Analytical and Bioanalytical Chemistry (v.404, #3)

Green analytical methods by Miguel de la Guardia; Sergio Armenta (625-626).
obtained his position in analytical chemistry at Valencia University in 1991. He has published around 500 papers in analytical chemistry regarding sample preparation and automation, molecular and atomic spectroscopy, and chromatography. He has supervised 33 PhD students from Spain, Morocco, Iraq, Algeria, Brazil, and Egypt and has given invited lectures at conferences at numerous Spanish universities and also in France, Germany, Italy, Brazil, Venezuela, Morocco, Turkey, China, Norway, the UK, the USA, Colombia, Egypt, Uruguay, Argentina, Switzerland, Estonia, the Netherlands, and Belgium. obtained his PhD degree in analytical chemistry at the Universitat de València in 2006. He is currently a senior researcher of analytical chemistry in the Applied Chemometrics Research Group at the Universitat Autònoma de Barcelona. His research topics included coupling of automated flow systems with IR and Raman spectroscopic detection and vibrational spectroscopy combined with chemometrics. His current research fields include the development of fast and sensitive analytical methods based on ion mobility spectrometry.

The colorimetric determination of the concentration of polyphenols, flavonoids, and anthocyanins in wine samples, using a mobile phone camera for sample spot capture on a paper microzone and a remote computer with dedicated software for quantification, is presented as an illustrative application of green analytical chemistry. A comparison of the results with conventional spectrophotometry demonstrates that both methods yield similar results. Developing the assay took approximately 2 months, and the use of chemicals, compared with spectrophotometry, was reduced by about two orders of magnitude: the paper assay consumed 0.4 mL of reagent for 100 samples, whereas the spectrophotometric assay required 100 mL. The relative testing times for 100 samples were 7 h by spectrophotometry and 2 h for paper—a savings on the order of 3.5. No analytical instrumentation was used for the colorimetry on paper microzones. Instead, the assay took advantage of the existing communication technology and free software. The assay was found to be effective, with a nonlinear response at the concentration range of 0.2–5 g/L. The detection limit of the proposed method is in sub-grams per liter.
Keywords: Green analytical chemistry: paper microzone; Colorimetry; Antioxidants; Wine

The capabilities of ion mobility spectrometry (IMS) as a high throughput and green analytical tool in the occupational health and safety control, using pyrethroids as models has been evidenced. The method used for dermal and inhalation exposure assessment is based on the passive pyrethroid sampling using Teflon membranes, direct thermal extraction of the pyrethroids, and measurement of the vaporized analytes by IMS without reagent and solvent consumption. The IMS signatures of the studied synthetic pyrethroids under atmospheric pressure chemical ionization by investigating the formed negative ion products have been obtained. The main advantages of the proposed procedure are related to the obtained limits of detection, ranging from 0.08 to 5 ng, the simplicity of measurement, the lack of sample treatment, and therefore, solvent consumption and waste generation, and finally, the speed of analysis. Figure Schematic diagram of the fast and green IMS procedure for the occupational pyrethroid exposure assessment. Step 1. Sampling; Step 2. IMS measurement; Step 3. Data treatment
Keywords: Ion mobility spectrometry; Green analytical chemistry; Pyrethroids; Occupational exposure; Dermal; Respiratory

Protein determination in serum and whole blood by attenuated total reflectance infrared spectroscopy by D. Perez-Guaita; J. Ventura-Gayete; C. Pérez-Rambla; M. Sancho-Andreu; S. Garrigues; M. de la Guardia (649-656).
Attenuated total reflectance mid-infrared spectra of serum and blood samples were obtained from 4,000 to 600 cm−1. Models for the determination of albumin, immunoglobulin, total globulin, and albumin/globulin coefficients were established for serum samples, using reference data obtained by capillary electrophoresis. Based on the use of the amide bands I and II regions, the relative root mean square error of prediction (RRMSEP) was 4.9, 14.9, 4.5, and 7.1 % for albumin, immunoglobulin, total globulin, and albumin/globulin coefficients, respectively, determined in an independent validation set of 120 samples using 200 samples for calibration. Additionally, the use of Kennard–Stone method for the selection of a representative calibration subset of samples provided comparable results using only 60 samples. For whole blood analysis, hemoglobin was determined in 40 validation samples using models built from 40 calibration independent samples with RRMSEP of 8.3, 5.5, and 4.9 % with models built from direct spectra in the first case and from sample spectra recorded after lysis by sodium dodecyl sulfate and freezing, respectively, for the last two ones. The developed methodologies offer green alternatives for patient diagnosis in a few minutes, minimizing the use of reagents and residues and being adaptable for its use as a point-of-care method.
Keywords: Serum protein determination; ATR-FTIR; PLS; Point-of-care; Variable subset selection; Green analytical chemistry

Ecofriendly in-line process monitoring: a case study. Anthracene photodegradation in the presence of refuse-derived soluble bio-organics by A. Bianco Prevot; P. Avetta; D. Fabbri; E. Montoneri; A. Morales-Rubio; M. de la Guardia (657-664).
Photodegradation of anthracene has been studied in aqueous solutions containing soluble bio-organic substances isolated from urban refuse. To perform a preliminary rapid feasibility study of this process while reducing the amount of analytical effort and reagents, an experimental set-up was developed comprising a Teflon coil surrounding a UV-lamp and coupled with an in-line spectrofluorimeter. In this fashion only few millilitres of solution are needed to study the degradation process. Furthermore, the in-line spectroscopic approach enables monitoring of the process without consumption of reagents. Additional studies by liquid chromatography and use of toxicity tests clearly indicated that the apparent inhibition effect of bio-organic compounds on anthracene degradation is not relevant. The results imply that urban refuse may be used as an auxiliary in the recovery of polycyclic aromatic hydrocarbons from contaminated soil by washing, without deleterious effects on the photodegradation of anthracene and other aromatic pollutants.
Keywords: Anthracene degradation; Soluble bio-organics; Green analytical chemistry; In-line monitoring

HF-LPME as a green alternative for the preconcentration of nickel in natural waters by Cristina Vergel; Rocío Montoya; Carolina Mendiguchía; Manuel García-Vargas; Carlos Moreno (665-670).
In the last years, some analytical methodologies have been identified as a source of pollution, receiving increasing attention to decrease their impact on the environment. In this sense, the so-called solvent-less methodologies appear as a green alternative to reduce the volume of solvents used in many sample treatment procedures and, consequently, the volume of toxic wastes produced. Among these techniques, analytical methodologies based on liquid-phase microextraction are being continuously developed, although most applications are focused on organic compounds. In this work, a three-phase hollow-fibre liquid-phase microextraction (HF-LPME) system has been developed for the preconcentration of nickel in natural waters, prior to the analysis by atomic absorption spectrometry. Under optimum conditions, the new system allowed an enrichment factor of 29.80 to be obtained after 60 min of experiment, and it was successfully applied to the determination of nickel in both saline and non-saline water samples, at ppb and ppt levels. The results were compared with those obtained using a well-established methodology based on liquid solvent extraction showing no significant differences (α = 0.05) between both values. In addition, the new HF-LPME presents the advantages of a green analytical technique, as its greenness profile shows, with the additional reduction of sample manipulation and time cost.
Keywords: Green chemistry; Liquid microextraction; Hollow fibre; Nickel; Natural water

The optimization of a clean procedure based on ultrasound-assisted emulsification liquid–liquid microextraction for the sensitive determination of four bisphenols is presented. The miniaturized technique was coupled with gas chromatography–mass spectrometry after derivatization by in situ acetylation. The Taguchi experimental method, an orthogonal array design, was applied to find the optimal combination of seven factors (each factor at three levels) influencing the emulsification, extraction and collection efficiency, namely acetic anhydride volume, sodium phosphate concentration, carbon tetrachloride volume, aqueous sample volume, sodium chloride concentration and ultrasound power and application time. A second factorial design was applied with four factors and five levels for each factor, 25 experiments being performed in this instance. The matrix effect was evaluated, and it was concluded that sample quantification can be done by calibration with aqueous standards. The detection limits ranged from 0.01 to 0.03 ng mL-1 depending on the compound. The environmentally friendly sample pretreatment procedure was applied to study the migration of the bisphenols from different types of samples: thermal printer paper, compact discs, digital versatile discs, small tight-fitting waistcoats, baby’s bottles, baby bottle nipples of different materials and children’s toys. Figure USAEME is a useful sample pretreatment procedure for controlling the small amounts of bisphenols released from samples
Keywords: Ultrasound-assisted emulsification liquid–liquid microextraction; Gas chromatography–mass spectrometry; Taguchi design; Bisphenols; Baby utensils; Migration studies

One of the principal objectives of sustainable and green processing development remains the dissemination and teaching of green chemistry in colleges, high schools, and academic laboratories. This paper describes simple glassware that illustrates the phenomenon of extraction in a conventional microwave oven as energy source and a process for green analytical chemistry. Simple glassware comprising a Dean–Stark apparatus (for extraction of aromatic plant material and recovery of essential oils and distilled water) and a Vigreux column (as an air-cooled condenser inside the microwave oven) was designed as an in-situ extraction vessel inside a microwave oven. The efficiency of this experiment was validated for extraction of essential oils from 30 g fresh orange peel, a by-product in the production of orange juice. Every laboratory throughout the world can use this equipment. The microwave power is 100 W and the irradiation time 15 min. The method is performed at atmospheric pressure without added solvent or water and furnishes essential oils similar to those obtained by conventional hydro or steam distillation. By use of GC–MS, 22 compounds in orange peel were separated and identified; the main compounds were limonene (72.1 %), β-pinene (8.4 %), and γ-terpinene (6.9 %). This procedure is appropriate for the teaching laboratory, does not require any special microwave equipment, and enables the students to learn the skills of extraction, and chromatographic and spectroscopic analysis. They are also exposed to a dramatic visual example of rapid, sustainable, and green extraction of an essential oil, and are introduced to successful sustainable and green analytical chemistry. Figure In situ microwave extraction of essential oils.
Keywords: Microwave extraction; Dean–Stark; Vigreux; Essential oil; Green analytical chemistry; Teaching laboratory

The 7th international conference on instrumental methods of analysis: modern trends and applications (IMA 2011) by Maria Ochsenkühn-Petropoulou; Panagiotis Kefalas; Nikolaos Kallithrakas-Kontos (683-684).
is a Professor at the School of Chemical Engineering of the National Technical University of Athens (NTUA) and head of the Instrumental Methods of Analysis Environment Group within the Laboratory of Inorganic and Analytical Chemistry. Since 1976, she has been Professor of Instrumental Methods of Analysis at the NTUA and teaches instrumental methods of analysis, environmental control and advanced inorganic chemistry to graduate and postgraduate students of the NTUA and has published ten educational books. Her research interests include speciation analysis; investigation into airborne particulate composition and particulates emitted from catalytic vehicles; trace-element analysis by voltammetric, spectrometric and hyphenated chromatographic techniques; utilization of industrial byproducts in the laboratory and on a pilot-plant scale; and the production and characterization of superconducting powders and coatings. She has published more than 220 scientific papers in international journals and conference proceedings and she has been the coordinator of more than 40 European and Greek research projects. She is the founder of the IMA International Conference series. is a Professor at the Technical University of Crete (Chania) and the head of the Analytical and Environmental Chemistry Laboratory. His main research interests are in the area of X-ray analysis, improving the minimum detection limits of the method, total reflection X-ray fluorescence, speciation, surface analysis, archaeometry and environmental analysis. He has published 45 original papers and book chapters, with more than 300 citations, and he acts as a referee for many of the most important journals of his scientific area. is presently coordinator of studies and research for the department of Food Quality and Chemistry of Natural Products with the Mediterranean Agronomic Institute of Chania/Centre International de Hautes Etudes Agronomiques Méditerranéennes (MAICh/CIHEAM). His main interests are the isolation and identification of natural products, exploitation of agricultural waste, antioxidant activity and chemiluminescence, authentication, ESI fragmentation mechanisms and synthetic approaches to natural products. He currently has 90 peer-reviewed publications, 3 book chapters, and 1 EU patent on the isolation of high-purity oleuropein from the leaves of the olive tree.

A novel, automatic on-line sequential injection dispersive liquid–liquid microextraction (SI-DLLME) method, based on 1-hexyl-3-methylimidazolium hexafluorophosphate ([Hmim][PF6]) ionic liquid as an extractant solvent was developed and demonstrated for trace thallium determination by flame atomic absorption spectrometry. The ionic liquid was on-line fully dispersed into the aqueous solution in a continuous flow format while the TlBr 4 complex was easily migrated into the fine droplets of the extractant due to the huge contact area of them with the aqueous phase. Furthermore, the extractant was simply retained onto the surface of polyurethane foam packed into a microcolumn. No specific conditions like low temperature are required for extractant isolation. All analytical parameters of the proposed method were investigated and optimized. For 15 mL of sample solution, an enhancement factor of 290, a detection limit of 0.86 μg L−1 and a precision (RSD) of 2.7% at 20.0 μg L−1 Tl(I) concentration level, was obtained. The developed method was evaluated by analyzing certified reference materials while good recoveries from environmental and biological samples proved that present method was competitive in practical applications. Figure An automatic sequential injection dispersive liquid-liquid microextraction (SI-DLLME) system for thallium determination
Keywords: Sequential injection; Dispersive liquid–liquid microextraction; Atomic spectrometry; Thallium; Ionic liquid; Polyurethane foam

A high-performance liquid chromatographic method with electrochemical detection (HPLC-ED) at a boron-doped diamond film electrode with preliminary separation and preconcentration by solid-phase extraction (SPE) has been developed for the determination of 1-hydroxypyrene (1-HP) in human urine. 1-HP is among the most widely used biomarkers of exposure to polycyclic aromatic hydrocarbons. Optimal HPLC-ED conditions have been found: mobile phase methanol–0.05 mol L−1 phosphate buffer pH 5.0 (80:20, v/v), detection potential +1,000 mV versus Ag/AgCl (3 mol L−1 KCl), and flow rate 0.8 mL min−1. For SPE, LiChrolut® RP-18 E cartridges were used. The extraction yield was (87.0 ± 5.8) % (n = 5). The concentration dependence of 1-HP was measured in the concentration range from 0.01 to 10 μmol L−1 (2.18–2,180 μg L−1) using methanolic solutions resulting from the SPE pretreatment of spiked human urine samples. The limit of detection (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) of the biomarker were 0.013 μmol L−1 (2.84 μg L−1) and 0.043 μmol L−1 (9.39 μg L−1), respectively, which is sufficient for its determination in the urine of persons exposed to polycyclic aromatic hydrocarbons. Figure Chromatograms of 1-hydroxypyrene (injected 20 μL of methanolic solution resulting from the solid phase extraction pretreatment of spiked human urine sample); concentrations of the analyte in urine: 0 (1), 0.2 (2), 0.4 (3), 0.6 (4), 0.8 (5) and 1.0 μmol L−1 (6). Measured by HPLC with electrochemical detection at the boron-doped diamond film electrode; detection potential +1,000 mV vs. Ag/AgCl (3 mol L−1 KCl); mobile phase: MeOH:0.05 mol L−1 phosphate buffer pH 5.0 (80:20, v/v); flow rate 0.8 mL min−1
Keywords: 1-Hydroxypyrene; High-performance liquid chromatography with electrochemical detection; Boron-doped diamond film electrode; Solid-phase extraction; Human urine

Hydrophilic interaction ultra performance liquid chromatography retention prediction under gradient elution by Helen Gika; Georgios Theodoridis; Fulvio Mattivi; Urska Vrhovsek; Adriani Pappa-Louisi (701-709).
The development and application of new separation mechanisms such as hydrophilic interaction chromatography (HILIC) is of high importance for the simultaneous analysis of polar molecules such as primary metabolites. However the retention mechanism in HILIC is not fully understood and as a result retention prediction tools are not at hand for this chromatographic approach. In the present report we study the utility of a simple algorithm, based on a simple linear and/or a simple logarithmic retention model, for retention prediction in HILIC gradient separation of a mixture of 23 selected compounds including (poly)amines, amino acids, saccharides, and other molecules. Utilizing two types of gradient elution programs with or without an isocratic part, retention data were collected in order to build prediction models. Starting from at least three gradient runs the prediction of analyte retention was very satisfactory for all gradient programs tested, providing useful evidence of the value of such retention time prediction methodologies. Figure
Keywords: Hydrophilic interaction chromatography (HILIC); Gradient elution; Retention prediction; Polar analytes; Metabolomics

Highly sensitive detection and discrimination of LR and YR microcystins based on protein phosphatases and an artificial neural network by O. I. Covaci; A. Sassolas; G. A. Alonso; R. Muñoz; G. L. Radu; B. Bucur; J.-L. Marty (711-720).
The inhibition characteristics of three different protein phosphatases by three microcystin (MC) variants—LR, YR, and RR—were studied. The corresponding K I for each enzyme–MC couple was calculated. The toxicity of MC varies in the following order: MC-LR > MC-YR > MC-RR. The sensitivity of the enzymes increased in the following order: mutant PP2A < mutant PP1 < natural PP2A. The best limit of detection obtained was 21.2 pM MC-LR using the most sensible enzyme. Methanol, ethanol, and acetonitrile up to 2 % (v/v) may be used in inhibition measurements. An artificial neural network (ANN) was used to discriminate two MC variants—LR and YR—using the differences in inhibition percentages measured with mutant PP1 and natural PP2A. The ANN is able to analyze mixtures with concentrations ranging from 8 to 98 pM MC-LR and 31 to 373 pM MC-YR.
Keywords: Protein phosphatase; Microcystin; Artificial neural network; Influence of organic solvents

The performance of microbore columns with polypropylene (PP) capillary-channeled polymer (C-CP) fibers as the support/stationary phase for separation of macromolecules has been investigated. Polypropylene C-CP fibers (40 μm diameter) were packed in fluorinated ethylene propylene (FEP) tubing of inner diameter 0.8 mm and lengths of 40, 60, 80, and 110 cm. The performance of PP fiber packed microbore columns (peak width, peak capacity, and resolution) was evaluated for separation of a three-protein mixture of ribonuclease A, cytochrome c, and transferrin under reversed-phase gradient conditions. The low backpressure characteristics of C-CP fiber columns enable operation at high linear velocities (up to 75 mm s−1 at 1.5 mL min−1). In contrast with the performance of other phases, such velocities enable enhanced resolution of the three-protein mixture, because peak widths decrease with velocity. Increased column length resulted in increased resolution, because the peak widths remained essentially constant, although retention times increased. In addition, it was found that the peak capacity increased with column length and linear velocity. Radial compression of the microbore tubing enhanced the homogeneity of the packing and, thereby, separation efficiency and resolution. Radial compression of columns resulted in a decrease in the interstitial fraction (~5 %), but increased resolution of ~14 % between ribonuclease A and cytochrome c. Even so, a linear velocity of 75 mm s−1 required a backpressure of 9.5 MPa only. It is clear that the fluid and solute-transport properties of the C-CP fiber microbore columns afford far better performance than is obtainable by use of standard format columns. The ability to achieve high separation efficiencies, rapidly and with low volume flow rates, holds promise for high-capacity protein separations in proteomics applications.
Keywords: Microbore; Capillary-channeled polymer; Fiber; Radial compression; Protein chromatography

A new type of bonded stationary phase for liquid chromatography, with the properties of immobilized artificial membranes, has been synthesized. Alkyl-phosphate adsorbents were obtained by modification of aminopropyl silica gel. The structures of the synthesized materials were confirmed by use of instrumental techniques—elemental analysis, infrared spectroscopy (FTIR), and 13 C and 29Si CP/MAS NMR. Analysis revealed that the adsorbents mimic the phospholipids present in natural cell membranes. The new synthesized alkyl-phosphate stationary phases may be used for liquid chromatographic separation of biologically active compounds of different polarity.
Keywords: Alkyl-phosphate bonded phase; Immobilized artificial membrane; Liquid chromatography

Acylcarnitine profiling in dried blood spots (DBS) is a useful method for high-throughput newborn screening of metabolic disorders, but differentiation of isobaric and isomeric compounds is not achievable. Chromatographic methods for separation have already been reported but are specific for short-chain acylcarnitines or time-consuming. The aim of this work was to develop a fast ultraperformance liquid chromatography (UPLC)–tandem mass spectrometry (MS/MS) method for separation and quantification of a large number of acylcarnitines, including dicarboxylic acylcarnitines and hydroxyacylcarnitines, in DBS and plasma samples. Acylcarnitines from DBS and plasma were converted to their butyl esters and analyzed by electrospray ionization MS/MS. Chromatographic separation was achieved using a UPLC system equipped with an ethylene-bridged hybrid C18 column. The correlation coefficients of the calibration curves (r 2) ranged from 0.990 to 0.999. The limit of detection ranged from 0.002 and 0.063 μM for all compounds, and the limit of quantification ranged from 0.004 and 0.357 μM. Precision ranged from 0.8 to 8.8 % and the mean recovery was 103 %. Profiles of acylcarnitine isomers were investigated in specimens obtained from patients diagnosed with different inborn errors of metabolism. Acylcarnitine concentrations were also measured in 58 term newborns and compared with flow injection analysis measurements. With this newly developed UPLC–MS/MS method, the simultaneous detection of 61 (13 of these labeled) acylcarnitines in DBS and plasma can be achieved in 15 min including postrun equilibration. The method has been validated and can be used as an important component of newborn screening methods as a second-tier test for discrimination and to confirm diagnosis. Figure From LC-MS/MS to UPLC-MS/MS: scheme of the method for simultaneous separation of acylcarnitine in dried blood spot and plasma.
Keywords: Acylcarnitines; Newborn screening; Second-tier test; Ultraperformance liquid chromatography; Mass spectrometry; Metabolic disease

A simple cellulose acetate membrane-based small lanes technique for protein electrophoresis by Na Na; Tingting Liu; Xiaojun Yang; Binjie Sun; Jenny Ouyang; Jin Ouyang (753-762).
Combining electrophoresis with a cellulose acetate membrane-based technique, we developed a simple and low-cost method, named cellulose acetate membrane-based small lanes (CASL), for protein electrophoresis. A home-made capillary plotter controlled by a 3D moving stage was used to create milli-to-micro channels by printing poly(dimethylsiloxane) on to a hydrophilic cellulose acetate membrane. In the hydrophilic channels, 5 nL protein mixture was separated on the basis of electro-migration under an electric field. Compared with polyacrylamide gel electrophoresis (PAGE), CASL resulted in higher protein signal intensity for separation of mixtures containing the same mass of protein. The platform was easily fabricated at low cost (approx. $0.005 for each 1-mm-wide channel), and separation of three protein mixtures was completed in 15 min. Both electrophoresis time and potential affected the separation. Rather than chromatographic separation, this method accomplished application of microchannel techniques for cellulose acetate membrane-based protein electrophoresis. It has potential in proteomic analysis, especially for rapid, low-cost, and low-volume sample analysis in clinical diagnosis. Figure Cellulose Acetate Membrane-Based Small Lanes for Protein Electrophoresis. Left: The separation of protein mixture. Right: The picture of the device for the fabrication of hydrophilic channels
Keywords: Cellulose acetate membrane-based small lanes; Protein electrophoresis; Microchannel technique; Hydrophilic channel

Novel Fe3O4@La x Si y O5 affinity microspheres consisting of a superparamagnetic Fe3O4 core and an amorphous lanthanum silicate shell have been synthesized. The core–shell-structured Fe3O4@La x Si y O5 microspheres, with a mean size of ca. 480 nm, had rough lanthanum silicate surfaces and displayed relatively strong magnetism (47.2 emu g−1). This novel affinity material can be used for selective capture, rapid magnetic separation, and part dephosphorylation (which plays an important role in identifying phosphopeptides in MS) of the phosphopeptides in a peptide mixture. Its ability to selectively trap and magnetically isolate as well as label the phosphopeptides was evaluated using a standard phosphorylated protein (β-casein) and a real sample (human serum). Phosphopeptides and their corresponding label ions were detected for concentrations of β-casein as low as 1 × 10−9 M and in mixtures of β-casein and BSA with molar ratios as low as 1:50. In addition, this affinity material, with its labeling properties, is superior to commercial TiO2 beads in terms of interference from non-phosphopeptide molecules. These results reveal that the lanthanum silicate coated magnetic microspheres represent a promising affinity material for the rapid purification and recognition of phosphopeptides. Figure A novel lanthanum silicate coated magnetic microspheres core-shell microsphere consisting of a superparamagnetic Fe3O4 core and an amorphous lanthanum silicate shell are synthesized. This novel affinity material can be used for selective capture, rapid magnetic separation, and is well-suited to the role of identifying phosphopeptides in a peptide mixture
Keywords: Magnetic microspheres; Lanthanum silicate; Phosphopeptide; Affinity material; Mass spectrometry

We report the synthesis and characterization of a tamoxifen-tethered single-walled carbon nanotube (SWCNT) conjugate, in which tamoxifen is covalently attached to the single-walled carbon nanotube via oxidation and esterification reactions for the first time. The functionalized SWCNT derivative was characterized by using spectroscopic techniques: IR, UV–vis, Raman, and 1H NMR Spectroscopy. The attachment of the drug tamoxifen to SWCNTs is analogous to the gold conjugate, which provided an endocrine treatment for breast cancer. Figure Synthesis of tamoxifen-tethered SWCNT conjugate
Keywords: Single-walled carbon nanotubes; Tamoxifen; Breast cancer; Drug delivery; Photothermal therapy

Identification of isethionic acid and other small molecule metabolites of Fragilariopsis cylindrus with nuclear magnetic resonance by Arezue F. B. Boroujerdi; Peter A. Lee; Giacomo R. DiTullio; Michael G. Janech; Sarah B. Vied; Daniel W. Bearden (777-784).
Nuclear magnetic resonance (NMR) spectroscopy has been used to obtain metabolic profiles of the polar diatom Fragilariopsis cylindrus, leading to the identification of a novel metabolite in this organism. Initial results from an ongoing metabolomics study have led to the discovery of isethionic acid (2-hydroxyethanesulfonic acid, CAS: 107-36-8) as a major metabolite in F. cylindrus. This compound is being produced by the organism under normal culture conditions. This finding is the first report of a diatom producing isethionic acid. In addition to isethionic acid, four other metabolites, dimethylsulfoniopropionate (DMSP), betaine, homarine, and proline were present and may serve as osmoprotectants in F. cylindrus. NMR-based metabolite profiles of F. cylindrus were obtained along a growth curve of the organism. The relative concentration levels of the five metabolites were monitored over a growth period of F. cylindrus from 18 to 25 days. All showed an increase in relative concentration with time, except for proline, which began to decrease after day 21.
Keywords: Fragilariopsis cylindrus ; Nuclear magnetic resonance; Metabolite profiles; Isethionic acid; Metabolomics

Potential-modulated fluorescence spectroscopy of zwitterionic and dicationic membrane-potential-sensitive dyes at the 1,2-dichloroethane/water interface by Toshiyuki Osakai; Tatsuya Yoshimura; Daichi Kaneko; Hirohisa Nagatani; Sang-Hyun Son; Yutaka Yamagishi; Koji Yamada (785-792).
The previously introduced technique of potential-modulated fluorescence (PMF) spectroscopy was used to study the potential-induced fluorescence change of some different dyes at the polarized 1,2-dichloroethane (DCE)/water (W) interface. A zwitterionic dye (POLARIC 488PPS) showed a PMF response similar to that for the previously studied dye (di-4-ANEPPS) with the same ionic state, and the PMF response was likewise explained by the potential-dependent reorientation of the dye at the DCE/W interface. Though a monocationic dye (POLARIC 488PM) showed no distinct PMF signal, a dicationic dye (di-2-ANEPEQ) showed two relatively weak but detectable PMF signals at lower and higher potential. It has thus been found that the ionic state of a potential-sensitive dye strongly influences the potential-induced reorientation of the dye at the interface and consequently its PMF response. These results support the reorientation/solvatochromic mechanism proposed for “slow” dyes but do not necessarily exclude the electrochromic mechanism proposed for “fast” dyes. PMF spectroscopy would provide useful information on the design of slow dyes for the measurement of the resting potential of cell membranes Fig Potential-dependent reorientation of a zwitterionic membrane-potential-sensitive dye (POLARIC 488PPS) at the oil/water interface and its potential-modulated fluorescence signal (inset)
Keywords: Membrane-potential-sensitive dye; POLARIC™; Liquid/liquid interface; Potential modulated fluorescence

X-ray phase-contrast imaging based on grating interferometry is a technique with the potential to provide absorption, differential phase contrast, and dark-field signals simultaneously. The multi-line X-ray source used recently in grating interferometry has the advantage of high-energy X-rays for imaging of thick samples for most clinical and industrial investigations. However, it has a drawback of limited field of view (FOV), because of the axial extension of the X-ray emission area. In this paper, we analyze the effects of axial extension of the multi-line X-ray source on the FOV and its improvement in terms of Fresnel diffraction theory. Computer simulation results show that the FOV limitation can be overcome by use of an alternative X-ray tube with a specially designed multi-step anode. The FOV of this newly designed X-ray source can be approximately four times larger than that of the multi-line X-ray source in the same emission area. This might be beneficial for the applications of X-ray phase contrast imaging in materials science, biology, medicine, and industry. Figure Visibility map on the observation plane using five lines of the multi-step X-ray source
Keywords: X-ray phase contrast imaging; Grating interferometry; Multi-line X-ray source; Field of view

Mass spectrometry based analysis of nucleotides, nucleosides, and nucleobases—application to feed supplements by Stefan Neubauer; Ariana Rugova; Dinh Binh Chu; Hedda Drexler; Anja Ganner; Michael Sauer; Diethard Mattanovich; Stephan Hann; Gunda Koellensperger (799-808).
In this work, accurate MS-based methods for quantitative profiling of nucleotides, nucleosides, and nucleobases in yeast extracts used as additives in animal feedstuff are presented. Reversed-phase chromatography utilizing a stationary phase compatible with 100 % aqueous mobile phases resulted in superior analytical figures of merit than HILIC or ion-pair reversed-phase separation. The novel separation method was combined with both molecular and elemental mass spectrometry. By use of RP–LC–MS–MS, excellent limits of detection <1 μmol L−1 could be obtained for all the compounds investigated. The elemental speciation analysis approach enabled determination of nucleotides by phosphorus detection. Sensitivity of LC–ICP–MS was 1–2 orders of magnitude lower than that of LC–MS–MS. Quantitative analysis of yeast products using complementary MS detection furnished values in good agreement. Figure Reversed phase chromatographic separation of nucleotides, nucleosides and nucleobases combined with mass spectrometric detection
Keywords: Nucleotide; Nucleoside; Nucleobase; LC–MS–MS; LC–ICP–MS; Feed supplement

Current interest by the food industry in exploring reformulation options that lower the content of trans fat in edible fats and oils requires methods to accurately measure low levels of trans fat. In the present study, the quantitation of trans fat in 25 edible fat and oil samples was evaluated using two current analytical approaches, attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), and gas chromatography with flame ionization detection (GC-FID) according to Official Methods of the American Oil Chemists’ Society. Significant differences between the ATR-FTIR and reference GC-FID quantitations were found for samples with a trans fat content <2% of total fat. These discrepancies could be explained, in part, by the presence of certain oil constituents (e.g., vitamins, carotenoids, high levels of saturated fat) that produced absorbance bands at or near 966 cm−1 in the ATR-FTIR spectra, a region that was previously identified as being characteristic of isolated trans double bonds. Results demonstrate that the natural content of such oil constituents could result in significant overestimations of trans fat when ATR-FTIR is used to analyze edible fats and oils with a trans fat content <2% of total fat. Figure Quantitation of trans fat in edible fats and oils by ATR-FTIR and GC-FID analyses. The left y-axis represents the ATR-FTIR quantitation of trans fat or unidentified constituents (as percent of total fat) based on the total absorption at 966 cm −1. The right y-axis represents the GC-FID quantitation of trans fat (as percent of total fat). One-way analysis of variance was used to determine pairwise comparisons of the mean trans fat content in the fatand oil samples (*P<0.05).
Keywords: Trans fat; Attenuated total reflection; Fourier-transform infrared spectroscopy; Gas chromatography; Edible oils

Surface chemistry and surface reactivity of fibrous amphiboles that are not regulated as asbestos by M. Fantauzzi; A. Pacella; J. Fournier; A. Gianfagna; G. B. Andreozzi; A. Rossi (821-833).
Three fibrous amphiboles that are not regulated as asbestos—two from Biancavilla (Sicily, Italy) and one from Libby (MT, USA)—were studied in order to establish relationships between surface chemistry and surface reactivity. The three fibrous samples, plus one prismatic fluoro-edenite from Biancavilla that was used for comparison, were investigated by X-ray photoelectron spectroscopy (XPS) in order to obtain their quantitative surface compositions and to determine the chemical environment of the Fe in each case. In particular, the Fe 2p3/2 peak was fitted and, for the first for these materials, the binding energies of Fe(II) oxide, Fe(III) oxide and Fe(III) oxyhydroxide were identified. Bulk chemistries and Fe oxidation states were obtained from previous studies for the samples from Biancavilla, and were investigated in the present work by electron microprobe (EMP) and 57Fe Mössbauer spectroscopy (MS) for the sample from Libby. Comparison between surface and bulk data revealed that the sample with the lowest bulk Fe oxidation state was the one most affected by surface oxidation, while the samples with bulk highly-oxidised Fe were showing very high signal of Fe (III) oxy-hydroxide probably due to weathering. The surface reactivities of the fibrous amphiboles were investigated by measuring the production of the [DMPO, HO]• radical adduct using electron paramagnetic resonance (EPR) spectroscopy. Notably, significant chemical reactivity was observed; it was found to be comparable with—or, for the Libby sample, even higher than—that of fibrous tremolite (one of the six asbestos minerals). A positive linear correlation was observed when the production of HO• radical was plotted versus the Fe(II) content on the fibre surface. Data on fibrous tremolite obtained from previous studies were added to substantiate the correlation. These results provide evidence that Fe(II) at the fibre surface controls the production of radicals at the fibre surface. The observed relationship provides further confirmation that Fe topochemistry is strictly related to—though not solely responsible for—the toxicity of asbestos and other fibrous amphiboles that are not regulated as asbestos.
Keywords: Asbestos; Fibrous amphiboles; Crystal chemistry; Surface chemistry; Surface reactivity; HO• radical; X-ray photoelectron spectroscopy (XPS); Fe 2p3/2 peak fitting; Mössbauer spectroscopy (MS); Electron paramagnetic resonance (EPR)

Comparison of different methods for generation of single-stranded DNA for SELEX processes by M. Svobodová; A. Pinto; P. Nadal; C. K. O’ Sullivan (835-842).
Single-stranded DNA (ssDNA) generation is a crucial step in several molecular biology applications, such as sequencing or DNA chip and microarray technology. Molecules of ssDNA also play a key role in the selection of ssDNA aptamers through Systematic Evolution of Ligands by EXponential enrichment (SELEX). With particular interest for this application, herein we present a comparative study of the most used methods for generation of ssDNA used in SELEX, such as asymmetric PCR, enzyme digestion and magnetic separation with streptavidin beads. In addition, we evaluate a new technique that combines asymmetric PCR and enzyme digestion with the aim to achieve the maximum efficiency in ssDNA generation. The methods studied were compared in terms of quality of ssDNA using electrophoretic analysis and generated ssDNA yields were quantitatively measured using an Enzyme-Linked OligoNucleotide Assay (ELONA).
Keywords: Single stranded DNA (ssDNA); SELEX; Exonuclease digestion; Magnetic beads; Asymmetric PCR; ELONA

Francisella tularensis are very small, gram-negative bacteria which are capable of infecting a number of mammals. As a highly pathogenic species, it is a potential bioterrorism agent. In this work we demonstrate a fast immunological detection system for whole F. tularensis bacteria. The technique is based on a quartz crystal microbalance with dissipation monitoring (QCMD), which uses sensor chips modified by a specific antibody. This antibody is useful as a capture molecule to capture the lipopolysaccharide structure on the surface of the bacterial cell wall. The QCMD technique is combined with a microfluidic system and allows the label-free online detection of the binding of whole bacteria to the sensor surface in a wide dynamic concentration range. A detection limit of about 4 × 103 colony-forming units per milliliter can be obtained. Furthermore, a rather short analysis time and a clear discrimination against other bacteria can be achieved. Additionally, we demonstrate two possibilities for specific and significant signal enhancement by using antibody-functionalized gold nanoparticles or an enzymatic precipitation reaction. These additional steps can be seen as further proof of the specificity and validity. Figure Operating principle. Figure illustrates binding of F.tularensis bacteria onto the gold surface of a quartz crystal microbalance (QCM) sensor chip and two different approaches of signal enhancement using gold nanoparticles (Au-NPs)- or peroxidase (POD)-modified antibodies.
Keywords: Francisella tularensis ; Quartz crystal microbalance with dissipation monitoring; Bacteria detection; Signal enhancement

A novel strategy for phosphopeptide enrichment using lanthanide phosphate co-precipitation by Munazza Raza Mirza; Matthias Rainer; Yüksel Güzel; Iqbal M. Choudhary; Günther K. Bonn (853-862).
Reversible phosphorylation of proteins is a common theme in the regulation of important cellular functions such as growth, metabolism, and differentiation. The comprehensive understanding of biological processes requires the characterization of protein phosphorylation at the molecular level. Although, the number of cellular phosphoproteins is relatively high, the phosphorylated residues themselves are generally of low abundance due to the sub-stoichiometric nature. However, low abundance of phosphopeptides and low degree of phosphorylation typically necessitates isolation and concentration of phosphopeptides prior to mass spectrometric analysis. In this study, we used trivalent lanthanide ions (LaCl3, CeCl3, EuCl3, TbCl3, HoCl3, ErCl3, and TmCl3) for phosphopeptide enrichment and cleaning-up. Due to their low solubility product, lanthanide ions form stable complexes with the phosphate groups of phosphopeptides and precipitate out of solution. In a further step, non-phosphorylated compounds can easily be removed by simple centrifugation and washing before mass spectrometric analysis using Matrix-assisted laser desorption/ionisation-time of flight. The precipitation method was applied for the isolation of phosphopeptides from standard proteins such as ovalbumin, α-casein, and β-casein. High enrichment of phosphopeptides could also be achieved for real samples such as fresh milk and egg white. The technology presented here represents an excellent and highly selective tool for phosphopeptide recovery; it is easily applicable and shows several advantages as compared with standard approaches such as TiO2 or IMAC. Figure Highly selective co-precipitation of phosphopeptides by trivalent lanthanide ions
Keywords: Phosphoproteomics; Phosphopeptides; Precipitation; Enrichment; Lanthanides

On-line solid-phase extraction high-performance liquid chromatography-tandem mass spectrometry for the quantitative analysis of tacrolimus in whole blood hemolyzate by Volker Neu; Nathanaël Delmotte; Uwe Kobold; Thomas Dülffer; Rupert Herrmann; Herbert von der Eltz; Christian G. Huber (863-874).
Tacrolimus is an immunosuppressive drug essential for preventing organ rejection after transplantation. Since tacrolimus strongly binds to erythrocytes, therapeutic monitoring requires its quantification in whole blood lyzate, representing one of the most difficult to analyze biological fluids due to its high protein load. In this communication, we report on the successful combination of whole blood hemolysis employing ionic liquids, followed by sample preparation by means of on-line solid phase extraction (SPE) using restricted access materials (RAM), which permitted the efficient removal of hemoglobin and other large biomolecules. Among six different tested RAM columns, highest hemoglobin depletion and analyte extraction efficiency was obtained with a polymer-based, glycoprotein-coated RAM stationary phase (Biotrap 500 MS) operated at an alkaline pH of 10.7. Analyte quantification was performed by high-performance liquid chromatography-selected reaction monitoring tandem mass spectrometry (HPLC-SRM-MS/MS). The ability to quantify tacrolimus in therapeutically relevant concentrations in whole blood hemolyzates was demonstrated via external calibration with lower limits of detection and quantification of 2.00 and 7.23 ng mL−1, respectively. Moreover, the investigation of heparin-pretreated blood samples during blood sampling led to an increase in sensitivity for the analyte, while the method appeared to be more robust with ethylenediaminetetraacetic acid as anticoagulant.
Keywords: Tacrolimus; Whole blood analysis; Liquid chromatography; Electrospray ionization mass spectrometry; On-line solid phase extraction

Terbuthylazine (TBA) is a widely applied herbicide and an environmental contaminant. Following its use, humans, such as agricultural workers and rural residents, may be exposed. An isotope-dilution liquid chromatography coupled to electrospray-tandem mass spectrometry method for the determination of TBA, and its metabolite desethylterbuthylazine (DET) in human urine and hair was developed and validated. Under the optimised conditions, analytes were extracted from urine using a solid phase cartridge or from hair by sonication in methanol. Analytes were separated using a C18 reversed-phase chromatographic column and quantified, after positive ionization using a heated electrospray source, by a triple quadrupole mass detector in the selected reaction monitoring mode. Validation showed linear dynamic ranges up to 100 μg/L or 5.00 ng/mg hair, inter- and intra-run precisions <7%, and accuracies within 12% of spiked concentrations. Limits of quantification were 0.25 μg/L in urine and 0.01 ng/mg hair for both TBA and DET. Matrix effect evaluation showed that the isotope dilution approach allowed for the control of bias sources. TBA and DET were determined in specimens of agriculture workers exposed to TBA using the validated method. Hair samples contained TBA levels in the low nanogram per milligram range, and urine samples contained DET levels in the low microgram per liter range. Conversely, TBA levels in urine samples and DET levels in hair samples were always below the limit of quantification. Figure TBA absorption and elimination routes
Keywords: Terbuthylazine; Desethylterbuthylazine; Urine; Hair; Biological monitoring

GC–MS-based metabolic profiling reveals metabolic changes in anaphylaxis animal models by Xia Hu; Gong-ping Wu; Meng-hui Zhang; Shan-qing Pan; Rong-rong Wang; Jie-hu Ouyang; Jun-ge Liu; Zi-yuan Chen; Hong Tian; Dong-bo Liu (887-893).
Clinical definition and appropriate management of anaphylaxis is a clinical challenge because there is large variability in presenting clinical signs and symptoms. Monitoring of the metabolic status of anaphylaxis may be helpful in understanding its pathophysiological processes and diagnosis. The purpose of this study was to conduct GC–MS serum metabolic profiling of anaphylaxis animal models and search for potential biomarkers of anaphylaxis. Thirty-six guinea pigs were randomly divided into an ovalbumin group (n = 12), a cattle albumin group (n = 12), and a control group (n = 12). The IgE level in the serum of the guinea pigs was evaluated by use of ELISA kits and the major metabolic changes in serum were detected by gas chromatography–mass spectrometry. Typical clinical symptoms appeared after the animals had been challenged with ovalbumin or cattle albumin. The IgE levels in serum of both model groups were significantly higher than those of the control group. Clustering trend of the three groups based on variables was observed and nine out of 858 metabolomic features were found to be significantly different between control group and model groups. Among the nine features, six features were tentatively identified as metabolites related to energy metabolism and signal transduction in anaphylaxis. In conclusion, GC–MS-based metabolic profiling analysis might be an effective auxiliary tool for investigation of anaphylaxis. Figure Chromatographic profile of the derivatized serum
Keywords: Anaphylaxis; Allergen; ELISA; Gas chromatography-mass spectrometry; Metabolic profiling

Assay of whole blood (6S)-5-CH3-H4folate using ultra performance liquid chromatography tandem mass spectrometry by Susanne H. Kirsch; Wolfgang Herrmann; Jürgen Geisel; Rima Obeid (895-902).
Folates act as essential coenzymes in many biological pathways, including the synthesis and methylation of DNA. Low folate concentration in serum and whole blood (WB) is associated with several disease conditions. We describe a stable-isotope-dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantification of (6S)-5-CH3-H4folate (where H4folate is tetrahydrofolate) and non-CH3-H4folate [sum of HCO-H4folate, (6R)-5,10-CH+-H4folate, (6R)-5,10-CH2-H4folate, (6S)-H4folate, dihydrofolate, and folic acid] in WB. The assay includes a solid-phase extraction procedure after the hemolysis and deconjugation. The method was linear over the concentration range from 0.2 to 200 nmol/L. The limits of detection were 0.40 nmol/L or lower for the folate forms. The interassay coefficients of variation were 7.4 % for (6S)-5-CH3-H4folate and 15.4 % for non-CH3-H4folate. For the folate forms, the recoveries were between 97.1 % and 102.7 %. Sample preparation caused the generation of artificial folic acid in WB and serum in a dose-dependent manner, which can lead to misinterpretation of the results. The use of antioxidants could not prevent the formation of folic acid. The median fasting WB folate concentrations from 42 nonsupplemented and nonfortified adults were 576 nmol/L (6S)-5-CH3-H4folate and 73.6 nmol/L non-CH3-H4folate, and 1,206 nmol/L (6S)-5-CH3-H4folate and 155 nmol/L non-CH3-H4folate for 35 adults who had taken 500 μg of folic acid, 50 mg of vitamin B6, and 500 μg of vitamin B12 per day orally for 6 months. In conclusion, the UPLC-MS/MS method is fast and has a good sensitivity and selectivity for WB folates. We observed a dose-dependent oxidation of (6S)-H4folate, which resulted in the formation of artificial folic acid in serum and WB. To minimize this effect, we recommend a fast sample preparation.
Keywords: Folate; Red blood cells; 5-Methyltetrahydrofolate; Folic acid; Homocysteine; Tandem mass spectrometry

Coiled carbon nanotubes combined with ionic liquid: a new soft material for SPE by M. L. Polo-Luque; B. M. Simonet; M. Valcárcel (903-907).
For the first time a soft material formed from coiled carbon nanotubes and 1-hexyl-3-methylimidazolium hexafluorophosphate has been used as sorbent material. The soft material has high stability as well as a high capacity to adsorb analytes. In this work we propose using a natural cotton fiber impregnated with the soft material to miniaturize the system. The system was tested for the analysis of polycyclic aromatic hydrocarbons in spiked river water samples. The absolute recovery ranged between 97.5 and 105.5 %, demonstrating the usefulness of the soft material. The limit of detection ranged from 2.5 to 6.1 μg/L and the precision expressed as the relative standard deviation for the analysis of five consecutive analyses ranged between 2.5 and 5.8 %.
Keywords: Soft material; Coiled carbon nanotubes; Polycyclic aromatic hydrocarbons; Solid phase microextraction

Lab in a syringe: fully automated dispersive liquid–liquid microextraction with integrated spectrophotometric detection by Fernando Maya; Burkhard Horstkotte; José Manuel Estela; Víctor Cerdà (909-917).
A new approach for the integration of various analytical steps inside a syringe (Lab in a Syringe) is presented. Fully automated dispersive liquid–liquid microextraction with integrated spectrophotometric detection is carried out in-syringe using a very simple instrumental setup. The lighter-than-water organic droplets released in the extraction step accumulate at the head of the syringe, where two optical fibers are placed on both sides of the syringe, facing each other and enabling the in situ quantification of the extracted compounds. By this, monitoring of the progressively accumulating droplet in the head of the syringe was further possible. In this first report, the developed instrumental setup has been applied to the determination of the dye rhodamine B in water samples and soft drinks. The main parameters influencing the extraction such as the selection of the extractant and disperser solvents, extractant/disperser and organic/water phase ratios, pH of the aqueous phase, extraction flow rates, and extraction time were investigated. Under the selected conditions, rhodamine B was quantified in a working range of 0.023–2 mg L−1 with a limit of detection of 0.007 mg L−1. Good repeatability values of up to 3.2 % (RSD) were obtained for ten consecutive extractions. The enrichment factor for a 1 mg L−1 rhodamine B standard was 23, and up to 51 extractions were accomplished in 1 h. Figure The dispersive liquid-liquid microextraction technique is fully automated based on in-syringe extraction followed by in-situ spectrophotometric detection
Keywords: Dispersive liquid–liquid microextraction; Sequential injection analysis; Sample treatment automation; Fatty alcohols; Rhodamine B