20: 1535–1542, 1974). Blood was drawn from 148 age-matched chickens starting at 2.5 years of age every 3 months for 1 year. At the conclusion of the 1 year sample collection period, the 73 birds that remained alive were euthanized, necropsied, and tissues were collected. Pathological assessment of resected tissues from these 73 birds confirmed that five birds (6.8%) developed EOC. A proteomics workflow including in-gel digestion, nanoLC coupled to high-performance mass spectrometry, and label-free (spectral counting) quantification was used to measure the biological intra-individual variability (CVW) of the chicken plasma proteome. Longitudinal plasma sample sets from two birds within the 73-bird biorepository were selected for this study; one bird was considered “healthy” and the second bird developed late-stage EOC. A total of 116 proteins from un-depleted plasma were identified with 80 proteins shared among all sample sets. Analytical variability (CVA) of the label-free proteomics workflow was measured using a single plasma sample analyzed five times and was found to be ≥CVW in both birds for 16 proteins (20%) and in either bird for 25 proteins (31%). Ovomacroglobulin (ovostatin) was found to increase (p < 0.001) over a 6 month period in the late-stage EOC bird providing an initial candidate protein for further investigation. Figure Experimental workflow for using quantitative LC-MS/MS proteomics to measure the intra-individual variability of the chicken plasma proteome as it relates to health and spontaneous onset of epithelial ovarian cancer in an effort to identify diagnostic protein biomarkers, some of which may be specific to individuals (i.e., personalized reference ranges).
Keywords: Genomics/proteomics; Mass spectrometry/ICP-MS; Biological samples; Bioanalytical methods; Clinical/biomedical analysis

Evaluation of nucleic acid duplex formation on gold over layers in biosensor fabricated using Czochralski-grown single-crystal silicon substrate by Subash C. B. Gopinath; Ramanujam Kumaresan; Koichi Awazu; Makoto Fujimaki; Minoru Mizuhata; Junji Tominaga; Penmetcha K. R. Kumar (751-758).
With a view to developing an economical and elegant biosensor chip, we compared the efficiencies of biosensors that use gold-coated single-crystal silicon and amorphous glass substrates. The reflectivity of light over a wide range of wavelengths was higher from gold layer coated single-crystal silicon substrates than from glass substrates. Furthermore, the efficiency of reflection from gold layers of two different thicknesses was examined. The thicker gold layer (100 nm) on the single-crystal silicon showed a higher reflectivity than the thinner gold film (10 nm). The formation of a nucleic acid duplex and aptamer–ligand interactions were evaluated on these gold layers, and a crystalline silicon substrate coated with the 100-nm-thick gold layer is proposed as an alternative substrate for studies of interactions of biomolecules. Figure Reflection based biosensor. Thin gold-layer coated on Czochralski-grown single-crystal silicon. Thiolated DNA hybridized with RNA-aptamer followed by protein was attached to obtain the reflection changes.
Keywords: Spectrophotometry; Crystal; Amorphous; Au layer; DNA hybridization

FRET detection of Octamer-4 on a protein nanoarray made by size-dependent self-assembly by Phat L. Tran; Jessica R. Gamboa; David J. You; Jeong-Yeol Yoon (759-768).
An alternative approach for fabricating a protein array at nanoscale is suggested with a capability of characterization and/or localization of multiple components on a nanoarray. Fluorescent micro- and nanobeads each conjugated with different antibodies are assembled by size-dependent self-assembly (SDSA) onto nanometer wells that were created on a polymethyl methacrylate (PMMA) substrate by electron beam lithography (EBL). Antibody-conjugated beads of different diameters are added serially and electrostatically attached to corresponding wells through electrostatic attraction between the charged beads (confirmed by zeta potential analysis) and exposed p-doped silicon substrate underneath the PMMA layer. This SDSA method is enhanced by vibrated-wire-guide manipulation of droplets on the PMMA surface containing nanometer wells. Saturation rates of antibody-conjugated beads to the nanometer patterns are up to 97% under one component and 58–70% under two components nanoarrays. High-density arrays (up to 40,000 wells) could be fabricated, which can also be multi-component. Target detection utilizes fluorescence resonance energy transfer (FRET) from fluorescent beads to fluorescent-tagged secondary antibodies to Octamer-4 (Oct4), which eliminates the need for multiple steps of rinsing. The 100 nm green beads are covalently conjugated with anti-Oct4 to capture Oct4 peptides (39 kDa); where the secondary anti-Oct4 and F(ab)2 fragment of anti-gIgG tagged with phycoerythrin are then added to function as an indicator of Oct4 detection. FRET signals are detected through confocal microscopes, and further confirmed by Fluorolog3 spectrofluorometer. The success rates of detecting Oct4 are 32% and 14% of the beads in right place under one and two component nanoarrays, respectively. Ratiometric FRET is used to quantify the amount of Oct4 peptides per each bead, which is estimated about 2 molecules per bead.
Keywords: E-beam lithography; Nanometer pattern generation system; Fluorescence resonance energy transfer (FRET); Wire-guide droplet manipulation

Acetaldehyde adducts of hemoglobin have been regarded as potential biochemical markers of alcohol exposure. In this study a novel sensitive method using liquid chromatography coupled to time-of-flight mass spectrometry (LC–TOF MS) has been used to investigate changes in adduct levels in alcohol detoxification patients. Hemoglobin and authentic blood samples from 66 adults with an alcohol-dependence syndrome and from 12 children were analyzed for acetaldehyde modifications with and without trypsin digestion using LC–TOF MS. After in-vitro incubation of hemoglobin with increasing concentrations of acetaldehyde, followed by tryptic digestion, 21 modified peptide fragments could be identified from their accurate mass and retention time shift. Eight of these could also be detected in authentic human blood samples. Trace amounts in children’s blood were indicative of an endogenous source. Modified peptide levels in patients’ samples with and without ethanol were significantly different, as also were levels in samples from admission and from five days later. Samples obtained 5, 10, or 15 days after admission did not differ in adduct levels. The LC–TOF MS method was sensitive enough to detect acetaldehyde-modified hemoglobin peptides in blood samples from patients with an alcohol-dependence syndrome. However, elevated levels were only observed after recent ethanol consumption and decreased during five days of abstinence, suggesting that acetaldehyde-modified tryptic peptides of hemoglobin are potential biomarkers only for short-term ethanol ingestion. Figure Deconvoluted mass peaks resulting from mass spectra of human hemoglobin chains incubated with acetaldehyde
Keywords: Forensics/toxicology; Mass spectrometry; HPLC; Ethanol; Hemoglobin

Quantitative determination of vitamin D metabolites in plasma using UHPLC-MS/MS by Shujing Ding; Inez Schoenmakers; Kerry Jones; Albert Koulman; Ann Prentice; Dietrich A. Volmer (779-789).
Vitamin D is an important determinant of bone health at all ages. The plasma concentrations of 25-hydroxy vitamin D (25-OH D) and other metabolites are used as biomarkers for vitamin sufficiency and function. To allow for the simultaneous determination of five vitamin D metabolites, 25-OH D3, 25-OH D2, 24,25-(OH)2 D3, 1,25-(OH)2 D3, and 1,25-(OH)2 D2, in low volumes of human plasma, an assay using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) was established. Plasma samples were spiked with isotope-labeled internal standards and pretreated using protein precipitation, solid-phase extraction (SPE) and a Diels–Alder derivatization step with 4-phenyl-1,2,4-triazoline-3,5-dione. The SPE recovery rates ranged from 55% to 85%, depending on the vitamin D metabolite; the total sample run time was <5 min. Mass spectrometry was conducted using positive ion electrospray ionization in the multiple reaction monitoring mode on a quadrupole–quadrupole-linear ion trap instrument after pre-column addition of methylamine to increase the ionization efficiency. The intra- and inter-day relative standard deviations were 1.6–4.1% and 3.7–6.8%, respectively. The limit of quantitation for these compounds was determined to be between 10 and 20 pg/mL. The 25-OH D results were compared with values obtained for reference materials (DEQAS). In addition, plasma samples were analyzed with two additional Diasorin antibody assays. All comparisons with conventional methods showed excellent correlations (r 2 = 0.9738) for DEQAS samples, demonstrating the high degree of comparability of the new UHPLC-MS/MS technique to existing methods.
Keywords: Vitamin D; Metabolites; Plasma; UHPLC; Tandem mass spectrometry; Quantification; Multiple reaction monitoring (MRM)

Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization by Maggie P. Y. Lam; S. O. Siu; Edward Lau; Xiuli Mao; H. Z. Sun; Philip C. N. Chiu; William S. B. Yeung; David M. Cox; Ivan K. Chu (791-804).
We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC—with only a single sample injection event—as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.
Keywords: RP-HILIC; UPLC; Hydrophilic analytes; Glycan separation; Full-component analysis

A new detection format for multiplexed analysis based on fluorescent encoded magnetic composite nanoparticles is presented. Two kinds of virus were analyzed by this new method: equine influenza virus (EIV) and equine infectious anemia virus (EIAV). Firstly, EIV antigen and EIAV antigen were conjugated to two kinds of fluorescent encoded magnetic composite nanoparticles, while the green-emitting CdTe quantum dots (QDs) were attached to the antibody of EIV and EIAV. Then both green-emitting CdTe QD-labeled antibodies and antigens labeled with fluorescent encoded magnetic composite nanoparticles were used to form an immunoassay system for the detection of EIV and EIAV antigens. The method is time-saving and has higher sensitivity (1.3 ng mL−1 for EIV antigens and 1.2 ng mL−1 for EIAV antigens) than the conventional methods. A competitive immunoassay method based on this analysis system was used to detect EIV and EIAV antigens in spiked serum samples with satisfactory results. Figure Schematic diagram of the whole process of the immunoassay method based on the flourescent encoded magnetic composite nanoparticles.
Keywords: Fluorescent encoded magnetic composite nanoparticles; Multiplex immunoassays equine influenza virus; Equine infectious anemia virus

Ability of a salivary intrinsically unstructured protein to bind different tannin targets revealed by mass spectrometry by Francis Canon; Alexandre Giuliani; Franck Paté; Pascale Sarni-Manchado (815-822).
Astringency is thought to result from the interaction between salivary proline-rich proteins (PRP) that belong to the intrinsically unstructured protein group (IUP), and tannins, which are phenolic compounds. IUPs have the ability to bind several and/or different targets. At the same time, tannins have different chemical features reported to contribute to the sensation of astringency. The ability of both electrospray ionization mass spectrometry and tandem mass spectrometry to investigate the noncovalent interaction occurring between a human salivary PRP, IB5, and a model tannin, epigallocatechin 3-O-gallate (EgCG), has been reported. Herein, we extend this method to study the effect of tannin chemical features on their interaction with IB5. We used five model tannins, epigallocatechin (EgC), epicatechin 3-O-gallate (ECG), epigallocatechin 3-O-gallate (EgCG), procyanidin dimer B2 and B2 3′-O-gallate, which cover the main tannin chemical features: presence of a gallate moiety (galloylation), the degree of polymerization, and the degree of B ring hydroxylation. We show the ability of IB5 to bind these tannins. We report differences in stoichiometries and in stability of the IB5•1 tannin complexes. These results demonstrate the main role of hydroxyl groups in these interactions and show the involvement of hydrogen bonds. Finally, these results are in line with sensory analysis, by Vidal et al. (J Sci Food Agric 83:564–573, 2003) pointing out that the chain length and the level of galloylation are the main factors affecting astringency perception. Figure CID MS/MS approach to monitor the stability of noncovalent complexes between a human salivary proline-rich protein and model tannins that cover the main chemical features of tannins
Keywords: Polyphenol; Noncovalent interaction; Proline-rich protein; Saliva; Astringency; Intrinsically unstructured protein

Peroxyoxalate chemiluminescence detection for the highly sensitive determination of fluorescence-labeled chlorpheniramine with Suzuki coupling reaction by Lawrence Asamoah Adutwum; Naoya Kishikawa; Kaname Ohyama; Shiro Harada; Kenichiro Nakashima; Naotaka Kuroda (823-829).
A sensitive and selective high performance liquid chromatography-peroxyoxalate chemiluminescence (PO-CL) method has been developed for the simultaneous determination of chlorpheniramine (CPA) and monodesmethyl chlorpheniramine (MDCPA) in human serum. The method combines fluorescent labeling with 4-(4,5-diphenyl-1H-imidazole-2-yl)phenyl boronic acid using Suzuki coupling reaction with PO-CL detection. CPA and MDCPA were extracted from human serum by liquid–liquid extraction with n-hexane. Excess labeling reagent, which interfered with trace level determination of analytes, was removed by solid-phase extraction using a C18 cartridge. Separation of derivatives of both analytes was achieved isocratically on a silica column with a mixture of acetonitrile and 60 mM imidazole-HNO3 buffer (pH 7.2; 85:15, v/v) containing 0.015% triethylamine. The proposed method exhibited a good linearity with a correlation coefficient of 0.999 for CPA and MDCPA within the concentration range of 0.5–100 ng/mL. The limits of detection (S/N = 3) were 0.14 and 0.16 ng/mL for CPA and MDCPA, respectively. Using the proposed method, CPA could be selectively determined in human serum after oral administration.
Keywords: Suzuki coupling reaction; Peroxyoxalate chemiluminescence; Chlorpheniramine; Monodesmethyl chlorpheniramine; HPLC

2-Hydroxy-4-methoxybenzophenone (HMB), which is one of the most commonly used UV filters in sunscreen cosmetics to protect skin from the deleterious effects of the sun, can be percutaneously absorbed, further metabolized, and finally excreted or bioaccumulated. An analytical method for the sensitive determination of HMB and its three metabolites in both human urine and semen is developed. The presented analytical method is based on a solid-phase extraction (SPE) procedure to clean-up and preconcentrate the target analytes from the urine and semen samples followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) detection. The methodology was fully validated and the standard addition calibration method was used to quantify the target analytes in order to correct the matrix effects observed. Considering this approach, the accuracy of the method was evaluated and the recoveries ranged from 98% to 115% and from 86% to 111% in urine and semen samples, respectively, depending on the analyte. For urine samples, the limits of detection ranged between 0.027 and 0.103 ng mL−1 and the repeatability of the method, expressed as relative standard deviation, was in the range of 7.2–9.2%, depending on the analyte. In the case of semen samples, the limits of detection ranged between 1 and 3 ng mL−1 whereas the repeatability was in the range of 2.2–6.4%, depending on the analyte. The described SPE-LC-MS/MS method was satisfactorily applied to both urine and semen samples from a male volunteer who applied a sunscreen cosmetic product containing HMB. HMB and its metabolites were found and quantified in the low ng mL−1 range in both urine and semen samples, although at a different extent.
Keywords: Liquid chromatography–tandem mass spectrometry (LC-MS/MS); Solid-phase extraction (SPE); 2-Hydroxy-4-methoxybenzophenone; Metabolites; Urine; Semen

Human AGP is an acidic glycoprotein mainly produced by liver that presents a high degree of heterogeneity. It can present different amino acid sequences and has five N-glycosylation sites leading to a wide range of different protein isoforms. AGP structure and composition has been widely studied due to its drug-binding behavior and relation with disease. However, so far, the characterization has been performed only on protein fragments, i.e., the peptide or glycan level. Here, the analysis of intact human AGP purified from human serum is performed by capillary electrophoresis–time-of-flight mass spectrometry. In this way, it is possible to characterize more than 150 human AGP isoforms, differing both in the amino acid sequence and in the glycosylation. The detected masses could be attributed unequivocally to an overall composition based on the combination of the analysis of the released glycans and the characterization of the deglycosylated protein. Different AGP samples purified from human serum were characterized and compared. High inter-individual variability among AGP isoforms expression was observed. The presented method enables for the first time clinical studies based on detailed isoform distribution of intact glycoproteins.
Keywords: Capillary electrophoresis–mass spectrometry; AGP; Glycoprotein; Glycan analysis; Isoform

The first liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of p-aminohippuric acid and inulin, both typical biomarkers of kidney function. 5-(Hydroxymethyl)furfural, generated from inulin by acid and heat preparation, was used as an inulin substitute for the quantification. Acetaminophen was used as the internal standard. Solid-phase extraction was carried out with 5% methanol as the washing solution to optimize the retention of the analytes and to avoid obstruction of the orifice plate of the mass spectrometer caused by any unreacted inulin residue remaining from the sample preparation process. Chromatography separation was performed on a Symmetry C18 column and a mobile phase composed of 2 mM ammonium formate and 0.1% formic acid in water (solvent A) and 2 mM ammonium formate and 0.1% formic acid in acetonitrile (solvent B) (30:70, v/v). Detection was performed with a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization in the multiple reaction monitoring mode. The selected transitions were m/z 195.2 → 120.2, 127.1 → 109.1, and 152.1 → 110.0 for p-aminohippuric acid, inulin [measured as 5-(hydroxymethyl)furfural], and acetaminophen, respectively. The linearity ranged from 10 to 140 μg/mL and from 100 to 1,400 μg/mL for p-aminohippurric acid and inulin (r > 0.99), respectively. The precisions and accuracies were all within 12 and 11% for the lower limit of quantification and quality control samples, respectively. This application was proven to be reliable and accurate and was successfully applied to a renal function study.
Keywords: p-Aminohippuric acid; Inulin; Solid-phase extraction; Liquid chromatography–tandem mass spectrometry; Renal function study; Validation

Glucosamine-functionalized silver glyconanoparticles: characterization and antibacterial activity by Murugan Veerapandian; Suk Kyung Lim; Hyang Mi Nam; Gobianand Kuppannan; Kyusik S. Yun (867-876).
We report the analytical and in vitro antibacterial activity of glucosamine-functionalized silver glyconanoparticles. Morphological characterization ensured the surface topography and particle size distribution of both silver and glucosamine–silver nanoparticles. Surface plasmon resonance of both types of nanoparticle was determined from UV–visible spectroscopy using four different sample concentrations (10–40 μL). The resulting functionalized glyconanoparticles show maximum absorbance with a red shift of 30 ± 5 nm (390–400 nm) from their initial absorbance (425–430 nm). FT-Raman and 1H-NMR spectroscopic measurement confirmed the surface functionalization of glucosamine on the silver surface through the carbonyl group of a secondary amide linkage (–NH–CO–), elucidated by the conjugation of N-hydroxysuccinimide (NHS)-terminated silver nanoparticles and the amino group of glucosamine. Antimicrobial experiments with well-characterized silver nanoparticles (AgNPs) and glucosamine-functionalized silver nanoparticles (GlcN-AgNPs) demonstrate that GlcN-AgNPs have similar and enhanced minimum inhibitory concentration (MIC) against eight gram-negative and eight gram-positive bacteria compared with AgNPs. MIC data shows that Klebsiella pneumoniae (ATCC 700603) and Bacillus cereus isolate express high levels of inhibition, with the quantity and magnitude of inhibition being higher in the presence of GlcN-AgNPs. Figure Glucosamine-functionalized silver glyconanoparticles as antibacterial agent
Keywords: Nanoparticle; Silver; Glucosamine; Glyconanoparticle; Antibacterial agent

Rapid and simple UPLC-MS/MS method for precise phytochelatin quantification in alga extracts by Anja Bräutigam; Dirk Wesenberg; Hugues Preud’homme; Dirk Schaumlöffel (877-883).
Quantitative phytochelatin (PC) analysis is, due to oxidation sensitivity of the PCs, matrix effects, and time consuming sample preparation, still a challenging analytical task. In this study, a rapid, simple, and sensitive method for precise determination of native PCs in crude extracts of the green alga Chlamydomonas reinhardtii was developed. Algae were exposed 48 h to 70 μM Cd. Coupling of ultra performance liquid chromatography and electrospray ionization tandem mass spectrometry with multi-reaction mode transitions for detection permitted the required short-time, high-resolution separation and detection specificity. Thus, under optimized chromatographic conditions, 10 thiol peptides were baseline-separated within 7 min. Relative detection limits in the nanomolar range in microliter sample volumes were achieved (corresponding to absolute detection limits at femtomol level). Next to glutathione (GSH), the most abundant cadmium-induced PCs in C. reinhardtii, namely CysGSH, PC2, PC3, CysPC2, and CysPC3, were quantified with high reproducibility at concentrations between 15 and 198 nmol g−1 fresh weight. The biological variation of PC synthesis of nine independently grown alga cultures was determined to be on average 13.7%. Figure A rapid UPLC-MS/MS method was developed for thiol peptide quantification in micro litre sample volumes at the nanomol level. For the first time reproducible quantification of six thiol peptides (GSH, CysGSH, PC2, PC3, CysPC2 and CysPC3) in crude extracts of Clamydomonas reinhardtii was possible
Keywords: Phytochelatin quantification; Chlamydomonas ; UPLC; ESI-MS/MS

Inkjet-printed paperfluidic immuno-chemical sensing device by Koji Abe; Kaori Kotera; Koji Suzuki; Daniel Citterio (885-893).
This paper reports on an inkjet printing method for the fabrication of lateral flow immunochromatographic devices made from a single piece of filter paper by patterning microfluidic channels and dispensing immunosensing inks, requiring only a single printing apparatus. This “paperfluidic” immunosensing device allows for a less time-consuming and more low-cost fabrication compared with the conventional immunochromatographic strips requiring multiple pads, plastic or nylon backing, and a plastic case. A sandwich immunoreaction was performed on the patterned immunosensing paper device, and the sensitivity of the device was optimized with an IgG model analyte. Inkjet-printed antibodies on the test line and the control line were immobilized by physical adsorption, resulting in a very simple fabrication method applicable for pure cellulose surfaces. The color intensity in the test line and the control line was determined both by naked eye and by means of a color scanner in combination with a simple computer program. With the resulting paperfluidic immunosensing device, human IgG concentrations at least down to 10 μg/l could be detected within 20 min. Additionally, in order to demonstrate the feasibility of a total multianalyte sensing system, a combined immuno-chemical sensing device was also fabricated by patterning an additional microfluidic channel for a chemical assay onto the same paper substrate. This low-cost multianalyte paperfluidic sensing device thus demonstrates the feasibility of simple, portable, and disposable tools for pathogen detection in the field of medical, environmental, and food analyses, possibly resulting in useful devices in remote settings and less-industrialized countries. Figure This study describes a semiquantitative microfluidic multianalyte immuno-chemical sensing device fabricated by inkjet-printing from a single piece of filter paper
Keywords: Immunochromatography; Chemical sensor; Microfluidic device; Paper; Color analysis

For any analytical system the population mean (μ) number of entities (e.g., cells or molecules) per tested volume, surface area, or mass also defines the population standard deviation $$ (sigma = sqrt {mu } ) $$ . For a preponderance of analytical methods, σ is very small relative to μ due to their large limit of detection (>102 per volume). However, in theory at least, DNA-based detection methods (real-time, quantitative or qPCR) can detect ≈ 1 DNA molecule per tested volume (i.e., μ ≈ 1) whereupon errors of random sampling can cause sample means ( $$ overline x $$ ) to substantially deviate from μ if the number of samplings (n), or “technical replicates”, per observation is too small. In this work the behaviors of two measures of sampling error (each replicated fivefold) are examined under the influence of n. For all data (μ = 1.25, 2.5, 5, 7.5, 10, and 20) a large sample of individual analytical counts (x) were created and randomly assigned into N integral-valued sub-samples each containing between 2 and 50 repeats (n) whereupon N × n = 322 to 361. From these data the average μ-normalized deviation of σ from each sub-sample’s standard deviation estimate $$left( {s_j ;;j = 1;{hbox{to}};N;;N = 7;left[ {n = 50} ight];{hbox{to}};180;left[ {n = 2} ight]} ight)$$ was calculated (Δ). Alternatively, the average μ-normalized deviation of μ from each sub-sample’s mean estimate ( $$ {overline x_{ m{j}}} $$ ) was also evaluated (Δ′). It was found that both of these empirical measures of sampling error were proportional to $$ sqrt[{ - 2}]{{n cdot mu }} $$ . Derivative (∂/∂n · Δ or Δ′) analyses of our results indicate that a large number of samplings $$ (n approx {33}pm {3}.{1}) $$ are requisite to achieve a nominal sampling error for samples with a μ ≈ 1. This result argues that pathogen detection is most economically performed, even using highly sensitive techniques such as qPCR, when some form of organism cultural enrichment is utilized and which results in a binomial response. Thus, using a specific gene PCR-based (+ or −) most probable number (MPN) assay one could detect anywhere from 0.2 to 105 CFU mL−1 using 6 to 48 reactions (i.e., 8 dilutions × 6 replicates per dilution) depending on the initial concentration of the pathogen and volume sampled.
Keywords: Sampling error; Poisson distribution; Analytical data; MPN

HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals by Michael Melmer; Thomas Stangler; Mark Schiefermeier; Werner Brunner; Hansjörg Toll; Alfred Rupprechter; Wolfgang Lindner; Andreas Premstaller (905-914).
In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC–MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.
Keywords: 2-Aminobenzamide; Biopharmaceutical; Fluorescence labeling; HILIC; N-glycans; Sample preparation

A method for simultaneous determination of buprenorphine (BUP), norbuprenorphine (NBUP), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine, benzoylecgonine (BE), ecgonine methyl ester (EME), anhydroecgonine methyl ester (AEME), morphine, codeine, 6-acetylmorphine (6AM), heroin, 6-acetylcodeine (6AC), nicotine, cotinine, and trans-3′-hydroxycotinine (OH-cotinine) by liquid chromatography tandem mass spectrometry in oral fluid (OF) was developed and extensively validated. Acetonitrile (800 μL) and OF (250 μL) were added to a 96-well Isolute-PPT+protein precipitation plate. Reverse-phase separation was achieved in 16 min and quantification was performed by multiple reaction monitoring. The assay was linear from 0.5 or 1 to 500 μg/L. Intraday, interday, and total imprecision were less than 13% (n = 20), analytical recovery was 92–114% (n = 20), extraction efficiencies were more than 77% (n = 5), and process efficiencies were more than 45% (n = 5). Although ion suppression was detected for EME, cocaine, morphine, 6AC, and heroin (less than 56%) and enhancement was detected for BE and nicotine (less than 316%), deuterated internal standards compensated for these effects. The method was sensitive (limit of detection 0.2–0.8 μg/L) and specific (no interferences) except that 3-hydroxy-4-methoxyamphetamine interfered with AEME. No carryover was detected, and all analytes were stable for 24 h at 22 °C, for 72 h at 4 °C, and after three freeze–thaw cycles, except cocaine, 6AC, and heroin (22–97% loss). The method was applied to 41 OF specimens collected throughout pregnancy with a Salivette® OF collection device from an opioid-dependent BUP-maintained pregnant woman. BUP ranged from 0 to 7,400 μg/L, NBUP from 0 to 71 μg/L, methadone from 0 to 3 μg/L, nicotine from 32 to 5,020 μg/L, cotinine from 125 to 508 μg/L, OH-cotinine from 11 to 51 μg/L, cocaine from 0 to 419 μg/L, BE from 0 to 351 μg/L, EME from 0 to 286 μg/L, AEME from 0 to 7 μg/L, morphine from 0 to 22 μg/L, codeine from 0 to 1 μg/L, 6AM from 0 to 4 μg/L, and heroin from 0 to 2 μg/L. All specimens tested negative for EDDP and 6AC. This method permits a fast and simultaneous quantification of 16 drugs and metabolites in OF, with good selectivity and sensitivity.
Keywords: Oral fluid; Liquid chromatography tandem mass spectrometry; Buprenorphine; Bioanalytical methods; Forensics/toxicology

Broad-spectrum drug screening of meconium by liquid chromatography with tandem mass spectrometry and time-of-flight mass spectrometry by Johanna Ristimaa; Merja Gergov; Anna Pelander; Erja Halmesmäki; Ilkka Ojanperä (925-935).
Analysis of the major drugs of abuse in meconium has been established in clinical practice for detecting fetal exposure to illicit drugs, particularly for the ready availability of the sample and ease of collection from diapers, compared with neonatal hair and urine. Very little is known about the occurrence and detection possibilities of therapeutic and licit drugs in meconium. Meconium specimens (n = 209) were collected in delivery hospitals, from infants of mothers who were suspected to be drug abusers. A targeted analysis method by liquid chromatography–triple quadrupole mass spectrometry (LC-MS/MS) was developed for abused drugs: amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, morphine, codeine, 6-monoacetylmorphine, oxycodone, methadone, tramadol, buprenorphine, and norbuprenorphine. A separate LC-MS/MS method was developed for 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid. A screening method based on LC coupled to time-of-flight MS was applied to a broad spectrum of drugs. As a result, a total of 77 different compounds were found. The main drug findings in meconium were as follows: local anesthetics 82.5% (n = 172), nicotine or its metabolites 61.5% (n = 129), opioids 48.5% (n = 101), stimulants 21.0% (n = 44), hypnotics and sedatives 19.0% (n = 40), antidepressants 18.0% (n = 38), antipsychotics 5.5% (n = 11), and cannabis 3.0% (n = 5). By revealing drugs and metabolites beyond the ordinary scope, the present procedure helps the pediatrician in cases where maternal denial is strong but the infant seems to suffer from typical drug-withdrawal symptoms. Intrapartum drug administration cannot be differentiated from gestational drug use by meconium analysis, which affects the interpretation of oxycodone, tramadol, fentanyl, pethidine, and ephedrine findings.
Keywords: Pharmaceuticals; Biological samples; Clinical/biomedical analysis; Drug monitoring/drug screening; Forensics/toxicology; Mass spectrometry; Meconium

The analysis is described for the first time for separating eight alpha-adrenergic blocking agents (oxymetazoline, 5-methylurapidil, prazosin, phentolamine, RS-17053, methoxamine, yohimbine, and BMY7378) by capillary electrophoresis (CE) with UV detection. Optimum separation of the analytes was obtained on a 50 cm × 75 μm i.d. capillary using a buffer containing 20% acetonitrile, 60 mM ammonium acetate, and 1.0% glacial acetic in methanol medium, with applied voltage and capillary temperature of 23 kV and 25 °C, respectively. The relative standard deviations of the migration times and the peak areas of the eight analytes were in the ranges of 0.12–1.29% and 1.02–2.53%, respectively. Detection limits of oxymetazoline, 5-methylurapidil, prazosin, phentolamine, RS-17053, methoxamine, yohimbine, and BMY7378 were 0.5–1.0 μg mL−1. In the tested concentration range, good linear relationships (correlation coefficients >98%) between peak areas and concentrations of the analytes were observed. This method has been successfully applied for determination of prazosin and phentolamine with recoveries of 97.30% and 98.12%, respectively. The proposed method was successfully applied for the rapid CE determination of the frequently applied alpha-adrenergic blocking compounds phentolamine and prazosin in general pharmaceutical preparation. Figure Structures of α-adrenergic agonist drugs and internal standard used in this study
Keywords: α-Adrenergic blocking agent; Nonaqueous capillary electrophoresis; Simultaneous determination; Separation

Development of radiation indicators to distinguish between irradiated and non-irradiated herbal medicines using HPLC and GC-MS by Min Jung Kim; Hyeon A. Ki; Won Young Kim; Sukdeb Pal; Byeong Keun Kim; Woo Suk Kang; Joon Myong Song (943-953).
The effects of high dose γ-irradiation on six herbal medicines were investigated using gas chromatography–mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC). Herbal medicines were irradiated at 0–50 kGy with 60Co irradiator. HPLC was used to quantify changes of major components including glycyrrhizin, cinnamic acid, poncirin, hesperidin, berberine, and amygdalin in licorice, cinnamon bark, poncirin immature fruit, citrus unshiu peel, coptis rhizome, and apricot kernel. No significant differences were found between gamma-irradiated and non-irradiated samples with regard to the amounts of glycyrrhizin, berberine, and amygdalin. However, the contents of cinnamic acid, poncirin, and hesperidin were increased after irradiation. Volatile compounds were analyzed by GC/MS. The relative proportion of ketone in licorice was diminished after irradiation. The relative amount of hydrocarbons in irradiated cinnamon bark and apricot kernel was higher than that in non-irradiated samples. Therefore, ketone in licorice and hydrocarbons in cinnamon bark and apricot kernel can be considered radiolytic markers. Three unsaturated hydrocarbons, i.e., 1,7,10-hexadecatriene, 6,9-heptadecadiene, and 8-heptadecene, were detected only in apricot kernels irradiated at 25 and 50 kGy. These three hydrocarbons could be used as radiolytic markers to distinguish between irradiated (>25 kGy) and non-irradiated apricot kernels.
Keywords: γ-irradiation; Medicinal herbs; Gas chromatography/mass spectrometry (GC/MS); High-performance liquid chromatography (HPLC)

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography–electrospray tandem mass spectrometry by Yu Li; Junjian Fang; Shengming Wu; Kunpeng Ma; Haijing Li; Xianzhong Yan; Fangting Dong (955-961).
Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra ® MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r 2 = 0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.
Keywords: Salinomycin; Human plasma; LC-MS/MS

Methods for the detection of central nervous tissue (CNT) are urgently needed in food control as a means for controlling strict adherence to both food labeling and banning of specified BSE risk material. Here, we report data on heat stability of the CNT markers neuron-specific enolase (NSE) in western blotting, glial fibrillary acidic protein (GFAP) in an enzyme linked immunoassay, mRNAGFAP in a real-time PCR assay, and several fatty acids (C22:6, C24:0-OH, C24:1ω9/ω7, C24:1ω9-OH/ω7-OH, and C24:0) in gas chromatography mass spectrometry (GC/MS). The sample matrix, a standard material of emulsion-type sausage with varied contents of CNT (brain), was heat-treated in three studies: (1) routine meat technological heat treatment with low (85 °C, 30 min), medium (115 °C, 30 min), and high (133 °C, 30 min, 3 bar) heating of 72 anonymous samples from a blind trial; (2) heat treatment under experimental conditions (100, 110, …, 200 °C, 45 min); and (3) fractionized heating of central nervous system (up to three times) under moderate routine technological conditions (85, 100, and 115 °C, 30 min). The markers of the immunochemical methods showed a low GFAP or very low NSE temperature stability at medium and high temperature conditions. The real-time PCR assay gave inconsistent, non-quantitative results, which indicated an uncontrollable matrix effect. The relevant GC/MS markers (C24:0-OH, C24:1ω9/ω7, and C24:1ω9-OH/ω7-OH) proved to be extremely stable. Neither meat and bone meal conditions (133 °C) nor experimental heating (up to and above 140 °C) showed any reduction of GC/MS CNT quantification. On the contrary, a slight but significant increase was noted over a certain temperature range (120–140 °C) for most fatty acids, possibly due to an improved extractability of the fatty acids. We conclude that a quantitative approach is highly unreliable when using immunochemical methods; moreover, these methods might be basically prone to false-negative results depending on heat treatment and matrix composition. Therefore, antibodies with higher affinity to heat-treated CNT marker epitopes are needed. Relevant amounts of CNT (≥0.5%) in low- and medium-heated products would still be reliably detectable by the GFAP ELISA, which justifies its use as a screening method in official food control. The results obtained by the real-time PCR assay were contradictory to recently published data, indicating a need for further protocol optimization and collaborative trials. Up to date, the analytical approach using GC/MS is the only valid procedure as pertaining to heat stability and quantitative analysis; consequently, it should be recommended as the reference procedure in official food control for CNT detection in heat-treated meat products. Figure The introduction of central nervous tissue from bovines into the food chain probably caused a new variant of Creutzfeldt-Jacob disease in humans. Analytical control of meat products by immunochemical CNT detection can be hindered by so far unknown severe heat induced losses. In contrast the CNT-specific fatty acids detected by GC/MS turned out to be remarkably stable up to temperatures of 160 °C
Keywords: Bovine spongiform encephalopathy; Central nervous tissue; Specified risk material; GC/MS; ELISA; Western blot; RT-PCR; Fatty acids; NSE; GFAP

Hormones are among the highest-impact endocrine disrupters affecting living organisms in aquatic environments. These molecules have been measured in both wastewater and sewage sludge. Analytical techniques for such matrices are well described in the literature. In contrast, there is little information about the analysis of hormones in animal waste. The objectives of this study were, first, to propose a method for conditioning swine manure samples (addition of formaldehyde, separation of the solid and liquid phases, and duration of storage) in order to determine hormones in the liquid fraction of manure by solid-phase extraction (SPE) coupled with gas chromatography–mass spectrometry (GC–MS). Our results showed that analysis of hormones was affected by matrix changes which occurred during freezing and thawing and after addition of formaldehyde, an additive frequently used to preserve environmental samples. Thus, our results argue for the conditioning of samples without formaldehyde and for separating the solid and liquid fractions of manure before freezing. Second, this study reports on the use of a liquid extraction method coupled with SPE and GC–MS analysis for determination of hormones in the solid fraction of manure. Under the conditions selected, hormone recoveries were between 80 and 100%. Finally, the optimized method was used to quantify hormones in both liquid and solid fractions of swine manure from different breeding units. High levels of estrone and α-estradiol were found in samples whereas β-estradiol was detected in smaller amounts. Estriol and progesterone were mainly found in manure from the gestating sow building whereas testosterone was detected in manure from male breeding buildings.
Keywords: Steroid hormones analysis; Swine manure; Endocrine disruptors; Agricultural wastes; Gas chromatography–mass spectrometry

Fractionation of persistent organic pollutants in fish oil by high-performance liquid chromatography on a 2-(1-pyrenyl)ethyl silica column by X. Ortiz; R. Martí; M. J. Montaña; M. Gasser; L. Margarit; F. Broto; J. Díaz-Ferrero (985-994).
The analysis of persistent organic pollutants in foodstuffs has become necessary for control of their levels in products for human and animal consumption. These analytical procedures usually require a fractionation step in order to separate the different families of pollutants to avoid interferences during the instrumental determination. In this study the separation was carried out on a 2-(1-pyrenyl)ethyl silica column, where analyte fractionation was based on differences in planarity and aromaticity. The fractionation of several types of persistent organic pollutants found in fish oil samples was studied; the pollutants included polychlorinated dibenzo-p-dioxins and dibenzofurans, polychlorinated biphenyls, polybrominated diphenyl ethers, and some organochlorine pesticides. Fractions were analyzed by high-resolution gas chromatography with electron-capture detection and high-resolution gas chromatography–high resolution mass spectroscopy. Finally, the whole method (including the purification, fractionation, and instrumental determination steps) was validated and successfully applied to the analysis of several samples of fish oil.
Keywords: Persistent organic pollutants; Dioxins; Polychlorinated biphenyls; Polybrominated diphenyl ethers; Organochlorine pesticides; Fish oil

The performance of the dispersive liquid–liquid microextraction (DLLME) technique for the determination of eight UV filters and a structurally related personal care species, benzyl salicylate (BzS), in environmental water samples is evaluated. After extraction, analytes were determined by gas chromatography combined with mass spectrometry detection (GC-MS). Parameters potentially affecting the performance of the sample preparation method (sample pH, ionic strength, type and volume of dispersant and extractant solvents) were systematically investigated using both multi- and univariant optimization strategies. Under final working conditions, analytes were extracted from 10 mL water samples by addition of 1 mL of acetone (dispersant) containing 60 μL of chlorobenzene (extractant), without modifying either the pH or the ionic strength of the sample. Limits of quantification (LOQs) between 2 and 14 ng L−1, inter-day variability (evaluated with relative standard deviations, RSDs) from 9% to 14% and good linearity up to concentrations of 10,000 ng L−1 were obtained. Moreover, the efficiency of the extraction was scarcely affected by the type of water sample. With the only exception of 2-ethylhexyl-p-dimethylaminobenzoate (EHPABA), compounds were found in environmental water samples at concentrations between 6 ± 1 ng L−1 and 26 ± 2 ng mL−1. Figure 1 GC-MS chromatogram corresponding to a spiked (50 ng L-1) ultrapure water sample
Keywords: UV filters; Dispersive liquid–liquid microextraction; GC-MS; Water samples

A microwave-assisted extraction method followed by clean-up with solid-phase extraction (SPE) combined with large-volume injection gas chromatography–tandem mass spectrometry (LVI-GC-MS/MS) for the analysis of 17 pesticides in wild and aquaculture edible seaweeds has been developed. An experimental central composite design was employed to evaluate the effects of the main variables potentially affecting the extraction (temperature, time, and solvent volume) and to optimize the process. The most effective microwave extraction conditions were achieved at 125 °C and 12 min with 24 mL of hexane/ethyl acetate (80:20). SPE clean-up of the extracts with graphitized carbon and Florisil, optimized by means of the experimental design, proved to be efficient in the removal of matrix interferences. The analytical recoveries were close to 100% for all the analytes, with relative standard deviations lower than 13%. The limits of detection ranged from 0.3 to 23.1 pg g−1 and the limits of quantification were between 2.3 and 76.9 pg g−1, far below the maximum residue levels established by the European Union for pesticides in seaweed. The results obtained prove the suitability of the microwave-assisted extraction for the routine analysis of pesticides in aquaculture and wild seaweed samples.
Keywords: Multiresidue pesticide; Microwave-assisted extraction; GC-MS/MS; Seaweed

A specific, sensitive and robust liquid chromatography tandem mass spectrometry method for determining oxytetracycline, tetracycline, chlortetracycline and doxycycline in royal jelly and honey samples is presented. Extraction of drug residues was performed by ammonium acetate buffer as extractant followed by a clean-up with metal chelate affinity chromatography and solid-phase extraction. Tetracycline analysis was performed using liquid chromatography–electrospray ionisation–tandem mass spectrometry. The presented method is the first validated for royal jelly and in accordance with the requirements set by Commission Decision 2002/657/EC. Recoveries of the methods, calculated spiking the samples at 5.0, 10.0, 20.0 and 30.0 μg kg−1, were 79% to 90% for honey and 77% to 90% for royal jelly. The intra-day precision (RSD) ranged between 8.1% and 15.0% for honey and from 9.1% to 16.3% for royal jelly, while inter-day precision values were from 10.2% to 17.6% and from 10.6% to 18.4% respectively for honey and royal jelly. Linearity for the four analytes was calculated from 5.0 to 50.0 μg kg−1. The decision limits (CCα) ranged from 6.2 to 6.4 μg kg−1 and from 6.1 to 6.5 μg kg−1 for honey and royal jelly, respectively. Detection capabilities values (CCβ) ranged between 7.2 and 7.7 μg kg−1 and from 7.3 to 7.9 μg kg−1 respectively for honey and royal jelly. The developed method is currently in use for confirmation of the official control analysis of honey and royal jelly samples.
Keywords: Tetracyclines; Royal jelly; Honey; Mass spectrometry; Commission Decision 2002/657/EC

A fast liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method was developed to study five endocrine-disrupting compounds (4-n-nonylphenol, bisphenol A, estrone, 17β-estradiol and 17α-ethinylestradiol) in water. Different columns were tested; the chromatographic separation of the analytes was optimized on a Pinnacle DB biphenylic column with a water–acetonitrile gradient elution, which allowed the separation of the selected endocrine-disrupting compounds (EDCs) in less than 6 min. Quantitative analysis was performed in selected reaction monitoring (SRM) mode; two transitions were chosen for each compound, using the most abundant for quantitation. Calibration curves using bisphenol A-d 16 as internal standard were drawn, showing good correlation coefficients (0.9993–0.9998). All figures of merit of the method were satisfactory; limits of detection were in the low pg range for all analytes. The method was then applied to the determination of the analytes in real water samples: to this aim, polar organic chemical integrative samplers (POCIS) were deployed in the influent and in the effluent of a drinking water treatment plant in Liguria (Italy). The EDC level was rather low in the influent and negligible in the outlet, reflecting the expected function of the treatment plant.
Keywords: Endocrine-disrupting compounds (EDCs); LC-MS-MS; POCIS; Water

Development of a certified reference material for the determination of acrylamide in potato chips by Byungjoo Kim; Sunyoung Park; Insun Lee; Youngran Lim; Euijin Hwang; Hun-Young So (1035-1042).
A certified reference material (CRM), KRISS CRM 108-10-003, has been developed for analysis of acrylamide in potato chips, as a representative of carbohydrate-rich food cooked in high-temperature oil. The material was prepared by grinding commercially available potato chips to a paste which was then homogenized, bottled in 15-g units, and stored at −70 °C. Certification, homogeneity and stability testing, were carried out by liquid chromatography–isotope-dilution mass spectrometry (ID-LC–MS). A single ID-LC–MS measurement was performed for each of 10 selected units for certification and homogeneity assessment. The mean measurement result for the 10 bottles, 0.455 ± 0.012 mg kg−1, was assigned as the certified value of the CRM. The between-bottle homogeneity was 0.8% of the certified value. The within-bottle homogeneity, tested by measuring three replicate sub-samples from each of three randomly selected bottles, was similar to the between-bottle homogeneity. The stability of the CRM under storage conditions (−70 °C) was tested for 21 months and no change in the acrylamide content was observed within the measurement uncertainty. Stability of the CRM at –20 °C (storage at user’s site) and room temperature (for regular use and transportation) was also tested. Also presented is the newly designed procedure for evaluating the uncertainty of the certified value for the characterization scheme used in this study. Figure
Keywords: Certified reference material; Acrylamide; Potato chips; Isotope-dilution mass spectrometry

Determination of trace chromium(VI) in drinking water using X-ray fluorescence spectrometry after solid-phase extraction by Pedro R. Aranda; Susana Moyano; Luis D. Martinez; Irma E. De Vito (1043-1048).
A new, simple, and selective method for preconcentration and determination of Cr(VI) in aqueous samples. After adsorption in “batch mode” on Aliquat 336-AC, determinations were made directly on the solid by X-ray fluorescence spectrometry, which had the advantage of not requiring the step of elution of the chromium retained. The enrichment factor was calculated considering that the tablets obtained from 10 mL solution of Cr(VI) (1000 μg L-1) had a final thickness of 0.64 mm and a diameter of 16.7 mm; the volume deposited on the pellet was 0.14 cm3. The preconcentration factor obtained was 71-fold, which was highly satisfactory for chromium trace analysis by XRF. Finally, the method was successfully applied to the determination of Cr(VI) in drinking water samples. Figure Latin America is blessed with an abundance of fresh water. Although use of groundwater is significant in some regions, knowledge about its relationship to surface water, its quantity, its management, and its economic and social value, is quite limited. Hexavalent chromium is a heavy metal that is commonly used in industrial processes, for example the manufacture of stainless steel, textile dyes, and anti-corrosive coatings. At some levels it is a known carcinogen.
Keywords: Chromium; Preconcentration; Aliquat 336; Activated carbon (AC); X-ray fluorescence

Solid-phase microextraction (SPME) is a widely used sampling technique that has been proved to enable efficient extraction of a broad range of analytes. Generally, SPME achieves non-exhaustive extraction, and therefore the analyte mass transfer distribution in the sampled multiphase system should be considered while developing a calibration method. Here, a new method, aimed at quantifying the extracted analytes without the need to consider their mass distribution, is proposed. This method relies on the generation of mass response curves by loading a known analyte mass onto the absorbent phase of a SPME fiber, and then conducting analysis by the preferred technique. Precise and accurate deposition of analyte over the restricted dimension of a fiber is demonstrated for the first time by utilizing a drop-on-demand microdrop printer. This system enables direct, non-contact deposition of micron-sized drops containing negligible solvent volumes (<1 nL), on the center of the extraction phase of the fiber which enables immediate analysis. Printed fiber response curves were determined herein, with three model compounds of different volatility—2,4-dinitrotoluene (2,4-DNT), diphenylamine (DPA), and 1,3 diethyl-1,3-diphenylurea (ethyl centralite, EC), using two analytical techniques, gas chromatography–mass spectrometry (GC–MS) and ion mobility spectrometry (IMS). Quantification of the absolute amounts extracted by headspace SPME yielded comparable results between the two methods of analysis with only less than 10% variation for 2,4-DNT and EC and less than 30% for DPA. In comparison, quantification by the traditional liquid injection/spike response curves determined by each technique led to mass estimates that were significantly greater by hundreds of percent. Figure Microdrop printing method for loading known mass of analyte on to the absorbent phase of an SPME fiber for absolute mass quantification.
Keywords: Solid phase microextraction (SPME); Inkjet microdrop printing technology; Calibration method; Ion mobility spectrometer (IMS); Gas chromatography–mass spectrometry (GC–MS); Smokeless powders

Evaluation of laboratory powder X-ray micro-diffraction for applications in the fields of cultural heritage and forensic science by Silvie Švarcová; Eva Kočí; Petr Bezdička; David Hradil; Janka Hradilová (1061-1076).
The uniqueness and limited amounts of forensic samples and samples from objects of cultural heritage together with the complexity of their composition requires the application of a wide range of micro-analytical methods, which are non-destructive to the samples, because these must be preserved for potential late revision. Laboratory powder X-ray micro-diffraction (micro-XRD) is a very effective non-destructive technique for direct phase analysis of samples smaller than 1 mm containing crystal constituents. It compliments optical and electron microscopy with elemental micro-analysis, especially in cases of complicated mixtures containing phases with similar chemical composition. However, modification of X-ray diffraction to the micro-scale together with its application for very heterogeneous real samples leads to deviations from the standard procedure. Knowledge of both the limits and the phenomena which can arise during the analysis is crucial for the meaningful and proper application of the method. We evaluated basic limits of micro-XRD equipped with a mono-capillary with an exit diameter of 0.1 mm, for example the size of irradiated area, appropriate grain size, and detection limits allowing identification of given phases. We tested the reliability and accuracy of quantitative phase analysis based on micro-XRD data in comparison with conventional XRD (reflection and transmission), carrying out experiments with two-phase model mixtures simulating historic colour layers. Furthermore, we demonstrate the wide use of micro-XRD for investigation of various types of micro-samples (contact traces, powder traps, colour layers) and we show how to enhance data quality by proper choice of experiment geometry and conditions.
Keywords: Powder X-ray micro-diffraction; Quantitative phase analysis; Limits; Forensic; Artwork; Micro-samples

Analytical characterization of BCxNy films generated by LPCVD with triethylamine borane by Olaf Baake; Peter S. Hoffmann; Marina L. Kosinova; Andreas Klein; Beatrix Pollakowski; Burkhard Beckhoff; Nadeshda I. Fainer; Valentina A. Trunova; Wolfgang Ensinger (1077-1084).
Triethylamine borane (TEAB) and He, N2 or NH3 were applied as additional reaction gases in the production of BCxNy layers by low-pressure chemical vapor deposition (LPCVD). These layers were deposited on Si(100) wafers and characterized chemically by X-ray photoelectron spectroscopy (XPS) and synchrotron radiation-based total-reflection X-ray fluorescence analysis combined with near-edge X-ray absorption fine-structure spectroscopy (TXRF-NEXAFS). The composition of the material produced without NH3 was found to be dominated by B–C bonds with the stoichiometric formula B2C3N. B–N bonds with the formula B2CN3 were preferred when NH3 was added. A first attempt was made to compare the results obtained by applying trimethylamine borane and TEAB as single-source precursors.
Keywords: LPCVD; Boron carbonitride films; XPS; TXRF-NEXAFS

Determination of additives in an electrolytic zinc bath by q1H-NMR spectroscopy by Ainara Barriola; José I. Miranda; Miren Ostra; Carlos Ubide (1085-1094).
The use of proton nuclear magnetic resonance (1H-NMR) for the quantification of additives in an electrolytic Zn bath is reported. A simple and quick method is described that does not need any prior sample preparation. Contrary to other analytical methods, the three additives in the bath, benzylidene acetone (BDA), benzoic acid (BA) and poly(ethylene glycol) (PE400), can be quantified. Two calibration methods were tried: integration of NMR signals with the use of an internal standard and partial least squares (PLS) regression applied to the characteristic NMR peaks. Both methods are compared and the univariate method was preferred because of simplicity, accuracy and precision. The following limits of detection were found: 0.30 g L−1 BA, 0.08 g L−1 BDA and 0.7 g L−1 PE400 with dynamic ranges of at least 1.0–6.0, 0.1–0.6 and 3.0–18.0 g L−1 respectively. Those concentration ranges are suitable to follow the concentration of additives in the bath in real time. 1H-NMR spectra provide evidence for the BDA degradation pattern. Figure
Keywords: qNMR; Multivariate calibration; Zn electroplating; Additives

Investigation of the osteitis deformans phases in snake vertebrae by double-pulse laser-induced breakdown spectroscopy by M. Galiová; J. Kaiser; K. Novotný; M. Ivanov; M. Nývltová Fišáková; L. Mancini; G. Tromba; T. Vaculovič; M. Liška; V. Kanický (1095-1107).
Double-pulse laser-induced breakdown spectroscopy (DP-LIBS) was optimized for microspatial analyses of fossil and recent snake vertebrae. As complimentary techniques, solution analysis by inductively coupled plasma mass spectrometry and synchrotron radiation X-ray microtomography was utilized in order to determine the overall concentration of the selected elements in the samples and to visualize nondestructively the fossil sample microstructure, respectively. Elemental mapping of pathological bony tissue by DP-LIBS has been proven as a powerful tool for considering the osteitis deformans phases in fossil vertebrae.
Keywords: Laser ablation; LIBS; Mapping; X-ray microtomography; Fossil; Snake vertebra

A spectroscopic method to estimate the binding potency of amphiphile assemblies by D. R. Gauger; V. V. Andrushchenko; P. Bouř; W. Pohle (1109-1123).
A fast and convenient spectroscopic methodology to determine the water uptake capacity of amphiphile assemblies studied in multilayer films is presented. This method was developed to provide a reliable but relatively simple tool for estimating the binding potency of such complex systems. The water-binding potency represents a general propensity of higher-order systems to bind or embed relevant ligands, such as various non-lipid effectors in the case of artificial lipid membranes. In this sense, the binding potency might contribute to a specific functional role of certain lipids. The essence of the new method is that the calibration of data measured by infrared (IR) spectroscopy against those directly obtained by Karl–Fischer titration (KFT) enables one to replace the expensive chemical–analytical technique by a more comfortable and efficient IR-spectroscopic protocol. This approach combines the easy handling, versatility, and availability of IR spectroscopy with the high accuracy of KFT. The usefulness of the procedure is demonstrated on an example set of six amphiphiles with a common chain length of 18 carbon atoms. Despite this similarity, the binding potency data differ tremendously in a way which can be correlated with the systematic variations introduced into the amphiphile structure. Going further beyond the methodical aspect, the scientific relevance of the data is comprehensively discussed especially in terms of the structural factors that govern the binding potency of amphiphiles. That is favored mainly by fluidity and disfavored mainly by inter-amphiphile binding networks. For phosphatidylcholine, our data are strongly in favor of a particular hydration model that involves primary water binding to phosphate as well as the formation of water semi-clathrates hosting the trimethylammonium moiety. Interestingly, stearylamine and diolein assemblies did not take up any water at all. This unexpected hydrophobicity is due to the unusual structures formed in these latter cases: rigid ammonium amide with a strong hydrogen-bonding/salt bridge network in stearylamine, and patches of inverted micelles in diolein, as revealed by molecular dynamics simulations. Figure 1 Snapshots of six unit cells from the molecular dynamics simulations performed for the neat DOG60 system under periodic boundary conditions; the snapshots were taken at different times: a) 0 ns; b) 5 ns; c) 7 ns; d) 20 ns. The pictures demonstrate the formation of inverted micelles (b) from the bilayers (a) taken as an arbitrary starting configuration and the successive coalescence of these micelles into “patches” (c,d).
Keywords: Binding potency; Infrared spectroscopy; Karl–Fischer titration; Molecular dynamics; Amphiphiles (lipids, surfactants); Hydration

Iodine determination in food by inductively coupled plasma mass spectrometry after digestion by microwave-induced combustion by Márcia F. Mesko; Paola A. Mello; Cezar A. Bizzi; Valderi L. Dressler; Guenter Knapp; Érico M. M. Flores (1125-1131).
Iodine determination in food samples was performed by inductively coupled plasma mass spectrometry (ICP-MS) after digestion by microwave-induced combustion (MIC). Sample masses up to 500 mg of bovine liver, corn starch, milk powder, or wheat flour were completely combusted using the MIC system. Ammonium nitrate (6 mol l−1 solution, 50 μl) was used as an aid for ignition and vessels were charged with 15 bar of O2. The use of H2O, 0.9 mmol l−1 H2O2, 10 to 50 mmol l−1 (NH4)2CO3 and 56 mmol l−1 tetramethylammonium hydroxide was investigated as absorbing solutions, as well as the suitability of performing a reflux step after the combustion process. Digestion of food samples by pressurized microwave-assisted acid digestion, microwave-assisted extraction and conventional extraction of iodine in alkaline solution were also evaluated. Iodine recoveries higher than 99% were obtained using MIC and 50 mmol l−1 (NH4)2CO3 or 56 mmol l−1 tetramethylammonium hydroxide as absorbing solution and with 5 min for the reflux step. Accuracy was evaluated using certified reference materials (bovine muscle, corn bran, and milk powder) and agreement better than 97% was obtained. The limit of quantification by MIC and further ICP-MS determination was 0.002 µg g−1. Blanks were always low and no memory effects were observed. Digestion by MIC allowed the processing of up to eight samples by each run in 25 min with high efficiency of digestion (residual carbon content lower than 1%) providing a suitable medium for further iodine determination by ICP-MS.
Keywords: Iodine determination; Food samples; Microwave-induced combustion; Inductively coupled plasma mass spectrometry

Erratum to: Proteomic biomarkers in plasma that differentiate rapid and slow decline in lung function in adult cigarette smokers with chronic obstructive pulmonary disease (COPD) by Gaurav S. J. B. Rana; Timothy P. York; Jeffery S. Edmiston; Barbara K. Zedler; Joel G. Pounds; Joshua N. Adkins; Susan M. Varnum; Richard D. Smith; Zaigang Liu; Guoya Li; Bradley T. Webb; Edward L. Murrelle; Jason W. Flora (1133-1133).