Analytical and Bioanalytical Chemistry (v.397, #7)

Raman spectroscopy by Peter Vandenabeele (2629-2630).
is research professor in archaeometry at Ghent University. He studied chemistry, focusing in particular on analytical chemistry and Raman spectroscopy. He has authored and co-authored ca. 70 research papers about different applications of Raman spectroscopy, and has given several oral and poster presentations at international conferences.

Applications of Raman spectroscopy to gemology by Danilo Bersani; Pier Paolo Lottici (2631-2646).
Being nondestructive and requiring short measurement times, a low amount of material, and no sample preparation, Raman spectroscopy is used for routine investigation in the study of gemstone inclusions and treatments and for the characterization of mounted gems. In this work, a review of the use of laboratory Raman and micro-Raman spectrometers and of portable Raman systems in the gemology field is given, focusing on gem identification and on the evaluation of the composition, provenance, and genesis of gems. Many examples are shown of the use of Raman spectroscopy as a tool for the identification of imitations, synthetic gems, and enhancement treatments in natural gemstones. Some recent developments are described, with particular attention being given to the semiprecious stone jade and to two important organic materials used in jewelry, i.e., pearls and corals. Figure Pictures of a pyrope-almandine garnet and of a needle-shaped inclusion. The Raman spectrum of the inclusion is characteristic of rutile
Keywords: Raman spectroscopy; Gems; Minerals; Jade; Pearls; Corals

Rapid Raman mapping of a fulgurite by Elizabeth A. Carter; Matthew A. Pasek; Tim Smith; Terence P. Kee; Peter Hines; Howell G. M. Edwards (2647-2658).
A fulgurite is a naturally occurring glass formed when lightning hits sand, rock, or soil. The formation of fulgurites is accompanied by mineralogical and sometimes compositional changes, and may record information about the environment in which they were formed. A previous investigation using Raman point spectroscopy discovered the presence of anatase, a low-temperature polymorph of TiO2, and polyaromatic hydrocarbons within a fulgurite. These findings indicate that there were regions within the sample that were not subjected to temperatures of 2,000 K or more that the matrix is reported to attain when struck by lightning. This paper seeks to expand the previous research by utilizing the capabilities of a new Raman spectroscopic technological development that enables rapid mapping. The entire surface area of a cross-sectioned fulgurite (∼40 mm × 23 mm) sample was mapped allowing several regions of polyaromatic hydrocarbons and anatase to be located. Furthermore, shocked quartz was found within the boundary regions of the fulgurite, and is proposed to have resulted from contact with vaporized material during the lightning strike. Shocked quartz is typically indicative of extraterrestrial impact, yet its discovery here suggests that its formation is not exclusive to the impact process. Figure Raman map illustrating the distribution of normal and shocked quartz in a fulgurite
Keywords: Raman spectroscopy; Rapid mapping; Fulgurite; Shocked quartz

Investigation of inclusions trapped inside Libyan desert glass by Raman microscopy by Marcel Swaenen; Elżbieta Anna Stefaniak; Ray Frost; Anna Worobiec; René Van Grieken (2659-2665).
Several specimens of Libyan desert glass (LDG), an enigmatic natural glass from Egypt, were subjected to investigation by micro-Raman spectroscopy. The spectra of inclusions inside the LDG samples were successfully measured through the layers of glass and the mineral species were identified on this basis. The presence of cristobalite as typical for high-temperature melt products was confirmed, together with co-existing quartz. TiO2 was determined in two polymorphic species rutile and anatase. Micro-Raman spectroscopy proved also the presence of minerals unusual for high-temperature glasses such as anhydrite and aragonite.
Keywords: Libyan desert glass; Raman spectroscopy; Inclusions; Cristobalite; Anatase; Zircon; Rutile

A survey of gilts applied to stucco surfaces that specifically focuses on the compositions of their colored grounds is reported. Gilt samples of a common geographical (Lombardy in Italy) and temporal provenance (17th–18th century) were studied in the form of polished cross-sections by optical and electron microscopy (SEM-EDS), micro-Raman (μRaman) spectroscopy and Fourier-transform infrared microspectroscopy (μFTIR). Comparing samples with superimposed grounds and gilts enabled light to be shed on the choice of specific materials, their stratigraphic functions, decorative effects, and technological performances. Iron oxide pigments were found in the older grounds, sometimes in the presence of lead white (2PbCO3·Pb(OH)2) or minium (Pb3O4). In more recent grounds, chrome yellow (PbCrO4), chrome orange (PbCrO4·PbO), cinnabar (α-HgS) and barium white (BaSO4), invariably mixed with lead white, were encountered. Evidence for the use of organic mordants (colophony and wax, or siccative oil) was obtained by μFTIR. This combined μFTIR and μRaman spectroscopic and elemental (SEM-EDS) analytical approach enhances knowledge of the composition of gold grounds, their variability and their chronological evolution.
Keywords: Gilding technique; Stucco; Raman spectroscopy; Colored grounds; Chrome yellow and orange; Mordant gilding

The vibrational infrared spectroscopic analysis of an important historical necklace of 102 beads that are purported to be made of amber indicated strong signal characteristics of cellulose nitrate with dark green-coloured areas of a naphthylamine dye. Confocal Raman depth-profiling spectroscopy using a 785-nm laser excitation, a novel application first applied here for the analysis of inclusions in amber resin, confirmed that the beads were amber resin and that residues of cellulose nitrate, camphor plasticiser and a naphthylamine dyestuff were present in surface cracks and inclusions in the bead matrix. The bead stringing material was confirmed as cellulose, which was stained green in part with the dyestuff. Comparison of the Raman spectra of the amber beads with a resin database suggested that the amber was sourced from Northern England. The scientific evidence supports the stylistic opinion that the necklace is an important example that could date from the 19th Century and that efforts had been made to coat it with a synthetic dyed polymer; this provides a rather unusual example of the chemical masking of a genuine article—a procedure that renders the article of particular interest.
Keywords: Raman spectroscopy; Amber; Necklace; Museum artefact; Disguise

FT-Raman spectroscopic analysis of pigments from an Augustinian friary by Howell G. M. Edwards; Emma M. Newton; Sonia O’Connor; D. Evans (2685-2691).
The Raman spectroscopic analysis of several stone samples with applied red pigments obtained from an archaeological excavation of an Augustinian friary discovered during the construction of an extension to Hull Magistrates Court in 1994 has revealed a surprising diversity of composition. Cinnabar, red lead and haematite have all been identified alone or in admixture; the cinnabar is exceptional in that it has only been found heavily adulterated with red ochre and red lead, as the other two pigments are found alone. There are signatures of limewash putty, which has been applied to the stone substrate prior to the painting, which is characteristic of the Roman method of wall painting, and there are no traces of gypsum found in the specimens studied. This evidence indicates an early mediaeval method of stone decoration.
Keywords: Red pigments; Stone substrate; Raman spectroscopy; Archaeological excavation; Limewash putty

Determination of amylose content in starch using Raman spectroscopy and multivariate calibration analysis by Mariana R. Almeida; Rafael S. Alves; Laura B. L. R. Nascimbem; Rodrigo Stephani; Ronei J. Poppi; Luiz Fernando C. de Oliveira (2693-2701).
Fourier transform Raman spectroscopy and chemometric tools have been used for exploratory analysis of pure corn and cassava starch samples and mixtures of both starches, as well as for the quantification of amylose content in corn and cassava starch samples. The exploratory analysis using principal component analysis shows that two natural groups of similar samples can be obtained, according to the amylose content, and consequently the botanical origins. The Raman band at 480 cm−1, assigned to the ring vibration of starches, has the major contribution to the separation of the corn and cassava starch samples. This region was used as a marker to identify the presence of starch in different samples, as well as to characterize amylose and amylopectin. Two calibration models were developed based on partial least squares regression involving pure corn and cassava, and a third model with both starch samples was also built; the results were compared with the results of the standard colorimetric method. The samples were separated into two groups of calibration and validation by employing the Kennard-Stone algorithm and the optimum number of latent variables was chosen by the root mean square error of cross-validation obtained from the calibration set by internal validation (leave one out). The performance of each model was evaluated by the root mean square errors of calibration and prediction, and the results obtained indicate that Fourier transform Raman spectroscopy can be used for rapid determination of apparent amylose in starch samples with prediction errors similar to those of the standard method. Figure Raman spectroscopy has been successfully applied to the determination of the amylose content in cassava and corn starches by means of multivariate calibration analysis.
Keywords: Corn starch; Cassava starch; Fourier transform Raman; Principal component analysis; Partial least squares

Raman spectroscopic study of the uranyl sulphate mineral zippeite: low wavenumber and U–O stretching regions by Jakub Plášil; Elena Buixaderas; Jiří Čejka; Jiří Sejkora; Jan Jehlička; Milan Novák (2703-2715).
The uranyl sulphate mineral zippeite was studied by Raman spectroscopy. The phase purity of the sample was initially checked by X-ray powder diffraction and its chemical composition was defined by electron microprobe (wavelength dispersive spectroscopy, WDS) analysis. The Raman spectroscopy research focused on the low wavenumber and uranyl stretching vibration regions. Vibration bands down to 50 cm–1 were tentatively assigned. The U–O bond lengths were calculated based on empirical relations. Inferred values are consistent with those obtained from the crystal structure analysis of synthetic zippeite. Number of bands was interpreted on the basis of factor group analysis.
Keywords: Raman spectroscopy; Zippeite; Potassium uranyl sulphate; Factor group analysis

Portable Raman monitoring of modern cleaning and consolidation operations of artworks on mineral supports by I. Martínez-Arkarazo; A. Sarmiento; M. Maguregui; K. Castro; J. M. Madariaga (2717-2725).
Any restoration performed on cultural heritage artworks must guarantee a low impact on the treated surfaces. Although completely risk-free methods do not exist, the use of tailor-made procedures and the continuous monitoring by portable instrumentation is surely one of the best approaches to conduct a modern restoration process. In this work, a portable Raman monitoring, combined sometimes with spectroscopic techniques providing the elemental composition, is the key analysis technique in the three-step restoration protocol proposed: (a) in situ analysis of the surface to be treated (original composition and degradation products/pollutants) and the cleaning agents used as extractants, (b) the thermodynamic study of the species involved in the treatment in order to design a suitable restoration method and (c) application and monitoring of the treatment. Two cleaning operations based on new technologies were studied and applied to two artworks on mineral supports: a wall painting affected by nitrate impact, and a black crusted stone (chalk) altarpiece. Raman bands of nitrate and gypsum, respectively, decreased after the step-by-step operations in each case, which helped restorers to decide when the treatment was concluded, thus avoiding any further damage to the treated surface of the artworks.
Keywords: Artworks; Mineral support; Monitoring; Portability; Raman spectroscopy; Restoration

Raman spectral imaging of single cancer cells: probing the impact of sample fixation methods by Florence Draux; Cyril Gobinet; Josep Sulé-Suso; Aurélie Trussardi; Michel Manfait; Pierre Jeannesson; Ganesh D. Sockalingum (2727-2737).
Raman spectroscopy has proven its potential for the analysis of cell constituents and processes. However, sample preparation methods compatible with clinical practice must be implemented for collection of accurate spectral information. This study aims at assessing, using micro-Raman imaging, the effects of some routinely used fixation methods such as formalin-fixation, formalin-fixation/air drying, cytocentrifugation, and air drying on intracellular spectral information. Data were compared with those acquired from single living cells. In parallel to these spectral information, cell morphological modifications that accompany sample preparation were compared. Spectral images of isolated cells were first analyzed in an unsupervised way using hierarchical cluster analysis (HCA), which allowed delimitation of the cellular compartments. The resulting nuclei cluster centers were compared and revealed at the molecular level that fixation induced changes in spectral information assigned to nucleic acids and proteins. In a second approach, a supervised fitting procedure using model spectra of DNA, RNA, and proteins, chemically extracted from living cells, revealed very small modifications at the level of the localization and quantification of these macromolecules. Finally, HCA and principal components analysis (PCA) performed on individual spectra randomly selected from the nuclear regions showed that formalin-fixation and cytocentrifugation are sample preparation methods that have little impact on the biochemical information as compared to living conditions. Any step involving cell air drying seems to accentuate the spectral deviations from the other preparation methods. It is therefore important in a future context of spectral cytology to take into account these variations. Figure Raman spectral imaging in cytology: label-free cell imaging showing distribution of major macromolecules like DNA, RNA, and proteins
Keywords: Raman microimaging; Cell fixation; Statistical analysis; Spectral cytology

Raman-based geobarometry of ultrahigh-pressure metamorphic rocks: applications, problems, and perspectives by Andrey V. Korsakov; Vladimir P. Zhukov; Peter Vandenabeele (2739-2752).
Raman-based geobarometry has recently become increasingly popular because it is an elegant way to obtain information on peak metamorphic conditions or the entire pressure-temperature-time (P-T-t) path of metamorphic rocks, especially those formed under ultrahigh-pressure (UHP) conditions. However, several problems need to be solved to get reliable estimates of metamorphic conditions. In this paper we present some examples of difficulties which can arise during the Raman spectroscopy study of solid inclusions from ultrahigh-pressure metamorphic rocks.
Keywords: Raman spectroscopy; Thermoelastic model; Coesite; Quartz; Aragonite; Calcite

Raman spectra of pure biomolecules obtained using a handheld instrument under cold high-altitude conditions by Jan Jehlička; Peter Vandenabeele; Howell G. M. Edwards; Adam Culka; Tomáš Čapoun (2753-2760).
A handheld Raman spectrometer (Ahura First Defender) was tested for the unambiguous identification of biomolecules (pure amino acids, carboxylic acids, saccharides and trehalose) in the solid state under outdoor conditions (including moderate climate conditions as well as cold temperatures and high altitudes). The biomolecules investigated represent important objects of interest for future exobiological missions. Repetitive measurements carried out under identical instrumental setups confirmed the excellent reliability of the Raman spectrometer. Raman bands are found at correct wavenumbers ±3 cm−1 compared with reference values. This testing represents the first step in a series of studies. In a preliminary, challenging investigation to determine the detection limit for glycine dispersed in a powdered gypsum matrix, 10% was the lowest content confirmed unambiguously. Clearly there is a need to investigate further the detection limits of Raman spectroscopic analyses of biomolecules in more complex samples, to demonstrate the usefulness or disqualify the use of this technique for more realistic outdoor situations, such as eventual future missions to Mars. Figure Application of a portable Raman spectrometer at 3,300 m near the Corvatsch glacier
Keywords: Raman spectroscopy; Handheld instrumentation; In situ examination; Low-temperature conditions; Exobiology; Amino acids

Mass Spectrometry: Fourth conference of the Spanish Society of Mass Spectrometry (SEEM) by Félix Hernández; Juan V. Sancho; Damià Barceló (2761-2762).
is Professor of Analytical Chemistry at University Jaume I, Castellón, Spain. As Director of the Research Institute for Pesticides and Water, he leads a 30-strong research group in the field of analytical chemistry. His work is mainly focused on the development of advanced analytical methodology for pesticide residue analysis in a variety of sample matrices. He is also Director of the good laboratory practice (GLP)-certified Laboratory of Pesticide Residue Analysis (LARP) at the same university. LARP is the reference laboratory for GLP studies related to pesticide registration for the Spanish Ministry of Agriculture. The development of analytical strategies for rapid screening of organic micropollutants in the aquatic environment, making use of full-scan accurate mass spectrometry, is one of the latest research developments within his group. is Professor of Analytical Chemistry at University Jaume I, Castellón, Spain ,and is responsible for the Mass Spectrometry University Facility. His work is mainly focused on the application of advanced mass spectrometry techniques in environmental analysis and food safety. Current research is focused on the applications of ultra-high-performance liquid chromatography/tandem mass spectrometry using both triple quadrupole and hybrid quadrupole time of flight) for screening, quantitation, confirmation, and elucidation of organic contaminants in different fields. The development of metabolomic approaches in food authenticity and doping analysis is one of his latest research interests. is a full Research Professor in the Environmental Chemistry Department at IDAEA-CSIC in Barcelona, Spain, and Director of the Catalan Institute for Water Studies (ICRA) in Girona, Spain. He is also President of the Spanish Society of Mass Spectrometry. His research focuses on method development and the monitoring and fate of priority, new and emerging pollutants, using gas chromatography and liquid chromatography techniques coupled with advanced tandem and hybrid mass spectrometry analysis combined with effect studies using bioassays and biosensors.

The presence of a wide variety of organic pollutants with different physicochemical characteristics has been investigated in wastewater samples from a municipal solid-waste-treatment plant in Castellón, Spain. An advanced analytical strategy was applied—combined used of two powerful and complementary techniques, GC and LC, both hyphenated with tandem mass spectrometry with triple-quadrupole analyzers. The GC–MS–MS method was based on sample extraction using C18 SPE cartridges and enabled the determination of approximately 60 compounds from different chemical families, for example PAHs, octyl/nonylphenols, PCBs, organochlorine compounds, insecticides, herbicides, and PBDEs. Most of the compounds selected are included as priority contaminants in the European Union (EU) Water Directive. The UHPLC–MS–MS method, which provided high chromatographic resolution and sensitivity and short analysis time, used sample extraction with Oasis HLB SPE cartridges and enabled the determination of 37 (more polar) pesticides. The methodology developed was applied to the analysis of 41 water samples (20 untreated raw leachates and 21 treated samples) collected between March 2007 and February 2009. Amounts of the contaminants investigated rarely exceeded 0.5 μg L−1 in the treated (reverse osmosis) water samples analyzed. As expected, in untreated leachates the number of compounds detected and the concentrations found were notably higher than in treated waters. The most commonly detected pollutants were herbicides (simazine, terbuthylazine, terbutryn, terbumeton, terbacil, and diuron), fungicides (thiabendazole and carbendazim), and 4-t-octylphenol. The results obtained proved that use of reverse osmosis for water treatment was efficient and notably reduced the amounts of organic contaminants found in raw leachate samples. In order to investigate the presence of other non-target contaminants, water samples were also analyzed by using GC–TOF MS and LC–QTOF MS. Several organic pollutants that did not form a part of the previous list of target contaminants were identified in the samples, because of the high sensitivity of TOF MS in full-spectrum acquisition mode and the valuable accurate-mass information provided by these instruments. The insecticide diazinon, the fungicide diphenylamide, the UV filter benzophenone, N-butylbenzenesulfonamide (N-BBSA), the insect repellent diethyltoluamide, caffeine, and the pharmaceuticals erythromycin, benzenesulfonanilide, ibuprofen, atenolol, and paracetamol were some of the compounds identified in the water samples analyzed.
Keywords: Organic pollutants; Wastewater; UHPLC; GC; Tandem MS; TOF MS

Development of fast screening methods for the analysis of veterinary drug residues in milk by liquid chromatography-triple quadrupole mass spectrometry by José Luis Martínez Vidal; Antonia Garrido Frenich; María M. Aguilera-Luiz; Roberto Romero-González (2777-2790).
Two rapid multi-residue screening methods for the determination of 21 veterinary drugs in milk by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) have been developed and compared. For both methods, veterinary drugs were extracted from milk samples using a rapid extraction procedure based on the modification of the well-known QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, and no further clean-up steps were necessary. One screening method was based on the selection of a characteristic neutral loss or product ion of the various families of compounds, whereas another one was based on the choice of a selected reaction monitoring (SRM) for each compound. These methods were compared with regards to false negatives, cut-off values and the unreliability region. The total run time for both methods was 3 min, allowing quick selection of samples that contained veterinary drugs. Non-negative samples were re-analyzed by the UHPLC-MS/MS confirmation/quantification method, which consisted in the monitoring of two SRM for each compound. The methods were validated according to international guides. The proposed analytical methods allow for the identification and confirmation of the target veterinary drugs at trace levels employing quick analysis time.
Keywords: Veterinary drugs; Milk; Screening methods; Triple quadrupole; UHPLC

Simultaneous determination of triazines and their main transformation products in surface and urban wastewater by ultra-high-pressure liquid chromatography–tandem mass spectrometry by Federica Benvenuto; José M. Marín; Juan V. Sancho; Sergio Canobbio; Valeria Mezzanotte; Félix Hernández (2791-2805).
This work describes the optimization, validation and application of an ultra-high-pressure liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method for the quantification and confirmation of 11 compounds (atrazine, simazine, terbuthylazine, terbumeton, terbutryn and their main transformation products) in surface and wastewater samples. Several of these analytes are included in the list of priority substances in the framework on European Water Policy. The application of this method to water samples reveals that the most relevant transformation products (TPs) should be incorporated into current analytical methods to obtain a more realistic knowledge of water quality regarding pesticide contamination. TPs are generally more polar and mobile than parents and can be transported to the aquatic environment more easily than their precursors. This can explain their concentrations found in water, which in many cases are much higher than intact triazines. To efficiently combine UHPLC with MS/MS, a fast-acquisition triple quadrupole mass analyser was used. Working in selected reaction monitoring mode, up to three simultaneous transitions per compound were acquired, allowing a reliable quantification and confirmation at nanogram per litre levels. The method developed includes a pre-concentration step based on solid-phase extraction (OASIS HLB cartridges). Satisfactory recoveries (70–120%) and relative standard deviations (<20%) were obtained for all compounds in different water sample types spiked at two concentrations (0.025 and 0.1 µg/L in surface water; 0.25 and 1.0 µg/L in effluent wastewater; 0.5 and 2.0 μg/L in influent wastewater). The optimized method was found to have excellent sensitivity with instrumental detection limits as low as 0.03 pg. In addition, the influence of the matrix constituents on the ionization efficiency and the extraction recovery was studied in different types of Italian and Spanish surface and urban wastewater. Signal suppressions were observed for all compounds, especially for influent wastewater. The use of isotope-labelled internal standards was found to be the best approach to assure an accurate quantification in all matrix samples.
Keywords: Pesticide transformation products; Ultra-high-pressure liquid chromatography; Tandem mass spectrometry; Matrix effects; Surface and wastewater; Triazines

This paper describes the use of the relative isotopic mass defect, which is the mass defect between the monoisotopic mass of an element and the mass of its A + 1 or its A + 2 isotopic cluster. The relative isotopic mass defect is combined with the intensity of the isotopic cluster and a formula generator to find the correct molecular formula for unknown pesticides, using accurate mass measurements. This paper introduces the concept of the relative mass defect of isotopes and the isotopic mass average (IMA), especially for C, H, N, O, S, Cl, and Br, and how to correlate these measurements to the correct molecular formula of an unknown compound. A heuristic rule of ±3 × 10−3 u (+3 millimass units) is developed as a simple observational tool for viewing accurate mass data with four-decimal-place mass accuracy. This heuristic rule allows one to rapidly scan data “by eye” without the use of sophisticated software, and is a useful and rapid way of examining a molecular formula.
Keywords: Liquid chromatography/mass spectrometry; Accurate mass; Mass defect; Isotopologs; Pesticides; Unknown identification

This paper describes the development of a methodology for the simultaneous determination and quantification of hexabromocyclododecane (HBCD), tetrabromobisphenol A (TBBPA), and related compounds (bisphenol A, monobromobisphenol A, dibromobisphenol A, and tribromobisphenol A) in sludge and sediment samples. The selected method is based on an extraction with dichloromethane: methanol followed by purification via SPE C18 cartridges. Instrumental determination was carried out by liquid chromatography–quadrupole linear ion trap mass spectrometry (LC-QqLIT-MS), with quantification based on isotopic dilution method. Analyte recoveries were in the range of 39–120% and 88–126% for spiked sewage and sediment, respectively. Repeatability of replicate extractions was better than 13% relative standard deviation. Linearity was checked in the range of 0.05 and 25 injected nanograms. Limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.6 and 2.7 ng/g and 1.4 and 66 ng/g for sediment and sludge samples, respectively. The developed method was applied to sewage sludge and sediment samples collected along the Ebro River and Cinca River, one of its tributaries (northeast of Spain). TBBPA levels in sewage sludge ranged from not quantified to 1,329 ng/g dw, whereas levels in sediment samples were lower, between not detected and 15 ng/g dw. As regards HBCD, concentrations were between not detected and 375 ng/g for sludge samples and 0.8 and 1850 ng/g for sediments.
Keywords: Waste/sludge; HPLC; Organic compounds/trace organic compounds

Monitoring the large-scale production of the antihypertensive peptides RYLGY and AYFYPEL by HPLC-MS by María del Mar Contreras; Beatriz Gómez-Sala; Pedro Jesús Martín-Álvarez; Lourdes Amigo; Mercedes Ramos; Isidra Recio (2825-2832).
This work reports the quantitative analysis of two novel antihypertensive peptides αs1-CN f(90-94), with sequence RYLGY, and αs1-CN f(143-149), with sequence AYFYPEL, by high-performance liquid chromatography–mass spectrometry in food-grade hydrolysates of milk proteins. The method was validated and showed sufficient specificity, reproducibility, linearity and recovery. Linear calibrations of the molecular ions m/z 671.2 and 902.3 were selected for the determination of the peptides RYLGY and AYFYPEL, respectively, and showed good statistical results (R 2 ≥ 0.995 and with no significant lack-of-fit). The simplicity of RP-HPLC-MS method allowed the automated quantification of both antihypertensive peptides without any sample pretreatment. The application of this method permitted the evaluation of some hydrolysis variables, i.e., substrate, temperature, hydrolysis time or enzyme/substrate ratio, on the formation of antihypertensive peptides. The quantitative analysis of RYLGY and AYFYPEL showed that ultrafiltration was not effective to improve the content in active peptides, containing the hydrolysates and their respective permeates similar peptide amounts. Figure HPLC-MS allows the specific quantitative determination of the antihypertensive peptides RYLGY and AYFYPEL in milk casein hydrolysates.
Keywords: Antihypertensive peptides; Milk protein hydrolysate; Mass spectrometry; Functional ingredient; Quantitative analysis

Stir-bar-sorptive extraction (SBSE) with liquid desorption (LD) and ultra-high-performance liquid chromatography–electrospray ionization triple-quadrupole tandem mass spectrometry (UHPLC–(ESI)MS–MS) were used for analysis of six personal care products in environmental water: four UV filters (2,2-dihydroxy-4-methoxybenzophenone, benzophenone-3, octocrylene, and octyldimethyl-p-aminobenzoic acid) and two antimicrobial agents (triclocarban and triclosan). Experimental conditions that affect SBSE-LD sorption efficiency (extraction time and temperature, sample pH, and ionic strength) and desorption efficiency (solvent, temperature, and time) were optimized. The method proved to be sensitive—a 50-mL sample was used to determine these compounds in environmental waters at trace levels. The detection limits of the analytical method were 2.5 ng L−1 for river water and 5–10 ng L−1 for effluent and influent sewage water. In river waters, benzophenone-3 was found at levels from 6 ng L−1 to 28 ng L−1 and triclosan at levels −1 in influent sewage, whereas concentrations of benzophenone-3 and triclosan were commonly below 25 ng L−1 in effluent sewage.
Keywords: UV filters; PCPs; UHPLC–MS–MS; SBSE; Surface waters; Wastewaters

Fully automated screening of veterinary drugs in milk by turbulent flow chromatography and tandem mass spectrometry by Alida A. M. Stolker; Ruud J. B. Peters; Richard Zuiderent; Joseph M. DiBussolo; Cláudia P. B. Martins (2841-2849).
There is an increasing interest in screening methods for quick and sensitive analysis of various classes of veterinary drugs with limited sample pre-treatment. Turbulent flow chromatography in combination with tandem mass spectrometry has been applied for the first time as an efficient screening method in routine analysis of milk samples. Eight veterinary drugs, belonging to seven different classes were selected for this study. After developing and optimising the method, parameters such as linearity, repeatability, matrix effects and carry-over were studied. The screening method was then tested in the routine analysis of 12 raw milk samples. Even without internal standards, the linearity of the method was found to be good in the concentration range of 50 to 500 µg/L. Regarding repeatability, RSDs below 12% were obtained for all analytes, with only a few exceptions. The limits of detection were between 0.1 and 5.2 µg/L, far below the maximum residue levels for milk set by the EU regulations. While matrix effects—ion suppression or enhancement—are obtained for all the analytes the method has proved to be useful for screening purposes because of its sensitivity, linearity and repeatability. Furthermore, when performing the routine analysis of the raw milk samples, no false positive or negative results were obtained.
Keywords: Veterinary drugs; Milk; Turbulent flow; Screening; Tandem mass spectrometry

SPE and LC-MS/MS determination of 14 illicit drugs in surface waters from the Natural Park of L’Albufera (València, Spain) by Pablo Vazquez-Roig; Vicente Andreu; Cristina Blasco; Yolanda Picó (2851-2864).
A simple and robust method using solid-phase extraction (SPE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of 14 drugs of abuse and their metabolites (cocainics, amphetamine-like compounds, cannabinoids, and opiates) in surface waters has been developed. Seven SPE adsorbents (Oasis HLB, Oasis MCX, Oasis Wax, Supelselect HLB, Strata-X, Strata-XCW), amount of sorbent bed, water volume, and pH were investigated. The highest recoveries, as well as the simplest protocol, were obtained for Oasis HLB cartridges (6 mL/200 mg) using 250 mL of water. The proposed method was linear in a concentration range from 0.03–6 to 300–60,000 ng/L depending on the compound, with correlation coefficients higher than 0.998. Matrix effects have been studied in surface water samples, and several isotope-labeled internal standards have been evaluated as a way to compensate the signal suppression observed. Limits of detection (LODs) and quantification (LOQs) ranged from 0.01 to 1.54 ng/L and from 0.03 to 5.13 ng/L, respectively. Recoveries were 71–102% at the LOQ level and 77–104 at 50 ng/L. The intra-day and intermediate precisions were from 1% to 8% and from 2% to 11%, respectively. The present work reports for the first time the occurrence of drugs of abuse residues in surface water samples from the Natural Park of L’Albufera (Valencia, Spain). Codeine, cocaine, benzoylecgonine, ecgonine methylester, amphetamine, 3,4-methylendioxy methamphetamine, morphine, and methadone were quantified with median values of 11.10, 0.02, 5.59, 0.08, 0.21, 0.75 and 0.14 ng/L respectively, and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol was detected in one sample at levels
Keywords: Drugs of abuse; Liquid chromatography–mass spectrometry; Triple quadrupole; Solid-phase extraction; Environmental analysis; Surface water

Secondary ion mass spectrometry (SIMS) depth profiling has been applied to the study of the thermal annealing of ohmic contacts for high electron mobility transistors. The metallic stacks (Ti/Al/Ni/Au) were deposited over the Al0.28Ga0.72N/GaN/sapphire heterostructures and subjected to a rapid thermal annealing (850 °C for 30 s under N2 atmosphere) to improve the contact performance. The surface morphology and the in-depth chemical distribution of the layered contacts were severely modified due to the treatment. These modifications have been analyzed by SIMS depth profiling and scanning electron microscopy–energy-dispersive X-ray microanalysis. The SIMS analysis conditions have been optimized to achieve simultaneously good sensitivity and to avoid ion-induced mixing effects produced by the primary beam sputtering.
Keywords: SIMS; Depth profiling; Ohmic contacts; Metallic diffusion; HEMT

Multi-residue determination of 130 multiclass pesticides in fruits and vegetables by gas chromatography coupled to triple quadrupole tandem mass spectrometry by M. I. Cervera; C. Medina; T. Portolés; E. Pitarch; J. Beltrán; E. Serrahima; L. Pineda; G. Muñoz; F. Centrich; F. Hernández (2873-2891).
A multi-residue method has been developed and validated for the simultaneous quantification and confirmation of around 130 multiclass pesticides in orange, nectarine and spinach samples by GC-MS/MS with a triple quadrupole analyzer. Compounds have been selected from different chemical families including insecticides, herbicides, fungicides and acaricides. Three isotopically labeled standards have been used as surrogates in order to improve accurate quantitation. Samples were extracted by using accelerated solvent extraction (ASE) with ethyl acetate. In the case of spinach, an additional clean-up step by gel permeation chromatography was applied. Determination was performed by GC-MS/MS in electron ionization mode acquiring two MS/MS transitions for each analyte. The intensity ratio between quantitation transition (Q) and identification transition (q) was used as confirmatory parameter (Q/q ratio). Accuracy and precision were evaluated by means of recovery experiments in orange, nectarine, and spinach samples spiked at two concentration levels (0.01 and 0.05 mg/kg). Recoveries were, in most cases, between 70% and 120% and RSD were below 20%. The limits of quantification objective for which the method was satisfactorily validated in the three samples matrices were for most pesticides 0.01 mg/kg. Matrix effects over the GC-MS/MS determination were tested by comparison of reference standards in pure solvent with matrix-matched standards of each matrix. Data obtained showed enhancement of signal for the majority of analytes in the three matrices investigated. Consequently, in order to reduce the systematic error due to this effect, quantification was performed using matrix-matched standard calibration curves. The matrix effect study was extended to other food matrices such as raisin, paprika, cabbage, pear, rice, legume, and gherkin, showing in all cases a similar signal enhancement effect.
Keywords: Pesticides; Gas chromatography tandem mass spectrometry; Triple quadrupole; Fruits and vegetables; Matrix effect; Accelerated solvent extraction; Multi-residue analysis

In this work a fast liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method was developed for the analysis of toltrazuril, a coccidiostatic drug, and its metabolites in meat food products. The applicability of atmospheric pressure chemical ionization (APCI) and heated electrospray ionization in both positive and negative modes was studied. APCI in negative mode provided the best results and the base peak originated from the loss of CF3 (toltrazuril and toltrazuril sulfone) and CHF3• (toltrazuril sulfoxide) was used as the precursor ion in MS/MS. A fast LC separation on a C18 Fused-Core™ column was used together with the APCI-MS/MS method developed using enhanced mass resolution mode (highly selective selected reaction monitoring, H-SRM) to improve the sensitivity and selectivity for the analysis of these compounds in food samples. A simple sample treatment based on an extraction with acetonitrile and a cleanup with a C18 cartridge was used. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation lower than 10%), accuracy, and linearity and allowed the determination of these compounds in food samples down to the parts per billion level (limits of detection between 0.5 and 5 µg kg-1). Figure In this work a fast liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method was developed for the analysis of toltrazuril, a coccidiostatic drug, and its metabolites in meat food products. The applicability of atmospheric pressure chemical ionization (APCI) and heated electrospray ionization (HESI) in both positive and negative modes was studied. APCI in negative mode provided the best results and the base peak originated from the loss of CF3 (toltrazuril and toltrazuril sulfone) and CHF3· (toltrazuril sulfoxide) was used as the precursor ion in MS/MS. A fast LC separation on a C18 Fused-Core™ column was used together with the APCI-MS/MS method developed using enhanced mass resolution mode (highly selective selected reaction monitoring, H-SRM) to improve the sensitivity and selectivity for the analysis of these compounds in food samples. A simple sample treatment based on an extraction with acetonitrile and a clean-up with a C18 cartridge was used. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation lower than 10%), accuracy, and linearity and allowed the determination of these compounds in food samples down to the parts per billion level (limits of detection between 0.5 and 5 μg kg-1).
Keywords: Mass spectrometry; Liquid chromatography; Coccidiostats

Identification of free and conjugated metabolites of mesocarb in human urine by LC-MS/MS by C. Gómez; J. Segura; N. Monfort; T. Suominen; A. Leinonen; M. Vahermo; J. Yli-Kauhaluoma; R. Ventura (2903-2916).
The objective of the present study was to investigate mesocarb metabolism in humans. Samples obtained after administration of mesocarb to healthy volunteers were studied. The samples were extracted at alkaline pH using ethyl acetate and salting-out effect to recover metabolites excreted free and conjugated with sulfate. A complementary procedure was applied to recover conjugates with glucuronic acid or with sulfate consisting of the extraction of the urines with XAD-2 columns previously conditioned with methanol and deionized water; the columns were then washed with water and finally eluted with methanol. In both cases, the dried extracts were reconstituted and analyzed by ultra-performance liquid chromatography–tandem mass spectrometry. Chromatographic separation was carried out using a C18 column (100 mm × 2.1 mm i.d., 1.7 µm particle size) and a mobile phase consisting of water and acetonitrile with 0.01% formic acid with gradient elution. The chromatographic system was coupled to a mass spectrometer with an electrospray ionization source working in positive mode. Metabolic experiments were performed in multiple-reaction monitoring mode by monitoring one transition for each potential mesocarb metabolite. Mesocarb and 19 metabolites were identified in human urine, including mono-, di-, and trihydroxylated metabolites excreted free as well as conjugated with sulfate or glucuronic acid. All metabolites were detected up to 48 h after administration. The structures of most metabolites were proposed based on data from reference standards available and molecular mass and product ion mass spectra of the peaks detected. The direct detection of mesocarb metabolites conjugated with sulfate and glucuronic acid without previous hydrolysis has been described for the first time. Finally, a screening method to detect the administration of mesocarb in routine antidoping control analyses was proposed and validated based on the detection of the main mesocarb metabolites in human urine (p-hydroxymesocarb and p-hydroxymesocarb sulfate). After analysis of several blank urines, the method demonstrated to be specific. Extraction recoveries of 100.3 ± 0.8 and 105.9 ± 10.8 (n = 4), and limits of detection of 0.5 and 0.1 ng mL−1 were obtained for p-hydroxymesocarb sulfate and p-hydroxymesocarb, respectively. The intra- and inter-assay precisions were estimated at two concentration levels, 50 and 250 ng mL−1, and relative standard deviations were lower than 15% in all cases (n = 4).
Keywords: Mesocarb metabolites; Sulfated metabolites; Glucuronoconjugated metabolites; Doping control; Ultra-performance liquid chromatography–tandem mass spectrometry; Urine

Liquid analysis dielectric capillary barrier discharge by Sven Tombrink; Saskia Müller; Richard Heming; Antje Michels; Peter Lampen; Joachim Franzke (2917-2922).
In this study a simple micro-tube-based system for analysis of metal-containing liquids is introduced and its analytical performance is evaluated. It is based on a miniaturised dielectric barrier discharge driven at atmospheric pressure. The emission lines of various elements are observed. The system is developed for quantitative measurements and the limits of detection are determined. Because of very low flow rates of just μL min−1 the approach requires extremely low sample volumes.
Keywords: Dielectric capillary barrier discharge; Element spectrometry; Liquid

A novel dot-blot DNAzyme-linked aptamer assay for protein detection by Jinbo Zhu; Tao Li; Jiming Hu; Erkang Wang (2923-2927).
In this work, a novel dot-blot DNAzyme-linked aptamer assay (DLAA) for protein detection is developed with thrombin as a model protein. A peroxidase-like DNAzyme which serves as the catalytic label is tethered to a 15-mer thrombin-binding aptamer to form a label-free DNAzyme-linked aptamer probe. Based on specific interaction of the aptamer with target protein immobilized on nitrocellulose membrane, a DNAzyme layer is introduced onto the membrane. The DNAzyme can catalyze the H2O2-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine to produce a colored insoluble product that is apt to be adsorbed onto the nitrocellulose membrane. As a result, blue dots appear on the membrane, in contrast to the colorless background. As the concentration of thrombin increases, the color of dots gets deep. Such a protein concentration-dependent color change can be quantified via an image-processing software, with a detection limit of 0.6 μM. Furthermore, this assay has been applied successfully to the detection of thrombin in biological samples (e.g., human serum), indicating its practicality for bioanalysis.
Keywords: DNAzyme; Aptamer; Thrombin; Dot-blot assay

The influence of intracellular storage material on bacterial identification by means of Raman spectroscopy by Valerian Ciobotă; Eva-Maria Burkhardt; Wilm Schumacher; Petra Rösch; Kirsten Küsel; Jürgen Popp (2929-2937).
Previous studies dealing with bacterial identification by means of Raman spectroscopy have demonstrated that micro-Raman is a suitable technique for single-cell microbial identification. Raman spectra yield fingerprint-like information about all chemical components within one cell, and combined with multivariate methods, differentiation down to species or even strain level is possible. Many microorganisms may accumulate high amounts of polyhydroxyalkanoates (PHA) as carbon and energy storage materials within the cell and the Raman bands of PHA might impede the identification and differentiation of cells. To date, the identification by means of Raman spectroscopy have never been tested on bacteria which had accumulated PHA. Therefore, the aim of this study is to investigate the effect of intracellular polymer accumulation on the bacterial identification rate. Combining fluorescence imaging and Raman spectroscopy, we identified polyhydroxybutyrate (PHB) as a storage polymer accumulating in the investigated cells. The amount of energy storage material present within the cells was dependent on the physiological status of the microorganisms and strongly influenced the identification results. Bacteria in the stationary phase formed granules of crystalline PHB, which obstructed the Raman spectroscopic identification of bacterial species. The Raman spectra of bacteria in the exponential phase were dominated by signals from the storage material. However, the bands from proteins, lipids, and nucleic acids were not completely obscured by signals from PHB. Cells growing under either oxic or anoxic conditions could also be differentiated, suggesting that changes in Raman spectra can be interpreted as an indicator of different metabolic pathways. Although the presence of PHB induced severe changes in the Raman spectra, our results suggest that Raman spectroscopy can be successfully used for identification as long as the bacteria are not in the stationary phase. Figure Stained bacteria with or without PHB within the cells, and the corresponding Raman spectra.
Keywords: Raman spectroscopy; Polyhydroxybutyrate; Bacterial identification; Fluorescence staining

Reversibly sealed multilayer microfluidic device for integrated cell perfusion and on-line chemical analysis of cultured adipocyte secretions by Anna M. Clark; Kyle M. Sousa; Claire N. Chisolm; Ormond A. MacDougald; Robert T. Kennedy (2939-2947).
A three-layer microfluidic device was developed that combined perfusion of cultured cells with on-line chemical analysis for near real-time monitoring of cellular secretions. Two layers were reversibly sealed to form a cell chamber that allowed cells grown on coverslips to be loaded directly into the chip. The outlet of the chamber was in fluidic contact with a third layer that was permanently bonded. Perfusate from the cell chamber flowed into this third layer where a fluorescence enzyme assay for non-esterified fatty acid (NEFA) was performed on-line. The device was used to monitor efflux of NEFAs from ∼6,200 cultured adipocytes with 83 s temporal resolution. Perfusion of murine 3T3-L1 cultured adipocytes resulted in an average basal concentration of 24.2 ± 2.4 μM NEFA (SEM, n = 6) detected in the effluent corresponding to 3.31 × 10−5 nmol cell−1 min−1. Upon pharmacological treatment with a β-adrenergic agonist to stimulate lipolysis, a 6.9 ± 0.7-fold (SEM, n = 6) sustained increase in NEFA secretion was observed. This multilayer device provides a versatile platform that could be adapted for use with other cell types to study corresponding cellular secretions in near real-time.
Keywords: Adipocytes; Microfluidics; Enzyme assay; Integration; Fatty acids

Molecular beacons (MBs) are sensitive probes for many DNA sequence-specific applications, such as DNA damage detection, but suffer from technical and cost limitations. We have designed smart probes with self-quenching properties as an alternative to molecular beacons to monitor sequence-specific UV-induced photodamage of oligonucleotides. These probes have similar stem-loop structural characteristics as molecular beacons, but quenching is achieved instead via photoinduced intramolecular electron transfer by neighboring guanosine residues. Our results indicate that the probes are sensitive enough to detect nanomolar target concentrations and are specific enough to discriminate single-base damage. When the probes were used to monitor UV-induced photodamage in oligonucleotide sequences that differ by a single-base mismatch, the photodamage time constant was higher for the perfectly complementary target sequences than for the mismatch sequences, indicating that these probes are specific for each target sequence. In addition, time constants obtained for oligonucleotide target sequences with both stem and loop base mismatches are lower than those with only loop mismatches, suggesting that these sequences are also specifically distinguished by the smart probes. These probes thus constitute robust, sensitive, specific, and cheaper alternatives to MBs for sequence-specific DNA damage detection.
Keywords: Smart DNA probes; Self-quenching DNA probes; Photoinduced intramolecular electron transfer; Molecular beacons; DNA damage; Fluorescence

Direct methylation and solid-phase microextraction (SPME) were used as a sample preparation technique for classification of bacteria based on fatty acid methyl ester (FAME) profiles. Methanolic tetramethylammonium hydroxide was applied as a dual-function reagent to saponify and derivatize whole-cell bacterial fatty acids into FAMEs in one step, and SPME was used to extract the bacterial FAMEs from the headspace. Compared with traditional alkaline saponification and sample preparation using liquid–liquid extraction, the method presented in this work avoids using comparatively large amounts of inorganic and organic solvents and greatly decreases the sample preparation time as well. Characteristic gas chromatography/mass spectrometry (GC/MS) of FAME profiles was achieved for six bacterial species. The difference between Gram-positive and Gram-negative bacteria was clearly visualized with the application of principal component analysis of the GC/MS data of bacterial FAMEs. A cross-validation study using ten bootstrap Latin partitions and the fuzzy rule building expert system demonstrated 87 ± 3% correct classification efficiency. Figure
Keywords: Bacterial fatty acid methyl esters; Solid-phase microextraction; Tetramethylammonium hydroxide; Multivariate analysis; Chemometrics

On-chip photoactivation of heterologously expressed rhodopsin allows kinetic analysis of G-protein signaling by surface plasmon resonance spectroscopy by Konstantin E. Komolov; Mònica Aguilà; Darwin Toledo; Joan Manyosa; Pere Garriga; Karl-Wilhelm Koch (2967-2976).
Surface plasmon resonance spectroscopy allows the study of protein interaction dynamics in real-time. Application of this technique to G-protein coupled receptors, the largest family of receptors involved in signal transduction, has been complicated by their low level of expression and the critical dependence of their native conformation on the hydrophobic transmembrane lipid environment. Here, we investigate and compare three different strategies to immobilize rhodopsin, a prototypical G-protein coupled receptor on a sensor chip surface using antibodies and a lectin for receptor capturing. By further probing of different experimental conditions (pH, detergent type) we identified the optimal factors to maintain rhodopsin in a functional conformation and extended this approach to recombinant rhodopsin that was heterologously expressed in COS cells. Functional operation of rhodopsin on the sensor chip surface was proven by its activation and subsequent light-stimulated G-protein coupling. The influence of these experimental parameters on the association and dissociation kinetics of G-protein receptor coupling was determined. Thereby, we found that the kinetics of Gt interaction were not changed by the strategy of immobilization or the type of detergent. Regeneration of opsin directly on a chip allowed recycling of the immobilized native and recombinant receptor. Thus, the approach provides an experimental framework for choosing the most suitable conditions for the solubilization, immobilization, and for functional tests of rhodopsin on a biosensor surface. Figure Light-triggered binding of the G-protein transducin to recombinant rhodopsin on a biosensor surface. Rhodopsin is immobilized via a specific antibodies, interaction is monitored by surface plasmon resonance
Keywords: Surface plasmon resonance; G-protein coupled receptor; Rhodopsin; Signal transduction

Supramolecular recognition of estrogens via molecularly imprinted polymers by Bogusław Buszewski; Júlia Ričanyová; Renata Gadzała-Kopciuch; Michał Szumski (2977-2986).
The isolation and preconcentration of estrogens from new types of biological samples (acellular and protein-free simulated body fluid) by molecularly imprinted solid-phase extraction has been described. In this technique, supramolecular receptors, namely molecularly imprinted polymers (MIPs) are used as a sorbent material. The recognition sites of MIPs were prepared by non-covalent multiple interactions and formed with the target 17β-estradiol as a template molecule. High-performance liquid chromatography with spectroscopic UV, selective, and a sensitive electrochemical CoulArray detector was used for the determination of 17β-estradiol, estrone, and estriol in simulated body fluid which mimicked human plasma.
Keywords: Estrogens; MIPs; SPE; HPLC-UV/EC

Determination of different recreational drugs in hair by HS-SPME and GC/MS by Gustavo Merola; Stefano Gentili; Franco Tagliaro; Teodora Macchia (2987-2995).
A simple procedure combining headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC/MS) to detect and quantify amphetamines, ketamine, methadone, cocaine, cocaethylene and ∆9-tetrahydrocannabinol (THC) in hair is described. This procedure allows, in a single sample, even scant, analysis of drugs requiring different analytical conditions. A hair sample (10 mg) is washed and subjected to acidic hydrolysis. Then the HS-SPME is carried out (10 min at 90 °C) for amphetamines, ketamine, methadone, cocaine and cocaethylene. For derivatization of analytes, the fibre is introduced into the headspace of another closed vial containing acetic anhydride. After a chromatographic run, an alkaline hydrolysis for THC analysis is carried out in the same vial containing the hair sample previously used. For adsorption, the solid-phase microextraction needle is inserted into the headspace of the vial and the fibre is exposed for 30 min at 150 °C. For derivatization of analytes, the fibre is introduced into the headspace of another closed vial containing N-methyl-N-(trimethylsilyl)trifluoroacetamide. The GC/MS parameters were the same for both chromatographic runs. The linearity was proved to be between 0.01 and 10.00 ng/mg. The repeatability (intra- and interday precision) was below 10% as the coefficient of variation for all compounds. The accuracy, as the relative recovery, was 96.2–103.5% (spiked samples) and 88.6–101.7% (quality control sample). The limit of detection ranged from 0.01 to 0.12 ng/mg, and the limit of quantification ranged from 0.02 to 0.37 ng/mg. Application of the procedure to real hair samples is described. To the best of our knowledge, the proposed procedure combining HS-SPME and GC/MS is the first one be to successfully applied to the simultaneous determination of most of the common recreational drugs, including THC, in a single hair sample.
Keywords: Amphetamines; Cocaine; Hair; Headspace solid-phase microextraction and gas chromatography–mass spectrometry; Ketamine; ∆9-Tetrahydrocannabinol

Development of a novel enzyme reactor and application as a chemiluminescence flow-through biosensor by Chunyan Yang; Zhujun Zhang; Zuolong Shi; Pan Xue; Pingping Chang; Ruifang Yan (2997-3003).
A novel enzyme reactor was prepared using calcium alginate fiber (CAF) and amine-modified nanosized mesoporous silica (AMNMS) as a support. Combination of the adsorption of the enzyme on AMNMS with the cage effect of the polymer greatly increases the catalytic activity and the stability of the immobilized enzyme. It was shown that the lifetime, stability, and catalytic activity of the enzyme reactor were greatly improved by incorporating AMNMS into CAF to efficiently encapsulate the enzyme. Glucose oxidase was chosen as a model enzyme to explore the possibility of using CAF–AMNMS as a matrix for enzyme immobilization in the design of a chemiluminescence (CL) flow-through biosensor. The sensitivity of the flow-through biosensor combined with a novel luminol-diperiodatonickelate CL system was higher than for other reported CL biosensors. The proposed biosensor exhibits short response time, easy operation, long lifetime, high catalytic activity, high sensitivity, and simple assembly.
Keywords: Chemiluminescence; Flow-through biosensor; Calcium alginate fiber; Amine-modified nanosized mesoporous silica; Glucose oxidase; Glucose

Fully automatic flow method for the determination of scavenging capacity against nitric oxide radicals by Joana P. N. Ribeiro; Luís M. Magalhães; Marcela A. Segundo; Salette Reis; José L. F. C. Lima (3005-3014).
In the present work, a fluorimetric automatic method based on multisyringe flow injection analysis (MSFIA) was developed for in vitro evaluation of scavenging capacity against nitric oxide (NO) using 4,5-diaminofluorescein (DAF-2) as an NO-selective fluorogenic probe. The MSFIA manifold was assembled to perform the in-line generation of NO and the competitive reaction of putative scavenger molecules and DAF-2 with NO at conditions close to those found in vivo regarding temperature (37°C), pH (7.4), and concentration of NO (less than 1 μM). This approach allowed the evaluation of scavenging capacity against NO by endogenous antioxidant molecules, pharmaceutical compounds, and human plasma. IC50 values were calculated for rutin (1.30 ± 0.02 μM, positive control), cysteine (321 ± 8 μM), reduced glutathione (1106 ± 93 μM), uric acid (134 ± 12 μM), dipyrone (1.36 ± 0.06 μM), and captopril (363 ± 28 μM). A high degree of automation was attained as the successive dilution of antioxidant standard solutions required for IC50 assessment was performed automatically, in a dilution chamber placed in the flow system. A determination throughput of 16 h-1 and a good precision were attained (relative standard deviation between 1.6 and 9.0%), fostering the application of the proposed method to routine/screening analysis of scavenging capacity against NO. Figure Multisyringe flow injection system developed for in vitro evaluation of scavenging capacity against nitric oxide radical, incorporating a dilution chamber to automatically assess the IC50 values. MS, multisyringe; MV, multiposition selection valve; DC, dilution chamber; RC, reaction coil; C1, phosphate buffer solution (pH 7.4); C2, NaOH solution; DAF-2, fluorogenic probe; NOC-9, nitric oxide donor; AO, antioxidant standard solution; D, fluorimetric detector; W, waste.
Keywords: Nitric oxide; Scavenging capacity; Antioxidants; Human plasma; Multisyringe flow injection analysis; Fluorimetry

A fluorescence sensor was fabricated using R-phycoerythrin (R-PE) immobilized on eggshell membrane as the fluorescence probe, and salbutamol was determined based on the decrease in fluorescence intensity of R-phycoerythrin. The scanning electron and fluorescence micrographs showed the microstructure of the eggshell membrane and indicated that the R-PE was successfully immobilized on the eggshell membrane surface. The effects of some experimental parameters on the response of the biosensor were investigated in detail. The fluorescence sensor has a linear response to salbutamol concentrations ranging from 5.00 to 100 ng mL−1. The detection limit for the salbutamol is 3.50 ng mL−1 (S/N = 3). The reproducibility of fabricating the biosensors using six different membranes was good with a relative standard deviation (RSD) of 3.28%. The fluorescence sensor showed extremely good stability with a shelf life of at least 50 days and reversible response to salbutamol. Some common potential interferents showed little effect on the response of the salbutamol fluorescence sensor. The proposed method was successfully applied to the determination of the salbutamol in urine samples. Figure Immobilization of R-PE on the eggshell membrane (right). Fluorescence micrographs of fresh eggshell membrane (upper left) and eggshell membrane with immobilized R-PE (lower left).
Keywords: R-phycoerythrin; Eggshell membrane; Salbutamol; Fluorescence quenching

Dynamics of hyaluronan aqueous solutions as assessed by fast field cycling NMR relaxometry by Alena Průšová; Pellegrino Conte; Jiří Kučerík; Giuseppe Alonzo (3023-3028).
Fast field cycling (FFC) NMR relaxometry has been used to study the conformational properties of aqueous solutions of hyaluronan (HYA) at three concentrations in the range 10 to 25 mg mL–1. Results revealed that, irrespective of the solution concentration, three different hydration layers surround hyaluronan. The inner layer consists of water molecules strongly retained in the proximity of the HYA surface. Because of their strong interactions with HYA, water molecules in this inner hydration layer are subject to very slow dynamics and have the largest correlation times. The other two hydration layers are made of water molecules which are located progressively further from the HYA surface. As a result, decreasing correlation times caused by faster molecular motion were measured. The NMRD profiles obtained by FFC-NMR relaxometry also showed peaks attributable to 1H–14N quadrupole interactions. Changes in intensity and position of the quadrupolar peaks in the NMRD profiles suggested that with increasing concentration the amido group is progressively involved in the formation of weak and transient intramolecular water bridging adjacent hyaluronan chains. In this work, FFC-NMR was used for the first time to obtain deeper insight into HYA–water interactions and proved itself a powerful and promising tool in hyaluronan chemistry.
Keywords: FFC-NMR; Relaxometry; Correlation time; Quadrupole interactions; Hydration layer

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most common groups of pharmaceuticals detected in environmental matrices. Although several derivatization procedures have been employed in the gas chromatographic analysis of NSAIDs, the application of trimethylsilyldiazomethane has never yet been reported. This work has studied the derivatization of widely used NSAIDs (ibuprofen, ketoprofen and naproxen) by trimethylsilyldiazomethane. Special emphasis was placed on the influence of temperature and reaction time on the reaction yield, and on the determination of the instrumental detection limit. The results are compared with those obtained by methylation using boron trifluoride methanol solution, and by silylation with a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (99:1, v/v) and with N-methyl-N-[tert-butyldimethylsilyl]trifluoroacetamide. The derivatization of ibuprofen, ketoprofen and naproxen by trimethylsilyldiazomethane was shown to be simple, fast, efficient, and suitable for trace analysis (the respective instrumental detection limits for ibuprofen naproxen, and ketoprofen were 2, 4, and 4 ng). Trimethylsilyldiazomethane can be used as an alternative reagent for determining acidic drugs in environmental matrices.
Keywords: NSAIDs; Gas chromatography; Derivatization; Trimethylsilyldiazomethane; Pharmaceutical residues

An innovative enzyme-linked immunosorbent assay (ELISA) format based on antibody-coated magnetic micro-particles (MPs) for the sensitive detection of Ara h3/4 allergen in food is described. The immunosupport is suspended in the incubation solutions and the MPs with the captured allergen can be easily harvested on a magnet, separated from the solutions, and washed using an easy-to-use, fast and selective approach that allows its detection and quantification. Two differently coated MPs, ProteinA-Pn-b and MP-NH2-PAMAM G 1.5 -Pn-b immunosupports, were tested. The functionalization of the MPs with PAMAM-sodium carboxylate dendrimers elicits a major stability on the immunoglobulin activity resulting in a threefold enhancement of the analytical sensitivity for the assay with respect to a ProteinA immobilization. Validation was carried out on two different matrices: corn flakes and biscuits. In the case of MP-NH2-PAMAM G 1.5 -Pn-b immunosupport, limit of detection was found to be 0.2 mg peanuts/kg matrix in both matrices; the linear response range was demonstrated from 2.5 to 15 mg peanuts/kg matrix by performing statistical tests (homoscedasticity and Mandel fitting tests). Good accuracy and recovery (>80 ± 2%) were obtained. Different food samples were tested and the results were compared with those obtained with a commercially available ELISA kit. The results obtained in this work demonstrated the applicability of the immunomagnetic ELISA methods on real samples and the possibility to perform the assay with significantly reduced reagent and sample consumption.
Keywords: ELISA assay; Magnetic particles; Dendrimers; Hidden peanut allergens

Application of an electronic tongue based on FT-MIR to emulate the gustative mouthfeel “tannin amount” in red wines by L. Vera; L. Aceña; R. Boqué; J. Guasch; M. Mestres; O. Busto (3043-3049).
In this work, the ability of an electronic tongue based on Fourier-Transform Mid Infrared (FT-MIR) spectroscopy as a gustative sensor is assessed by emulating the responses of a tasting panel for the gustative mouthfeel “tannin amount”. The FT-MIR spectra were modeled against the sensory responses evaluated in 37 red wines by means of partial least squares (PLS) regression models. In order to find the wavenumbers more correlated with the sensorial attribute and thus providing the best predictive models, six different variable selection techniques were tested. The iterative predictor weighting IPW-PLS technique showed the best results with the smallest RMSEC and RMSECV values (0.07 and 0.13, respectively) using 20 selected wavenumbers. The coincident wavenumbers selected by the six variable selection techniques were interpreted based on the absorption bands of tannin and then a calibration model using these wavenumbers was built to validate the interpretation made. Coincident wavenumbers selected by the six different variable selection techniques
Keywords: FT-MIR; e-tongue; Variable selection; Sensorial analysis; Tannins; Calibration model

Instrumental measurement of bitter taste in red wine using an electronic tongue by Alisa Rudnitskaya; Hélène H. Nieuwoudt; Nina Muller; Andrey Legin; Maret du Toit; Florian F. Bauer (3051-3060).
An electronic tongue (ET) based on potentiometric chemical sensors was assessed as a rapid tool for the quantification of bitterness in red wines. A set of 39 single cultivar Pinotage wines comprising 13 samples with medium to high bitterness was obtained from the producers in West Cape, South Africa. Samples were analysed with respect to a set of routine wine parameters and major phenolic compounds using Fourier transform infrared-multiple internal reflection spectroscopy (WineScan) and high-performance liquid chromatography, respectively. A trained sensory panel assessed the bitterness intensity of 15 wines, 13 of which had a bitter taste of medium to high intensity. Thirty-one wine samples including seven bitter-tasting ones were measured by the ET. Influence of the chemical composition of wine on the occurrence of the bitter taste was evaluated using one-way analysis of variance. It was found that bitter-tasting wines had higher concentrations of phenolic compounds (catechin, epicatechin, gallic and caffeic acids and quercetin) than non-bitter wines. Sensitivity of the sensors of the array to the phenolic compounds related to the bitterness was studied at different pH levels. Sensors displayed sensitivity to all studied compounds at pH 7, but only to quercetin at pH 3.5. Based on these findings, the pH of wine was adjusted to 7 prior to measurements. Calibration models for classification of wine samples according to the presence of the bitter taste and quantification of the bitterness intensity were calculated by partial least squares-discriminant analysis (PLS-DA) regression. Statistical significance of the classification results was confirmed by the permutation test. Both ET and chemical analysis data could discriminate between bitter and control wines with the correct classification rates of 94% and 91%, respectively. Prediction of the bitterness intensity with good accuracy (root mean square error of 2 and mean relative error of 6% in validation) was possible only using ET data.
Keywords: Red wine; Bitterness; Electronic tongue; Potentiometric chemical sensors; Phenolics

The obligation for accredited laboratories to participate in proficiency tests under ISO 17025, performing multiresidue methods (MRMs) for pesticide residues, involves the reporting of a large number of individual z scores making the evaluation of the overall performance of the laboratories difficult. It entails, time and again, the need for ways to summarise the laboratory’s overall assessment into a unique combined index. In addition, the need for ways to continually evaluate the performance of the laboratory over the years is equally acknowledged. For these reasons, following 14 years of the European Union Reference Laboratory for Pesticide Residues in Fruits and Vegetables (EUPT-FV), useful formulas have been designed to globally evaluate the assessment of the participating laboratories. The aim is to achieve a formula which is easy to understand, which can be applied and which fits the purposes of long-term evaluation detecting positive and negative trends. Moreover, consideration is needed for a fair compensation of bad results in MRM, taking into account the large number of compounds that are covered. It is therefore important to be aware of the difficulties in getting satisfactory values from a wide range of compounds. This work presents an evaluation of the main well-established combined z score formulas together with those new ones developed here which have been applied to the European proficiency test results (EUPTs) over the years. Previous formulas such as the rescaled sum of z score (RSZ), the sum squared of z score (SSZ) and the relative laboratory performance (RLP) are compared with the newer ones: the sum of weighted z scores (SWZ) and the sum of squared z scores (SZ2). By means of formula comparisons, conclusions on the advantages, drawbacks and the most fit-for-purpose approach are achieved.
Keywords: Z score; Proficiency test; Multiresidue method; Combined z score; Pesticide; Fruit and vegetables; Performance; Weighted

Size-fractions from a soil humic acid were separated by preparative size-exclusion chromatography (SEC), desalted, and concentrated by ultrafiltration and vacuum centrifugation without being subjected to any freeze-drying process. After having assessed the lack of formation of any multiple-charged ions by high-resolution Fourier transform ion cyclotron resonance electrospray ionization (ESI) mass spectrometry (MS), the size-fractions were used by direct infusion to compare the molecular ion distribution by both atmospheric pressure chemical ionization (APCI)- and ESI-MS in negative mode. The weight- (Mw) and number-averaged (Mn) molecular weight obtained by ESI-MS were invariably larger than by APCI-MS for all size-fractions, thereby indicating that ESI is more efficient than APCI to evaluate the molecular mass distribution of humic samples. No substantial difference was observed when concentration and pH of unfreeze-dried humic size-fractions were varied. The negative mode was applied to assess the effect of cone voltage from −20 to −60 V on ESI of the humic size-fractions further separated through an on-line SEC column. The resulting mass spectra and Mw and Mn values suggested that the variation of cone voltage in ESI-MS affects the ionization potential of associated humic molecules more in solution rather than their fragmentation. These findings agree with previous observations which indicated a limitation of ESI in providing consistent mass detection for a complex mixture of heterogeneous humic molecules, especially when they are aggregated by a freeze-drying process.
Keywords: Soft ionization mass spectrometry; FT-ICR-ESI-MS; Soil humic acid; Size-fractions; Size-exclusion chromatography; Cone voltage

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of lipophilic marine toxins in shellfish extracts (mussel, oyster, cockle and clam) was validated in-house using European Union (EU) Commission Decision 2002/657/EC as a guideline. The validation included the toxins okadaic acid (OA), yessotoxin (YTX), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and 13-desmethyl spirolide-C (SPX1). Validation was performed at 0.5, 1 and 1.5 times the current EU permitted levels, which are 160 µg kg-1 for OA, AZA1 and PTX2 and 1,000 µg kg-1 for YTX. For SPX1, 400 µg kg-1 was chosen as the target level as no legislation has been established yet for this compound. The method was validated for determination in crude methanolic shellfish extracts and for extracts purified by solid-phase extraction (SPE). Extracts were also subjected to hydrolysis conditions to determine the performance of the method for OA and dinophysistoxin esters. The toxins were quantified against a set of matrix-matched standards instead of standard solutions in methanol. To save valuable standard, methanolic extract instead of the homogenate was spiked with the toxin standard. This was justified by the fact that the extraction efficiency is high for all relevant toxins (above 90%). The method performed very well with respect to accuracy, intraday precision (repeatability), interday precision (within-laboratory reproducibility), linearity, decision limit, specificity and ruggedness. At the permitted level the accuracy ranged from 102 to 111%, the repeatability from 2.6 to 6.7% and the reproducibility from 4.7 to 14.2% in crude methanolic extracts. The crude extracts performed less satisfactorily with respect to the linearity (less than 0.990) and the change in LC-MS/MS sensitivity during the series (more than 25%). SPE purification resulted in greatly improved linearity and signal stability during the series. Recently the European Food Safety Authority (EFSA) has suggested that to not exceed the acute reference dose the levels should be below 45 µg kg-1 OA equivalents and 30 µg kg-1 AZA1 equivalents. A single-day validation was successfully conducted at these levels. If the regulatory levels are lowered towards the EFSA suggested values, the official methods prescribed in legislation (mouse and rat bioassay) will no longer be sensitive enough. The validated LC-MS/MS method presented has the potential to replace these animal tests.
Keywords: Lipophilic marine toxins; Liquid chromatography tandem mass spectrometry; Method validation; Marine biotoxins

Determination of five polar herbicides in water samples by ionic liquid dispersive liquid-phase microextraction by Suli Wang; Liping Ren; Congyun Liu; Jing Ge; Fengmao Liu (3089-3095).
A simple, rapid and efficient ionic liquid based on dispersive liquid-phase microextraction (IL-DLPME) method was developed for the determination of three triazine and two phenylurea herbicides in water samples. IL (1-hexyl-3-methylimidazolium hexafluorophosphate [C6MIM][PF6]) that dispersed completely into the water solution under controlled temperature was used as the extraction solvent. The analytes were easily concentrated into the ionic liquid phase. This technique combined the process of extraction and concentration of the analytes into one step and avoided use of the more common, toxic organic solvents. The factors affecting the extraction efficiency such as the IL volume, sample pH, extraction time, centrifugal time, dissoluble temperature and ionic strength were optimized. The extracts were analyzed by high-performance liquid chromatography (HPLC) coupled with diode array detector (DAD). Under the optimized conditions, recoveries (50.5–109.1%) were obtained for the target analytes in water samples. The calibration curves were linear and the correlation coefficient ranged from 0.9947 to 0.9973 in the concentration levels of 5–100 μg L−1. The relative standard deviations (RSDs, n = 5) were 6.80–10.78%. The limit of detections (LODs) for the five polar herbicides were between 0.46 μg L−1 and 0.89 μg L−1.
Keywords: Ionic liquid dispersive liquid-phase microextraction; Triazine herbicide; Phenylurea herbicide; HPLC; Water

A method based on solid--phase extraction with a molecularly imprinted polymer (MIP) has been developed to determine five probable human carcinogenic polycyclic aromatic hydrocarbons (PAHs) in ambient air dust by gas chromatography-mass spectrometry (GC-MS). Molecularly imprinted poly(vinylpyridine-co-ethylene glycol dimethacrylate) was chosen as solid-phase extraction (SPE) material for PAHs. The conditions affecting extraction efficiency, for example surface properties, concentration of PAHs, and equilibration times were evaluated and optimized. Under optimum conditions, pre-concentration factors for MIP-SPE ranged between 80 and 93 for 10 mL ambient air dust leachate. PAHs recoveries from MIP-SPE after extraction from air dust were between 85% and 97% and calibration graphs of the PAHs showed a good linearity between 10 and 1000 ng L−1 (r = 0.99). The extraction efficiency of MIP for PAHs was compared with that of commercially available SPE materials—powdered activated carbon (PAC) and polystyrene-divinylbenzene resin (XAD)—and it was shown that the extraction capacity of the MIP was better than that of the other two SPE materials. Organic matter in air dust had no effect on MIP extraction, which produced a clean extract for GC-MS analysis. The detection limit of the method proposed in this article is 0.15 ng L−1 for benzo[a]pyrene, which is a marker molecule of air pollution. The method has been applied to the determination of probable carcinogenic PAHs in air dust of industrial zones and satisfactory results were obtained. Figa A multi-template imprinted polymer selectively extracted carcinogenic PAHs from air dust samples resulted in improvement of LOD of GC-MS
Keywords: Ambient air samples; Polycyclic aromatic hydrocarbons; Solid-phase extraction; Molecularly imprinted polymers; GC-MS; Environmental analysis

Dispersive liquid–liquid microextraction using a surfactant as disperser agent by Mohammad Saraji; Ali Akbar Hajialiakbari Bidgoli (3107-3115).
A novel and efficient surfactant-assisted dispersive liquid–liquid microextraction combined with high-performance liquid chromatography–photodiode array detection was developed for the determination of phenylurea herbicides in water samples. Based on this procedure, which is a dispersive-solvent-free technique, the extractant is dispersed in the aqueous sample using methyltrialkylammonium chloride. Compared with the conventional dispersive liquid–liquid microextraction, the new extraction method has many advantages such as higher extraction efficiency, low cost, reduced environmental hazards, and consumption of less extracting solvent. A few microliters of chloroform containing an appropriate amount of methyltrialkylammonium chloride (mixture of C8–C10) was used to extract the analytes from water samples. The main parameters relevant to the extraction process (namely, type of surfactant, selection of extractant solvent, extractant volume, surfactant concentration, ionic strength, and extraction time) were investigated. The performed analytical procedure showed limits of detection ranging from 2.3 to 18 ng/L, and precision ranges from 0.6% to 2.0% (as intra-day relative standard deviation, RSD) and from 1.3% to 8.3% (as inter-day RSD) depending on the analyte. The method showed good linearity between 0.04 and 40 µg/L with squared correlation coefficients better than 0.9920. This newly established approach was successfully applied to spiked real water samples. Figure Schematic representation of the surfactant assisted dispersive liquid-liquid microextraction.
Keywords: Surfactant-assisted dispersive liquid–liquid microextraction; Liquid chromatography; Water analysis; Phenylurea

Noninvasive depth profiling of walls by portable nuclear magnetic resonance by Bernhard Blümich; Agnes Haber; Federico Casanova; Eleonora Del Federico; Victoria Boardman; Gerhard Wahl; Antonella Stilliano; Licio Isolani (3117-3125).
A compact and mobile single-sided 1H NMR sensor, the NMR-MOUSE®, has been employed in the nondestructive characterization of the layer structure of historic walls and wall paintings. Following laboratory tests on a model hidden fresco, paint and mortar layers were studied at Villa Palagione and the Seminario Vescovile di Sant’ Andrea in Volterra, Italy. Different paint and mortar layers were identified, and further characterized by portable X-ray fluorescence spectroscopy where accessible. In the detached and restored fresco “La Madonna della Carcere” from the Fortezza Medicea in Volterra, paint and mortar layers were discriminated and differences in the moisture content of the adhesive that fixes the detached wall painting to its support were found in both restored and original sections. These investigations encourage the use of the portable and single-sided NMR technology for nondestructive studies of the layer structure and conservation state of historic walls. Figure A mobile NMR machine measuring a depth profile into a painted, old wall to unravel the layers from mortar and paint
Keywords: Nuclear magnetic resonance; X-ray fluorescence; Wall painting; Art conservation; Pigments; Nondestructive analysis

In the present study, the molecular chain changes and structural transitions of partially hydrolyzed poly(vinyl alcohol) (PVA) having a 12 mol% acetate unit were analyzed by moving-window two-dimensional (MW2D) correlation infrared spectroscopy combined with differential scanning calorimetry and thermogravimetric analysis. The results show the glass-transition temperature (T g ) of PVA is clearly distinguished by MW2D correlation infrared spectroscopy, and the acetate groups start to be eliminated around the melting temperature, whereas the free water molecules in PVA are eliminated above T g. The correlation movements of the O–H stretching modes, including the free hydroxyl groups and the hydrogen bonds, are clearly determined using MW2D correlation infrared spectroscopy. The spectral variations in the C=O stretching region caused by the elimination of the acetate unit from polymer chains are also discussed on the basis of the results of the MW2D correlation analysis. Such results cannot be obtained by traditional infrared spectroscopy owing to the complex overlapping peaks. Figure The structural variations of partially hydrolyzed poly(vinyl alcohol) studied by moving-window two-dimensional correlation infrared spectroscopy
Keywords: Moving-window two-dimensional correlation spectroscopy; Temperature-dependent infrared; Poly(vinyl alcohol); Partially hydrolyzed

Effect of some physico-chemical conditions on an immunoassay for viable Escherichia coli by Agnese Jurkevica; H. Brian Halsall; Carl J. Seliskar; William R. Heineman (3133-3136).
Viable Escherichia coli can be detected by an immunoassay in which live bacteria captured on antibody-coated paramagnetic beads are induced to synthesize the enzyme β-galactosidase, which catalyzes the hydrolysis of the slightly fluorescent substrate 4-methyl umbelliferyl-β-d-galactoside to the highly fluorescent product 7-hydroxy-4-methylcoumarin for detection. The effects of bacterial strain, presence of dead bacteria, and some environmental stresses on assay performance were evaluated.
Keywords: Escherichia coli ; Immunoassay; Fluorescence detection; Effect of physico-chemical conditions

Liquid-liquid extraction coupled with LC/MS/MS for monitoring of malonyl-CoA in rat brain tissue by Joelle M. Onorato; Luping Chen; Petia Shipkova; Zhengping Ma; Anthony V. Azzara; James J. Devenny; Ningning Liang; Tasir S. Haque; Dong Cheng (3137-3142).
The formation of malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of de novo fatty acid synthesis. Monitoring the changes of malonyl-CoA concentration in the brain in response to treatments such as pharmaceutical intervention (via ACC inhibitors) or different dietary conditions (such as varied feeding regimes) is of great interest and could help increase the understanding of how this molecule contributes to feeding behavior and overall energy balance. We have developed a sensitive analytical method for the determination of malonyl-CoA levels in rat brain tissue. The assay involved removal of tissue lipids by liquid-liquid extraction followed by LC/MS/MS analysis of the aqueous layer for malonyl-CoA. The method was sensitive enough (limit of quantitation = 50 ng/mL, or approximately 0.018 nmol/g brain tissue) to determine malonyl-CoA in individual rat brain preparations. The assay performance was sufficiently rugged to support drug discovery screening efforts and provided an additional analytical tool for monitoring brain malonyl-CoA levels.
Keywords: Malonyl-CoA; LC/MS/MS; Rat brain tissue; Liquid-liquid extraction

Patterned growth of vertically aligned silicon nanowire arrays for label-free DNA detection using surface-enhanced Raman spectroscopy by Changqing Yi; Cheuk-Wing Li; Huayang Fu; Mingliang Zhang; Suijian Qi; Ning-Bew Wong; Shuit-Tong Lee; Mengsu Yang (3143-3150).
Patterning is of paramount importance in many areas of modern science and technology. As a good candidate for novel nanoscale optoelectronics and miniaturized molecule sensors, vertically aligned silicon nanowire (SiNW) with controllable location and orientation is highly desirable. In this study, we developed an effective procedure for the fabrication of vertically aligned SiNW arrays with micro-sized features by using single-step photolithography and silver nanoparticle-induced chemical etching at room temperature. We demonstrated that the vertically aligned SiNW arrays can be used as a platform for label-free DNA detection using surface-enhanced Raman spectroscopy (SERS), where the inherent “fingerprint” SERS spectra allows for the differentiation of closely related biospecies. Since the SiNW array patterns could be modified by simply varying the mask used in the photolithographic processing, it is expected that the methodology can be used to fabricate label-free DNA microarrays and may be applicable to tissue engineering, which aims to create living tissue substitutes from cells seeded onto 3D scaffolds. Figure 1 Schematic illustration of fabrication procedures of SiNWs patterns
Keywords: Silicon nanowire; Single-step photolithography; Surface-enhanced Raman spectroscopy

Prostate-specific antigen (PSA) is a serum glycoprotein overproduced by the prostate in prostate cancer (≥4 ng/mL in the bloodstream). An immunoassay for total PSA (tPSA) was developed using the ALYGNSA method to enhance capture antibody orientation and a limit of detection of 0.63 ng/mL was reported, a limit 15-fold lower than a commercial tPSA ELISA assay. This ALYGNSA assay, however, was performed using only buffer-based proteins and blocking agents (Mackness et al., Anal Bioanal Chem 396:681–686, 2010). To improve the clinical application of this system, a serum-based tPSA ALYGNSA was developed employing human serum. This assay also resulted in a limit of detection of 0.63 ng/mL of tPSA protein. The findings reported here provide support for the clinical application of this assay for diagnosis, progression, treatment, and possible recurrence of prostate cancer.
Keywords: Total prostate-specific antigen; Cancer biomarker; ALYGNSA; Serum-based; Immunoassay

Enantioselective piezoelectric quartz crystal sensor for d-methamphetamine based on a molecularly imprinted polymer by Leveriza F. Arenas; Benilda S. Ebarvia; Fortunato B. Sevilla III (3155-3158).
A piezoelectric quartz crystal (PQC) sensor based on a molecularly imprinted polymer (MIP) has been developed for enantioselective and quantitative analysis of d-(+)-methamphetamine (d(+)-MA). The sensor was produced by bulk polymerization and the resulting MIP was then coated on the gold electrode of an AT-cut quartz crystal. Conditions such as volume of polymer coating, curing time, type of PQC, baseline solvent, pH, and buffer type were found to affect the sensor response and were therefore optimized. The PQC-MIP gave a stable response to different concentrations of d(+)-MA standard solutions (response time = 10 to 100 s) with good repeatability (RSD = 0.03 to 3.09%; n = 3), good reproducibility (RSD = 3.55%; n = 5), and good reversibility (RSD = 0.36%; n = 3). The linear range of the sensor covered five orders of magnitude of analyte concentration, ranging from 10−5 to 10−1 μg mL−1, and the limit of detection was calculated as 11.9 pg d(+)-MA mL−1 . The sensor had a highly enantioselective response to d(+)-MA compared with its response to l(−)-MA, racemic MA, and phentermine. The developed sensor was validated by applying it to human urine samples from drug-free individuals spiked with standard d(+)-MA and from a confirmed MA user. Use of the standard addition method (SAM) and samples spiked with d(+)-MA at levels ranging from 1 × 10−3 to 1 × 10−2 μg mL−1 showed recovery was good (95.3 to 110.9%).
Keywords: Piezoelectric quartz crystal; Molecularly imprinted polymer; Methamphetamine; Enantioselective sensor