Analytical and Bioanalytical Chemistry (v.400, #9)

is an Associate Professor of Chemistry at Concordia College in Moorhead, MN where his primary teaching responsibilities are focused on analytical and general chemistry. He has been actively involved in redesigning Concordia’s undergraduate laboratories toward a more inquiry-based approach, and has recently been awarded a National Science Foundation grant to incorporate student-designed projects involving analytical instrumentation into both the analytical and general chemistry laboratories. His research interests include applications of the scanning electrochemical microscope in corrosion science, and electrochemical methods of detection in HPLC.

Living cell spectroscopy challenge by Allison Stelling; Gerald Steiner (2681-2682).

Solution to nuclear Overhauser effect challenge by Reinhard Meusinger (2683-2684).

Analytical biophotonics by Jürgen Popp; Reiner Salzer (2685-2686).
Is a full professor at the Friedrich-Schiller University of Jena, Germany, where he holds a chair of physical chemistry. In June 2006 he also became the Scientific Director at the Institute of Photonic Technology. His work has been awarded the Faculty Prize of Chemistry (1995) by the “Bayerischer Habilitationsförderpreis” (1997), the “Förderpreis der Würzburger Korporationen” (2001), and the Kirchhoff–Bunsen Award (2002).J. Popp’s research interests are mainly centred around the development and application of frequency-resolved, time-resolved, and spatially resolved innovative laser spectroscopic methods and techniques ranging from the UV into the NIR region for the derivation of structure–activity or dynamic relationships. This type of investigative approach is essential in resolving important questions in fields of biology, medicine, pharmacy, and astronomy, and in the environmental and material sciences. In particular his expertise in the field of Raman spectroscopy and in the development of innovative Raman techniques should be emphasized. The results obtained by J. Popp have been published in more than 200 scientific articles in premier peer-reviewed journals. He is inventor named on eight patents in the field of spectroscopic instrumentation. Since 2008 he has been coordinator of the Network of Excellence for Biophotonics—“Photonics4Life”. Retired as Professor of Analytical Chemistry at the Technische Universität Dresden, Germany, in 2007. He obtained his academic degrees from the University of Leipzig, before taking up his appointment at the TU Dresden. His main scientific interests include molecular monitoring for early diagnosis of diseases, integration of biologically active functions into polymers, and electronic media in university education. Professor Salzer has authored 14 patents in different fields of analytical chemistry, and over 250 books and scientific publications.Professor Salzer has been elected on to a variety of national and international boards. He is a member of the Norwegian Academy of Science, a recipient of the Emich Plaque of the Austrian Society of Analytical Chemistry (2007) and of the Clemens Winkler Medal of the Division Analytical Chemistry of the German Chemical Society (2011). Professor Salzer is a long-time advisory board member for analytical and spectroscopic journals. He served as President of the Division Analytical Chemistry of the German Chemical Society, and is National Delegate to the Division of Analytical Chemistry of EuCheMS, where he is Head of the Study Group Education. He was elected as Vice-Chairman of the ECTNA Label Committee for the Chemistry Eurobachelor and Chemistry Euromaster and is currently involved in the development of a quality scheme for a Eurodoctorate label.

Non-linear fluorescence lifetime imaging of biological tissues by Riccardo Cicchi; Francesco Saverio Pavone (2687-2697).
In recent years fluorescence microscopy has become a widely used tool for tissue imaging and spectroscopy. Optical techniques, based on both linear and non-linear excitation, have been broadly applied to imaging and characterization of biological tissues. Among fluorescence techniques used in tissue imaging applications, in recent years two and three-photon excited fluorescence have gained increased importance because of their high-resolution deep tissue imaging capability inside optically turbid samples. The main limitation of steady-state fluorescence imaging techniques consists in providing only morphological information; functional information is not detectable without technical improvements. A spectroscopic approach, based on lifetime measurement of tissue fluorescence, can provide functional information about tissue conditions, including its environment, red-ox state, and pH, and hence physiological characterization of the tissue under investigation. Measurement of the fluorescence lifetime is a very important issue for characterizing a biological tissue. Deviation of this property from a control value can be taken as an indicator of disorder and/or malignancy in diseased tissues. Even if much work on this topic has still to be done, including the interpretation of fluorescence lifetime data, we believe that this methodology will gain increasing importance in the field of biophotonics. In this paper, we review methodologies, potentials and results obtained by using fluorescence lifetime imaging microscopy for the investigation of biological tissues. Figure
Keywords: Clinical/biomedical analysis; Fluorescence/luminescence; Laser spectroscopy

Optical coherence tomography—current technology and applications in clinical and biomedical research by Sebastian Marschall; Birgit Sander; Mette Mogensen; Thomas M. Jørgensen; Peter E. Andersen (2699-2720).
Optical coherence tomography (OCT) is a noninvasive imaging technique that provides real-time two- and three-dimensional images of scattering samples with micrometer resolution. By mapping the local reflectivity, OCT visualizes the morphology of the sample. In addition, functional properties such as birefringence, motion, or the distributions of certain substances can be detected with high spatial resolution. Its main field of application is biomedical imaging and diagnostics. In ophthalmology, OCT is accepted as a clinical standard for diagnosing and monitoring the treatment of a number of retinal diseases, and OCT is becoming an important instrument for clinical cardiology. New applications are emerging in various medical fields, such as early-stage cancer detection, surgical guidance, and the early diagnosis of musculoskeletal diseases. OCT has also proven its value as a tool for developmental biology. The number of companies involved in manufacturing OCT systems has increased substantially during the last few years (especially due to its success in opthalmology), and this technology can be expected to continue to spread into various fields of application.
Keywords: Optical coherence tomography; Optical imaging; Clinical/biomedical analysis

Optical coherence tomography in biomedical research by Julia Walther; Maria Gaertner; Peter Cimalla; Anke Burkhardt; Lars Kirsten; Sven Meissner; Edmund Koch (2721-2743).
Optical coherence tomography (OCT) is a noninvasive, high-resolution, interferometric imaging modality using near-infrared light to acquire cross-sections and three-dimensional images of the subsurface microstructure of biological specimens. Because of rapid improvement of the acquisition speed and axial resolution of OCT over recent years, OCT is becoming increasingly attractive for applications in biomedical research. Therefore, OCT is no longer used solely for structural investigations of biological samples but also for functional examination, making it potentially useful in bioanalytical science. The combination of in vivo structural and functional findings makes it possible to obtain thorough knowledge on basic physiological and pathological processes. Advanced applications, for example, optical biopsy in visceral cavities, have been enabled by combining OCT with established imaging modalities. This report gives an outline of the state of the art and novel trends of innovative OCT approaches in biomedical research in which the main focus is on applications in fundamental research and pre-clinical utilization.
Keywords: Optical coherence tomography; Instrumentation; Application; Biomedical imaging

Intra-operative optical diagnostics with vibrational spectroscopy by Allison Stelling; Reiner Salzer; Matthias Kirsch; Stephan B. Sobottka; Kathrin Geiger; Edmund Koch; Gabriele Schackert; Gerald Steiner (2745-2753).
Established methods for characterization of tissue and diagnostics, for example histochemistry, magnetic resonance imaging (MRI), X-ray tomography, or positron emission tomography (PET), are mostly not suitable for intra-operative use. However, there is a clear need for an intra-operative diagnostics especially to identify the borderline between normal and tumor tissue. Currently, vibrational spectroscopy techniques (both Raman and infrared) complement the standard methods for tissue diagnostics. Vibrational spectroscopy has the potential for intra-operative use, because it can provide a biochemically based profile of tissue in real time and without requiring additional contrast agents, which may perturb the tissue under investigation. In addition, no electric potential needs to be applied, and the measurements are not affected by electromagnetic fields. Currently, promising approaches include Raman fiber techniques and nonlinear Raman spectroscopy. Infrared spectroscopy is also being used to examine freshly resected tissue ex vivo in the operating theater. The immense volume of information contained in Raman and infrared spectra requires multivariate analysis to extract relevant information to distinguish different types of tissue. The promise and limitations of vibrational spectroscopy methods as intra-operative tools are surveyed in this review.
Keywords: Vibrational spectroscopy; Intra-operative; Tissue analysis

Detection of thiopurine methyltransferase activity in lysed red blood cells by means of lab-on-a-chip surface enhanced Raman spectroscopy (LOC-SERS) by Anne März; Bettina Mönch; Petra Rösch; Michael Kiehntopf; Thomas Henkel; Jürgen Popp (2755-2761).
In this contribution, the great potential of surface enhanced Raman spectroscopy (SERS) in a lab-on-a-chip (LOC) device for the detection of analyte molecules in a complex environment is demonstrated. Using LOC-SERS, the enzyme activity of thiopurine S-methyltransferase (TPMT) is analysed and identified in lysed red blood cells. The conversion of 6-mercaptopurine to 6-methylmercaptopurine catalysed by TPMT is observed as it gives evidence for the enzyme activity. Being able to determine the TPMT activity before starting a treatment using 6-mercaptopurine, an optimized dosage can be applied to each patient and serious toxicity appearing within thiopurine treatment will be prevented.
Keywords: SERS; Microfluidic lab-on-a-chip; Enzyme activity; Thiopurine methyltransferase; 6-Mercaptopurine

Raman spectroscopic detection of physiology changes in plasmid-bearing Escherichia coli with and without antibiotic treatment by Angela Walter; Martin Reinicke; Thomas Bocklitz; Wilm Schumacher; Petra Rösch; Erika Kothe; Jürgen Popp (2763-2773).
Bacterial resistances against antibiotics are increasingly problematic for medical treatment of pathogenic bacteria, e.g., in hospitals. Resistances are, among other genes, often encoded on plasmids which can be transmitted between bacteria not only within one species, but also between different species, genera, and families. The plasmid pDrive is transformed into bacteria of the model strain Escherichia coli DH5α. Within this investigation, we applied micro-Raman spectroscopy with two different excitation wavelengths in combination with support vector machine (SVM) and linear discriminant analysis (LDA) to differentiate between bacterial cultures according to their cultural plasmid content. Recognition rates of about 92% and 90% are achieved by Raman excitation at 532 and 244 nm, respectively. The SVM loadings reveal that the pDrive transformed bacterial cultures exhibit a higher DNA content compared to the untransformed cultures. To elucidate the influence of the antibiotic, ampicillin-treated cultures are also comprised within this study and are classified with rates of about 97% and 100% for 532 and 244 nm Raman excitation, respectively. The Raman spectra recorded with 532 nm excitation wavelength show differences of the secondary protein structure and enhanced stress-related respiration rates for the ampicillin-treated cultures. Independent cultural replicates of either ampicillin-challenged or non-challenged cultures are successfully identified with identification rates of over 90%. Figure The plasmid content of bacteria is directly evaluated by means of Raman spectroscopy
Keywords: Raman spectroscopy; Antibiotic resistance; Plasmids

Gender determination of fertilized unincubated chicken eggs by infrared spectroscopic imaging by Gerald Steiner; Thomas Bartels; Allison Stelling; Maria-Elisabeth Krautwald-Junghanns; Herbert Fuhrmann; Valdas Sablinskas; Edmund Koch (2775-2782).
Each year, billions of day-old layer chicks are produced in the world. Since only female chicks are reared for egg production, the chicks must be sexed and the unwanted male layer chicks are culled. The culling of male chicks is a serious problem, both in terms of animal welfare and waste disposal. The germinal disc in fertilized but unincubated eggs contains already several thousands of blastoderm cells. The cellular DNA in birds is different for male and female chicks. The difference in DNA content between male and female chicks is around 2% and is measurable by Fourier transform infrared (FT-IR) spectroscopy. In this study, small amounts of blastoderm cells from 22 chicken eggs were characterized by attenuated total reflection FT-IR spectroscopic imaging and classified by linear discriminant analysis. Polymerase chain reaction (PCR) was used as a reference method to determine the gender. The spectroscopic results demonstrate that male blastoderm cells exhibit a higher content of DNA than cells from female blastoderm. The spectroscopic-based gender determination led to the same result as the PCR analysis. FT-IR spectroscopic imaging allows the gender determination of unincubated eggs within a few seconds based on the accurate determination of the different DNA contents in blastoderm cells of both sexes.
Keywords: Spectroscopy; IR spectroscopy; Bioanalytical methods

A grating coupler system has been developed to measure refractive index gradients with high spatial (6.7 μm) and temporal (milliseconds) resolution. The system was applied to two-phase model systems consisting of water and non-aqueous pollution liquids. Refractive index gradients at the interfaces between the aqueous and organic phase of 1-butanol, hexane, and 1-heptanol were monitored under steady-state conditions. The temporal resolution was utilized in diffusion experiments with glycerol and sodium chloride in water, where the formation of a concentration gradient was studied. In a further application, the grating coupler system was modified to monitor low-level concentrations of aqueous pollution profiles as are caused by bacterial degradation in the aqueous phase. Toluene was selected as contaminant. The sensor sensitivity was improved by coating the sensor with the pre-concentrating polymers polydimethylsiloxane and Teflon® AF-2400. With the grating coupler setup, a multi-purpose instrument was created to measure high-resolution refractive index gradients with high temporal and spatial resolution in different fields of application. The new sensor system can be used to measure absolute refractive indices by covering parts of the sensing area with cover media of known refractive index. Coatings can be used for sensitivity improvement by pre-concentrating the sample, for selectivity by utilizing filtering properties of the coating, and as calibration standard for absolute refractive index measurements.
Keywords: Evanescent field; Planar waveguide; Grating coupler; Refractive index gradient; Sensor coating

We report the preparation of a kind of surface-enhanced Raman scattering (SERS) tags and explore their applications in multifunctional optical imaging of cancer cells. The proposed nanoparticles (SERS tags) are prepared by connecting dye molecules directly onto the surfaces of gold nanorods through Au–S or Au–N interactions. The dye molecules are used as Raman reporters, while gold nanorods are used as enhanced materials due to their localized surface plasmon resonance effect. Multilayered polymers are further coated onto the surfaces of the nanoparticles to reach better stability and biocompatibility. Gold nanorods with different aspect ratios and different dye molecules conjugated are compared in order to achieve the diversity of SERS tags and find out the optimized condition of SERS tags with the highest signal intensity. Our experiments show that the resulting nanoparticles, which are uptaken by cancer cells, can provide not only dark field cells images but also multiplexing SERS images. Figure Dark field image (a), transmission image (b), SERS spectrum (c) and fast SERS mapping of specific Raman bands (d) of HeLa cells marked with both anti-CEA-8 modified PAH-PSS-DTTC-643nm GNRs and Tf modified PAH-PSS-O170-643nm GNRs
Keywords: SERS tags; Dye molecules; Raman bands; Multiplexing image

Raman spectroscopic grading of astrocytoma tissues: using soft reference information by Claudia Beleites; Kathrin Geiger; Matthias Kirsch; Stephan B. Sobottka; Gabriele Schackert; Reiner Salzer (2801-2816).
Gliomas are the most frequent primary brain tumours. During neurosurgical treatment, locating the exact tumour border is often difficult. This study assesses grading of astrocytomas based on Raman spectroscopy for a future application in intra-surgical guidance. Our predictive classification models distinguish the surgically relevant classes “normal tissue” and “low” and “high grade astrocytoma” in Raman maps of moist bulk samples (80 patients) acquired with a fibre-optic probe. We introduce partial class memberships as a strategy to utilize borderline cases for classification. Borderline cases supply the most valuable training and test data for our application. They are (a) examples of the sought boundary and (b) the cases for which new diagnostics are needed. Besides, the number of suitable training samples increases considerably: soft logistic regression (LR) utilizes 85% more spectra and 50% more patients than linear discriminant analysis (LDA). The predictive soft LR models achieve ca. 85, 67 and 84% (normal, low and high grade) sensitivity and specificity. We discuss the different heuristics of LR and LDA in the light of borderline samples. While we focus on prediction, the spectroscopic interpretation of the predictive models agrees with previous descriptive studies. Unsaturated lipids are used to differentiate between normal and tumour tissues, while the total lipid content prominently contributes to the determination of the tumour grade. The high-wavenumber region above 2,800 cm−1 alone did not allow successful grading. We give a proof of concept for Raman spectroscopic grading of moist astrocytoma tissues and propose to include borderline samples into classifier training and testing.
Keywords: Gliomas; Astrocytomas; Grading; Classification; Tumour; Raman spectroscopy; Linear discriminant analysis; Logistic regression; Soft classification

The most prevalent molecular constituents of skin are spatially mapped by the use of nonlinear interferometric vibrational imaging, a coherent anti-Stokes Raman scattering (CARS)-based technique. Raman-like profiles over the range from 2,800 to 3,000 cm−1 are acquired by means of completely suppressing the non-resonant background, allowing the generation of images based on the molecule-specific spectral profiles over the probed region with high spatial resolution. A simple algorithm that maps spectral content to color allows the visualization of histology in a manner analogous to that obtained with more conventional staining procedures (e.g., hematoxylin-eosin), but faster and with the benefit of having access to localized spectra, which could further enhance the potential for diagnosis of diseases, especially during the early stages of development. Figure Identification of adipose and collagenous domains in skin with Nonlinear Interferometric Vibrational Imaging, a coherent anti-Stokes Raman scattering microscopy technique that excites vibrations over the bandwidth from 2800 to 3000 cm-1 and with suppression of the non-resonant background by means of detecting the anti-Stokes signals interferometrically.
Keywords: Optical imaging; Nonlinear interferometric vibrational imaging; Coherent anti-Stokes Raman scattering; Interferometry; Skin

Analysis of membrane and hydrophilic proteins simultaneously derived from the mouse brain using cloud-point extraction by Magnus Wetterhall; Ganna Shevchenko; Konstantin Artemenko; Marcus O. D. Sjödin; Jonas Bergquist (2827-2836).
In this study, a temperature-induced phase fractionation known as cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was used to simultaneously extract, concentrate, and fractionate hydrophobic and hydrophilic proteins from mouse brain tissue. Two bottom-up proteomic techniques were used to comprehensively identify the extracted proteins. The first “shotgun”-based approach included tryptic digestion of the proteins followed by reversed-phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). In the second approach, the extracted intact proteins were first separated by one-dimensional (1D) gel electrophoresis and then in-gel digested with trypsin and analyzed with nanoLC-MS/MS. In total, 1,825 proteins were unambiguously identified and the percentage of membrane proteins was 26% which is at the reported genome expression levels of 20–30%. The protein overlap between the two approaches was high. The majority (77%) of the identifications in the first approach was also found by the second method. The protein overlap between the CPE-extracted hydrophilic and hydrophobic fractions was rather small (16–23%) for both methods, which indicates a good phase separation. A quantitative evaluation of the CPE with iTRAQ labeling and nanoLC-ESI-MS/MS analysis gave iTRAQ ratios at the expected levels and an overall variation of the entire method at 17–31%. The results indicate very reproducible sample preparation and analysis methods that readily can be applied on large-scale sample sets.
Keywords: Cloud-point extraction (CPE); Brain; Proteomics; Membrane proteins (MPs); Mass spectrometry (MS); iTRAQ

The detection of corticosteroids and sex steroids in samples with no content indication, which are confiscated for forensic investigation, is a challenge in doping analysis. A screening method based on the identification of androgens, estrogens, gestagens, and their esters by means of a mass spectral library, along with a fast ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) method, was recently developed in our lab for the analysis of dietary supplements. However, for forensic investigations, it is important to extend the scope of the method to corticosteroids in various matrices. Therefore, 36 corticosteroids were added to the mass spectral library, and the sample preparation step was modified so that androgens, gestagens, corticosteroids, and their esters could be analyzed with only one injection with the UPLC-MS method. A complementary tool to the existing library identification was found in the extraction of common fragment ions out of the full scan data obtained for the library search. The fragment ion with m/z 147 was found to be a good marker for the detection of steroids. Extra confirmation was obtained from the fragment ions with m/z 135 (for all steroids) and 237 (specific for corticosteroids) or from the fragment ions with m/z 77, 91, and 105. The effectiveness of this approach was evaluated on some samples previously screened for forensic investigation with thin-layer chromatography and confirmed with a targeted gas chromatography–mass spectrometry method. This study shows that the combination of the library identification and the common fragment ions approach can be a valuable tool in the detection of steroids without defining any target at the start of the analysis.
Keywords: Corticosteroids; Library identification; Common fragment ions; UPLC-MS

Lanthanide-based time-resolved luminescence immunoassays by A. K. Hagan; T. Zuchner (2847-2864).
The sensitive and specific detection of analytes such as proteins in biological samples is critical for a variety of applications, for example disease diagnosis. In immunoassays a signal in response to the concentration of analyte present is generated by use of antibodies labeled with radioisotopes, luminophores, or enzymes. All immunoassays suffer to some extent from the problem of the background signal observed in the absence of analyte, which limits the sensitivity and dynamic range that can be achieved. This is especially the case for homogeneous immunoassays and surface measurements on tissue sections and membranes, which typically have a high background because of sample autofluorescence. One way of minimizing background in immunoassays involves the use of lanthanide chelate labels. Luminescent lanthanide complexes have exceedingly long-lived luminescence in comparison with conventional fluorophores, enabling the short-lived background interferences to be removed via time-gated acquisition and delivering greater assay sensitivity and a broader dynamic range. This review highlights the potential of using lanthanide luminescence to design sensitive and specific immunoassays. Techniques for labeling biomolecules with lanthanide chelate tags are discussed, with aspects of chelate design. Microtitre plate-based heterogeneous and homogeneous assays are reviewed and compared in terms of sensitivity, dynamic range, and convenience. The great potential of surface-based time-resolved imaging techniques for biomolecules on gels, membranes, and tissue sections using lanthanide tracers in proteomics applications is also emphasized.
Keywords: Bioassays; Fluorescence/luminescence; Rare earth elements; Genomics/proteomics; Bioanalytical methods

Antibody characterization and immunoassays for palytoxin using an SPR biosensor by Betsy Jean Yakes; Stacey L. DeGrasse; Mark Poli; Jonathan R. Deeds (2865-2869).
Palytoxin (PLTX), a polyether marine toxin originally isolated from the zoanthid Palythoa toxica, is one of the most toxic non-protein substances known. Fatal poisonings have been linked to ingestion of PLTX-contaminated seafood, and effects in humans have been associated with dermal and inhalational exposure to PLTX containing organisms and waters. Additionally, PLTX co-occurrence with other well-characterized seafood toxins (e.g., ciguatoxins, saxitoxins, tetrodotoxin) has hindered direct associations of PLTX to seafood-borne illnesses. There are currently no validated methods for the quantitative detection of PLTX(s). As such, a well-characterized, robust, specific analytical technique is needed for the detection of PLTX(s) in source organisms, surrounding waters, and clinical samples. Surface plasmon resonance (SPR) biosensors are ideally suited for antibody characterization and quantitative immunoassay detection. Herein, we describe a newly developed SPR assay for PLTX. An anti-mouse substrate was used to characterize the kinetic values for a previously developed monoclonal anti-PLTX. The characterized antibody was then incorporated into a sensitive, rapid, and selective PLTX assay. Buffer type, flow rate, analyte-binding time, and regeneration conditions were optimized for the antibody–PLTX system. Cross-reactivity to potentially co-occurring seafood toxins was also evaluated. We show that this optimized assay is capable of measuring low- to sub-ng/mL PLTX levels in buffer and two seafood matrices (grouper and clam). Preliminary results indicate that this SPR biosensor assay allows for (1) rapid characterization of antibodies and (2) rapid, sensitive PLTX concentration determination in seafood matrices. Method development information contained herein may be broadly applied to future PLTX detection and/or antibody characterization efforts.
Keywords: Palytoxin; Surface plasmon resonance; Biosensor; Antibody characterization; Immunoassay

Investigation of the specificity of Raman spectroscopy in non-invasive blood glucose measurements by Narahara Chari Dingari; Ishan Barman; Gajendra P. Singh; Jeon Woong Kang; Ramachandra R. Dasari; Michael S. Feld (2871-2880).
Although several in vivo blood glucose measurement studies have been performed by different research groups using near-infrared (NIR) absorption and Raman spectroscopic techniques, prospective prediction has proven to be a challenging problem. An important issue in this case is the demonstration of causality of glucose concentration to the spectral information, especially as the intrinsic glucose signal is smaller compared with that of the other analytes in the blood–tissue matrix. Furthermore, time-dependent physiological processes make the relation between glucose concentration and spectral data more complex. In this article, chance correlations in Raman spectroscopy-based calibration model for glucose measurements are investigated for both in vitro (physical tissue models) and in vivo (animal model and human subject) cases. Different spurious glucose concentration profiles are assigned to the Raman spectra acquired from physical tissue models, where the glucose concentration is intentionally held constant. Analogous concentration profiles, in addition to the true concentration profile, are also assigned to the datasets acquired from an animal model during a glucose clamping study as well as a human subject during an oral glucose tolerance test. We demonstrate that the spurious concentration profile-based calibration models are unable to provide prospective predictions, in contrast to those based on actual concentration profiles, especially for the physical tissue models. We also show that chance correlations incorporated by the calibration models are significantly less in Raman as compared to NIR absorption spectroscopy, even for the in vivo studies. Finally, our results suggest that the incorporation of chance correlations for in vivo cases can be largely attributed to the uncontrolled physiological sources of variations. Such uncontrolled physiological variations could either be intrinsic to the subject or stem from changes in the measurement conditions.
Keywords: Raman spectroscopy; Non-invasive glucose monitoring; Chance correlations; Causation; Animal model; Human subject

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone in human plasma is described. It was employed in a pharmacokinetic study. The analyte and internal standard diphenhydramine were extracted from plasma using liquid–liquid extraction, then separated on a Zorbax XDB C18 column using a mobile phase of methanol–water–formic acid (80:20:0.5, v/v/v). The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. Linearity was established in the concentration range of 10.0-5,000 pg/ml. The lower limit of quantification was 10.0 pg/ml. The intraday and interday relative standard deviation across three validation runs over the entire concentration range was less than 13%. Accuracy determined at three concentrations (25.0, 200, and 4,000 pg/ml for tandospirone) ranged from 94.4 to 102.1%. Each plasma sample was chromatographed within 3.4 min. The method proved to be highly selective and suitable for bioequivalence evaluation of different formulations containing tandospirone and clinical pharmacokinetic investigation of tandospirone.
Keywords: Tandospirone; Liquid chromatography–tandem mass spectrometry; Human plasma

In vitro phase I metabolism of the depsipeptide enniatin B by Lada Ivanova; Christiane Kruse Fæste; Silvio Uhlig (2889-2901).
The enniatins are a group of more than 20 cyclic depsipeptides from fungi with numerous biological effects. Enniatin B is commonly one of the principal analogues in species of the genus Fusarium, known to have ionophoric, antibiotic and insecticidal activity. In the present study, enniatin B was incubated with rat, dog and human liver microsomes. The compound was extensively metabolised, and 12 biotransformation products (M1–M12) were detected and their structures tentatively identified using a combination of mass spectrometric techniques and chemical derivatisation. Ion trap mass spectrometry, multiple-stage MS n fragmentation and high-resolution mass spectrometry were the instrumental backbone for structural determination, while acetylation, methylation and Jones oxidation were useful derivatisation techniques for the localisation of the site of biotransformation. Comparison of mass spectrometric data of the metabolism products with that of enniatin B suggested that M1–M5 are monohydroxylated species, while M8–M12 are the result of multiple oxidations (oxygenation and dehydrogenation). Metabolites M6 and M7 appeared to be enniatin B homologues and are the result of N-demethylation. Our findings show that oxidation and N-demethylation are the principal metabolic pathways in enniatin B phase I metabolism.
Keywords: Enniatins; Metabolism; Liver microsomes; Cytochrome P450; LC-MS

Sarcosine is an amino acid derivative of N-methylglycine and is involved in the amino acid metabolism and methylation processes that are enriched during prostate cancer progression. It could also serve as a new target to be measured during therapeutic interventions and help in the identification of aggressive tumors for radical treatment. In this study, we present a new urine test that can help early diagnosis of prostate cancer. The method for the quantification of sarcosine in urine consists of a solid-phase microextraction (SPME) step followed by gas chromatography–triple quadrupole mass spectrometry analysis. We used a preliminary derivatization step with ethyl chloroformate/ethanol and the corresponding ester was then extracted by SPME in immersion mode. Several fibers were evaluated and the optimization of the parameters affecting the SPME process was carried out using an experimental design. The optimal values were 20 min extraction time, 10% NaCl, and 270°C using a divinylbenzene/Carboxen/polydimethylsiloxane fiber. The triple quadrupole analyzer acquired data in selected reaction monitoring mode, allowing us to obtain reconstructed chromatograms with well-defined chromatographic peaks. The accuracy and precision of this method were evaluated at concentrations of 70, 250, and 800 ng/ml and were found to be acceptable. Very satisfactory values (0.10 and 0.16 ng/ml, respectively) were also achieved for the limit of detection and the limit of quantification. The proposed protocol represents a rapid, simple, selective, and sensitive tool to quantify sarcosine in urine samples for prostate cancer diagnosis and for a screening test.
Keywords: Sarcosine; Prostate cancer; Alkyl chloroformate; Gas chromatography; Solid-phase microextraction; Tandem mass spectrometry

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography by María Castro-Puyana; Ivonne Lammers; Joost Buijs; Cees Gooijer; Freek Ariese (2913-2919).
Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-β-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of l-MTX and its enantiomeric impurity d-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10−7 M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of l-MTX. Furthermore, l-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples. Figure A quenched phosphorescence method was developed and coupled to chiral EKC for the sensitive detection of the drug L-methotrexate and its impurity D-MTX; unspiked pharmaceutical formulation
Keywords: Capillary electrophoresis; Chiral analysis; Cyclodextrin; Enantiomeric purity

Damages induced by free radicals on human serum albumin (HSA), the most prominent protein in plasma, were investigated by Raman spectroscopy. HSA underwent oxidative and reductive radical stress. Gamma-irradiation was used to simulate the endogenous formation of reactive radical species such as hydrogen atoms (H), solvated electrons (e aq ) and hydroxyl radicals (OH). Raman spectroscopy was shown to be a useful tool in identifying conformational changes of the protein structure and specific damages occurring at sensitive amino acid sites. In particular, the analysis of the S–S stretching region suggested the radical species caused modifications in the 17 disulphide bridges of HSA. The concomitant action of e aq and H atoms caused the formation of cyclic disulphide bridges, showing how cystine pairs act as efficient interceptors of reducing species, by direct scavenging and electron transfer reactions within the protein. This conclusion was further confirmed by the modifications visible in the Raman bands due to Phe and Tyr residues. As regards to protein folding, both oxidative and reductive radical stresses were able to cause a loss in α-helix content, although the latter remains the most abundant secondary structure component. β-turns motifs significantly increased as a consequence of the synergic action of e aq and H atoms, whereas a larger increase in the β-sheet content was found following the exposure to OH and/or H attack. Schematic presentation of the procedure followed for the identification of damaging mechanisms on proteins undergone to oxidative and reductive radical stress. Step 1: generation of free radicals by gamma-radiolysis of water, mimicking an endogenous radical stress on human serum albumin (HSA); step 2: analysis of the Raman and IR spectra of the samples before and after radical stress exposure and step 3: identification of the main targets of the free radical attack and hypothesis of damaging radical-mediated mechanisms
Keywords: Human serum albumin; Raman spectroscopy; Gamma-irradiation; Radical-induced damage; Free radicals

Profiling of non-esterified fatty acids in human plasma using liquid chromatography-electron ionization mass spectrometry by Helga Trufelli; Giorgio Famiglini; Veronica Termopoli; Achille Cappiello (2933-2941).
This paper focuses on the development of a novel approach to analyze underivatized fatty acids in human plasma. The method is based on liquid–liquid extraction followed by reversed phase liquid chromatography coupled to direct-electron ionization mass spectrometry (LC-Direct-EI-MS). The assay is validated. Calibrations show satisfactory linearity and precision in the investigated range of linearity. Recoveries span from 75% to 104%. The method limits of detection, varying from 0.53 to 5.35 μM, are satisfactory for the quantitation of non-esterified fatty acids (NEFAs) in plasma at physiological levels. The method has been successfully applied to the NEFAs profiling of plasma samples from healthy adult volunteers and subjects affected by diabetes mellitus. Compared with published protocols based on gas chromatography–mass spectrometry and liquid chromatography coupled to electrospray ionization mass spectrometry, this method does not require derivatization and does not show matrix effects, thus simplifying sample preparation procedure and reducing the total time of analysis to approximately 90 min. In addition, Direct-EI-MS allows the acquisition of high-quality NIST library-matchable EI spectra, allowing an easy-to-obtain identification of the target NEFAs.
Keywords: Non-esterified fatty acids; Human plasma; LC-Direct-EI-MS; Diabetes

A new simple ultra-high-performance liquid chromatography method with diode array detection (UHPLC-DAD) was developed for chemical fingerprinting analysis of extracellular metabolites in fermentation broth of Geosmithia spp. The SPE method employing Oasis MCX strong cation-exchange mixed-mode polymeric sorbent was chosen for extraction of the metabolites. The analyses were performed on an Acquity UPLC BEH C18 column (100 × 2.1 mm i.d.; particle size, 1.7 μm; Waters) using a gradient elution program with an aqueous solution of trifluoroacetic acid and acetonitrile as the mobile phase. The applicability of the method was proved by analysis of 38 strains produced by different species and isolated from different sources (hosts). The results revealed the correlation of obtained UHPLC-DAD fingerprints with taxonomical identity.
Keywords: Ultra-high-performance liquid chromatography; Diode array detection; Solid-phase extraction; Extracellular secondary metabolites; Geosmithia spp.; Chromatographic fingerprinting

In this study, an analytical method for the simultaneous separation and characterization of various molecular species of lysophospholipids (LPLs) and phospholipids (PLs) is introduced by employing nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). Since LPLs and PLs in human plasma are potential biomarkers for cancer, development of a sophisticated analytical method for the simultaneous profiling of these molecules is important. Standard species of LPLs and PLs were examined to establish a separation condition using a capillary LC column followed by MS scans and data-dependent collision-induced dissociation (CID) analysis for structural identification. With nLC-ESI-MS/MS, regioisomers of each category of LPLs were completely separated and identified with characteristic CID spectra. It was applied to the comprehensive profiling of LPLs and PLs from a human blood plasma sample and yielded identifications of 50 LPLs (each regioisomer pair of 6 lysophosphatidylcholines (LPCs), 7 lysophosphatidylethanolamines (LPEs), 9 lysophosphatidic acid (LPAs), 2 lysophosphatidylglycerols (LPGs), and 1 lysophosphatidylserine (LPS)) and 62 PLs (19 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 3 phosphatidylserines (PSs), 16 phosphatidylinositols (PIs), 8 phosphatidylglycerols (PGs), and 5 phosphatidic acids (PAs)). Figure The study demonstrates that regioisomers of lysophospholipid can be completely separated and identified with characteristic CID spectra using nLC-ESI-MS-MS, along with the simultaneous profiling of phospholipids from human blood plasma.
Keywords: nLC-ESI-MS/MS; Lysophospholipids; Phospholipids; Human plasma; Tandem mass spectrometry

Cell–cell communication is often achieved via granular exocytosis, as in neurons during synaptic transmission or neuroendocrine cells during blood hormone control. Owing to its critical role in membrane properties and SNARE function, cholesterol is expected to play an important role in the highly conserved process of exocytosis. In this work, membrane cholesterol concentration is systematically varied in primary culture mouse chromaffin cells, and the change in secretion behavior of distinct vesicle pools as well as pool recovery following stimulation is measured using carbon-fiber microelectrode amperometry. Amperometric traces obtained from activation of the younger readily releasable and slowly releasable pool (RRP/SRP) vesicles at depleted cholesterol levels showed fewer sustained fusion pore features (6.1 ± 1.1% of spikes compared with 11.2 ± 1.0% for control), revealing that cholesterol content influences fusion pore formation and stability during exocytosis. Moreover, subsequent stimulation of RRP/SRP vesicles showed that cellular cholesterol level influences both the quantal recovery and kinetics of the later release events. Finally, diverging effects of cholesterol on RRP and the older reserve pool vesicle release suggest two different mechanisms for the release of these two vesicular pools. Figure Membrane cholesterol levels in chromaffin cells were modified, and changes in secretion behavior was determined using amperometry. Top trace obtained from single chromaffin cell stimulated with Ba2+ at high membrane cholesterol levels whereas the bottom trace illustrates the exocytosis of chromaffin cells at low cholesterol levels
Keywords: Chromaffin cell; Exocytosis; Cholesterol; Carbon-fiber microelectrode; Vesicle pools

A chemical signal generator for resolving temporal dynamics of single cells by Jian Sun; Jingjing Wang; Pu Chen; Xiaojun Feng; Wei Du; Bi-Feng Liu (2973-2981).
To investigate rapid cell signaling, analytical methods are required that can generate repeatable chemical signals for stimulating live cells with high temporal resolution. Here, we present a chemical signal generator based on hydrodynamic gating, permitting flexible stimulation of single adherent cells with a temporal resolution of 20 ms. Studies of adenosine triphosphate (ATP)-induced calcium signaling in HeLa cells were demonstrated using this developed method. Consecutive treatment of the cells with ATP pulses of 20 or 1 s led to an increase of latency, which might be another indicator of receptor desensitization in addition to the decrease in the amplitude of calcium spikes. With increasing duration of ATP pulses from milliseconds to a few seconds, the cellular responses transitioned from single calcium spikes to calcium oscillation gradually. We expected this method to open up a new avenue for potential investigation of rapid cell signaling. Fig. 1 A microfluidic chemical signal generator was developed for investigating rapid cell signaling based on hydrodynamic gating
Keywords: Single-cell analysis; Bioanalytical methods; Microfluidics; Calcium signaling; Chemical signal generator

GC–TOFMS analysis of metabolites in adherent MDCK cells and a novel strategy for identifying intracellular metabolic markers for use as cell amount indicators in data normalization by Bei Cao; Jiye Aa; Guangji Wang; Xiaolan Wu; Linsheng Liu; Mengjie Li; Jian Shi; Xinwen Wang; Chunyan Zhao; Tian Zheng; Sheng Guo; Jinao Duan (2983-2993).
Cultured cell lines are useful models in biomedical research that characterize metabolic responses to various stimuli (e.g., pathogens, toxins, or drugs/chemicals) and explore the underlying mechanisms. However, data from cell metabolomic studies must be normalized to the amount of cells, which is dependent on diverse treatments. The currently used methods of cell counting and protein assay involve extra work and delay the quenching of intracellular metabolism. To develop a convenient, alternative approach, in this study, intracellular metabolites were extracted from a series amount of cultured adherent cells and profiled by gas chromatography–time-of-flight mass spectrometry (GC–TOFMS). The GC–TOFMS signal intensities for 11 intracellular markers present in two different cell lines showed good linearity with the protein content, with inositol and pantothenate most promising (correlation coefficient > 0.970). Despite the various amounts of cells, the data normalized to the metabolic markers and protein amounts showed similar effectiveness, resulted in better separation of the two cell lines, closer clustering within each group(cell line) on a principal components analysis scores plot, and had lower relative standard deviations for intracellular metabolites than those of the non-normalized data, suggesting that these markers were effective indicators of cell amounts and independent of cell lines. Figure A schematic strategy for identifying intracellular metabolic markers for use as cell amount indicators in data normalization
Keywords: Cell amount indicator; Metabolic markers; GC–TOFMS; Inositol

Highly fluorescent CdTe quantum dots (QDs) stabilized by 3-mercaptopropionic acid were prepared by an aqueous solution approach and used as a fluorescent label to link substance P (SP) in studying the interaction of SP with NK-1 receptor, which was expressed on the AR4-2J cell line. Nonspecific adsorptions of CdTe QDs on the AR4-2J cell membrane were observed, whereas the QD–SP conjugates successfully crossed the cell membrane and entered the cytosol. SP is a neurotransmitter, and neurotransmitter-induced calcium concentration oscillation is a common phenomenon in diverse cells especially of secretory type. Cytosolic calcium concentration responses were studied in the AR4-2J cell line during stimulation with SP and QD–SP conjugates. The oscillations triggered by SP and QD–SP conjugates were dose-dependent and very similar. Such QD–SP conjugates readily internalized into the cytosol as would be expected of an active NK-1 ligand. Therefore QD–SP conjugates could be used successfully to study ligand and NK-1 receptor interactions in live cells. Our research may provide a meaningful reference for congener research. Figure Confocal microscopic images of AR4-2J cells after addition of (A) 3-MPA-capped CdTe QDs or (B) QD-SP conjugates. (A) 3-MPA-capped CdTe QDs did not induce any increases in cytosolic calcium concentration in AR4-2J cells, while (B) QD-SP conjugates triggered oscillatory intracellular calcium increases dose-dependent in AR4-2J cells
Keywords: Substance P; Quantum dots; Calcium concentration oscillations; Receptor internalization; AR4-2J cells

Imaging surface plasmon resonance for multiplex microassay sensing of mycotoxins by Denis Dorokhin; Willem Haasnoot; Maurice C. R. Franssen; Han Zuilhof; Michel W. F. Nielen (3005-3011).
A prototype imaging surface plasmon resonance-based multiplex microimmunoassay for mycotoxins is described. A microarray of mycotoxin–protein conjugates was fabricated using a continuous flow microspotter device. A competitive inhibition immunoassay format was developed for the simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN), using a single sensor chip. Initial in-house validation showed limits of detection of 21 and 17 ng/mL for DON and 16 and 10 ng/mL for ZEN in extracts, which corresponds to 84 and 68 μg/kg for DON and 64 and 40 μg/kg for ZEN in maize and wheat samples, respectively. Finally, the results were critically compared with data obtained from liquid chromatography-mass spectrometry confirmatory analysis method and found to be in good agreement. The described multiplex immunoassay for the rapid screening of several mycotoxins meets European Union regulatory limits and represents a robust platform for mycotoxin analysis in food and feed samples.
Keywords: Imaging surface plasmon resonance; Multiplex immunoassay; Mycotoxins; Deoxynivalenol; Zearalenone

Microbial genotoxicity bioreporters based on sulA activation by Alva Biran; Hadar Ben Yoav; Sharon Yagur-Kroll; Rami Pedahzur; Sebastian Buchinger; Yosi Shacham-Diamand; Georg Reifferscheid; Shimshon Belkin (3013-3024).
A bacterial genotoxicity reporter strain was constructed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bioreporter responded in a dose-dependent manner to three model DNA-damaging agents—hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)—detected 30–60 min after exposure. Detection thresholds were 0.15 μM for MMC, 7.5 μM for nalidixic acid, and approximately 50 μM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensitivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminoanthracene following metabolic activation with an S9 mammalian liver fraction.
Keywords: Genotoxicity detection; Whole-cell biosensors; Environmental monitoring; Electrochemistry; Bioluminescence; Escherichia coli

Anion-selective electrodes based on ionic liquid membranes: effect of ionic liquid anion on observed response by Rajani Gourishetty; Ann Marie Crabtree; William M. Sanderson; R. Daniel Johnson (3025-3033).
A systematic study of the behavior of ion-exchanger anion-selective electrodes prepared from seven different trihexyltetradecylphosphonium ionic liquids (ILs) was performed. The effective ion-exchange capacity of prepared ion-selective electrodes (ISEs) increased with decreasing IL anion lipophilicity, and analyte anion response slopes became more Nernstian concomitantly. With ILs having the most lipophilic constituent anions, incorporation of tridodecylmethylammonium chloride into membranes significantly enhanced responses toward all ions. However, ILs based on bis(trifluoromethylsulfonyl)imide and dodecylsulfate maintained sub-Nernstian responses upon such addition apparently due to their ability to coordinate cations. Electrodes prepared with high IL content displayed regions of super-Nernstian response, which could be eliminated by reducing percent of IL in the membrane; percentages at which optimal linear range was achieved also followed a trend with decreasing constituent IL anion lipophilicity. While selectivities of all electrodes followed the Hofmeister pattern, selectivity coefficient ranges generally were narrower than observed with traditionally plasticized ISEs, and selectivities for more hydrophilic analytes were improved slightly in ILs containing the most hydrophilic constituent anions.
Keywords: Anion-selective electrodes; Ionic liquids; Ion exchange; Potentiometry; Polymeric membranes; Plasticizers

A selective adenosine sensor derived from a triplex DNA aptamer by Mayurbhai Patel; Avishek Dutta; Haidong Huang (3035-3040).
The aim of this study is to develop a selective adenosine aptamer sensor using a rational approach. Unlike traditional RNA aptamers developed from SELEX, duplex DNA containing an abasic site can function as a general scaffold to rationally design aptamers for small aromatic molecules. We discovered that abasic site-containing triplex DNA can also function as an aptamer and provide better affinity than duplex DNA aptamers. A novel adenosine aptamer sensor was designed using such a triplex. The aptamer is modified with furano-dU in the binding site to sense the binding. The sensor bound adenosine has a dissociation constant of 400 nM, more than tenfold stronger than the adenosine aptamer developed from SELEX. The binding quenched furano-dU fluorescence by 40%. It was also demonstrated in this study that this sensor is selective for adenosine over uridine, cytidine, guanosine, ATP, and AMP. The detection limit of this sensor is about 50 nM. The sensor can be used to quantify adenosine concentrations between 50 nM and 2 μM. Fig. 1
Keywords: Adenosine sensor; DNA aptamer; Fluorescent nucleobase; DNA triplex; Fluorescence titration

Copolymerization of luminol on screen-printed cells for single-use electrochemiluminescent sensors by J. Ballesta-Claver; M. C. Valencia-Mirón; L. F. Capitán-Vallvey (3041-3051).
An efficient electrochemiluminescent (ECL) single-use sensor for H2O2 is presented based on an electropolymerized film prepared on screen-printed gold electrode (gold SPE). A study of the copolymerization of luminol in the presence of different monomers was carried out. The polymeric films were grown potentiodynamically with a potential interval between −0.2 and 1.0 V in 0.2 M H2SO4 and were characterized by their electrochemical, electrochemiluminescent, and superficial features. The polymer with the most efficient growth and ECL emission was poly(luminol-3,3′,5,5′-tetramethylbenzidine) at 1:5 ratio. These prepared SPE cells present good mechanical and photoemissive properties. A semi-logarithmic linearization shows a noticeable four decade-width concentration range with a limit of detection (LOD) of 2.6 × 10−9 M and a precision of 10.2% (n = 5; as relative standard deviation, RSD) in the medium range level. The described SPE ECL sensors will be useful for the determination of oxidase substrates in ECL single-use biosensors. Figure Schematic diagram of sensor preparation by luminol-TMB copolymer formation for electrochemiluminescent rainwater determination
Keywords: Electrochemiluminescence; Disposable optical sensor; Hydrogen peroxide; Poly(luminol-3,3′,5,5′-tetramethylbenzidine); Screen-printed electrodes

Chemical profile of meta-chlorophenylpiperazine (m-CPP) in ecstasy tablets by easy ambient sonic-spray ionization, X-ray fluorescence, ion mobility mass spectrometry and NMR by Wanderson Romão; Priscila M. Lalli; Marcos F. Franco; Gustavo Sanvido; Nicolas V. Schwab; Rafael Lanaro; José Luiz Costa; Bruno D. Sabino; Maria Izabel M. S. Bueno; Gilberto F. de Sa; Romeu J. Daroda; Vanderlea de Souza; Marcos N. Eberlin (3053-3064).
Meta-chlorophenylpiperazine (m-CPP) is a new illicit drug that has been sold as ecstasy tablets. Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) and X-ray fluorescence spectrometry (XRF) are shown to provide relatively simple and selective screening tools to distinguish m-CPP tablets from tablets containing amphetamines (mainly 3,4-methylenedioxymethamphetamine (MDMA)). EASI-MS detects the active ingredients in their protonated forms: [m-CPP + H]+ of m/z 197, [MDMA + H]+ of m/z 194, and [2MDMA + HCl + H]+ of m/z 423 and other ions from excipients directly on the tablet surface, providing distinct chemical fingerprints. XRF identifies Cl, K, Ca, Fe, and Cu as inorganic ingredients present in the m-CPP tablets. In contrast, higher Cl concentrations and a more diverse set of elements (P, Cl, Ca, Fe, Cu, Zn, Pt, V, Hf, Ti, Pt, and Zr) were found in MDMA tablets. Principal component analysis applied to XRF data arranged samples in three groups: m-CPP tablets (four samples), MDMA tablets (twenty three samples), and tablets with no active ingredients (three samples). The EASI-MS and XRF techniques were also evaluated to quantify m-CPP in ecstasy tablets, with concentrations ranging from 4 to 40 mg of m-CPP per tablets. The m-CPP could only be differentiated from its isomers (o-CPP and for the three isomers p-CPP) by traveling wave ion mobility mass spectrometry and NMR measurements.
Keywords: Drug monitoring/drug screening; Ambient mass spectrometry/EASI-MS; Forensics/toxicology; Metals/heavy metals; X-ray spectroscopy (XPS | XRF | EDX); NMR/ESR

Are these liquids explosive? Forensic analysis of confiscated indoor fireworks by Kepa Castro; Silvia Fdez-Ortiz de Vallejuelo; Izaskun Astondoa; Félix M. Goñi; Juan Manuel Madariaga (3065-3071).
Complete forensic analysis of several confiscated liquids and gels putatively used as firework components was achieved by combining Raman, FTIR spectroscopy, and scanning electron microscopy combined with energy-dispersive spectroscopy (SEM–EDS). The chemical composition of the liquids was consistent with their use as indoor fireworks. Alcohols (methanol and isopropyl alcohol) were used to solubilise compounds producing coloured flames. Boric acid, recently introduced in the list of Substances of Very High Concern (SVHC) for the REACH Regulation of the European Union, was found in one of the samples.
Keywords: Raman; SEM–EDS; FTIR; Fireworks; Forensic

Analytical characteristics and determination of major novel brominated flame retardants (NBFRs) in indoor dust by Nadeem Ali; Stuart Harrad; Dudsadee Muenhor; Hugo Neels; Adrian Covaci (3073-3083).
A new method was developed and optimized for the detection of major “novel” brominated flame retardants (NBFRs), which included decabromodiphenyl ethane (DBDPE), 1,2-bis(2,4,6-tribromophenoxy) ethane (BTBPE), tetrabromobisphenol A-bis(2,3-dibromopropylether) (TBBPA-DBPE), 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBPH) and hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO). Several solid phase sorbents were tested, and finally, a two-step cleanup procedure was established. The first step on activated silica was used to fractionate the dust extracts, while the second step on acidified silica (silica gel impregnated with sulphuric acid 44% w/w) and on Florisil®, respectively, was essential for advanced cleanup. High recoveries for NBFRs (range, 75–94%) were achieved. Analysis was performed by gas chromatography coupled with mass spectrometry in electron capture negative ionization using a DB-5ms (15 m × 0.25 mm × 0.1 μm) capillary column. Quantification of DBDPE, BTBPE and TBBPA-DBPE was based on ion m/z 79, while characteristic ions were used for quantification of TBB (m/z 359), HCDBCO (m/z 310) and TBPH (m/z 384). The method provided good repeatability; within- and between-day precision were ≤14% for all NBFRs. Method limits of quantification ranged between 1 and 20 ng g−1; dust and NBFRs were not detected in blanks. The method was further applied to indoor dust (n = 21) collected from e-waste facilities in Thailand. Except for HCDBCO, all NBFRs were detected in the e-waste dust with concentrations up to 44,000 and 22,600 ng g−1 DBDPE and BTBPE, respectively. The dust profile was dominated by DBDPE (50%) > BTBPE (45%) > TBBPA-DBPE (3%) > TBPH (1.9%) > TBB (0.1%). Significant correlations (p < 0.05) were found between the concentrations of BTBPE and BDE 183 or BDE 197 on the one hand, between TBPH and BDE 47 or BDE 99, and between DBDPE and BDE 209, on the other hand. Concentrations of TBB were not positively correlated with TBPH, which suggests different emission sources.
Keywords: NBFRs; Indoor dust; Analytical method; E-waste dust

Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants. Figure Microbead coated with antibody
Keywords: Flow cytometry; Immunoassay; Magnetic microbeads; Mycotoxins; Cereal; Mass spectrometry; Nano-LC

A rapid and simple miniaturized liquid–liquid extraction method has been developed for the determination of topramezone in soil, corn, wheat, and water samples using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-electrospray ionization (ESI)/MS/MS). The established method for the extraction and purification procedure was based on liquid–liquid partitioning into an aqueous solution at a low pH (pH ≈ 2.5), followed by back-partitioning into water at pH > 9. Two precursor, product ion transitions for topramezone were measured and evaluated to provide the maximum degree of confidence in the results. Under negative ESI conditions, quantitation was achieved by monitoring the fragment at m/z = 334 and the qualitative fragment at m/z = 318, whereas also collecting the corresponding parent ion at m/z = 362. Chromatographic separation was achieved using gradient elution with a mobile phase consisting of methanol and a 0.01% aqueous ammonium hydroxide solution. Recovery studies for soil, corn, wheat, and water were conducted at four different topramezone concentrations (5 or 10, 50, 100, and 1,000 μg kg−1); the overall average recoveries ranged from 79.9% to 98.4% with intra-day relative standard deviations (RSD) of 3.1~8.7% and inter-day RSD of 4.3~7.5%. Quantitative results were determined from calibration curves of topramezone standards containing 1–500 μg L−1 with an R 2 ≥ 0.9994. Method sensitivities expressed as limits of quantitation were typically 6, 8, 9, and 1 μg kg−1 in soil, corn, wheat, and water, respectively. The results of the method validation confirmed that this proposed method was convenient and reliable for the determination of topramezone residues in soil, corn, wheat, and water. Figure
Keywords: Topramezone; UPLC-MS/MS; Determination; LLE

A high-performance liquid chromatography (HPLC) method was established using an analytical reversed-phase column and gradient elution to achieve chromatographic separation of typical compounds in essential oils. For detection, a diode array detector monitoring different wavelengths simultaneously as well as a mass spectrometer (MS) were used. Atmospheric pressure chemical ionization operating in the positive mode turned out to be a suitable tool to detect volatiles of different chemical classes and to identify them in essential oil matrices. Characteristic fingerprints of eucalyptus, lavender, may chang, pine, rosemary, thyme, and turpentine essential oils monitored at a representative wavelength (220 nm) demonstrated the suitability of HPLC in essential oil analysis. Additional monitoring wavelengths (210, 250, and 280 nm) provided useful information about the identity of the specific component and opened the possibility to differentiate presumably coeluting compounds by means of their distinct absorption behavior. Finally, peak assignment in seven essential oils was performed on the basis of characteristic retention times and UV and MS data of a broad set of reference volatiles. Figure HPLC analysis of essential oils
Keywords: Essential oils; Terpenoids; Volatiles; HPLC/DAD; Mass spectrometry; LC/APCI-MS

Ultra-trace analysis of 36Cl by accelerator mass spectrometry: an interlaboratory study by S. Merchel; W. Bremser; V. Alfimov; M. Arnold; G. Aumaître; L. Benedetti; D. L. Bourlès; M. Caffee; L. K. Fifield; R. C. Finkel; S. P. H. T. Freeman; M. Martschini; Y. Matsushi; D. H. Rood; K. Sasa; P. Steier; T. Takahashi; M. Tamari; S. G. Tims; Y. Tosaki; K. M. Wilcken; S. Xu (3125-3132).
A first international 36Cl interlaboratory comparison has been initiated. Evaluation of the final results of the eight participating accelerator mass spectrometry (AMS) laboratories on three synthetic AgCl samples with 36Cl/Cl ratios at the 10−11, 10−12, and 10−13 level shows no difference in the sense of simple statistical significance. However, more detailed statistical analyses demonstrate certain interlaboratory bias and underestimation of uncertainties by some laboratories. Following subsequent remeasurement and reanalysis of the data from some AMS facilities, the round-robin data indicate that 36Cl/Cl data from two individual AMS laboratories can differ by up to 17%. Thus, the demand for further work on harmonising the 36Cl-system on a worldwide scale and enlarging the improvement of measurements is obvious.
Keywords: Accelerator mass spectrometry; Long-lived radionuclides; Cosmogenic nuclides; Exposure dating

The trace analysis of microorganisms in real samples by combination of a filtration microcartridge and capillary isoelectric focusing by Marie Horká; Jaroslav Horký; Anna Kubesová; Eva Zapletalová; Karel Šlais (3133-3140).
Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface. Figure The pre-concentration and separation of spores of Monilinia species and of Penicillium expansum from the real suspensions including combination of filtration and capillary isoelectric focusing were developed and the possibility of their application to real samples was verified. The coupling of the filtration cartridge with the separation capillary can improve the detection limit of the isoelectric focusing with the UV-detection by at least four orders of magnitude.
Keywords: Capillary isoelectric focusing; Preconcentration; Filtration microcartridge; Trace number of microorganisms; Real samples; UV detection

Identification strategy for unknown pollutants using high-resolution mass spectrometry: Androgen-disrupting compounds identified through effect-directed analysis by Jana M. Weiss; Eszter Simon; Gerard J. Stroomberg; Ronald de Boer; Jacob de Boer; Sander C. van der Linden; Pim E. G. Leonards; Marja H. Lamoree (3141-3149).
Effect-directed analysis has been applied to a river sediment sample of concern to identify the compounds responsible for the observed effects in an in vitro (anti-)androgenicity assay. For identification after non-target analysis performed on a high-resolution LTQ-Orbitrap, we developed a de novo identification strategy including physico-chemical parameters derived from the effect-directed analysis approach. With this identification strategy, we were able to handle the immense amount of data produced by non-target accurate mass analysis. The effect-directed analysis approach, together with the identification strategy, led to the successful identification of eight androgen-disrupting compounds belonging to very diverse compound classes: an oxygenated polyaromatic hydrocarbon, organophosphates, musks, and steroids. This is one of the first studies in the field of environmental analysis dealing with the difficult task of handling the large amount of data produced from non-target analysis. The combination of bioassay activity assessment, accurate mass measurement, and the identification and confirmation strategy is a promising approach for future identification of environmental key toxicants that are not included as priority pollutants in monitoring programs.
Keywords: LTQ-Orbitrap; Non-target analysis; Identification; (Anti-)androgenic; Effect-directed analysis (EDA); MODELKEY

NMR depth profiles as a non-invasive analytical tool to probe the penetration depth of hydrophobic treatments and inhomogeneities in treated porous stones by Valeria Di Tullio; Noemi Proietti; Donatella Capitani; Ilaria Nicolini; Anna Maria Mecchi (3151-3164).
Hydrophobic treatment is one of the most important interventions usually carried out in the conservation of stone artifacts and monuments. The analytical study reported in this paper was aimed at answering general questions such as the penetration depth of a hydrophobic treatment into a porous material, its capability to impair the water absorption, how the presence of a treatment may change the open porosity available to the water, and how a treatment may affect the diffusion of water inside a porous structure. Also, inhomogeneities in treated stones due to sharp variations of the amount of the absorbed product in the porous material were evidenced and scaled. The results of this fully non-invasive analytical study were rationalized in terms of new parameters obtained by a suitable process of nuclear magnetic resonance data. These analytical parameters reported here for the first time, namely the hydrophobic efficiency, the penetration depth, and angles describing changes in slope in depth profiles, gave important information in assessing the performance of a treatment. Figure NMR depth profiles encoding water penetration in calcarenite specimens untreated (yellow), and treated with a water repellent for different lengths of time (5 s blue, 600 s green, 1,800 s red). A calcarenite specimen is shown on the left side.
Keywords: Unilateral NMR; Depth profiles; Penetration depth; Hydrophobic treatment; Inorganic porous materials

Validation of an accelerated solvent extraction liquid chromatography–tandem mass spectrometry method for Pacific ciguatoxin-1 in fish flesh and comparison with the mouse neuroblastoma assay by Jia Jun Wu; Yim Ling Mak; Margaret B. Murphy; James C. W. Lam; Wing Hei Chan; Mingfu Wang; Leo L. Chan; Paul K. S. Lam (3165-3175).
Ciguatera fish poisoning (CFP) is a global foodborne illness caused by consumption of seafood containing ciguatoxins (CTXs) originating from dinoflagellates such as Gambierdiscus toxicus. P-CTX-1 has been suggested to be the most toxic CTX, causing ciguatera at 0.1 μg/kg in the flesh of carnivorous fish. CTXs are structurally complex and difficult to quantify, but there is a need for analytical methods for CFP toxins in coral reef fishes to protect human health. In this paper, we describe a sensitive and rapid extraction method using accelerated solvent extraction combined with high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) for the detection and quantification of P-CTX-1 in fish flesh. By the use of a more sensitive MS system (5500 QTRAP), the validated method has a limit of quantification (LOQ) of 0.01 μg/kg, linearity correlation coefficients above 0.99 for both solvent- and matrix-based standard solutions as well as matrix spike recoveries ranging from 49% to 85% in 17 coral reef fish species. Compared with previous methods, this method has better overall recovery, extraction efficiency and LOQ. Fish flesh from 12 blue-spotted groupers (Cephalopholis argus) was assessed for the presence of CTXs using HPLC-MS/MS analysis and the commonly used mouse neuroblastoma assay, and the results of the two methods were strongly correlated. This method is capable of detecting low concentrations of P-CTX-1 in fish at levels that are relevant to human health, making it suitable for monitoring of suspected ciguateric fish both in the environment and in the marketplace.
Keywords: Ciguatera fish poisoning; Ciguatoxin; HPLC-MS/MS; Accelerated solvent extraction; Mouse neuroblastoma assay

Erratum to: The effect of bias-temperature stress on Na+ incorporation into thin insulating films by Stefan Krivec; Michael Buchmayr; Thomas Detzel; Till Froemling; Juergen Fleig; Herbert Hutter (3179-3179).