Analytical and Bioanalytical Chemistry (v.400, #3)

is the Charles A. Dana Professor of Chemistry at Bates College in Lewiston, Maine. He currently carries out research with the aid of undergraduate students in the area of chiral NMR shift reagents. His research accomplishments were recognized with the 2010 American Chemical Society Award for Research at an Undergraduate Institution. He is active in efforts to reform the undergraduate analytical chemistry curriculum to include inquiry- and project-based experiences. His educational activities were recognized through receipt of the 1999 J.C. Giddings Award for Excellence in Education sponsored by the Analytical Division of the American Chemical Society. More information about his activities can be found at http://www.bates.edu/x51735.xml .

is the author or coauthor of over 130 research articles, reviews, and book chapters. He is a member of the International Advisory Board of Analytical and Bioanalytical Chemistry. He has worked for over 20 years as an analytical chemist for the Chevron Corporation and now runs his own consulting company, Fetzpahs Consulting, in Hercules, CA, USA. His book, “Career management for chemists—a guide to success in a chemistry career” was published by Springer.

Hofmeister effect challenge by Mahdi Hashemi; Thomas G. Chasteen (643-644).

Solution to green beans challenge by Hervé This (645-646).

Focus on surface analysis by Michael Kopnarski (647-647).
is currently a member of the Research Centre for Optics and Material Science OPTIMAS and the head of the Institute of Surface and Thin Film Analysis IFOS at the Technical University of Kaiserslautern. His main research interests are instrumental and applied surface and thin film analysis.

The effect of bias-temperature stress on Na+ incorporation into thin insulating films by Stefan Krivec; Michael Buchmayr; Thomas Detzel; Till Froemling; Juergen Fleig; Herbert Hutter (649-657).
The action of Na+ incorporation into thin insulating films and transport therein under influence of a bias voltage and temperature (BT stress) is the subject of this work. Deposited onto highly n-doped Si wafers, the insulators get BT stressed and subsequently investigated by means of time-of-flight–secondary ion mass spectrometry (ToF-SIMS). A thin PMMA film, spin-coated onto the insulator, serves as host matrix for a defined amount of Na+, provided via sodium triflate. Combining BT stress and ToF-SIMS depth profiling enables the unambiguous detection of Na+, incorporated into the insulating material. The insulators of interest vary in their nitride content: SiO2, SiOxNy, and Si3N4. For SiO2, it is shown that once a threshold BT stress is exceeded, Na+ gets quantitatively incorporated from PMMA into the underlying insulator, finally accumulating at the SiO2/Si interface. A quantitative assessment by combination of Butler–Volmer kinetics with hopping dynamics reveals activation energies of E a = 1.55 − 2.04 eV for Na+ transport in SiO2 with varying thickness. On the other hand, SiOxNy and Si3N4 films show a different Na+ incorporation characteristic in this type of experiment, which can be explained by the higher coordination of nitrogen and hence the reduced Na+ permeability within these insulators.
Keywords: Mobile ions; Bias-temperature stress; ToF-SIMS; Barrier layers

Oxygen diffusion in grain boundaries: a ToF-SIMS investigation on hot-rolled steel sheets by Markus Holzweber; Markus Kriegl; Arno Schintlmeister; Dieter Paesold; Herbert Danninger; Herbert Hutter (659-663).
A study of grain boundary diffusion of oxygen in hot-rolled steel sheets is carried out by means of time-of-flight secondary-ion-mass-spectrometry (ToF-SIMS). This involves polishing of the sample surface prior to the oxygen exposure. A nickel layer deposited after exposure ensures a homogeneous extraction field for ToF-SIMS measurements at the Ni–steel interface. The sample is bevelled at an angle of 11.5° to spread up the diffusion pathway by a factor of 5. The oxygen distribution is then acquired via ToF-SIMS in imaging mode from which diffusion parameters are calculated according to the Whipple–Le Claire’s approach.
Keywords: ToF-SIMS; Grain boundaries; Diffusion; Steel

Characterization of the interface interaction of cobalt on top of copper- and iron-phthalocyanine by Felix Schmitt; Jens Sauther; Stefan Lach; Christiane Ziegler (665-671).
The electronic structure of the interface between ferromagnetic cobalt and the organic semiconductors copper- (CuPc) and iron-phthalocyanine (FePc) was investigated by means of photoemission spectroscopy (UPS, IPES, and XPS). These metal-phthalocyanine (MePc) molecules have an open shell structure and are known to show promising properties for their use in organic spintronics. In spintronic devices, the interface between ferromagnetic electrode and the organic layer determines the spin injection properties and is hence important for the quality of, e.g., a possible spin-valve device. For this purpose, cobalt was deposited onto the MePcs, such as in devices with ferromagnetic top contacts. The reported investigations reveal a diffusion of cobalt into the organic layers and chemical reactions at the interface.
Keywords: Organic spintronics; Phthalocyanines; Photoemission spectroscopy; Interfaces

Photoionization cross-section weighted DFT simulations as promising tool for the investigation of the electronic structure of open shell metal-phthalocyanines by Michael Vogel; Felix Schmitt; Jens Sauther; Benedikt Baumann; Anna Altenhof; Stefan Lach; Christiane Ziegler (673-678).
The valence band structure of different metal-phthalocyanines was investigated by comparing ultraviolet photoelectron spectra at different excitation energies with simulated spectra that take the different photoionization cross-sections at these energies into account. The Kohn-Sham eigenvalue spectra, derived from density functional theory calculations, using hybrid exchange-correlation functionals, were weighted with the photoionization coefficients in accordance with the used excitation energy. By applying these techniques, the differences in the photoelectron spectra using He I and He II radiation can be reproduced and investigated. It will be shown that the 3d-orbitals of the used metal central atom of these molecules have a major influence. The changes at different excitation energies were studied for Fe, Co, and Cu central atoms to describe the chemical tailoring effects.
Keywords: Metal-phthalocyanine (MePc); Photoionization cross-section; UPS; FePc; CuPc; CoPc; DFT

Protein adhesion on dental surfaces—a combined surface analytical approach by Christine Müller; Johanna Wald; Wiebke Hoth-Hannig; Natalia Umanskaya; Daniel Scholz; Matthias Hannig; Christiane Ziegler (679-689).
Protein adsorption is a field of huge interest in a number of application fields. Information on protein adhesion is accessible by a variety of methods. However, the results obtained are significantly influenced by the applied technique. The objective of this work was to understand the role of adhesion forces (obtained by scanning force spectroscopy, SFS) in the process of protein adsorption and desorption. In SFS, the protein is forced to and retracted from the surface, even under unfavorable conditions, in contrast to the natural situation. Furthermore, adhesion forces are correlated with adhesion energies, neglecting the entropic part in the Gibbs enthalpy. In this context, dynamic contact angle (DCA) measurements were performed to identify the potential of this method to complement SFS data. In DCA measurements, the protein diffuses voluntarily to the surface and information on surface coverage and reversibility of adsorption is obtained, including entropic effects (conformational changes and hydrophobic effect). It could be shown that the surface coverage (by DCA) of bovine serum albumin on dental materials correlates well with the adhesion forces (by SFS) if no hydrophobic surface is involved. On those, the entropic hydrophobic effect plays a major role. As a second task, the reversibility of the protein adsorption, i.e., the voluntary desorption as studied by DCA, was compared to the adhesion forces. Here, a correlation between low adhesion forces and good reversibility could be found as long as no covalent bonds were involved. The comparative study of DCA and SFS, thus, leads to a more detailed picture of the complete adsorption/desorption cycle.
Keywords: Dynamic contact angle analysis; Wilhelmy plate method; Scanning force spectroscopy; Protein adsorption; Protein adhesion; Protein desorption; Bovine serum albumin; Dental material

XPS investigations of electrolyte/electrode interactions for various Li-ion battery materials by S. Oswald; D. Mikhailova; F. Scheiba; P. Reichel; A. Fiedler; H. Ehrenberg (691-696).
For future Li-ion battery applications the search for both new design concepts and materials is necessary. The electrodes of the batteries are always in contact with electrolytes, which are responsible for the transport of Li ions during the charging and discharging process. A broad range of materials is considered for both electrolytes and electrodes so that very different chemical interactions between them can occur, while good cycling behavior can only be obtained for stable solid-electrolyte interfaces. X-ray photoelectron spectroscopy (XPS) was used to study the most relevant interactions between various electrode materials in contact with different electrolyte solutions. It is shown how XPS can provide useful information on reactivities and thus preselect suitable electrode/electrolyte combinations, prior to electrochemical performance tests. Figure Characteristic changes of the Li1s XP-spectra at Li2O2 powder after storage in LiPF6 for various time point to a LiF formation
Keywords: Li ion battery; Electrolyte; Surface interaction; XPS

The adsorption properties of bovine serum albumin (BSA) on pure titanium (99.99%+) were studied by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). For this purpose, films consisting of BSA were prepared on cleaned titanium sheets in a phosphate buffered solution under different conditions and analysed by ToF-SIMS. The dependence of the surface coverage on the concentration of the protein solution could be determined from the secondary ion mass spectrometry signals. The results agree satisfactorily with the corresponding results obtained by XPS. The observed adsorption behaviour can be fitted by the well-known formula describing a Langmuir isotherm. The Langmuir constant for pH 7 was found to be $$ K = {174}pm {99}{lg^{{ - 1}}} $$ .
Keywords: ToF-SIMS; Quantitative SIMS; Protein adsorption kinetics; BSA; XPS; Organic layers

A competitive DNA hybridization assay based on the photoelectrochemistry of the semiconductor quantum dot-single stranded DNA conjugates (QD-ssDNA) was developed. Hybridization of QD-ssDNA with the capture probe DNA immobilized on the indium–tin oxide electrodes enables photocurrent generation when the electrochemical cell was illuminated with a light source. Upon the competition between QD-ssDNA and single-stranded target DNA, the photocurrent response decreased with the increase in the target DNA concentration. A linear relationship between the photocurrent and the target DNA concentration was obtained (R 2 = 0.991). The selectivity of system towards the target DNA was also demonstrated using non-complementary sample.
Keywords: Photoelectrochemistry; DNA; Quantum dots; Genosensor; Photocurrent

Determination of gallium originated from a gallium-based anticancer drug in human urine using ICP-MS by Darya G. Filatova; Irina F. Seregina; Lidia S. Foteeva; Vladimir V. Pukhov; Andrei R. Timerbaev; Mikhail A. Bolshov (709-714).
Urine analysis gives an insight into the excretion of the administered drug which is related to its reactivity and toxicity. In this work, the capability of inductively coupled plasma mass spectrometry (ICP-MS) to measure ultratrace metal levels was utilized for rapid assaying of gallium originating from the novel gallium anticancer drug, tris(8-quinolinolato)gallium(III) (GaQ3), in human urine. Sample dilution with 1% (v/v) HNO3 as the only required pre-treatment was shown to prevent contamination of the sample introduction system and to reduce polyatomic interferences from sample components. The origin of the blank signal at masses of gallium isotopes, 71 and 69, was investigated using high-resolution ICP-MS and attributed, respectively, to the formation of 36Ar35Cl+ and 40Ar31P+ ions and, tentatively, to a triplet of doubly charged ions of Ba, La, and Ce. The accuracy and precision performance was tested by evaluating a set of parameters for analytical method validation. The developed assay has been applied for the determination of gallium in urine samples spiked with GaQ3. The achieved recoveries (95–102%) and quantification limit of 0.2 μg L−1 emphasize the practical applicability of the presented analytical approach to monitor renal elimination of GaQ3 at all dose levels in clinical trials that are currently in progress.
Keywords: Anticancer metallodrugs; Biological fluids; Inductively coupled plasma mass spectrometry

A sensitive assay for the quantification of morphine and its active metabolites in human plasma and dried blood spots using high-performance liquid chromatography–tandem mass spectrometry by Claudia F. Clavijo; Keith L. Hoffman; James J. Thomas; Brendan Carvalho; Larry F. Chu; David R. Drover; Gregory B. Hammer; Uwe Christians; Jeffrey L. Galinkin (715-728).
Opioids such as morphine are the cornerstone of pain treatment. The challenge of measuring the concentrations of morphine and its active metabolites in order to assess human pharmacokinetics and monitor therapeutic drugs in children requires assays with high sensitivity in small blood volumes. We developed and validated a semi-automated LC-MS/MS assay for the simultaneous quantification of morphine and its active metabolites morphine 3β-glucuronide (M3G) and morphine 6β-glucuronide (M6G) in human plasma and in dried blood spots (DBS). Reconstitution in water (DBS only) and addition of a protein precipitation solution containing the internal standards were the only manual steps. Morphine and its metabolites were separated on a Kinetex 2.6-μm PFP analytical column using an acetonitrile/0.1% formic acid gradient. The analytes were detected in the positive multiple reaction mode. In plasma, the assay had the following performance characteristics: range of reliable response of 0.25–1000 ng/mL (r 2 > 0.99) for morphine, 1–1,000 ng/mL (r 2 > 0.99) for M3G, and 2.5–1,000 ng/mL for M6G. In DBS, the assay had a range of reliable response of 1–1,000 ng/mL (r 2 > 0.99) for morphine and M3G, and of 2.5–1,000 ng/mL for M6G. For inter-day accuracy and precision for morphine, M3G and M6G were within 15% of the nominal values in both plasma and DBS. There was no carryover, ion suppression, or matrix interferences. The assay fulfilled all predefined acceptance criteria, and its sensitivity using DBS samples was adequate for the measurement of pediatric pharmacokinetic samples using a small blood of only 20–50 μL.
Keywords: Morphine; Morphine 3β-glucoronide; Morphine 6β-glucuronide; LC-MS/MS; Human plasma; Dried blood spots

Kinetic analysis of the estrogen receptor alpha using RIfS by Peter Fechner; Florian Pröll; Christiane Albrecht; Günter Gauglitz (729-735).
The label-free time-resolved reflectometric interference spectroscopy has been used to study the interaction of the human estrogen receptor alpha (ERa) and different types of ligands. Different possible sensor surface coatings including various estrogen derivatives were evaluated for their suitability for detection of ERa. The determination of the kinetic and thermodynamic constants was carried out for the interaction in the heterogeneous phase as well as for the interaction in homogeneous phase. In addition, the affinity of 11 ligands ranging from natural hormones and pharmaceuticals to endocrine disrupting chemicals (EDCs) has been determined with this label-free assay format.
Keywords: Estrogen receptor alpha (ERa); Nuclear receptor (NR); Reflectometric interference spectroscopy (RIfS); Biosensor; Label-free; Kinetics; Endocrine disrupting chemical (EDC)

An LC-MS/MS method for the determination of the atypic neuroleptic clozapine and its two main metabolites norclozapine and clozapine-N-oxide has been developed and validated for serum and urine. After addition of d4-clozapine as deuterated internal standard a fast single-step liquid–liquid extraction under alkaline conditions and with ethyl acetate as organic solvent followed. The analytes were chromatographically separated on a Synergi Polar RP column using gradient elution with 1 mM ammonium formate and methanol. Data acquisition was performed on a QTrap 2000 tandem mass spectrometer in multiple reaction monitoring mode with positive electrospray ionization. Two transitions were monitored for each analyte in order to fulfill the established identification criteria. The validation included the determination of the limits of quantification (1.0 ng/mL for all analytes in serum and 2.0 ng/mL for all analytes in urine), assessment of matrix effects (77% to 92% in serum, 21 to 78% in urine) and the determination of extraction efficiencies (52% to 85% for serum, 59% to 88% for urine) and accuracy data. Imprecision was <10%, only the quantification of norclozapine in urine yielded higher relative standard deviations (11.2% and 15.7%). Bias values were below ±10%. Dilution of samples had no impact on the correctness for clozapine and norclozapine in both matrices and for clozapine-N-oxide in serum. For quantification of clozapine-N-oxide in urine a calibration with diluted calibrators has to be used. Calibration curves were measured from the LOQ up to 2,000 ng/mL and proved to be linear over the whole range with regression coefficients higher than 0.98. The method was finally applied to several clinical serum and urine samples and a cerebro-spinal fluid sample of an intoxicated 13-month-old girl.
Keywords: Clozapine; Norclozapine; Clozapine-N-oxide; Liquid–liquid extraction; LC-MS/MS

Novel multifunctional chitosan-GMA-IDA-Cu(II) nanospheres for high dynamic range characterization of the human plasma proteome by Xiajuan Zou; Lijun Zhong; Dan Liu; Bin Yang; Yaxin Lou; Jiarou Peng; Matthias Rainer; Isabel Feuerstein; Najam-ul-Haq Muhammad; Christian W. Huck; Günther K. Bonn; Yuxin Yin (747-756).
In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100 nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35–55 mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application. Figure The human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanosphere with MS spectrometry, results in a novel strategy which should make it possible to characterize the plasma proteome in a single test.
Keywords: Plasma; Nanosphere; Biomarkers; Chitosan-GMA-IDA-Cu(II); MALDI-TOF-MS

A method for determination of two relevant sphingoid precursors such as sphingosine and sphinganine and the corresponding conjugates sphingosine 1-phosphate and sphinganine 1-phosphate in human urine and serum is here presented. The method is characterized by a solid- phase extraction step with in situ derivatization of the sphingolipids in the eluate (o-phthaldialdehyde derivatives) to obtain fluorescent compounds. In this way, sample preparation was completely performed in a single automated step by means of a lab-on-valve system. Derivatized analytes were injected into a liquid chromatography system operating at micro regime and detected by laser-induced fluorescence. For determination of sphingoid phosphates, they were enzymatically converted to free sphingoids to obtain stable fluorescent derivatives. The detection limits were in the range 4.2–10.2 ng mL−1 for serum and 0.56–1.36 ng mL−1 for urine, with repeatability ranging from 3.9% to 6.2% expressed as relative standard deviation. The method was validated by direct infusion tandem mass spectrometry in multiple reaction monitoring to compare results provided by analysis of biofluids and to confirm the identity of the target compounds. Sensitivity and precision were better than or similar to those provided by the confirmatory method. The automation of sample preparation enables to scale-down this step and improves precision by minimization of human intervention, being thus suitable for clinical analysis.
Keywords: Sphingoids; Sphingosine; Laser-induced fluorescence; Solid-phase extraction; Lab-on-valve; Lipidomics

A new method of high-speed cellular protein separation and insight into subcellular compartmentalization of proteins by Evelyn Png; WanWen Lan; Melisa Lazaroo; Silin Chen; Lei Zhou; Louis Tong (767-775).
Transglutaminase (TGM)-2 is a ubiquitous protein with important cellular functions such as regulation of cytoskeleton, cell adhesion, apoptosis, energy metabolism, and stress signaling. We identified several proteins that may interact with TGM-2 through a discovery-based proteomics method via pull down of flag-tagged TGM-2 peptide fragments. The distribution of these potential binding partners of TGM-2 was studied in subcellular fractions separated by density using novel high-speed centricollation technology. Centricollation is a compressed air-driven, low-temperature stepwise ultracentrifugation procedure where low extraction volumes can be processed in a relatively short time in non-denaturing separation conditions with high recovery yield. The fractions were characterized by immunoblots against known organelle markers. The changes in the concentrations of the binding partners were studied in cells expressing short hairpin RNA against TGM-2 (shTG). Desmin, mitochondrial intramembrane cleaving protease (PARL), protein tyrosine kinase (NTRK3), and serine protease (PRSS3) were found to be less concentrated in the 8.5%, 10%, 15%, and 20% sucrose fractions (SFs) from the lysate of shTG cells. The Golgi-associated protein (GOLGA2) was predominantly localized in 15% SF fraction, and in shTG, this shifted to predominantly in the 8.5% SF and showed larger aggregations in the cytosol of cells on immunofluorescent staining compared to control. Based on the relative concentrations of these proteins, we propose how trafficking of such proteins between cellular compartments can occur to regulate cell function. Centricollation is useful for elucidating biological function at the molecular level, especially when combined with traditional cell biology techniques.
Keywords: Centricollation; Centrifugation; Cell function; Trafficking

Microbead-assisted PDA sensor for the detection of genetically modified organisms by Min-Cheol Lim; Yeo-Jae Shin; Tae-Joon Jeon; Hae-Yeong Kim; Young-Rok Kim (777-785).
A simple and sensitive approach for the detection of marker protein, phosphinothricin acetyltransferase, from genetically modified crops was developed based on the colorimetric transition of polydiacetylene (PDA) vesicles in combination with silica microbeads. PDAs have attracted a great deal of interests as a transducing material due to their special features that allow colorimetric response to sensory signals, as well as their inherent simplicity. However, most PDA-based biosensors require additional analytical equipment such as a fluorescence microscope or UV–Vis spectrometer. In this study, we report a new approach to increase the degree of color transition by coupling antibody-conjugated PDA vesicles with silica microbeads in an effort to monitor the results with the unaided eye or simple RGB analysis. By immobilizing PDA vesicles on silica microbeads, we were able to overcome the disadvantages of colloidal PDA-based sensors and increase the degree of colorimetric changes in response to target molecules to a concentration as low as 20 nM. The additional stresses were given to PDA vesicles by antigen–antibody bridging of PDA vesicles coupled with microbeads, resulting in enhanced blue–red color transition. All the results showed that PDA vesicles in conjunction with silica microbeads will be a promising transducing material for the detection of target proteins in diagnostic and biosensing applications.
Keywords: Colorimetric sensor; Polydiacetylene; Silica microbead; PAT; GMO

The activity of the α-l-fucosidase (AFU) enzyme represents an excellent test for diagnosis of hepatocellular carcinoma (HCC) and fucosidosis recognized in inborn disorder of metabolism and increases the sensitivity of detection to 95.5% in patients with HCC. Therefore, the determination of the activity of AFU enzyme is very important and can be used as a screening tool for the early diagnosis of tumors for HCC patients. A simple, accurate, and sensitive potentiometric method was developed for measuring the activity of AFU. The method was based upon measuring the concentration of 2-chloro-4-nitrophenol (2-chloro-4-NP) using a 2-chloro-4-NP-rhodamine B ion pair in a PVC membrane sensor. The electrode shows a linear, reproducible, and stable potentiometric response with an anionic Nernstian slope of −51.13 ± 0.6 mV/decade over a wide range of concentrations 10−5–10−2 M and a detection limit of 1.0 × 10−6 M of 2-chloro-4-NP. The membrane exhibits a fast response time of 30 s, over a pH range of 4.0–6.5. The selectivity coefficients indicate excellent selectivity for 2-chloro-4-NP over a number of interfering species, e.g., chloride, nitrate, sulfate, chromate urea, albumin, glucose, uric acid, and total protein. The prepared sensor has been used successfully for the determination of 2-chloro-4-NP produced from the hydrolysis of 2-chloro-4-NP-α-l-fucopyranoside substrate. It was also applied for the determination α-l-fucosidase enzyme of 33 serum samples of healthy subjects and patients. The average recoveries ± RSD for the healthy subjects, cirrhosis of chronic hepatitis C and B, and HCC serum samples were 102.6 ± 1.01%, 101.5 ± 0.95%, and 100.1 ± 1.1%, respectively. The results obtained are in good agreement with those obtained by standard methods. Figure Potentiometric response of 2-chloro-4-NP using 2-chloro-4-NP-Rd PVC membrane sensor in phosphate buffer of pH 5 at 25 °C
Keywords: Rhodamine B (Rd); Potentiometry; PVC membrane sensor; α-l-Fucosidase enzyme (AFU)

We investigated the electrochemical detection of aspartate transaminase (AST) and alanine transaminase (ALT) by using a multienzyme-modified electrode surface. Determination of the activities of transaminases in human serum is clinically significant because their concentrations and ratios indicate the presence of hepatic diseases or myocardial dysfunction. For electrochemical detection of AST and ALT, enzymes that participate in the reaction mechanism of AST and ALT, such as pyruvate oxidase (POX) and oxaloacetate decarboxylase, were immobilized on an electrode surface by using an amine-reactive self-assembled monolayer and a homobifunctional cross-linker. In the presence of suitable substrates such as l-aspartate (l-alanine) and α-ketoglutarate, AST and ALT generate pyruvate as an enzymatic end product. To determine the activities of AST and ALT, electroanalyses of pyruvate were conducted using a POX and ferrocenemethanol electron shuttle. Anodically generated oxidative currents from multienzyme-mediated reactions were correlated to AST and ALT levels in human plasma. On the basis of the electrochemical analysis, we obtained calibration results for AST and ALT concentrations from 7.5 to 720 units/L in human plasma-based samples, covering the required clinical detection range. Figure POX-OAC calatytic cycles for AST and ALT analysis
Keywords: Electrochemical biosensor; Immobilized enzyme surface; Clinical enzyme analysis; Multienzyme assay

This work reported for the first time the use of flow injection electrochemiluminescence (FI-ECL) sensor for the determination of durabolin in an aqueous system based on CdTe quantum dot (QD) films. Aqueous CdTe colloidal solutions were prepared using thioglycolic acid as a capping agent. Zetasizer Nano ZS (Malvern, UK) was employed to characterize the size of CdTe QDs. The UV–vis and photoluminescence spectra of samples were systematically characterized. Indium tin oxide (ITO) slide glass was modified with CdTe QDs by layer-by-layer self-assembly. CdTe QD films were packed into a homemade cell and used as a recognizer of the FI-ECL sensor to determine durabolin. The intensive anodic ECL emission was obtained at a starting potential of +1.3 V (vs. Ag/AgCl) in a carbonate bicarbonate buffer solution with a pH of 9.93 at an ITO electrode. The ECL intensity was correlated linearly with the concentration of durabolin over the range of 1.0 × 10−8–1.0 × 10−5 g mL−1, and the detection limit was 2.5 × 10−9 g mL−1. The relative standard deviation for the determination of 1.0 × 10−6 g mL−1 durabolin was 1.04% (n = 11). This simple and sensitive sensor revealed good reproducibility for ECL analysis. As a result, the new FI-ECL sensor had been successfully applied to the determination of durabolin in food samples. This strategy could be easily realized and opened new avenues for the applications of QDs in ECL biosensing. Figure This work reported for the first time the flow injection electrochemiluminescence (FI-ECL) sensor for the determination of durabolin in an aqueous system based on CdTe quantum dot (QD) films. Schematic diagram of the FI-CL system and the ECL cell was shown in online abstract figure.
Keywords: Quantum dots (QDs); Self-assembly; Flow injection electroluminescence (FI-ECL); Sensors; Durabolin

In situ laser-induced photochemical silver substrate synthesis and sequential SERS detection in a flow cell by Krisztian Herman; László Szabó; Loredana F. Leopold; Vasile Chiş; Nicolae Leopold (815-820).
A new, simple, and effective approach for multianalyte sequential surface-enhanced Raman scattering (SERS) detection in a flow cell is reported. The silver substrate was prepared in situ by laser-induced photochemical synthesis. By focusing the laser on the 320 μm inner diameter glass capillary at 0.5 ml/min continuous flow of 1 mM silver nitrate and 10 mM sodium citrate mixture, a SERS active silver spot on the inner wall of the glass capillary was prepared in a few seconds. The test analytes, dacarbazine, 4-(2-pyridylazo)resorcinol (PAR) complex with Cu(II), and amoxicillin, were sequentially injected into the flow cell. Each analyte was adsorbed to the silver surface, enabling the recording of high intensity SERS spectra even at 2 s integration times, followed by desorption from the silver surface and being washed away from the capillary. Before and after each analyte passed the detection window, citrate background spectra were recorded, and thus, no “memory effects” perturbed the SERS detection. A good reproducibility of the SERS spectra obtained under flow conditions was observed. The laser-induced photochemically synthesized silver substrate enables high Raman enhancement, is characterized by fast preparation with a high success rate, and represents a valuable alternative for silver colloids as SERS substrate in flow approaches. Figure Setup, in situ prepared silver substrate, and sequentially recorded SERS spectra.
Keywords: SERS; Flow cell; Silver substrate

A mussel tissue certified reference material for multiple phycotoxins. Part 1: design and preparation by Pearse McCarron; Håkan Emteborg; Cíara Nulty; Thomas Rundberget; Jared I. Loader; Katharina Teipel; Christopher O. Miles; Michael A. Quilliam; Philipp Hess (821-833).
The development of multi-analyte methods for lipophilic shellfish toxins based on liquid chromatography–mass spectrometry permits rapid screening and analysis of samples for a wide variety of toxins in a single run. Validated methods and appropriate certified reference materials (CRMs) are required to ensure accuracy of results. CRMs are essential for accurate instrument calibration, for assessing the complete analytical method from sample extraction to data analysis and for verifying trueness. However, CRMs have hitherto only been available for single toxin groups. Production of a CRM containing six major toxin groups was achieved through an international collaboration. Preparation of this material, CRM-FDMT1, drew on information from earlier studies as well as improved methods for isolation of toxins, handling bulk tissues and production of reference materials. Previous investigations of stabilisation techniques indicated freeze-drying to be a suitable procedure for preparation of shellfish toxin reference materials and applicable to a wide range of toxins. CRM-FDMT1 was initially prepared as a bulk wet tissue homogenate containing domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxin-2, yessotoxin and 13-desmethylspirolide C. The homogenate was then freeze-dried, milled and bottled in aliquots suitable for distribution and analysis. The moisture content and particle size distribution were measured, and determined to be appropriate. A preliminary toxin analysis of the final material showed a comprehensive toxin profile. Figure Bottling of CRM-FDMT1
Keywords: CRM-FDMT1; Certified reference material; Shellfish toxins; Phycotoxins; Accuracy; Precision; Liquid chromatography–mass spectrometry

A freeze-dried mussel tissue certified reference material (CRM-FDMT1) containing multiple groups of shellfish toxins has been prepared. Toxin groups present in the material include okadaic acid and the dinophysistoxins, azaspiracids, yessotoxins, pectenotoxins, spirolides and domoic acid. In this work, analytical methods have been examined for the characterisation of the candidate CRM. A comprehensive extraction procedure was developed, which gave good recovery (>98%) for all lipophilic toxins studied. A fast liquid chromatography–mass spectrometry (LC-MS) method was developed that separates the major toxins according to the MS ionisation mode of optimum sensitivity. Matrix effects associated with analysis of these extracts using the developed LC-MS method were assessed. Standard addition and matrix-matched calibration procedures were evaluated to compensate for matrix effects. The methods and approaches will be used for the precise characterisation of the homogeneity and stability of the various toxins in CRM-FDMT1 and for the accurate assignment of certified values. The developed methods also have excellent potential for application in routine regulatory monitoring of shellfish toxins. Figure LC-MS/MS chromatogram of CRM-FDMT1: a mussel tissue CRM containing multiple groups of phycotoxins
Keywords: Shellfish toxins; LC-MS; Sample preparation; Matrix effects; Standard addition; Matrix-matched calibration; CRM-FDMT1

A mussel tissue certified reference material for multiple phycotoxins. Part 3: homogeneity and stability by Pearse McCarron; Håkan Emteborg; Sabrina D. Giddings; Elliott Wright; Michael A. Quilliam (847-858).
A candidate certified reference material (CRM) for multiple shellfish toxins (domoic acid, okadaic acid and dinophysistoxins, pectenotoxins, yessotoxin, azaspiracids and spirolides) has been prepared as a freeze-dried powder from mussel tissues (Mytilus edulis). Along with the certified values, the most important characteristics for a reference material to be fit-for-purpose are homogeneity and stability. Acceptable between-bottle homogeneity was found for this CRM. Within-bottle homogeneity was assessed using domoic acid, and it was shown that repeated subsampling of the CRM can be performed precisely down to 0.35 g. Both short- and long-term stability studies carried out under isochronous conditions demonstrated excellent stability of the various toxins present in the material. While degradation of some analytes was observed at +60°C in short-term studies, it was determined that shipping at ambient temperature is adequate. No instability was detected in long-term stability studies, and it was shown that the material can be held at +18°C safely for up to 1 year. To guarantee stability of the CRM over its lifetime the stock will be maintained at −20°C. The results of the homogeneity and stability testing show that CRM–FDMT1 is appropriate for its intended use in quality assurance and quality control of shellfish toxin analysis methods. Figure Short-term stability of AZA3 in CRM–FDMT1
Keywords: Shellfish toxins; Phycotoxins; CRM–FDMT1; Stability; Homogeneity; LC–MS; HPLC

Solid-phase microextraction (SPME) is a powerful technique, easy to implement for on-site static sampling of indoor VOCs emitted by building materials. However, a major constraint lies in the establishment of calibration curves which requires complex generation of standard atmospheres. Thus, the purpose of this paper is to propose a model to predict adsorption kinetics (i.e., calibration curves) of four model VOCs. The model is based on Fick’s laws for the gas phase and on the equilibrium or the solid diffusion model for the adsorptive phase. Two samplers (the FLEC® and a home-made cylindrical emission cell), coupled to SPME for static sampling of material emissions, were studied. A good agreement between modeling and experimental data is observed and results show the influence of sampling rate on mass transfer mode in function of sample volume. The equilibrium model is adapted to quite large volume sampler (cylindrical cell) while the solid diffusion model is dedicated to small volume sampler (FLEC®). The limiting steps of mass transfer are the diffusion in gas phase for the cylindrical cell and the pore surface diffusion for the FLEC®. In the future, this modeling approach could be a useful tool for time-saving development of SPME to study building material emission in static mode sampling.
Keywords: Volatile organic compounds; Indoor air; Material; Emission; Solid-phase microextraction; Adsorption modeling; Calibration curves prediction

With more stringent legislation on brominated flame retardants, it is expected that increasing amounts of substitutes would replace polybrominated diphenylethers (PBDEs). Therefore, the development and optimization of analytical methodologies that allow their identification and quantification are of paramount relevance. This work describes the optimization of an analytical procedure to determine pentabromochlorocyclohexane, tetrabromo-o-chlorotoluene, 2,3,5,6-tetrabromo-p-xylene, tetrabromophthalic anhydride, 2,3,4,5,6-pentabromotoluene, tris(2,3-dibromopropyl)phosphate, decabromodiphenylethane and 1,2-bis(2,4,6-tribromophenoxy)ethane together with PBDEs in sediments and in suspended particulate matter. This method comprises a pressurized liquid extraction followed by three cleanup steps (gel permeation chromatography and solid phase extraction on Oasis™ HLB and on silica cartridges). Gas chromatography–mass spectrometry, using electron capture negative chemical ionization, is used for the final analysis. The proposed method provides recoveries >85%. The method was applied to sediment and suspended particulate matter samples from different locations in the Western Scheldt estuary (the Netherlands). To the best of our knowledge, this is the first time that the occurrence of the additive flame retardants 2,3,5,6-tetrabromo-p-xylene, 3,4,5,6-tetrabromo-o-chlorotoluene and 2,3,4,5,6-pentabromochlorocyclohexane is reported in the literature. The concentrations of these new flame retardants ranged from 0.05 to 0.30 μg/kg dry weight.
Keywords: Pentabromochlorocyclohexane; Tetrabromo-o-chlorotoluene; 2,3,5,6-Tetrabromo-p-xylene; 2,3,4,5,6-Pentabromotoluene; Decabromodiphenylethane; 1,2-bis(2,4,6-Tribromophenoxy) ethane

Matrix solid-phase dispersion extraction of organophosphorus pesticides from propolis extracts and recovery evaluation by GC/MS by Gloria M. Acosta-Tejada; Salvador Medina-Peralta; Yolanda B. Moguel-Ordóñez; David Muñoz-Rodríguez (885-891).
Five organophosphorus pesticides (dichlorvos, diazinon, malathion, methyl parathion and coumaphos) were extracted from propolis by matrix solid-phase dispersion (MSPD) extraction using octadecylsilica (C18, 1.0 g) as dispersant material. The kind of solvent elution (acetonitrile or ethyl acetate), volume (8 mL and 15 mL), and adsorbent used to clean-up the extracts (graphitized carbon, florisil™ and silica) were optimized using fortified propolis samples (5.0 μg g−1). Recovery was determined by gas chromatography with mass spectrometric detection in selected ion monitoring mode (GC/MS-SIM) and statistical analysis was done to determine better extraction conditions. Relatively high recovery and lower relative standard deviation values (3.1–14.6%) were obtained when analytes were eluted with ethyl acetate from the MSPD column. Diazinon, malathion, methyl parathion, and coumaphos show recoveries of 72.7%, 84.6%, 62.6%, and 78.3%, respectively. In contrast, the recovery for dichlorvos was 53.8%. Additional adsorbents tested for clean-up and increase in solvent elution did not affect recoveries positively and caused a high background in chromatograms. Thus, final conditions were 1 mL of sample, 1 g C18 and 8 mL of ethyl acetate.
Keywords: Organophosphorus pesticides; Propolis; MSPD; GC/MS-SIM