Analytical and Bioanalytical Chemistry (v.407, #12)

is Senior Professor at the Eberhard Karls University of Tübingen working in analytical and physical chemistry. He was chairman of the GDCh Division of Analytical Chemistry and chaired the Europt(r)ode VIII meeting. For more than 15 years his main scientific interest has been on research and development of chemical and biochemical sensors, with special focus on the characterization of interfaces of polymers and biomembrane surfaces, spectroscopy techniques, use of spectral interferometry to monitor changes in optical thickness of thin layers, and effects of Fresnel reflectivity at interfaces. He has been an editor of Analytical and Bioanalytical Chemistry (ABC) since 2002.

The highest melting point challenge by John Arblaster (3265-3265).

Solution to the left-handed DNA challenge by Juris Meija (3267-3267).

Surface-enhanced Raman spectroscopy of urine for prostate cancer detection: a preliminary study by Greta Del Mistro; Silvia Cervo; Elena Mansutti; Riccardo Spizzo; Alfonso Colombatti; Pietro Belmonte; Renzo Zucconelli; Agostino Steffan; Valter Sergo; Alois Bonifacio (3271-3275).
Surface-enhanced Raman scattering (SERS) spectra were obtained from urine samples from subjects diagnosed with prostate cancer as well as from healthy controls, using Au nanoparticles as substrates. Principal component analysis (PCA) of the spectral data, followed by linear discriminant analysis (LDA), leads to a classification model with a sensitivity of 100 %, a specificity of 89 %, and an overall diagnostic accuracy of 95 %. Even considering the very limited number of samples involved in this report, preliminary results from this approach are extremely promising, encouraging further investigation.
Keywords: SERS; Raman; Prostate cancer; Urine; Nanoparticles

Multi-color reflectance imaging of middle ear pathology in vivo by Tulio A. Valdez; Nicolas Spegazzini; Rishikesh Pandey; Kaitlyn Longo; Christopher Grindle; Donald Peterson; Ishan Barman (3277-3283).
Otoscopic examination using white-light illumination has remained virtually unchanged for well over a century. However, the limited contrast of white-light otoscopy constrains the ability to make accurate assessment of middle ear pathology and is subject to significant observer variability. Here, we employ a modified otoscope with multi-color imaging capabilities for superior characterization of the middle ear constituents in vivo and for enhanced diagnosis of acute otitis media and cholesteatoma. In this pilot study, five patients undergoing surgery for tympanostomy tube placement and congenital cholesteatoma excision were imaged using the custom-designed multi-color video-rate reflectance imaging system. We show that the multi-color imaging approach offers an increase in image contrast, thereby enabling clear visualization of the middle ear constituents, especially of the tympanic membrane vascularity. Differential absorption at the multiple wavelengths provides a measure of biochemical and morphological information, and the rapid acquisition and analysis of these images aids in objective evaluation of the middle ear pathology. Our pilot study shows the potential of using label-free narrow-band reflectance imaging to differentiate middle ear pathological conditions from normal middle ear. This technique can aid in obtaining objective and reproducible diagnoses as well as provide assistance in guiding excisional procedures.
Keywords: Otoscopy; Acute otitis media; Reflectance; Autofluorescence; Imaging; Medical device

Size and shape characterization of hydrated and desiccated exosomes by Vasiliy S. Chernyshev; Rakesh Rachamadugu; Yen Hsun Tseng; David M. Belnap; Yunlu Jia; Kyle J. Branch; Anthony E. Butterfield; Leonard F. Pease III; Philip S. Bernard; Mikhail Skliar (3285-3301).
is a PhD candidate in the Department of Chemical Engineering at the University of Utah and a National Science Foundation IGERT fellow. He received a BS degree in biomedical engineering from the University of Utah. His research is focused on bioanalytical chemistry, nanobiosensors, nanomaterials, and microfluidics. received a master’s degree in cell and molecular biology from the University of University of Arkansas. Currently he is a laboratory specialist in the Bernard laboratory, and his expertise is in cell culture, microRNA processing, and gene expression profiling. is a PhD candidate in the Department of Chemical Engineering at the University of Utah. is Director of the Electron Microscopy Core Laboratory and a research associate professor in the Department of Biochemistry at the University of Utah. He received training in cryogenic electron microscopy as a graduate student at Purdue University in the laboratory of Timothy Baker and as a postdoctoral researcher and staff scientist at the National Institutes of Health in the laboratory of Alasdair Steven. is a PhD candidate in the Department of Chemical Engineering at the University of Utah. Her current research focuses on noninvasive ultrasound methods to measure temperature distribution in solids. Her master’s degree project in the same department focused on characterization of nanoparticles. is a doctoral student and associate instructor in the Department of Chemical Engineering at the University of Utah. His main research interest is in engineering education, focusing on the creation and analysis of interactive simulations for undergraduate chemical engineering courses. is an Assistant Professor (lecturing) in the Chemical Engineering Department at the University of Utah. His research interests focus on undergraduate education, targeted drug delivery, data processing, and instrumentation. 's research interests include transport phenomena in the gastrointestinal tract, ultrasound and single photon emisssion computed tomography contrast agent development, tissue metabolomics, soft tissue replacement, pathogenesis of eosinophilic esophagitis, social network analysis in engineering education, and entrepreneurship. is an Associate Professor in the Department of Pathology at the University of Utah and an investigator at the Huntsman Cancer Institute. He is board certified in clinical pathology and serves as Medical Director of Molecular Oncology Diagnostics at ARUP Laboratories. His research interests include using comprehensive genomics to classify solid tumors into biologically and clinically relevant groups, and evaluating circulating tumor biomarkers. is Professor of Chemical Engineering at the University of Utah. He is a recipient of several national awards, including the National Science Foundation CAREER Award and the American Heart Association Established Investigator Award. His diverse research interests include characterization of nanoscale systems and biomarker devolvement based on biophysical properties of circulating nanovesicles Exosomes are stable nanovesicles secreted by cells into the circulation. Their reported sizes differ substantially, which likely reflects the difference in the isolation techniques used, the cells that secreted them, and the methods used in their characterization. We analyzed the influence of the last factor on the measured sizes and shapes of hydrated and desiccated exosomes isolated from the serum of a pancreatic cancer patient and a healthy control. We found that hydrated exosomes are close-to-spherical nanoparticles with a hydrodynamic radius that is substantially larger than the geometric size. For desiccated exosomes, we found that the desiccated shape and sizing are influenced by the manner in which drying occurred. Isotropic desiccation in aerosol preserves the near-spherical shape of the exosomes, whereas drying on a surface likely distorts their shapes and influences the sizing results obtained by techniques that require surface fixation prior to analysis.
Keywords: Hydrated and desiccated exosomes; Size and shape characterization

Quantitative assessment of the degree of lipid unsaturation in intact Mortierella by Raman microspectroscopy by Ute Münchberg; Lysett Wagner; Carsten Rohrer; Kerstin Voigt; Petra Rösch; Gerhard Jahreis; Jürgen Popp (3303-3311).
Fungi of the genus Mortierella can accumulate large amounts of unusual lipids depending on species, strain, and growth conditions. Fast and easy determination of key parameters of lipid quality for these samples is required. In this contribution, we apply Raman microspectroscopy to determine the degree of unsaturation for fungal lipids directly inside intact hyphae without elaborate sample handling. Six Mortierella species were grown under varying conditions, and Raman spectra of single lipid vesicles were acquired. From the spectra, we calculate a peak intensity ratio I(1270 cm−1)/I(1445 cm−1) from the signals of =CH and –CH2/–CH3 groups, respectively. This ratio is linked to the iodine value (IV) using spectra of reference compounds with known IV. IVs of fungal samples are compared to gas chromatography results. Values from both methods are in good accordance. Lipid composition is found to vary between the investigated species, with Mortierella alpina having the most unsaturated lipid (IV up to 280) and Mortierella exigua the least unsaturated (IV as low as 70). We find Raman microspectroscopy a suitable tool to determine the IV reliably, fast, and easily inside intact hyphae without extensive sample handling or treatment. The method can also be transferred to other microscopic samples. Graphical Abstract Raman spectroscopic access to the degree of unsaturation of lipid vesicles inside fungal hyphae
Keywords: Oleaginous fungi; Mortierellales ; Mortierellomycotina ; Lipid bodies; Polyunsaturated fatty acids; Arachidonic acid production

Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis by Ursula Eisold; Frank Sellrie; Jörg A. Schenk; Christine Lenz; Walter F. M. Stöcklein; Michael U. Kumke (3313-3323).
Fluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRA antibody either a strong fluorescence quenching (G71-DC7) or a distinct fluorescence enhancement (G71-BE11) upon formation of the immune complex was observed. Additional rhodamine derivatives were used to gain further information on the binding interaction. The data reveal that such haptens as 5-TAMRA could generate different paratopes with equal binding affinities but different binding interactions, which provide the opportunity to adapt bioanalysis methods including immunoassays for optimized detection principles for the same hapten depending on the specific requirements. Graphical Abstract The fluorescence of 5-TAMRA is altered upon antibody binding. Depending on the antibody used the fluorescence is heavily quenched (left) or enhanced (right) by the binding interaction
Keywords: mAb; Fluorescence; Anisotropy; Exciplex; Energy-transfer probe

Vibrational algorithms for quantitative crystallographic analyses of hydroxyapatite-based biomaterials: I, theoretical foundations by Giuseppe Pezzotti; Wenliang Zhu; Marco Boffelli; Tetsuya Adachi; Hiroaki Ichioka; Toshiro Yamamoto; Yoshinori Marunaka; Narisato Kanamura (3325-3342).
The Raman spectroscopic method has quantitatively been applied to the analysis of local crystallographic orientation in both single-crystal hydroxyapatite and human teeth. Raman selection rules for all the vibrational modes of the hexagonal structure were expanded into explicit functions of Euler angles in space and six Raman tensor elements (RTE). A theoretical treatment has also been put forward according to the orientation distribution function (ODF) formalism, which allows one to resolve the statistical orientation patterns of the nm-sized hydroxyapatite crystallite comprised in the Raman microprobe. Close-form solutions could be obtained for the Euler angles and their statistical distributions resolved with respect to the direction of the average texture axis. Polarized Raman spectra from single-crystalline hydroxyapatite and textured polycrystalline (teeth enamel) samples were compared, and a validation of the proposed Raman method could be obtained through confirming the agreement between RTE values obtained from different samples.
Keywords: Hydroxyapatite; Polarized Raman spectroscopy; Crystallographic analyses; Tooth enamel

Vibrational algorithms for quantitative crystallographic analyses of hydroxyapatite-based biomaterials: II, application to decayed human teeth by Tetsuya Adachi; Giuseppe Pezzotti; Toshiro Yamamoto; Hiroaki Ichioka; Marco Boffelli; Wenliang Zhu; Narisato Kanamura (3343-3356).
A systematic investigation, based on highly spectrally resolved Raman spectroscopy, was undertaken to research the efficacy of vibrational assessments in locating chemical and crystallographic fingerprints for the characterization of dental caries and the early detection of non-cavitated carious lesions. Raman results published by other authors have indicated possible approaches for this method. However, they conspicuously lacked physical insight at the molecular scale and, thus, the rigor necessary to prove the efficacy of this spectroscopy method. After solving basic physical challenges in a companion paper, we apply them here in the form of newly developed Raman algorithms for practical dental research. Relevant differences in mineral crystallite (average) orientation and texture distribution were revealed for diseased enamel at different stages compared with healthy mineralized enamel. Clear spectroscopy features could be directly translated in terms of a rigorous and quantitative classification of crystallography and chemical characteristics of diseased enamel structures. The Raman procedure enabled us to trace back otherwise invisible characteristics in early caries, in the translucent zone (i.e., the advancing front of the disease) and in the body of lesion of cavitated caries.
Keywords: Polarized Raman spectroscopy; Tooth enamel; Dental caries; Carious lesions

The problem of interpretation of common provenance of the samples within the infrared spectra database of polypropylene samples from car body parts and plastic containers as well as Raman spectra databases of blue solid and metallic automotive paints was under investigation. The research involved statistical tools such as likelihood ratio (LR) approach for expressing the evidential value of observed similarities and differences in the recorded spectra. Since the LR models can be easily proposed for databases described by a few variables, research focused on the problem of spectra dimensionality reduction characterised by more than a thousand variables. The objective of the studies was to combine the chemometric tools easily dealing with multidimensionality with an LR approach. The final variables used for LR models' construction were derived from the discrete wavelet transform (DWT) as a data dimensionality reduction technique supported by methods for variance analysis and corresponded with chemical information, i.e. typical absorption bands for polypropylene and peaks associated with pigments present in the car paints. Univariate and multivariate LR models were proposed, aiming at obtaining more information about the chemical structure of the samples. Their performance was controlled by estimating the levels of false positive and false negative answers and using the empirical cross entropy approach. The results for most of the LR models were satisfactory and enabled solving the stated comparison problems. The results prove that the variables generated from DWT preserve signal characteristic, being a sparse representation of the original signal by keeping its shape and relevant chemical information. Graphical Abstract The schematic procedure of expressing the evidential value of multidimensional data (FTIR and Raman spectra) by means of the likelihood ratio approach supported by discrete wavelet transform for reducing data dimensionality
Keywords: Raman spectroscopy of blue car paints; FTIR spectrometry of polymers; Data dimensionality reduction; Wavelet transform; Comparison problem; Likelihood ratio

Laser ablation-ICP-MS depth profiling to study ancient glass surface degradation by Serena Panighello; Johannes T. Van Elteren; Emilio F. Orsega; Ligia M. Moretto (3377-3391).
In general the analysis of archeological glass represents a challenge for a wide variety of objects because of the presence of physical and/or chemical damage on the surface of the artifact, also known as weathering or corrosion. To retrieve accurate bulk elemental information by laser ablation-inductively coupled plasma-mass spectrometry (ICP-MS), the original, pristine glass needs to be “reached”, thereby penetrating the alteration layer which is often more than 10 μm thick. To study this alteration layer the laser was operated in the drilling mode, either with a low (1 Hz) or a high (10 Hz) pulse repetition rate for a period of 50 s yielding detailed spatial information for ca. 20 elements over a shallow depth (ca. 5 μm) or less-detailed spatial information for 50–60 elements over a greater depth (ca. 50 μm). Quantitative elemental depth profiles (in wt%) were obtained with the so-called sum normalization calibration protocol, based on summation of the elements as their oxides to 100 wt%. We were able to associate the increase of SiO2 (in wt%) in the alteration layer to the volumetric mass density change in the glass as a result of depletion of Na2O and K2O. Also the interaction of the number of laser shots with the alteration layer is shown experimentally via depth measurements using profilometry. Chemical and physical changes in four ancient glass artifacts, directly and indirectly measureable by laser drilling, were studied as a function of internal and external factors such as age, composition, and exposure conditions.
Keywords: Laser ablation; Microanalysis; Depth profiling; Bulk composition; Internal standardization; Glass density

Quantitative urinary proteomics using stable isotope labelling by peptide dimethylation in patients with prostate cancer by Chunhui Li; Tuo Zang; Karolina Wrobel; Jeffrey T.-J. Huang; Ghulam Nabi (3393-3404).
Prostate cancer (PCa) is the most commonly diagnosed malignancy in men. The current prevalent diagnosis method, prostate-specific antigen (PSA) screening test, has low sensitivity, specificity and is poor at predicting the grade of disease. Thus, new biomarkers are urgently needed to improve the PCa diagnosis and staging for the management of patients. The aim of this study is to investigate the first voided urinary sample after massage for biomarker discovery for PCa. In this work, untargeted metabolomic profiling of the first voided urinary sample after massage from 28 confirmed prostate cancer patients, 20 benign enlarged prostate patients and 6 healthy volunteers was performed using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Single and multiple peptide protein and cross-linking molecules were identified using PEAKS software. Analytical and diagnostic performance was tested using the Student’s t test, Benjamini Hochberg correction and the receiver operating characteristic (ROC) curves. Using differential display analysis to compare peptides and cross-linking molecules of urinary samples between patients with benign, enlarged prostate and malignant cancer, we identified multiple peptides derived from osteopontin (SPP1) and prothrombin (F2) that are lower in PCa patients than in benign and enlarged prostate. The diagnosis accuracies of SPP1 and F2 peptides are 0.65–0.77 and 0.68–0.72, respectively. In addition to this, there are significant differences between PCa and benign/enlarged prostate patients in pyridinoline (PYD) and deoxypyridinoline (DPD) (p value = 0.001). Differences also, as shown in the excretion of these molecules for different stages of PCa (p value = 0.04) as the level of DPD and DPD/PYD ratio, were high in patients with locally advanced tumours. The study underscores the importance of proteomics analysis, and our results demonstrate that a urinary-based in depth proteomic approach allows the potential identification of dysregulated pathways and diagnostic biomarkers.
Keywords: Prostate cancer; Cancer diagnostic markers; Urine; Proteomics analysis; Expressed prostatic secretions

The application of HPLC and microprobe NMR spectroscopy in the identification of metabolites in complex biological matrices by Zhaoxia Miao; Mengxia Jin; Xia Liu; Wei Guo; Xiangju Jin; Hongyue Liu; Yinghong Wang (3405-3416).
Nuclear magnetic resonance (NMR)-based metabolomics can be used directly to identify a variety of metabolites in biological fluids and tissues. Metabolite analysis is an important part of life science and metabolomics research. However, the identification of some metabolites using NMR spectroscopy remains a big challenge owing to low abundance or signal overlap. It is important to develop a method to measure these compounds accurately. Two-dimensional NMR spectroscopy, metabolite prediction software packages, and spike-in experiments with authentic standards are often used to solve these problems, but they are costly and time-consuming. In this study, methods were developed to identify metabolites in complex biological mixtures using both high-performance liquid chromatography (HPLC) and off-line microprobe NMR spectroscopy. With use of these methods, 83 and 73 metabolites were identified in Sprague Dawley rat urine and feces, respectively. Among them, 40 and 45 metabolites, respectively, could not be identified with traditional NMR methods. Our research revealed that the combination of HPLC and NMR techniques could significantly improve the accuracy of trace and overlapped metabolite identification, while offering an effective and convenient approach to identify potential biomarkers in complex biological systems.
Keywords: High-performance liquid chromatography; NMR; Urine; Feces; Metabolite identification

2-Mercaptobenzothiazole (MBT) is one of the most important vulcanization accelerators in the industrial production of rubber, especially car tires. Given its wide use in household articles and industrial rubber products it has a high potential to migrate into the environment. Humans can be exposed by dermal, oral, or inhalative routes. Incorporated MBT is excreted in urine, mainly as conjugates to glucuronide, sulfate, and mercapturic acid. On the basis of these facts MBT has been selected as a substance of high interest in the large scale 10-year German project on human biomonitoring (HBM); a cooperation between the German Federal Ministry for the Environment (BMUB) and the German Chemical Industry Association (VCI) with the objective of developing new analytical methods for relevant chemicals. The presented method was developed to determine MBT in human urine to reliably investigate the internal human MBT dose. Total MBT is measured after enzymatic hydrolysis followed by application of high-pressure liquid chromatography tandem mass spectrometry (HPLC–MS–MS) in positive-electrospray-ionization mode (ESI+) using isotope-dilution quantification. High sample throughput could be obtained by use of the column-switching technique. Optimization yielded an analytical method with a low and reproducible limit of detection (LOD) of 0.4 μg L−1 and a limit of quantification (LOQ) of 1 μg L−1, and low relative standard deviations in the range 1.6–5.8 %. A small biomonitoring study covering unexposed humans and occupationally exposed workers was performed to establish the feasibility and reliability of the method. MBT was found in only one urine sample from the unexposed humans, at a value of 10.8 μg MBT per liter, whereas it was found in all samples from the tested workers at values of up to 6210 μg MBT per liter.
Keywords: 2-Mercaptobenzothiazole; MBT; Urine; Conjugates; Excretion; LC–MS–MS; Biomonitoring

The intracellular distribution of the antiancer drug doxorubicin (DOX) was followed qualitatively by fluorescence confocal spectral imaging (FCSI) and quantitatively by capillary electrophoresis (CE). FCSI permits the localization of the major fluorescent species in cell compartments, with spectral shifts indicating the polarity of the respective environment. However, distinction between drug and metabolites by FCSI is difficult due to their similar fluorochromes, and direct quantification of their fluorescence is complicated by quantum yield variation between different subcellular environments. On the other hand, capillary electrophoresis with fluorescence detection (CE-LIF) is a quantitative method capable of separating doxorubicin and its metabolites. In this paper, we propose a method for determining drug and metabolite concentration in enriched nuclear and cytosolic fractions of cancer cells by CE-LIF, and we compare these data with those of FCSI. Significant differences in the subcellular distribution of DOX are observed between the drug administered as a molecular solution or as a suspension of drug-loaded iron oxide nanoparticles coated with polyethylene glycol. Comparative analysis of the CE-LIF vs FCSI data may lead to a tentative calibration of this latter method in terms of DOX fluorescence quantum yields in the nucleus and more or less polar regions of the cytosol.
Keywords: Doxorubicin; Nanovectors; Capillary electrophoresis; Cancer

Using dielectrophoresis to study the dynamic response of single budding yeast cells to Lyticase by Shi-Yang Tang; Pyshar Yi; Rebecca Soffe; Sofia Nahavandi; Ravi Shukla; Khashayar Khoshmanesh (3437-3448).
Budding yeast cells are quick and easy to grow and represent a versatile model of eukaryotic cells for a variety of cellular studies, largely because their genome has been widely studied and links can be drawn with higher eukaryotes. Therefore, the efficient separation, immobilization, and conversion of budding yeasts into spheroplast or protoplast can provide valuable insight for many fundamentals investigations in cell biology at a single cell level. Dielectrophoresis, the induced motion of particles in non-uniform electric fields, possesses a great versatility for manipulation of cells in microfluidic platforms. Despite this, dielectrophoresis has been largely utilized for studying of non-budding yeast cells and has rarely been used for manipulation of budding cells. Here, we utilize dielectrophoresis for studying the dynamic response of budding cells to different concentrations of Lyticase. This involves separation of the budding yeasts from a background of non-budding cells and their subsequent immobilization onto the microelectrodes at desired densities down to single cell level. The immobilized yeasts are then stimulated with Lyticase to remove the cell wall and convert them into spheroplasts, in a highly dynamic process that depends on the concentration of Lyticase. We also introduce a novel method for immobilization of the cell organelles released from the lysed cells by patterning multi-walled carbon nanotubes (MWCNTs) between the microelectrodes.
Keywords: Microfluidics; Dielectrophoresis; Yeast

Liquid chromatography tandem mass spectrometry quantitation of intracellular concentrations of ganciclovir and its phosphorylated forms by Pierre-André Billat; François-Ludovic Sauvage; Nicolas Picard; Naїma Tafzi; Sophie Alain; Marie Essig; Pierre Marquet; Franck Saint-Marcoux (3449-3456).
Ganciclovir (GCV) is prescribed for cytomegalovirus infection which is a major issue in immunodepressed patients. It is however characterized by hematological toxicity. A better understanding of GCV concentration-effects relationships implies the measurement of intracellular forms. The objective of this study was to develop a method to measure GCV and its derivatives in cells. A four-stage procedure was developed with the following strategy: (1) to separate into different fractions the different intracellular forms of GCV (GCV itself and its phosphorylated forms) by solid-phase extraction (SPE) from blood cells, (2) to dephosphorylate the different phosphorylated forms into GCV, (3) to perform a second SPE to desalt samples and concentrate GCV, and (4) to measure GCV concentrations in the different extracts using a triple-quadrupole, linear ion trap mass spectrometer. Finally, the procedure was tested in 17 patients receiving GCV. From lysed cells, the different forms of GCV were fractionated, the phosphorylated forms were eluted with different KCl solutions, and the obtained fractions were treated with acid phosphatase to transform the phosphorylated metabolites back into GCV. The method was validated from 5 to 500 μg L−1 with a limit of detection of 1 μg L−1. The whole procedure was validated according to the US Food and Drug Administration guidelines and successfully applied in 17 patients receiving GCV. The method liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowing the measurement of GCV and its phosphorylated forms in blood cells was developed and can be used in developing clinical studies to explore the role of these biomarkers in the event of toxicity.
Keywords: Ganciclovir; Intracellular; LC-MS/MS; Metabolites

The number of so-called new psychoactive substances (NPS) is still increasing by modification of the chemical structure of known (scheduled) drugs. As analogues of amphetamines, 2-aminopropyl-benzofurans were sold. They were consumed because of their euphoric and empathogenic effects. After the 5-(2-aminopropyl)benzofurans, the 6-(2-aminopropyl)benzofuran isomers appeared. Thus, the question arose whether the metabolic fate, the mass spectral fragmentation, and the detectability in urine are comparable or different and how an intake can be differentiated. In the present study, 6-(2-aminopropyl)benzofuran (6-APB) and its N-methyl derivative 6-MAPB (N-methyl-6-(2-aminopropyl)benzofuran) were investigated to answer these questions. The metabolites of both drugs were identified in rat urine and human liver preparations using GC-MS and/or liquid chromatography-high resolution-mass spectrometry (LC-HR-MSn). Besides the parent drug, the main metabolite of 6-APB was 4-carboxymethyl-3-hydroxy amphetamine and the main metabolites of 6-MAPB were 6-APB (N-demethyl metabolite) and 4-carboxymethyl-3-hydroxy methamphetamine. The cytochrome P450 (CYP) isoenzymes involved in the 6-MAPB N-demethylation were CYP1A2, CYP2D6, and CYP3A4. An intake of a common users’ dose of 6-APB or 6-MAPB could be confirmed in rat urine using the authors’ GC-MS and the LC-MSn standard urine screening approaches with the corresponding parent drugs as major target allowing their differentiation. Furthermore, a differentiation of 6-APB and 6-MAPB in urine from their positional isomers 5-APB and 5-MAPB was successfully performed after solid phase extraction and heptafluorobutyrylation by GC-MS via their retention times.
Keywords: Designer drugs; 6-APB; 6-MAPB; Metabolism; GC-MS; LC-(HR)-MSn

Development of a LC-MS/MS method for the simultaneous screening of seven water-soluble vitamins in processing semi-coarse wheat flour products by Eric Nurit; Bernard Lyan; Agnès Piquet; Gérard Branlard; Estelle Pujos-Guillot (3471-3479).
Wheat is the second largest crop cultivated around the world and constitutes a major part of the daily diet in Europe. It is therefore important to determine the content of micronutrient in wheat and wheat-based food products to define the contribution of wheat-based foods to the nutrition of the consumers. The aim of the present work was to develop a simple and rapid method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of seven water-soluble vitamins in various wheat-based food materials. The vitamins present in the test material were separated in less than 15 min by using a reverse-phase C18 column, and analyzed by positive ion electrospray selected reaction monitoring MS/MS. The MS response for all the vitamins was linear over the working range (0.05 to 9 μg/mL) with correlation coefficients ranging between 0.991 and 1. Limits of quantification in the different food materials ranged from 0.09 to 3.5 μg/g. Intra-day and inter-day precision were found satisfactory. The developed method was applied for the simultaneous analysis of the water-soluble vitamin natural content of different semi-coarse wheat flours and in their corresponding baking products. Graphical Abstract Simultaneous screening of 7 water-soluble vitamins in semi-coarse wheat flour, dough, bread and toasted bread
Keywords: LC-MS/MS; Water-soluble vitamins; Food processing; Wheat

Hairpin-based DNA electrochemical sensor for selective detection of a repetitive and structured target codifying a gliadin fragment by Begoña Martín-Fernández; Noemí de-los-Santos-Álvarez; María Jesús Lobo-Castañón; Beatriz López-Ruiz (3481-3488).
High selectivity of genosensors is crucial for certain applications such as those involving species with high genetic variability. This is an unresolved problem when dealing with long target sequences that is further complicated when the target contains repetitive sequence domains. As a model for this situation, the problem of detecting gluten in food with identification of the source is studied. In order to discriminate the specific DNA sequence that encodes the wheat prolamin (gliadin) from rye and barley prolamins, the exquisite selectivity of a rationally designed hairpin capture probe is proposed and compared to a nonstructured capture probe. An electrochemical sandwich assay is proposed, involving capture probes chemisorbed on Au surfaces and biotinylated-signaling probes in combination with streptavidin-peroxidase labeling conjugates. As a result, a genosensor with similar sensitivity to that observed with linear probes but with complete specificity against closely related species was achieved. The surface-attached DNA stem-loop yields a device capable of accurately discriminating wheat DNA from rye and barley with a limit of detection of 1 nM. Graphical Abstract Hairpin-based DNA sensor for the highly selective detection of wheat sequence
Keywords: Hairpin-DNA probe; Electrochemical genosensor; Self-assembled monolayers; Enzyme amplification; Screen-printed gold electrodes; Specificity

Automated solid-phase extraction coupled online with HPLC-FLD for the quantification of zearalenone in edible oil by Sarah S. Drzymala; Stefan Weiz; Julia Heinze; Silvia Marten; Carsten Prinz; Annett Zimathies; Leif-Alexander Garbe; Matthias Koch (3489-3497).
Established maximum levels for the mycotoxin zearalenone (ZEN) in edible oil require monitoring by reliable analytical methods. Therefore, an automated SPE-HPLC online system based on dynamic covalent hydrazine chemistry has been developed. The SPE step comprises a reversible hydrazone formation by ZEN and a hydrazine moiety covalently attached to a solid phase. Seven hydrazine materials with different properties regarding the resin backbone, pore size, particle size, specific surface area, and loading have been evaluated. As a result, a hydrazine-functionalized silica gel was chosen. The final automated online method was validated and applied to the analysis of three maize germ oil samples including a provisionally certified reference material. Important performance criteria for the recovery (70–120 %) and precision (RSDr <25 %) as set by the Commission Regulation EC 401/2006 were fulfilled: The mean recovery was 78 % and RSDr did not exceed 8 %. The results of the SPE-HPLC online method were further compared to results obtained by liquid–liquid extraction with stable isotope dilution analysis LC-MS/MS and found to be in good agreement. The developed SPE-HPLC online system with fluorescence detection allows a reliable, accurate, and sensitive quantification (limit of quantification, 30 μg/kg) of ZEN in edible oils while significantly reducing the workload. To our knowledge, this is the first report on an automated SPE-HPLC method based on a covalent SPE approach. Graphical Abstract SPE-HPLC online method for automatic analysis of the mycotoxin zearalenone in edible oils.
Keywords: Dynamic covalent hydrazine chemistry (DCHC); Maize germ oil; Food; Mycotoxin

A simple and reliable method for the simultaneous determination of chiral pesticide flufiprole enantiomers using high-performance liquid chromatography has been established. The separation and determination were performed using reversed-phase chromatography on a carbamoyl–cellulose-type chiral stationary phase, a Lux Cellulose-2 column. The effects of different mobile phase composition on separation were discussed. The absolute configuration of flufiprole enantiomers was measured through the combination of experimental and predicted ECD spectra. An Alumina-N solid-phase extraction (SPE) column was used in the cleanup of the vegetables, fruits, and soil samples. The method was evaluated by the specificity, matrix effect, linearity, precision, accuracy and stability. The mean recoveries of two enantiomers ranged from 86.8 to 98.9 %, with 1.1–6.4 % intra-day relative standard deviation (RSD) and 1.2 to 5.8 % inter-day RSD. Good linearity (R 2 > 0.998) was obtained for all analyte matrix calibration curves within the range of 0.2–20 mg L−1. The limit of detection for two enantiomers in the six matrices was 0.007–0.008 mg kg−1, whereas the limit of quantification of two enantiomers in fruits, vegetables, and soil was 0.021–0.025 mg kg−1. The results confirmed that this method was convenient and accurate for the simultaneous determination of flufiprole enantiomers in food and environmental samples.
Keywords: Flufiprole; Enantioseparation; HPLC; Enantiomer analysis; Absolute configuration

Polycyclic aromatic hydrocarbons (PAHs) associated with two minerals (silica sand and bentonite) presenting opposite retention properties were analyzed with a thermodesorption (Td)-GC-MS coupling in order to validate this technique as a new and rapid way to evaluate the solid sorption properties. Two analysis modes were used, evolved gas analysis (EGA) and Td with cryo-trap. EGA allowed a real-time monitoring of the compounds desorbed during a temperature program and gave a first screening of the samples while Td gave more precise indications on compound abundances for selected temperature ranges. When associated with silica sand, PAHs were released at relatively low temperatures (<300 °C) close to corresponding boiling point, whereas for the PAH/bentonite mixture, PAHs were desorbed at much higher temperatures; they were also present in much lower abundance and were associated with mono-aromatic compounds. With bentonite, the PAH abundances decreased and the mono-aromatics increased with the increasing PAH molecular weight. These results indicated a clear PAH retention by the bentonite due to polymerization, followed by a thermal cracking at higher temperatures. The Td-GC-MS was proven to efficiently underline differences in retention properties of two minerals, and this study highlights the great potential of this technique to evaluate compound/matrix bond strength and interaction.
Keywords: Evolved gas analysis; Thermal desorption; Bentonite; Silica sand; Polycyclic aromatic hydrocarbon; Sorption

An approach for identification and determination of arsenic species in the extract of kelp by Lee L. Yu; Chao Wei; Rolf Zeisler; Junting Tong; Rabia Oflaz; Haixia Bao; Jun Wang (3517-3524).
The National Institute of Standards and Technology is developing a kelp powder standard reference material (SRM) in support of dietary supplement measurements. Edible seaweeds such as kelp and laver consumed as diet or dietary supplement contain tens of mg/kg arsenic. The speciation information of arsenic in the seaweed should be provided because the total arsenic alone does not fully address the safety issue of the dietary supplement as the value assignment is originally intended. The inability to avail all arsenic species for value assignment measurements prevented the certification of arsenic species in the candidate SRM; however, approximately 70 % of total arsenic extracted with a 1:1 volume fraction of methanol:water mixture allowed arsenic speciation values to be assigned to a procedure-defined extract, which may be used for method validation in research to improve upon current extraction and measurement practices. Arsenic species in kelp and laver were identified using electrospray ionization ion trap time of flight mass spectrometry (ESI-IT-TOF). Arsenosugars As(328), As(482), and As(392) were found in the kelp candidate SRM while As(328) and As(482) were found in GBW 08521, a certified reference material (CRM) of laver produced by the National Institute of Metrology of China (NIM). A discovery that the digests of kelp and laver contained only dimethylarsinic acid led to the conclusion that the seaweeds did not contain detectible levels of arsenobetaine, arsenocholine or trimethylarsine oxide that could overlap with the peaks of arsenosugars in the separation. The mean ± s of (5.68 ± 0.28) mg/kg and (13.43 ± 0.31) mg/kg found for As(482) and As(392) in kelp, respectively, using instrumental neutron activation analysis (INAA) demonstrated that value assignment measurement of arsenosugars was possible without arsenosugar calibration standards.
Keywords: Arsenic species; Arsenosugar; Microwave; Digestion; INAA; IT-TOF; LC-ICP-MS

Synthesis of boronic-acid-functionalized magnetic attapulgite for selective enrichment of nucleosides by Ting Cheng; Huihui Li; Ying Ma; Xiaoyan Liu; Haixia Zhang (3525-3529).
2,4-Difluoro-3-formyl-phenylboronic acid (DFFPBA)-modified magnetic attapulgite (ATP-Fe3O4-NH2-DFFPBA) was synthesized and employed to capture and enrich cis-diol-containing biomolecules. The resulting material exhibited a high saturation magnetization value of 20.71 emu/g, allowing the absorbent to be conveniently magnetically separated. Combining the Fe3O4 nanoparticles with the high specific surface area of attapulgite yielded a material with a high capture capacity (13.78 mg/g) for adenosine. Furthermore, ATP-Fe3O4-NH2-DFFPBA was found to possess remarkable selectivity for adenosine at a low molar ratio of adenosine/2-deoxyadenosine (1:500). The potential applications of this material were explored by using it to extract five nucleosides from urine samples, and the results demonstrate that it can decrease matrix interference and selectively enrich analytes. Graphical Abstract Boronic-acid-functionalized magnetic attapulgite could selectively enrich the nucleosides in urine samples with the help of an external magnet
Keywords: Boronate affinity chromatography; Nucleosides; Magnetic; Attapulgite; Enrichment

Preliminary study on suitability of ionic liquids as potential passive-sampling media of polyaromatic-hydrocarbon (PAH) analyses in water by Joanna Maszkowska; Elżbieta Synak; Aleksandra Fabiańska; Magda Caban; Stefan Stolte; Pascale Husson; Margarida F. Costa Gomes; Roman Synak; Piotr Stepnowski (3531-3536).
Recently, ionic liquids (ILs) have been regarded as an attractive water-immiscible phase in liquid–liquid extraction. Because ILs have a wide range of polarity irrespective of their miscibility with water, the possibility of using them as an effective extraction phase for a broad range of contaminants means they are starting to be of particular interest. In this study we investigated a wide variety of ionic liquids, which are known to be hydrolytically stable and of a hydrophobic character, for their potential suitability as passive-sampling media for monitoring selected polyaromatic hydrocarbons. Preliminary research in this field has indicated very promising results using these novel extraction media. Because there is an enormous number of possible cation–anion combinations offering tuneable properties of ionic liquids with the potential for effective passive extraction, we hope this paper will encourage the scientific community to undertake further studies verifying the undoubted usefulness of these alternative solvents as passive samplers for many other groups of analytes. Additionally, because of the unusual solubility properties that have already been proved for ILs, it is very probable that it would soon be possible to deliver a very effective system able to extract analytes differing widely in polarity.
Keywords: Ionic liquids; Passive sampling; Water analysis; PAH