Analytical and Bioanalytical Chemistry (v.396, #8)

Depth profile analysis: coatings and thin layers by Beatriz Fernandez; Rosario Pereiro (2723-2724).
carried out her Ph.D. research at University of Oviedo (Spain), working on new quantification methods based on glow discharges for nonconducting materials and liquids. This was followed by a two-year postdoctoral appointment in the group of Olivier Donard at the Multidisciplinary Institute of Environmental Science and Materials in Pau (France) to work on new methods for the analysis of trace elements in solids using laser ablation inductively coupled plasma mass spectrometry in combination with isotope dilution. Since September 2008, she has had a prestigious Juan de la Cierva research contract at University of Oviedo. Her main scientific interests are related to optical and mass spectrometry techniques for the direct analysis of materials. has been Associate Professor at University of Oviedo (Spain) since 1996. Following the completion of her Ph.D. degree in 1991, she was awarded a postdoctoral fellowship allowing her to spend 14 months working in Gary Hieftje’s research group at Indiana University (USA). Her current research interests include developing novel analytical strategies for elemental and molecular in-depth profile analysis of thin layers, and investigation of new strategies to improve the selectivity and sensitivity of molecular luminescence by using nanostructured sensing phases, such as molecularly imprinted polymers and quantum dots. She has coauthored more than 100 scientific papers and several patents.

Towards nanometric resolution in multilayer depth profiling: a comparative study of RBS, SIMS, XPS and GDOES by Ramón Escobar Galindo; Raul Gago; David Duday; Carlos Palacio (2725-2740).
An increasing amount of effort is currently being directed towards the development of new functionalized nanostructured materials (i.e., multilayers and nanocomposites). Using an appropriate combination of composition and microstructure, it is possible to optimize and tailor the final properties of the material to its final application. The analytical characterization of these new complex nanostructures requires high-resolution analytical techniques that are able to provide information about surface and depth composition at the nanometric level. In this work, we comparatively review the state of the art in four different depth-profiling characterization techniques: Rutherford backscattering spectroscopy (RBS), secondary ion mass spectrometry (SIMS), X-ray photoelectron spectroscopy (XPS) and glow discharge optical emission spectroscopy (GDOES). In addition, we predict future trends in these techniques regarding improvements in their depth resolutions. Subnanometric resolution can now be achieved in RBS using magnetic spectrometry systems. In SIMS, the use of rotating sample holders and oxygen flooding during analysis as well as the optimization of floating low-energy ion guns to lower the impact energy of the primary ions improves the depth resolution of the technique. Angle-resolved XPS provides a very powerful and nondestructive technique for obtaining depth profiling and chemical information within the range of a few monolayers. Finally, the application of mathematical tools (deconvolution algorithms and a depth-profiling model), pulsed sources and surface plasma cleaning procedures is expected to greatly improve GDOES depth resolution.
Keywords: Depth profiling; XPS; SIMS; GDOES; RBS; Multilayer; High resolution

Depth profiling of nanostructures is of high importance both technologically and fundamentally. Therefore, many different methods have been developed for determination of the depth distribution of atoms, for example ion beam (e.g. O 2 + , Ar+) sputtering, low-damage C60 cluster ion sputtering for depth profiling of organic materials, water droplet cluster ion beam depth profiling, ion-probing techniques (Rutherford backscattering spectroscopy (RBS), secondary-ion mass spectroscopy (SIMS) and glow-discharge optical emission spectroscopy (GDOES)), X-ray microanalysis using the electron probe variation technique combined with Monte Carlo calculations, angle-resolved XPS (ARXPS), and X-ray photoelectron spectroscopy (XPS) peak-shape analysis. Each of the depth profiling techniques has its own advantages and disadvantages. However, in many cases, non-destructive techniques are preferred; these include ARXPS and XPS peak-shape analysis. The former together with parallel factor analysis is suitable for giving an overall understanding of chemistry and morphology with depth. It works very well for flat surfaces but it fails for rough or nanostructured surfaces because of the shadowing effect. In the latter method shadowing effects can be avoided because only a single spectrum is used in the analysis and this may be taken at near normal emission angle. It is a rather robust means of determining atom depth distributions on the nanoscale both for large-area XPS analysis and for imaging. We critically discuss some of the techniques mentioned above and show that both ARXPS imaging and, particularly, XPS peak-shape analysis for 3D imaging of nanostructures are very promising techniques and open a gateway for visualizing nanostructures.
Keywords: Nanostructures; XPS peak-shape analysis; Depth profiling; 3D imaging

Non-destructive depth compositional profiles by XPS peak-shape analysis by M. C. López-Santos; F. Yubero; J. P. Espinós; A. R. González-Elipe (2757-2768).
The measured peak shape and intensity of the photoemitted signal in X-ray photoelectron spectroscopy (XPS) experiments (elastic and inelastic parts included) are strongly correlated, through electron-transport theory, with the depth distribution of photoelectron emitters within the analyzed surface. This is the basis of so-called XPS peak-shape analysis (also known as the Tougaard method) for non-destructive determination of compositional in-depth (up to 6–8 nm) profiles. This review describes the theoretical basis and reliability of this procedure for quantifying amounts and distributions of material within a surface. The possibilities of this kind of analysis are illustrated with several case examples related to the study of the initial steps of thin-film growth and the modifications induced in polymer surfaces after plasma treatments. Figure Photoemitted spectra and in-depth concentration profiles (blue: oxygen; orange: carbon), obtained by means of XPS peak shape analysis, of a PET plasma activated surface. The shown topography corresponds to an atomic force microscopy image of the treated surface.
Keywords: XPS; Surface analysis; Depth profiling; Plasma treatment; Film growth

Acoustics and atomic force microscopy for the mechanical characterization of thin films by Daniele Passeri; Andrea Bettucci; Marco Rossi (2769-2783).
The science and technology of thin films require the development of nondestructive methods for their quantitative mechanical characterization with nanometric spatial resolution. High-frequency ultrasonic techniques—especially acoustic microscopy—and atomic force microscopy (AFM) have been demonstrated to represent versatile tools for developing such methods. In particular, in the last 15 years, the combination of AFM, which can probe the surface of a sample by applying ultralow loads (from micronewtons down to piconewtons) with a micromachined tip having an apex radius of a few nanometers, and ultrasonics techniques led researchers to develop some unique tools which allow one to perform not only spot measurements of the sample elastic modulus, but also to obtain both the qualitative imaging of mechanical properties and the quantitative mapping of the elastic modulus of the sample surface with nanometric lateral resolution. In the present review, firstly a brief overview of the main ultrasound-based techniques for thin film characterization is reported. Then, some of the ultrasonic AFM techniques are described, emphasizing their capability of retrieving maps of both the tip–sample contact stiffness and the sample elastic modulus. Although these techniques are less affected by the mechanical properties of the substrates than standard indentation tests, a method for the correction of the substrate effect in ultrathin films is reported in detail. Finally, by probing the mechanical properties of a small portion of the sample volume underneath the tip, we illustrate the techniques as tools for the qualitative and quantitative characterization of variations in the adhesion between a thin film and a buried interface, as well as for detecting subsurface defects, voids, cracks, and dislocations. Figure Indentation modulus map of a 88 nm thick aluminum finger (right) on a glass substrate (left) obtained by atomic force acoustic microscopy.
Keywords: Atomic force microscopy; Thin film; Interface/surface analysis; Ultrasonics; Mechanical properties

Structural and surface analysis of unsupported and alumina-supported La(Mn,Fe,Mo)O3 perovskite oxides by M. G. Rosmaninho; J. C. Tristão; F. C. C. Moura; R. M. Lago; M. H. Araújo; J. L. G. Fierro (2785-2795).
A series of bulk and Al2O3-supported perovskite oxides of the type LaMn1 − x − y Fe x Mo y O3 (x = 0.00−0.90 and y = 0.00–0.09) were synthesized by the citric acid complexation–gelation method followed by annealing in air at 800 °C. For all samples, the local environment and the chemical state and concentration of surface species were determined. Mössbauer spectra revealed the only presence of octahedral Fe3+ ions dispersed in the perovskite structure, however well-crystallized together with a poorly crystalline LaFeO3 phases were detected for larger substitutions (x = 0.90). A similar picture was obtained for Mo-loaded (y = 0.02 and 0.05) samples but a new phase most likely related to Fe3+ ions dispersed aside from the perovskite structure was found for larger substitutions (y = 0.09). Together with these structures, supported samples showed the presence of LaFeO3 nanoparticles. Finally, photoelectron spectroscopy indicated that the chemical state and composition of the samples in the surface region (2–3 nm) approaches that of the bulk. For the unsupported substituted samples, iron (and molybdenum) enters into the perovskite structure while manganese tends to be slightly segregated. Moreover, in supported perovskites, a fraction of Mo and La atoms interact with the alumina surface. All these oxides were active in methane combustion and best performance was recorded for the Fe-rich composition (x = 0.9) in which both Mn3+ and Mo3+ ions were in the same proportion (y = 0.05).
Keywords: ABO3 oxides; Perovskite oxides; Surface composition; Mössbauer spectroscopy; XPS; Photoelectron spectroscopy; Catalysts; Methane combustion

Using a combination of TEM and XPS, we made an analysis of the complex high-temperature annealing effect on ultrathin titanium deuteride (TiD y ) films evaporated on a Si(100) substrate and covered by an ultrathin palladium layer. Both the preparation and annealing of the TiD y /Pd bi-layer films were performed in situ under UHV conditions. It was found that the surface and bulk morphology of the bi-layer film as well as that of the Si substrate material undergo a microstructural and chemical conversion after annealing and annealing-induced deuterium evolution from the TiD y phase. Energy-filtered TEM (EFTEM) mapping of cross-section images and argon ion sputter depth profiling XPS analysis revealed both a broad intermixing between the Ti and Pd layers and an extensive inter-diffusion of Si from the substrate into the film bulk area. Segregation of Ti at the Pd top layer surface was found to occur by means of angle-resolved XPS (ARXPS) and the EFTEM analyses. Selected area diffraction (SAD) and XPS provided evidence for the formation of a new PdTi2 bimetallic phase within the top region of the annealed film. Moreover, these techniques allowed to detect the initial stages of TiSi phase formation within the film–substrate interlayer.
Keywords: Palladium; Titanium deuteride; Silicon; TEM; XPS

X-ray photoelectron spectroscopy (XPS) is one of the main methods for elemental and bonding-state characterization in the near-surface region. Applying it as angle-resolved XPS (ARXPS), it is promising for non-destructive depth profiling in the nanometre-thickness range. The challenges for the application of ARXPS are discussed for the early film growth of Ti and Ta on SiO2 produced by in situ magnetron sputtering. The measurements showed several phase-formation processes at the interfaces, which were used as the basis for the ARXPS data analysis and resulted in different interlayer growth information for Ti and Ta. From a systematic ARXPS data interpretation in combination with simulations, limits of the method are critically discussed. Figure Scheme of an ARXPS experiment of a layer structure
Keywords: Angle-resolved X-ray photoelectron spectroscopy; Film growth; Phase formation; Simulation

On optical depth profiling using confocal Raman spectroscopy by N. A. Freebody; A. S. Vaughan; A. M. Macdonald (2813-2823).
Until 2006 the performance of confocal Raman spectroscopy depth profiling was typically described and modeled through the application of geometrical optics, including refraction at the surface, to explain the degree of resolution and the precise form of the depth profile obtained from transparent and semicrystalline materials. Consequently a range of techniques, physical and analytical, was suggested to avoid the errors thus encountered in order to improve the practice of Raman spectroscopy, if not the understanding of the underlying mechanisms. These approaches were completely unsuccessful in accounting for the precise form of the depth profile, the fact that spectra obtained from laminated samples always contain characteristic peaks from all materials present both well above and below the focal point and that spectra can be obtained when focused some 40 μm above the sample surface. This paper provides further evidence that the physical processes underlying Raman spectroscopy are better modeled and explained through the concept of an extended illuminated volume contributing to the final Raman spectrum and modeled through a photon scattering approach rather than a point focus ray optics approach. The power of this numerical model lies in its ability to incorporate, simultaneously, the effects of degree of refraction at the surface (whether using a dry or oil objective lens), the degree of attenuation due to scatter by the bulk of the material, the Raman scattering efficiency of the material, and surface roughness effects. Through this we are now able to explain why even removing surface aberration and refraction effects through the use of oil immersion objective lenses cannot reliably ensure that the material sampled is only that at or close to the point of focus of the laser. Furthermore we show that the precise form of the depth profile is affected by the degree of flatness of the surface of the sample. Perhaps surprisingly, we show that the degree of flatness of the material surface is, in fact, more important than obtaining a precise refractive index match between the immersion oil and the material when seeking a high-quality depth profile or Raman spectrum from within a transparent or semicrystalline material, contrary to accepted norms that samples for interrogation by Raman spectroscopy require little preparation.
Keywords: Confocal Raman spectroscopy; Depth profiling; Photon scattering; Oil immersion; Refraction; Surface roughness; Numerical modeling; Polymers; Oil immersion lenses

Depth profile characterization of ultra shallow junction implants by Philipp Hönicke; Burkhard Beckhoff; Michael Kolbe; Damiano Giubertoni; Jaap van den Berg; Giancarlo Pepponi (2825-2832).
A need for analysis techniques, complementary to secondary ion mass spectrometry (SIMS), for depth profiling dopants in silicon for ultra shallow junction (USJ) applications in CMOS technologies has recently emerged following the difficulties SIMS is facing there. Grazing incidence X-ray fluorescence (GIXRF) analysis in the soft X-ray range is a high-potential tool for this purpose. It provides excellent conditions for the excitation of the B-K and the As-L iii,ii shells. The X-ray standing wave (XSW) field associated with GIXRF on flat samples is used here as a tunable sensor to obtain information about the implantation profile because the in-depth changes of the XSW intensity are dependent on the angle of incidence. This technique is very sensitive to near-surface layers and is therefore well suited for the analysis of USJ distributions. Si wafers implanted with either arsenic or boron at different fluences and implantation energies were used to compare SIMS with synchrotron radiation-induced GIXRF analysis. GIXRF measurements were carried out at the laboratory of the Physikalisch-Technische Bundesanstalt (PTB) at the electron storage ring BESSY II using monochromatized undulator radiation of well-known radiant power and spectral purity. The use of an absolutely calibrated energy-dispersive detector for the acquisition of the B-Kα and As-Lα fluorescence radiation enabled the absolute determination of the total retained dose. The concentration profile was obtained by ab initio calculation and comparison with the angular measurements of the X-ray fluorescence.
Keywords: Ultra shallow junctions; Elemental depth profile; Grazing incidence X-ray fluorescence analysis

The development of highly ordered and self-assembled magnetic nanostructures such as arrays of Fe or Ni nanowires and their alloys is arousing increasing interest due to the peculiar magnetic properties of such materials at the nanoscale. These nanostructures can be fabricated using nanoporous anodic alumina membranes or self-assembled nanotubular titanium dioxide as templates. The chemical characterization of the nanostructured layers is of great importance to assist the optimization of the filling procedure or to determine their manufacturing quality. Radiofrequency glow discharge (RF-GD) coupled to optical emission spectrometry (OES) is a powerful tool for the direct analysis of either conducting or insulating materials and to carry out depth profile analysis of thin layers by multi-matrix calibration procedures. Thus, the capability of RF-GD-OES is investigated here for the in-depth quantitative analysis of self-aligned titania nanotubes and self-ordered nanoporous alumina filled with arrays of metallic and magnetic nanowires obtained using the template-assisted filling method. The samples analysed in this work consisted of arrays of Ni nanowires with different lengths (from 1.2 up to 5 µm) and multilayer nanowires of alternating layers with different thicknesses (of 1–2 µm) of Ni and Au, or Au and FeNi alloy, deposited inside the alumina and titania membranes. Results, compared with other techniques such as scanning electron microscopy and energy-dispersive X-ray spectroscopy, show that the RF-GD-OES surface analysis technique proves to be adequate and promising for this challenging application. Figure SEM image of nanoporous alumina template with in-depth profile obtained by RF-GD-OES
Keywords: Glow discharge; Optical emission spectrometry; Nanostructures; In-depth profile; Metal nanowires

Investigation of glow-discharge-induced morphology modifications on silicon wafers and chromium conversion coatings by AFM and rugosimetry by Julien Malherbe; Hervé Martinez; Beatriz Fernández; Olivier F. X. Donard (2841-2853).
The effect of radiofrequency glow-discharge sputtering on the sample surface in terms of modifications in the surface morphology were investigated in this work by using atomic force microscopy (AFM) and rugosimetry measurements. The influence of GD operating parameters (e.g. rf power, discharge pressure and sputtering time) on surface roughening was investigated using two different types of samples: mirror-polished and homogeneous silicon wafers and chromate conversion coatings (CCCs). Surface morphology changes produced by GD sputtering into the sample surface were carefully investigated by AFM and rugosimetry, both at the original sample surface and at the bottom of GD craters using different GD experimental conditions, such as the sputtering time (from 1 s to 20 min), rf forward power (20–60 W for the Si wafer and 10–60 W for the CCC), and discharge pressure (400–1,000 Pa for the Si wafer and 500–1000 Pa for the CCC). In the present study, GD-induced morphology modifications were observed after rf-GD-OES analysis, both for the silicon wafers and the CCC. Additionally, the changes observed in surface roughness after GD sputtering were found to be sample-dependent, changing the proportion, shape and roughness of the micro-sized patterns and holes with the sample matrix and the GD conditions. Figure Analysis of the morphology using AFM in craters formed after Glow Discharge sputtering
Keywords: Glow discharge spectrometry; Atomic force microscopy; Rugosimetry; Morphology; In-depth profile analysis; Semiconductor materials; Interface/surface analysis

Characterisation of tribocorrosion behaviour of multilayer PVD coatings by R. Bayón; R. Nevshupa; C. Zubizarreta; U. Ruiz de Gopegui; J. Barriga; A. Igartua (2855-2862).
The effect of repassivation on tribocorrosion behaviour of two multilayer coatings of different structures is studied experimentally by measuring the variation of instantaneous open-circuit potential during friction. One coating consists of alternating Cr and CrN layers, while another consists of alternated layers of CrN and ZrN. Analysis of the results showed that friction force, i.e. the rate of the mechanical energy supplied to the material in the contact zone, has no direct influence on the tribocorrosion behaviour; however, the wear rate does strongly influence the tribocorrosion. A simple phenomenological model of repassivation of the multilayer coating is developed assuming “surface coverage” approach. This model establishes the relationship between the rate of mechanical activation of material by friction and the behaviour of the open-circuit potential.
Keywords: Electroanalytical methods; Interface/surface analysis; Modelling

Polymer screening by radiofrequency glow discharge time-of-flight mass spectrometry by L. Lobo; N. Tuccitto; N. Bordel; R. Pereiro; J. Pisonero; A. Licciardello; A. Tempez; P. Chapon; A. Sanz-Medel (2863-2869).
The aim of this work is to optimise and evaluate radiofrequency glow discharge (RF GD) time-of-flight mass spectrometry (TOFMS) for identification of organic polymers. For this purpose, different polymers including poly[methylmethacrylate], poly[styrene], polyethylene terephthalate-co-isophthalate and poly[alpha-methylstyrene] have been deposited on silicon wafers and the RF GD-TOFMS capabilities for qualitative identification of these polymeric layers by molecular depth profiling have been investigated. Although some molecular information using the RF continuous mode is available, the pulsed mode offers a greater analytical potential to characterise such organic coatings. Some formed polyatomic ions have proved to be useful to identify the different polymer layers, confirming that layers having similar elemental composition but different polymer structure could be also differentiated and identified. Figure Radiofrequency glow discharge time-of-flight mass spectrometry can be used for qualitative identification of polymers.
Keywords: Glow discharge; Mass spectrometry; Polymers; Coatings; Depth profiles

Detection of negative ions in glow discharge mass spectrometry for analysis of solid specimens by Stela Canulescu; Igor S. Molchan; C. Tauziede; Agnes Tempez; J. A. Whitby; George E. Thompson; Peter Skeldon; P. Chapon; Johann Michler (2871-2879).
A new method is presented for elemental and molecular analysis of halogen-containing samples by glow discharge time-of-flight mass spectrometry, consisting of detection of negative ions from a pulsed RF glow discharge in argon. Analyte signals are mainly extracted from the afterglow regime of the discharge, where the cross section for electron attachment increases. The formation of negative ions from sputtering of metals and metal oxides is compared with that for positive ions. It is shown that the negative ion signals of F and TaO2F are enhanced relative to positive ion signals and can be used to study the distribution of a tantalum fluoride layer within the anodized tantala layer. Further, comparison is made with data obtained using glow-discharge optical emission spectroscopy, where elemental fluorine can only be detected using a neon plasma. The ionization mechanisms responsible for the formation of negative ions in glow discharge time-of-flight mass spectrometry are briefly discussed.
Keywords: RF glow discharge mass spectrometry; Negative ions; Fluorine ions; Pulsed discharge; Anodic tantala; Mass spectrometry/ICP-MS; Thin films; (XPS|XRF|EDX)

Direct chemical in-depth profile analysis and thickness quantification of nanometer multilayers using pulsed-rf-GD-TOFMS by R. Valledor; J. Pisonero; N. Bordel; J. I. Martín; C. Quirós; A. Tempez; A. Sanz-Medel (2881-2887).
Nanometer depth resolution is investigated using an innovative pulsed-radiofrequency glow discharge time-of-flight mass spectrometer (pulsed-rf-GD-TOFMS). A series of ultra-thin (in nanometers approximately) Al/Nb bilayers, deposited on Si wafers by dc-magnetron sputtering, is analyzed. An Al layer is first deposited on the Si substrate with controlled and different values of the layer thickness, t Al. Samples with t Al = 50, 20, 5, 2, and 1 nm have been prepared. Then, a Nb layer is deposited on top of the Al one, with a thickness t Nb = 50 nm that is kept constant along the whole series. Qualitative depth profiles of those layered sandwich-type samples are determined using our pulsed-rf-GD-TOFMS set-up, which demonstrated to be able to detect and measure ultra-thin layers (even of 1 nm). Moreover, Gaussian fitting of the internal Al layer depth profile is used here to obtain a calibration curve, allowing thickness estimation of such nanometer layers. In addition, the useful yield (estimation of the number of detected ions per sputtered atom) of the employed pulsed-rf-GD-TOFMS system is evaluated for Al at the selected operating conditions, which are optimized for the in-depth profile analysis with high depth resolution.
Keywords: Glow discharge; Mass spectrometry; Depth profile analysis; Nanometer layers; Useful yield

Combined use of X-ray photoelectron and Mössbauer spectroscopic techniques in the analytical characterization of iron oxidation state in amphibole asbestos by Marzia Fantauzzi; Alessandro Pacella; Davide Atzei; Antonio Gianfagna; Giovanni B. Andreozzi; Antonella Rossi (2889-2898).
Asbestos fibers are an important cause of serious health problems and respiratory diseases. The presence, structural coordination, and oxidation state of iron at the fiber surface are potentially important for the biological effects of asbestos because iron can catalyze the Haber–Weiss reaction, generating the reactive oxygen species ⋅OH. Literature results indicate that the surface concentration of Fe(III) may play an important role in fiber-related radical formation. Amphibole asbestos were analyzed by X-ray photoelectron spectroscopy (XPS) and Mössbauer spectroscopy, with the aim of determining the surface vs. bulk Fe(III)/Fetot ratios. A standard reference asbestos (Union Internationale Contre le Cancer crocidolite from South Africa) and three fibrous tremolite samples (from Italy and USA) were investigated. In addition to the Mössbauer spectroscopy study of bulk Fe(III)/Fetot ratios, much work was dedicated to the interpretation of the XPS Fe2p signal and to the quantification of surface Fe(III)/Fetot ratios. Results confirmed the importance of surface properties because this showed that fiber surfaces are always more oxidized than the bulk and that Fe(III) is present as oxide and oxyhydroxide species. Notably, the highest difference of surface/bulk Fe oxidation was found for San Mango tremolite—the sample that in preliminary cytotoxicity tests (MTT assay) had revealed a cell mortality delayed with respect to the other samples.
Keywords: Asbestos amphiboles; Mössbauer spectroscopy; X-ray photoelectron spectroscopy; Oxidized iron; Crocidolite; Tremolite

Quantification of urinary AICAR concentrations as a matter of doping controls by Andreas Thomas; Simon Beuck; Jens Christian Eickhoff; Sven Guddat; Oliver Krug; Matthias Kamber; Wilhelm Schänzer; Mario Thevis (2899-2908).
Influencing the endurance in elite sports is one of the key points in modern sports science. Recently, a new class of prohibited substances reached in the focus of doping control laboratories and their misuse was classified as gene doping. The adenosine monophosphate activated protein kinase activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) was found to significantly enhance the endurance even in sedentary mice after treatment. Due to endogenous production of AICAR in healthy humans, considerable amounts were present in the circulation and, thus, were excreted into urine. Considering these facts, the present study was initiated to fix reference values of renally cleared AICAR in elite athletes. Therefore a quantitative analytical method by means of isotope-dilution liquid chromatography (analytical column: C6-phenyl) coupled to tandem mass spectrometry, after a sample preparation consisting of a gentle dilution of native urine, was developed. Doping control samples of 499 athletes were analysed, and AICAR concentrations in urine were determined. The mean AICAR value for all samples was 2,186 ng/mL with a standard deviation of 1,655 ng/mL. Concentrations were found to differ depending on gender, type of sport and type of sample collection (in competition/out of competition). The method was fully validated for quantitative purposes considering the parameters linearity, inter- (12%, 7% and 10%) and intraday precision (14%, 9% and 12%) at low, mid and high concentration, robustness, accuracy (approx. 100%), limit of quantification (100 ng/mL), stability and ion suppression effects, employing an in-house synthesised 13C5-labelled AICAR as internal standard. Urinary AICAR concentrations (ng/mL)
Keywords: Gene doping; Sports drug testing; LC-MS/M; Mass spectrometry

We introduce a simplified sample preparation method using bare TiO2 nanoparticles (NPs) to serve as multifunctional nanoprobes (desalting, accelerating, and affinity probes) for effective enrichment of phosphopeptides from microwave-assisted tryptic digestion of phosphoproteins (α-casein, β-casein and milk) in Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). The results demonstrate that TiO2 NPs can effectively enrich and accelerate the digestion reactions of phosphoproteins in aqueous solutions and also from complex real samples. After the microwave experiments, we directly injected the resulting solutions into the ESI-MS and MALDI-MS systems for analysis, and excellent sensitivity was achieved without the need for any washing procedure or separation process. The reasons are attributed to the high binding affinity and selectivity of TiO2 NPs toward phosphopeptides. Thus, phosphopeptides can be adsorbed onto the TiO2 NP surface. The digested or partially digested phosphoproteins can be concentrated onto the TiO2 NP surface. This results in the effective or complete digestion of phosphoproteins in a short period of time (45 s). In addition, high sensitivity and sequence coverage of phosphopeptide can be obtained using TiO2 NPs as microwave absorbers and affinity probes in MALDI-MS and ESI-MS. This is due to the photocatalytic nature of the TiO2 NPs because the absorption of microwave radiation that can accelerate the activation of trypsin for efficient digestion of phosphoproteins and enhances the ionization of phosphopeptides. The lowest concentrations detected for ESI-MS and MALDI-MS were 0.1 µM and 10 fmol, respectively, for α-casein. Comparing the two-step approach of TiO2 NPs with microscale TiO2 particles, the microscale TiO2 particles shows no effect on the microwave-assisted tryptic digestion of phosphoproteins. The current approach offers multiple advantages, such as great simplicity, high sensitivity and selectivity, straightforward and separation/washing-free technique for phosphorpeptide enrichment analysis.
Keywords: Microwave-assisted tryptic digestion; TiO2 nanoparticles; Protein; Phosphopeptide enrichment; Electrospray ionization mass spectrometry; Matrix-assisted laser desorption ionization time of flight mass spectrometry

In the present article, a novel microfluidic immunosensor coupled with electrochemical detection for anti-gliadin IgG antibody quantification is proposed. This device represents an important tool for a fast, simple, sensitive, and automated diagnostic for celiac disease, which is carried out through detection of anti-gliadin IgG antibodies present in human serum samples. Celiac disease (CD) is an autoimmune disease generated by gluten protein fractions called prolamins. This pathology affects about one in 250 people around the world, produces intestinal inflammation, villous atrophy, and crypt hyperplasia, which causes a range of symptoms including altered bowel habits, malnutrition and weight loss. Our immunosensor consists of a Plexiglas device coupled to a gold electrode, with a central channel containing 3-aminopropyl-modified controlled pore glass (AP-CPG). The quantification of anti-gliadin IgG antibodies was carried out using a heterogeneous, non-competitive enzyme-linked immunosorbent assay (ELISA) in which IgG antibodies bound to gliadin protein, immobilized on AP-CPG, were determined by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP, and the electroactive product was quantified on a gold electrode at 0.250 V. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.52 and 2.72 UR mL−1, respectively, and the within- and between-assay coefficients of variation were below 5.8%. The optimized procedure was applied to the determination of anti-gliadin IgG antibodies in human serum samples.
Keywords: Celiac disease; Microfluidic system; Enzyme immunoassays; Gold electrode; Gliadin; Immunosensor

Tetracycline antibiotics in hospital and municipal wastewaters: a pilot study in Portugal by A. Pena; M. Paulo; L. J. G. Silva; M. Seifrtová; C. M. Lino; P. Solich (2929-2936).
This study investigated the occurrence of tetracyclines (TCs), namely minocycline (MIN), TC, and its epimer epitetracycline (ETC), and doxycycline (DC), in four hospital wastewater effluents and its fate in municipal wastewater treatment plants (WWTPs), in Coimbra, Portugal. Analytical determination was carried out by solid-phase extraction followed by liquid chromatography with fluorescence detection. A gradient system with a mobile phase containing oxalic acid 0.02 M and acetonitrile was used. After postcolumn derivatization with magnesium reagent, TCs were detected at λ exc 386 nm and λ em 500 nm. The proposed method allowed good sensitivity, accuracy, and precision. LOQs were 0.5 μg l−1 for ETC and TC and 15 and 5 μg l−1 for MIN and DC, respectively. The recovery values ranged between 66.4% and 117.1%, and intraday and interday repeatability was lower than 6.8%. The method was successfully used to determine the presence of the above-mentioned TCs in 24 wastewater composite samples obtained from hospital effluents and from influent and effluent of the WWTP located in Coimbra, Portugal. MIN and TC were found in 41.7% of the samples; ETC and DC were found in 25% and 8.3% of the samples, respectively. The levels found ranged from 6 to 531.7 μg l−1 in hospital effluents, while its concentrations in WWTP ranged from 95.8 to 915.3 μg l−1. A seasonal influence in the concentrations found has also been observed, the levels found in samples collected during spring being higher than those observed in samples collected during autumn; however, these are only preliminary results. The WWTP removal rate ranged between 89.5% and 100%.
Keywords: Tetracyclines; SPE; LC–FD; Postcolumn derivatization; Environmental wastewaters

A hydrophilic interaction ultraperformance liquid chromatography (HILIC–UPLC) method for genomic DNA methylation assessment by UV detection by Salvatore Sotgia; Angelo Zinellu; Elisabetta Pisanu; Luciano Murgia; Gerard Aime Pinna; Leonardo Gaspa; Luca Deiana; Ciriaco Carru (2937-2941).
A hydrophilic interaction chromatography-based method, in combination with 1.7 µm ethylene bridged hybrid particle packed column (100 mm × 2.1 mm I.D.) and ultraperformance liquid chromatography, has been developed to measure cytosine (C) and methylcytosine (mC) in order to evaluate the extent of DNA methylation. Separation of cytosine and methylcytosine was achieved with good resolution and in fairly short times (5.5 min) by using isocratic elution with a mixture of 97:3 (v/v) acetonitrile/10 mM ammonium acetate as a mobile phase. The determination coefficients of C and mC were high (R 2 > 0.999) within the range tested. The %RSD for intraday and interday were respectively 2.2% and 2.5% for C and 3.5% and 3.8% for mC. The limit of detection was 0.52 µM (0.52 fmol on-column) both for C and mC while the limit of quantification was 1.72 µM (1.72 fmol on-column) both for C and mC. The smallest amount of purified DNA that yielded a measurable level of C and mC was 10 µg. On the whole, this method is simple, rapid, sensitive, and precise.
Keywords: DNA methylation; Hydrophilic interaction chromatography (HILIC); Ultraperformance liquid chromatography (UPLC); 1.7 µm ethylene bridged hybrid (BEH) particle

Ultra-low-angle microtomy and static secondary ion mass spectrometry for molecular depth profiling of UV-curable acrylate multilayers at the nanoscale by Yannick Vercammen; Roel De Mondt; Jaymes Van Luppen; Frank Vangaever; Luc Van Vaeck (2943-2954).
Development of sustainable materials requires methods capable of probing the molecular composition of samples not only at the surface but also in depth. Static secondary ion mass spectrometry (S-SIMS) characterises the distribution of organic and inorganic compounds at the surface. Ultra-low-angle microtomy (ULAM) has been studied as an alternative or complementing method to the molecular depth profiling with, e.g. C 60 + projectiles. Acrylate-based multilayers relevant to industrial inkjet printing have been sectioned at a cutting angle below 1°. In this way, analysis of the section over a distance of 1 µm allows a depth range in the order of a few nm in the original sample to be achieved. Adequate procedures to optimise the ULAM step and minimise or control the cutting artefacts have been developed. The combination of ULAM with S-SIMS has allowed a depth resolution of 10 nm to be obtained for components at a distance of 35 μm from the surface.
Keywords: Polymers; Interface/surface analysis; Mass spectrometry/ICP-MS; Organic compounds/trace organic compounds

Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin by Seiichi Sakamoto; Futoshi Taura; Benyakan Pongkitwitoon; Waraporn Putalun; Ryota Tsuchihashi; Junei Kinjo; Hiroyuki Tanaka; Satoshi Morimoto (2955-2963).
A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly4Ser)2 between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL−1) was improved to eightfold higher than that in conventional ELISA (0.2 μg mL−1), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.
Keywords: Enzyme-linked immunosorbent assay; Fluorescent single-domain antibody; Fluorescence-linked immunosorbent assay; Green fluorescent protein; Plumbagin; Single-chain variable fragment

A zirconia (ZrO2)-modified solid-phase extraction sorbent has been evaluated for selective extraction of phosphatidylcholines from biological samples, followed by analysis of the isolated solutes by reversed-phase liquid chromatography–electrospray ionization–tandem mass spectrometry. The clean-up process was optimized using seven standard phosphatidylcholines including two lyso derivatives. Different acidic conditions were tested for the bonding and washing steps; for elution, various aqueous or methanolic bases were studied. Experiments were conducted hydrodynamically using extraction cartridges, and statically in batch mode; the performance of the sorbent was significantly better when used in the flow-through mode. The developed clean-up procedure was used to selectively enrich phosphatidylcholines from whole milk, human plasma, and mouse plasma, to show the wide applicability of the method. For the preceding extraction of total lipids from the matrix, different solvent mixtures (methanol–chloroform, methanol–methyl tert-butyl ether, and ethanol–ethyl acetate) were compared. Accuracy and reproducibility of the proposed sample-preparation procedure were evaluated. Matrix effects possibly affecting mass spectrometric analysis were studied before and after the solid-phase extraction. They were found to be significant for several analytes, stressing the importance of a sample clean-up procedure. Under identical experimental conditions, recovery of bound phosphatidylcholines by zirconia was superior to that by other metal oxides, for example titania (TiO2) and stannia (SnO2).
Keywords: Phosphatidylcholines; Zirconium dioxide; Titanium dioxide; Tin dioxide; Tandem mass spectrometry; Electrospray ionization

A comprehensive method was developed for the simultaneous trace analysis of ten hormone antagonist pharmaceuticals (raloxifene, exemestane, letrozole, anastrozole, mifepristone, finastride, tamoxifen, N-desmethyltamoxifen, clomiphene, and toremifene) in municipal sewage and hospital wastewater samples. The target compounds were firstly extracted using an Oasis HLB cartridge, followed by purification by an aminopropyl cartridge, and were then analyzed by liquid chromatography electrospray ionization tandem mass spectrometry in positive ion mode. The recoveries for the analytes based on internal standard calibration in different test matrices ranged from 67.6 to 118.6% (with the exception of mifepristone in clinical wastewater samples), with relative standard deviations less than 20%. The method quantification limits of the ten pharmaceuticals were in the range 0.10–2.0 ng/L. Excluding exemestane and N-desmethyltamoxifen, eight drugs were detected at 0.20–195.0 ng/L in hospital wastewater and municipal wastewater samples from Beijing. Figure Analysis of hormone antagonists in clinical and municipal wastewater by liquid chromatography tandem mass spectrometry
Keywords: Hormone antagonists; Solid-phase extraction; Liquid chromatography–tandem mass spectrometry; Wastewater samples

Evaluation of potential breath biomarkers for active smoking: assessment of smoking habits by Monica Alonso; Mar Castellanos; Juan M. Sanchez (2987-2995).
Different compounds have been reported as biomarkers of a smoking habit, but, to date, there is no appropriate biomarker for tobacco-related exposure because the proposed chemicals seem to be nonspecific or they are only appropriate for short-term exposure. Moreover, conventional sampling methodologies require an invasive method because blood or urine samples are required. The use of a microtrap system coupled to gas chromatography–mass spectrometry analysis has been found to be very effective for the noninvasive analysis of volatile organic compounds in breath samples. The levels of benzene, 2,5-dimethylfuran, toluene, o-xylene, and m- p-xylene have been analyzed in breath samples obtained from 204 volunteers (100 smokers, 104 nonsmokers; 147 females, 57 males; ages 16 to 53 years). 2,5-Dimethylfuran was always below the limit of detection (0.005 ppbv) in the nonsmoker population and always detected in smokers independently of the smoking habits. Benzene was only an effective biomarker for medium and heavy smokers, and its level was affected by smoking habits. Regarding the levels of xylenes and toluene, they were only different in heavy smokers and after short-term exposure. The results obtained suggest that 2,5-dimethylfuran is a specific breath biomarker of smoking status independently of the smoking habits (e.g., short- and long-term exposure, light and heavy consumption), and so this compound might be useful as a biomarker of smoking exposure. Figure Extracted GC-MS chromatograms (m/z=78, 91, and 96) from a smoker and a healthy nonsmoker person.
Keywords: Breath biomarkers; Smoking; 2,5-Dimethylfuran; Benzene

This study demonstrates the use of solid-phase microextraction (SPME) to extract and pre-concentrate volatile signatures from static air above plastic explosive samples followed by detection using ion mobility spectrometry (IMS) optimized to detect the volatile, non-energetic components rather than the energetic materials. Currently, sample collection for detection by commercial IMS analyzers is conducted through swiping of suspected surfaces for explosive particles and vapor sampling. The first method is not suitable for sampling inside large volume areas, and the latter method is not effective because the low vapor pressure of some explosives such as RDX and PETN make them not readily available in the air for headspace sampling under ambient conditions. For the first time, headspace sampling and detection of Detasheet, Semtex H, and C-4 is reported using SPME-IMS operating under one universal setting with limits of detection ranging from 1.5 to 2.5 ng for the target volatile signatures. The target signature compounds n-butyl acetate and the taggant DMNB are associated with untagged and tagged Detasheet explosives, respectively. Cyclohexanone and DMNB are associated with tagged C-4 explosives. DMNB is associated with tagged Semtex H explosives. Within 10 to 60 s of sampling, the headspace inside a glass vial containing 1 g of explosive, more than 20 ng of the target signatures can be extracted by the SPME fiber followed by IMS detection.
Keywords: Plastic explosives; C-4; Detasheet; Semtex; Solid-phase microextraction; Ion mobility spectrometry

Magnesium (Mg) as a biodegradable metal has potential advantages as an implant material. This paper studies the effect of magnesium ions on osteoblast (U2-OS) behavior since magnesium implants mainly dissolve as divalent magnesium ions (Mg2+). A real-time monitoring technique based on electric cell-substrate impedance sensing (ECIS) was used for measuring cell proliferation, migration, adhesion, and cytotoxicity in magnesium-conditioned media. The impedance results show that U2-OS proliferation and adhesion were inhibited in not only a magnesium-free medium but also in a medium with a high concentration of magnesium. The impedance method produced more sensitive results than the output of an MTT assay. Other standard bioanalytical tests were conducted for comparison with the ECIS method. Immunochemistry was carried out to study cell adhesion in magnesium-conditioned media by staining using F-actin and α-tubulin and correlated cell density on the electrode with impedance. Bone tissue formation was studied using von Kossa staining and indicated the mineralization level of cells in magnesium-conditioned media decreased with the increase of magnesium ion concentration. Real-time PCR provided gene expression indicators of cell growth, apoptosis, inflammation, and migration. Compared to the bioanalytical methods of immunochemistry and MTT assays, which need preparation time and post-washing step, ECIS was able to measure cell activity in real time without any cell culture modification. In summary, ECIS might be an effective way to study biodegradable magnesium implants. Figure Principle of ECIS for analyzing U2-OS cell behavior under different concentrations of magnesium: a osteoblast cells are floating in the medium and the electrode impedance is small; and b osteoblast cells create a monolayer on the electrode which increases the impedance
Keywords: Biodegradable magnesium implant; U2-OS cells; Electric cell-substrate impedance sensing (ECIS); Cytotoxicity; MTT assay; Real-time PCR

Validated quantification for selective cellular uptake of ginsenosides on MCF-7 human breast cancer cells by liquid chromatography–mass spectrometry by Young Wan Ha; Kwang Seok Ahn; Jang-Choon Lee; Sung-Hoon Kim; Bong Chul Chung; Man Ho Choi (3017-3025).
The cellular behavior of ginsenosides on cancer cells has not been measured directly despite their potent anticancer activities and biological actions. A liquid chromatography–mass spectrometry (LC-MS) method was developed to measure the selective cellular uptake of ginsenosides in both cell lysates and culture media. Fifteen ginsenosides were separated within 17 min with good peak shapes using a 2-μm sub-particle size C18 column. Quantification was performed by triple-quadrupole MS with electrospray ionization in negative ion mode. The sample preparation containing the solid-phase extraction was linear (correlation coefficient, r 2 > 0.992) for all analytes, while the limit of quantification ranged from 0.5 to 2.0 ng/mL in both matrices. The assay precision (%CV) and accuracy (%bias) at three different concentrations (5, 20, and 100 ng/mL) were 1.4% to 11.6% and 94.9% to 106.4%, respectively. When this method was used to examine the selective cellular uptake of ginsenosides, the relative non-polar and protopanaxadiol class ginsenosides, such as Rg3, Rk1, Rg5, Rh2, compound-K, and protopanaxadiol (PPD), showed cellular uptake in the MCF-7 cells, but the relative polar and protopanaxatriol class of ginsenosides did not accumulate in the cells. The most non-polar ginsenoside PPD, which is an aglycone of the protopanaxadiol type, resulted in the highest uptake rate. These results show that the different anticancer activities are due to the selective uptake of ginsenosides based on their chemical structures. This LC-MS-based method can be used to estimate the biological activity of ginsenosides on cells from their structural diversity. Figure The structure and the ratios of cellular uptake of ginsenosides evaluated by liquid chromatography–mass spectrometry
Keywords: Ginsenoside; Cellular uptake; Breast cancer cell; LC-MS

Quantitative analysis of penicillins in porcine tissues, milk and animal feed using derivatisation with piperidine and stable isotope dilution liquid chromatography tandem mass spectrometry by Frédérique van Holthoon; Patrick P. J. Mulder; Eric O. van Bennekom; Henri Heskamp; Tina Zuidema; Hans (J.) A. van Rhijn (3027-3040).
Penicillins are used universally in both human and veterinary medicine. The European Union (EU) has established maximum residue levels (MRLs) for most ß-lactam antibiotics in milk and animal tissues and included them in the National Residue Monitoring Programs. In this study, a novel method is described for the determination and confirmation of eight penicillins in porcine tissues, milk and animal feed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). To prevent degradation of penicillin residues during workup, a derivatisation procedure was developed, by which penicillins were converted to stable piperidine derivatives. Deuterated piperidine derivatives were synthesised for all relevant penicillins, enabling the use of isotope dilution for accurate quantification. Penicillin residues were derivatised in the crude extract with piperidine and isolated using solid-phase extraction. The penicillin piperidine derivatives were determined by LC–MS/MS. The method was validated at the current MRLs, which range from 25–300 µg kg−1 in muscle and kidney to 4–30 µg kg−1 in milk as well as at the target value of 100 µg kg−1 chosen for animal feed, according to the EU requirements for a quantitative confirmatory method. Accuracy ranged from 94–113% (muscle), 83–111% (kidney) and 87–103% (milk) to 88–116% (animal feed). Intra-day precision (relative standard deviation (RSD)r) ranged from 5–13% (muscle, n = 18), 4–17% (kidney, n = 7) and 5–18% (milk, n = 7) to 11–32% (animal feed, n = 18). Inter-day precision (RSDRL, n = 18) ranged from 6–23% (muscle) to 11–36% (animal feed). From the results, it was concluded that the method was fit for purpose at the target MRLs in animal tissue and target levels for animal feed.
Keywords: Penicillins; Piperidine; Derivatisation; LC–MS/MS; Isotope dilution; Validation

Two independent liquid chromatography inductively coupled plasma-mass spectrometry (LC/ICP-MS) methods for the separation of arsenic species in urine have been developed with quantification by standard additions. Seven arsenic species have been quantified in a new NIST frozen human urine Standard Reference Material (SRM) 2669 Arsenic Species in Frozen Human Urine, Levels 1 and 2. The species measured were: arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA), dimethylarsinate (DMA), arsenobetaine (AB), arsenocholine (AC), and trimethylarsine oxide (TMAO). The purity of each arsenic standard used for quantification was measured as well as the arsenic species impurities determined in each standard. Analytical method limits of detection (L D) for the various species in both methods ranged from 0.2 to 0.8 μg L−1 as arsenic. The results demonstrate that LC/ICP-MS is a sensitive, reproducible, and accurate technique for the determination of low-level arsenic species in urine. Measurements of the arsenic species 3 years after initial production of the SRM demonstrate the stability of the arsenic species in the urine reference material. Figure SRM 2669 Arsenic Species in Frozen Human Urine
Keywords: ICP mass spectrometry; Arsenic speciation; Reference materials; Urine

Certification of reference materials for Cd, Cr, Hg and Pb in polypropylene by Liuxing Feng; Liandi Ma; Jun Wang; Hai Lu (3051-3057).
Two reference materials, at relatively low and high concentrations (GBW08404 and GBW08405), for analysis of the mass fractions of Cd, Cr, Hg and Pb in polypropylene were developed. The reference materials were prepared by doping blank polypropylene base material with Cd, Cr, Hg and Pb in the form of oxides, salts or pigments. Homogeneity and stability studies were performed by inductively coupled plasma mass spectrometry. The certification of the four analytes was carried out by isotope-dilution mass spectrometry (IDMS) with microwave-assisted digestion. Combined uncertainties were calculated from the IDMS uncertainty evaluation budget and the uncertainty of the homogeneity. The mass fractions of Cd, Cr, Hg and Pb of the two certified reference materials (CRMs) were from 8 to 1,000 mg kg−1. The two samples were also used in an interlaboratory comparison scheme in which National Institute of Metrology, China, National Metrological Institute of Japan and Korea Research Institute of Standards and Science participated. The agreement of the comparison results proved that the certification procedure of the CRMs is valid and that the certified values of Cd, Cr, Hg and Pb are accurate and reliable. Figure Certified reference materials for Cd, Cr, Hg and Pb in polypropylene (GBW08404 and GBW08405)
Keywords: Certified reference material; Element; Polypropylene; Isotope dilution mass spectrometry; Interlaboratory comparison

A simple procedure has been developed and validated for the qualitative and quantitative analysis of several opiates (morphine, 6-acetylmorphine, codeine, 6-acetylcodeine) and tramadol in hair. The analytes were extracted from within the matrix via an overnight incubation with methanol at 65 °C, and afterwards the samples were cleaned up by mixed-mode solid-phase extraction. The extracts were derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide with 5% trimethylchlorosilane and analyzed by gas chromatography–mass spectrometry in the selected ion monitoring mode. The method was linear from 0.05 (lower limit of quantitation) to 50 ng/mg (40 ng/mg for tramadol), with correlation coefficients higher than 0.99 for all compounds, accomplishing the cut-off values proposed by the Society of Hair Testing for the detection of these substances in hair (0.2 ng/mg). Intra- and interday precision and trueness were in conformity with the criteria normally accepted in bioanalytical method validation, and the sample cleanup step presented a mean efficiency higher than 90% for all analytes. Furthermore, using these incubation conditions, 6-acetylmorphine did not significantly hydrolyze to morphine. For these reasons, and because of its simplicity, the proposed method can be successfully applied in the determination of these compounds in hair samples, and is suitable for application in routine analysis with forensic purposes.
Keywords: Opiates; Hair; Solid-phase extraction; GC-MS

Isolation of N-linked glycopeptides by hydrazine-functionalized magnetic particles by Shisheng Sun; Ganglong Yang; Ting Wang; Qinzhe Wang; Chao Chen; Zheng Li (3071-3078).
We introduce a novel combination of magnetic particles with hydrazine chemistry, dubbed as hydrazine-functionalized magnetic particles (HFMP) for isolation of glycopeptides. Four methods have been developed and compared for the production of HFMP by hydrazine modification of the surface of the carboxyl and epoxy-silanized magnetic particles, respectively. The evaluation of the capability and specificity of HFMP as well as the optimization of the coupling condition for capturing of glycoproteins were systematically investigated. The results showed that HFMP prepared by adipic dihydrazide functionalization from carboxyl-silanized magnetic particles (HFCA) displayed the maximum capture capacity and isolated efficiency for glycoprotein. When measured with glycoproteins, the capacity of the HFCA (1 g) for coupling bovine fetuin was 130 ± 5.3 mg. The capability of this method was also confirmed by successful isolation of all formerly glycosylated peptides from standard glycoproteins and identification of their glycosylation sites, which demonstrated the feasibility of the HFCA as an alternative solid support for isolation of glycoproteins/glycopeptides. Figure Schematic diagram for the preparation of hydrazine-functionalized magnetic particles (HFMP) and isolation of N-linked glycopeptides by HFMP from protein sample.
Keywords: Hydrazine-functionalized magnetic particles; Glycopeptides; Glycoproteins; Glycosylation sites; Mass spectrometry

Flow field-flow fractionation: a versatile approach for size characterization of α-tocopherol-induced enlargement of gold nanoparticles by Wimut Sermsri; Purim Jarujamrus; Juwadee Shiowatana; Atitaya Siripinyanond (3079-3085).
Flow field-flow fractionation (FlFFF) was used for size characterization of gold nanoparticles. The measured particle sizes obtained from FlFFF for the commercial 10 nm gold nanoparticle standard and the gold nanoparticles synthesized in the laboratory were in good agreement with those measured by transmission electron microscopy (TEM). Further, the capability of α-tocopherol to induce enlargement of gold nanoparticles by catalysis of the reduction of AuCl 4 by citrate was observed by monitoring the changes in particle size of gold nanoparticles using FlFFF. The effects of α-tocopherol and incubation time on enlargement of the gold nanoparticles were examined. Higher concentrations of α-tocopherol resulted in larger nanoparticles. At fixed α-tocopherol concentration, larger nanoparticles were formed at longer incubation times.
Keywords: Gold nanoparticles; Field-flow fractionation; α-Tocopherol; Particle size

Simultaneous chemical fingerprint and quantitative analysis of Ginkgo biloba extract by HPLC–DAD by Daoquan Tang; Dongzhi Yang; Anbang Tang; Yuanyuan Gao; Xianglan Jiang; Jie Mou; Xiaoxing Yin (3087-3095).
A reverse-phase liquid chromatography method with diode array detection was developed to evaluate the quality of Ginkgo biloba extract through establishing chromatographic fingerprint and simultaneous determination of eight flavonoid compounds, namely rutin, myricetin, quercitrin, quercetin, luteolin, kaempferol, apigenin, and isorhamnetin. The chromatographic separation was performed on an Agilent SB-C18 column (250 × 4.6 mm, 5.0 µm) with a gradient elution program using a mixture of methanol and 0.1% formic acid (v/v) as mobile phase within 55 min at 360-nm wavelength. The correlation coefficients of similarity for different batches of G. biloba extract from the same manufacturer and G. biloba extract from different manufacturers were determined from the LC fingerprints, and they shared a close similarity. The eight flavonoid compounds showed good regression (R 2 > 0.9995) within test ranges, and the recovery of the method was in the range of 94.1–101.4%. In addition, the content of those eight flavonoid compounds in G. biloba extract prepared by different manufacturers of China was determined to establish the effectiveness of the method. The results indicated that the developed method by having a combination of chromatographic fingerprint and quantification analysis could be readily utilized as a quality control method for G. biloba extract and its related traditional Chinese medicinal preparations. Figure Fingerprint chromatograph of GBE from different manufactures.
Keywords: Column liquid chromatography; Fingerprint analysis; Flavonoids compounds; Ginkgo biloba extract

A procedure for the determination of traces of mercury by liquid-phase microextraction based on solidification of a floating organic droplet for separation and electrothermal atomic absorption spectrometry for final measurement has been developed. For this purpose, 50 µL of pre-heated (50 °C) undecanoic acid (UA), are added to 25 mL of aqueous sample solution at pH 5. The mixture, maintained at 50 °C, is stirred for 10 min using a high stirring rate in order to fragment the UA drop into droplets, thus favoring the extraction process. Next, the vial is immersed in an ice bath, which results in the solidification of the UA drop that is easily separated. Injection into the atomizer is carried out after gentle heating. The pyrolytic atomizers are coated with electrolytically reduced palladium that acts as an effective chemical modifier for more than 500 firings. Under the optimized conditions, the detection limit was 70 ng L−1 mercury with an enrichment factor of 430. The relative standard deviation of the measurements was in the 2.1–3.5% range. Recovery studies applied to the determination of mercuric ions in bottled and tap water samples were in the 92–104% range. Figure LPME-SFO allows liquid phase and the solidified organic reagent to be easily separated
Keywords: Liquid-phase microextraction; LPME; LPME-SFO; Mercury; ETAAS; Waters

Cobalt phthalocyanine-modified screen-printed carbon electrodes (CoPC-SPCEs) have been investigated as disposable sensors for the measurement of citric acid. The analyte was found to undergo an electrocatalytic oxidation process involving the Co2+/Co3+ redox couple. Calibration plots were found to be linear in the range 2 mM to 2.0 M; replicate determinations of a 5.2 mM citric acid (n = 4) solution gave a coefficient of variation of 1.43%. Additions of metal ions, such as Ag+, Pb2+, Cu2+, Fe3+ and Ca2+, were found not to interfere. The effects of hesperidin, cysteine, ethylenediaminetetraacetic acid (EDTA), ascorbic, formic, malic, malonic, tartaric, oxalic and trichloroacetic acids on the determination of citric acid were examined and, under the conditions employed, only oxalic acid and EDTA were found to give any significant interference. The sensors were evaluated by carrying out citric acid determinations on spiked and unspiked samples of an acid citrate dextrose (ACD) formulation, lime flesh and juice. For lime juice, recoveries were calculated to be 96.8% (% CV = 2.7%) for a sample fortified with 5% citric acid and for ACD 99.4% (%CV = 2.6%) when fortified at 2.30% citric acid. Further studies showed the possibility of determining citric acid concentrations in lime juice and fruit directly, without the need for an added electrolyte. These performance characteristics indicate that reliable data may be obtained for citric acid measurements in such samples. To our knowledge, this is the first report on the electrocatalytic oxidation of citric acid and its application using a CoPC-SPCE. Figure Direct measurement of citric acid in limes based on the electrocatalytic oxidation of the analyte at a screen-printed carbon electrode modified with cobalt phthalocyanine.
Keywords: Citrate, cobalt phthalocyanine; Screen-printed carbon electrode; Lime; Pharmaceutical; Voltammetry

Erratum to: Current trends in the detection of peroxide-based explosives by Raychelle M. Burks; David S. Hage (3113-3114).