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Analytical and Bioanalytical Chemistry (v.388, #5-6)

Promotion from associate to full professor by Patricia Ann Mabrouk (pp. 987-991).

is Professor of Chemistry and Chemical Biology at Northeastern University (Boston, MA, USA). Her research interests are in chemical education (graduate education, active learning, and undergraduate research), materials science, and bioanalytical chemistry.

Half-titration challenge by Juris Meija; Janis Bisenieks (pp. 993-994).

Solution to the chemical uncertainty principle challenge by Juris Meija (pp. 995-996).

Igor A. Kaltashov, Stephen J. Eyles: Mass spectrometry in biophysics. Conformation and dynamics of biomolecules by Ralf Krüger (pp. 997-998).

Teresa Kowalska, Joseph Sherma (Eds.): Preparative Layer Chromatography by Wolfgang Schwack (pp. 999-1000).

Focus on sample handling by Cristina Nerín (pp. 1001-1002).

has been Professor of Analytical Chemistry at the Centro Politécnico Superior of the University of Zaragoza since 1998. Dr. Nerín’s present research include the study of food packaging materials, which involves developing the analytical methodology required for specific migration analysis as well as designing and developing new active packaging materials with enhanced properties for use as antioxidants, antimicrobials, or materials that are able to supply specific aromas to the food or the product in contact with them. Topics of interest include liquid- and solid-phase microextraction devices and hyphenated techniques that use mass spectrometry to identify the unknowns potentially present in food. Dr. Nerín was the chairwoman of the XII Symposium on Sample Handling in Environmental and Biological Analysis.

Combination of analytical and microbiological techniques to study the antimicrobial activity of a new active food packaging containing cinnamon or oregano against E. coli and S. aureus by R. Becerril; R. Gómez-Lus; P. Goñi; P. López; C. Nerín (pp. 1003-1011).

The aim of this work is the optimization and application of a group of analytical and microbiological techniques in the study of the activity of essential oils (EOs) incorporated in a new antimicrobial packaging material and the research in depth of the interaction between the microbial cells and the individual compounds present in the active material. For this purpose the antimicrobial activity of the active packaging containing cinnamon or oregano was evaluated against E. coli and S. aureus. The vapour phase activity and the direct contact between the antimicrobial agents themselves, or once incorporated in the packaging material, and the microbial cells have been studied. The direct contact was studied using a broth dilution method. The vapour phase was evaluated by using a new method which involves the use of a filter disk containing the EOs. Furthermore, the kill time assay was used to determine the exposure time for the maximum efficiency in packaging, and transmission electron microscopy was used to investigate the antimicrobial activity and the possible mechanism of action against E. coli and S. aureus. Finally, the compounds absorbed by cells were identified. The results showed that the techniques used provide relevant information about the antibacterial activity of cinnamon and oregano in direct contact as well as in the vapour phase. The antimicrobial packaging showed a fast efficiency which supports its likely application as a food packaging material. Bacteria treated with EOs exhibit a wide range of significant abnormalities; these include formation of blebs, coagulation of cytoplasmatic constituents, collapse of the cell structure and lack of cytoplasmatic material. Some of these observations are correlated to the ability of some of these substances to disrupt envelop structure, especially the inner membrane. After an extraction from dead cells, cinnamaldehyde was detected by GC-MS in E. coli exposed to the active packaging containing cinnamon.

Keywords: Cinnamon; Oregano; Active packaging; Antimicrobial plastic films; Microbiological tests; Cell damage; GC-MS analysis; Transmission electron microscopy

Testing organic solvents for the extraction from fish of sulfophenylcarboxylic acids, prior to determination by liquid chromatography-mass spectrometry by D. Álvarez-Muñoz; A. Gómez-Parra; E. González-Mazo (pp. 1013-1019).

The present paper describes the use of different solvent mixtures to extract from fish various sulfophenylcarboxylic acids (SPCs of C₆ to C₁₃), and their originating compounds, linear alkylbenzene sulfonates (LAS of C₁₀ to C₁₃). The analytical method utilized involves pressurized liquid extraction, followed by preconcentration of the samples, purification by solid-phase extraction, and finally identification and quantification of the target compounds by high-performance liquid chromatography-mass spectrometry using a system equipped with an electrospray interface operating in negative ion mode. The SPCs and LAS were extracted from spiked fish first with hexane to remove interference from fats, then with different mixtures of solvents: dichloromethane followed by methanol; 50:50 dichloromethane-methanol; and 30:70 dichloromethane-methanol. The LAS recoveries obtained with these three extraction options were high (between 68.5 and 80.8%); however, owing to the low percentages obtained for SPC homologues (13.5, 13.1, and 15.9%, respectively), another extraction procedure with methanol was developed in order to increase these recoveries. The percentage of recovery for total SPCs with the methanolic extraction was higher (90.1%), with a standard deviation of 9.9, and the LAS recoveries also increased (99.9%). Detection limits were between 1 and 22 ng g⁻¹ for LAS, and between 1 and 58 ng g⁻¹ for SPCs. Quantitation limits were between 4 and 73 ng g⁻¹ for LAS, and between 2 and 193 ng g⁻¹ for SPCs. This method has been applied to measure the biotransformation of 2ØC₁₀ LAS (where Ø is a sulfophenyl group) in fish exposed in a flow-through system, and enabled the separation and identification of SPCs from 5ØC₆ to 9ØC₁₀.

Keywords: Linear alkylbenzene sulfonates; Sulfophenylcarboxylic acids; Mass spectrometry; Environmental analysis; Fish

Microwave-assisted extraction followed by headspace solid-phase microextraction and gas chromatography with mass spectrometry detection (MAE-HSSPME-GC-MS/MS) for determination of polybrominated compounds in aquaculture samples by A. M. Carro; R. A. Lorenzo; F. Fernández; R. Phan-Tan-Luu; R. Cela (pp. 1021-1029).

This paper describes the first validated method for the extraction, purification and determination of trace levels of a number of pollutants of growing concern, including polybrominated biphenyls (PBBs) and polybrominated diphenyl ethers (PBDEs), in aquaculture feeds and products. The new procedure comprises microwave-assisted extraction (MAE; optimized, using a central composite experimental design, to 15 min at 85 °C in 14 mL of 1:1 hexane/dichloromethane), and concentration by headspace solid-phase microextraction (HSSPME), and separation/quantification by gas chromatography with mass spectrometry detection (GC-MS/MS). The method was validated on the reference materials IAEA-406 and WMF-01. Limits of detection for fourteen of the fifteen analytes considered range from 10 to 600 pg g⁻¹, and limits of quantification from 50 pg g⁻¹ to 1.9 ng g⁻¹. Linear ranges, accuracies and precisions are reported.

Keywords: MAE-HSSPME; Brominated flame retardants; Aquaculture feed and products; GC-MS/MS

UPLC–MS as a powerful technique for screening the nonvolatile contaminants in recycled PET by K. Bentayeb; R. Batlle; J. Romero; C. Nerín (pp. 1031-1038).

The possibility of using recycled polyethylene terephthalate as a food contact material is being seriously considered, but the potential migration of nonvolatile compounds from it must be assessed to ensure that it is safe to do so. In the study presented here, four samples of recycled PET were each exposed to three food simulants under the harsh extraction conditions stipulated by European legislation regarding migration tests. The nonvolatile compounds that migrated from them were determined by ultra performance liquid chromatography–mass spectrometry using three different cone voltages, and both positive and negative ionization modes. A total of 36 chemical compounds were detected, some of which were identified, including common additives such as N,N′-di-beta-naphthyl-p-phenylenediamine (antioxidant) and 2,4-di-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl)phenol (light stabilizer) as well as degradation compounds such as ethylene terephthalate dimers and trimers. In addition, specific migration values of three common components of polyethylene terephthalate (diethylene glycol, terephthalic acid, and isophthalic acid) were determined and found to occur at levels of <1 mg/kg—much lower than the specific migration limits stipulated by European legislation.

Keywords: Recycled PET; Nonvolatile; Residuals; Migration; UPLC–MS

A new molecularly imprinted polymer for the on-column solid-phase extraction of diethylstilbestrol from aqueous samples by J. C. Bravo; R. M. Garcinuño; P. Fernández; J. S. Durand (pp. 1039-1045).

The estrogenic compound diethylstilbestrol (DES) is widely studied because of its potential endocrine disruption effects. The prohibition of the use of diethylstilbestrol as a growth promoter has not been enough to ensure the total disappearance of this compound from environmental matrices. Due to the low levels of DES present in the environment, preconcentration and clean up methods are necessary for its analysis. This paper describes the synthesis and use of a molecularly imprinted polymer (MIP) as sorbent for on-column solid-phase extraction of DES from aqueous samples. The selectivity of the DES-MIP was evaluated towards several selected estrogens such as hexestrol (HEX), estrone (E1), estriol (E3), estradiol (E2) and ethynylestradiol (EE2). HPLC-DAD was used to quantify all analytes at 230-nm wavelength. The method has been successfully applied to the analysis of DES in spiked river and tap water samples, with recoveries of 72% and 83% respectively.

Keywords: SPE; Diethylstilbestrol; Molecularly imprinted polymers (MIPs); Water samples

The Ninth International Symposium on Kinetics in Analytical Chemistry (9th KAC) by Aziz Amine (pp. 1047-1047).

was Head of Department of Chemical Engineering and Environment of the University of Hassan II-Mohammedia from 1999 to 2003. Professor Amine’s research over the last 20 years has focused on sensors and biosensors. He is author of more than 100 papers and scientific contributions and has served as coordinator of several national and international research projects. He is also on the editorial board of Biosensors and Bioelectronics.

Fast, sensitive and cost-effective detection of nerve agents in the gas phase using a portable instrument and an electrochemical biosensor by Fabiana Arduini; Aziz Amine; Danila Moscone; Francesco Ricci; Giuseppe Palleschi (pp. 1049-1057).

The nerve agents are chemical warfare agents known to be used during terrorist attacks. An inexpensive and portable system to be used by first responders and military personnel is of interest owing to the continuing threat of possible terrorist attacks. Amperometric biosensors based on cholinesterase inhibition show such potentialities. In this work butyrylcholinesterase was immobilized onto screen-printed electrodes modified with Prussian blue and the nerve agent detection was performed by measuring the residual activity of enzyme. The optimized biosensor was tested with sarin and VX standard solutions, showing detection limits of 12 and 14 ppb (10% of inhibition), respectively. The enzymatic inhibition was also obtained by exposing the biosensors to sarin in gas phase. Two different concentrations of sarin gas (0.1 and 0.5 mg m⁻³) at different incubation times (from 30 s up to 10 min) were tested. It is possible to detect sarin at a concentration of 0.1 mg m⁻³ with 30-s incubation time, with a degree of inhibition of 34%, which match the legal limits (immediate danger to life and health).

Keywords: Biosensor; Nerve agents; Butyrylcholinesterase; Screen printed electrode

Bioanalytical device based on cholesterol oxidase-bonded SAM-modified electrodes by A. Parra; E. Casero; F. Pariente; L. Vázquez; E. Lorenzo (pp. 1059-1067).

A rapid, simple and reproducible two-step method for constructing cholesterol biosensors by covalently bonding cholesterol oxidase (ChOx) to a 3,3′-dithiodipropionic acid di(N-succinimidyl ester) (DTSP)-modified gold electrode is described. Exhaustive characterizations of both the immobilization process and the morphological properties of the resulting ChOx monolayer were performed via a quartz crystal microbalance (QCM) and atomic force microscopy (AFM) operated under liquid conditions, respectively. In addition, scanning electrochemical microscopy (SECM) measurements were performed in order to check that the immobilized enzyme retains its catalytic activity. The replacement of the natural electron acceptor (O₂) in the enzymatic reaction with an artificial mediator, hydroxymethylferrocene (HMF), was also studied. Finally, cholesterol was amperometrically determined by measuring the hydrogen peroxide produced during the enzymatic reaction at +0.5 V. The optimized cholesterol biosensor exhibited a sensitivity of 54 nA mM⁻¹ and a detection limit of 22 μM.

Keywords: Cholesterol oxidase biosensor; SAM-modified electrodes; QCM; AFM; SECM

Modelling of the impedimetric responses of an aflatoxin B₁ immunosensor prepared on an electrosynthetic polyaniline platform by Joseph H. O. Owino; Anna Ignaszak; Amir Al-Ahmed; Priscilla G. L. Baker; Hailemichael Alemu; Jane Catherine Ngila; Emmanuel I. Iwuoha (pp. 1069-1074).

Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. An electrochemical immunosensor for the determination of aflatoxin B₁ (AFB₁) was developed with AFB₁antibody (AFB₁-Ab) immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of the Pt/PANi–PSSA electrode, the Pt/PANi–PSSA/AFB₁-Ab immunosensor and Pt/PANi–PSSA/AFB₁-Ab incubated in bovine serum albumin (BSA) were 0.458, 720 and 1,066 kΩ, respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 kΩ for the Pt/PANi–PSSA electrode to 1,066 kΩ for the Pt/PANi–PSSA/AFB₁-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB₁ immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 kΩ L/mg.

Keywords: Aflatoxin B₁ ; Aflatoxin B₁ antibody; Electrochemical impedance spectroscopy; Differential pulse voltammetry; Immunosensor

Sensitive bioanalysis—combining single-molecule spectroscopy with mono-labeled self-quenching probes by Nicole Marmé; Jens-Peter Knemeyer (pp. 1075-1085).

Fluorescence single-molecule spectroscopy is an appropriate tool for modern bioanalysis. This technique enables the development of ultra sensitive assays, especially when combined with self-quenching probes. In this review we report novel DNA, enzyme, and antibody assays based on mono-labeled fluorescent probes that are quenched by photoinduced electron transfer (PET).

Keywords: Single-molecule spectroscopy; Photoinduced electron transfer (PET); Self-quenching probes; Ultra-sensitive bioanalysis

Development and evaluation of a candidate reference measurement procedure for the determination of testosterone in human serum using isotope dilution liquid chromatography/tandem mass spectrometry by Susan S.-C. Tai; Bei Xu; Michael J. Welch; Karen W. Phinney (pp. 1087-1094).

A candidate reference measurement procedure for total testosterone in human serum involving isotope dilution (ID) coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. The endogenous testosterone and its internal standard (testosterone-d ₃) were extracted from the serum matrix using a combination of solid-phase extraction and liquid–liquid extraction prior to reversed-phase LC/MS/MS. Accuracy of the measurements was evaluated by a recovery study using testosterone-spiked serum. The recovery of the added testosterone ranged from 100.0 to 100.3%. This method was applied to the determination of testosterone in frozen serum samples from three individual donors (one female and two males) with the testosterone concentrations ranging from 0.3 to 8.5 ng g⁻¹. Repeatability with within-set coefficients of variation (CVs) from 0.1 to 1.0% and intermediate precision with between-set CVs from 0.1 to 0.5% for both female and male serum materials were demonstrated. Excellent linearity was obtained for all linear regression lines. The detection limit at a signal-to-noise ratio of approximately 3 was 2 pg of testosterone in serum. Structural analogs as well as testosterone metabolites were tested and found to not interfere with the measurement of testosterone. This well-characterized LC/MS/MS method for serum testosterone, which demonstrates good accuracy and precision, and low susceptibility to interferences, qualifies as a reference measurement procedure that can be used to provide an accuracy base to which routine methods for testosterone can be compared and that will serve as a standard of higher order for measurement traceability.

Keywords: Testosterone; Reference measurement procedure; Electrospray ionization; Isotope dilution; Liquid chromatography/tandem mass spectrometry; LC/MS/MS; Solid-phase extraction; Liquid–liquid extraction

Factorial-design optimization of gas chromatographic analysis of tetrabrominated to decabrominated diphenyl ethers. Application to domestic dust by Jorge Regueiro; Maria Llompart; Carmen Garcia-Jares; Rafael Cela (pp. 1095-1107).

Gas chromatographic analysis of polybrominated diphenyl ethers (PBDEs) has been evaluated in an attempt to achieve better control of the separation process, especially for highly substituted congeners. Use of a narrow-bore capillary column enabled adequate determination of tetra, penta, hexa, hepta, octa, nona and decaBDE congeners in only one chromatographic run while maintaining resolution power similar to that of conventional columns. A micro electron-capture detector (GC–μECD) was used. Chromatographic conditions were optimized by multifactorial experimental design, with the objective of obtaining not only high sensitivity but also good precision. In this way two different approaches to maximizing response and minimizing variability were tested, and are fully discussed. These optimum chromatographic conditions were then used to determine PBDEs extracted from domestic dust samples by microwave-assisted solvent extraction (MASE). Quantitative recovery (90–108%) was achieved for all the PBDEs and method precision (RSD < 13%) was satisfactory. Accuracy was tested by use of the standard reference material SRM 2585, and sub-ng g⁻¹ limits of detection were obtained for all compounds except BDE-209 (1.44 ng g⁻¹). Finally, several samples of house dust were analysed by use of the proposed method and all the target PBDEs were detected in all the samples. BDE-209 was the predominant congener. Amounts varied from 58 to 1615 ng g⁻¹ and the average contribution to the total PBDE burden of 52%. The main congeners of the octaBDE mixture (BDE-183, BDE-197, BDE-207 and BDE-196) also made an important contribution (29%) to the total. These are the first data about the presence of these compounds in European house-dust samples. Finally, the sum of the main congeners in the pentaBDE commercial mixture (BDE-47, BDE-99, and BDE-100) contributed 14% to the total. Figure Polybrominated diphenyl ethers in House Dust

Keywords: Polybrominated diphenyl ethers; Flame retardants; Factorial design; Gas chromatography–μECD; Microwave-assisted solvent extraction; House dust

The dosage of small volumes for chromatographic quantifications using a drop-on-demand dispenser system by Matthias Englmann; Agnes Fekete; Istvan Gebefügi; Philippe Schmitt-Kopplin (pp. 1109-1116).

A commercially available piezo-driven drop-on-demand dispenser was tested for its suitability for the preparation of analytical calibration standards and in a standard addition approach prior to quantitative ultra performance liquid chromatography (UPLC) analysis of homoserines. The reproducibility of the drop-on-demand dosing system was tested and the verification of the droplet volume was performed by preparing a series of 1.0 mg/L caffeine standard solutions from a 1,000.0 mg/L stock solution and analysis of the concentrations obtained by UPLC. The reproducibility was better than 1% relative standard deviation from measurement to measurement and the highest was 1.6% from day to day. The results were compared with the conventional way of generating standard solutions (pipetting). A gravimetric method and a photography-based method for the determination of the average single droplet volume were compared and found to be in very good agreement. The system was employed for the quantification of N-decanoyl homoserine by standard addition in bacterial culture supernatants containing this analyte. The agreement with conventional quantification techniques was high. The paper shows the feasibility of the approach with advantages in low sample and solvent volume consumption and very good reproducibility and reliability combined with easy usage. Figure Ejected droplet, 60 μs after application of the pulse

Keywords: Piezo-driven drop-on-demand dispenser; Ultra performance liquid chromatography; Droplets; Dispenser; Calibration standards; N-Acyl homoserines

Biosensor-guided screening for macrolides by V. Möhrle; M. Stadler; G. Eberz (pp. 1117-1125).

Macrolides are complex polyketides of microbial origin that possess an extraordinary variety of pharmacological properties, paired with an impressive structural diversity. Bioassays for specific detection of such compounds will be of advantage for a class-specific drug screening. The current paper describes a cell-based microbial biosensor, assigning a luminescence response to natural or chemically modified macrolides, independent from their biological activity. This biosensor is based on the coupling of the structural luciferase genes of Vibrio fischeri to the regulatory control mechanism of a bacterial erythromycin resistance operon. The bioassays is easy to handle and can be applied to various screening formats. The feasibility of the test system for natural products screening is exemplified by the isolation and characterization of picromycin from a Streptomyces species. Biosensor-guided screening for macrolides is based on macrolide-promoted expression of lux genes and induction of luminescence (independent of macrolide antibiotic activity)

Keywords: Bioluminescence; Biosensor; Macrolide; Natural product; Reporter gene

Red blood cells do not attenuate the SPCE fluorescence in surface assays by Evgenia G. Matveeva; Ignacy Gryczynski; Anne Barnett; Nils Calander; Zygmunt Gryczynski (pp. 1127-1135).

We describe the positive effect of surface plasmon-coupled fluorescence emission (SPCE) on the detection of a signal from a surface immunoassay in highly absorbing or/and scattering samples. A model immunoassay using fluorescently labeled anti-rabbit antibodies that bind to rabbit immunoglobulin on a silver surface was performed, and the signal was detected in the presence of various highly absorbing and/or scattering solutions or suspensions, such as hemoglobin solution, plastic beads, and red blood cells. The results showed that a highly absorbing solution consisting of small molecules (dye, hemoglobin) attenuates the SPCE signal approximately 2–3-fold. In contrast, suspensions with the same absorption containing large particles (large beads, red blood cell suspension) attenuate the SPCE signal only slightly, approximately 5–10%. Also, a suspension of large undyed, highly scattering beads does not reduce the SPCE signal. The effects on the immunoassay signal of the sample background absorption and scattering, the size of the background particles, and the geometry of the experimental set-up are discussed. We believe that SPCE is a promising technique in the development of biosensors utilized for surface-based assays, as well as any assays performed directly in highly absorbing and/or scattering solutions without washing or separation procedures. Figure Red blood cells (unlike hemoglobin) do not attenuate the SPCE fluorescence in surface assays

Keywords: Fluorescence immunoassays; Surface plasmon-coupled emission; Background suppression; Hemoglobin; Red blood cells

Recognition of oxytocin by capillary electrochromatography with monolithic tetrapeptide-imprinted polymer used as the stationary phase by Chao Zheng; Zhaosheng Liu; Ruyu Gao; Lihua Zhang; Yukui Zhang (pp. 1137-1145).

Using YPLG (Tyr-Pro-Leu-Gly), a tetrapeptide, as the template, an imprinted monolithic column was prepared and applied to the selective recognition of oxytocin based on the epitope approach and capillary electrochromatography (CEC). By optimizing the polymerization solution in terms of functional monomer, cross-linking reagent, porogen, and imprinted template via CEC evaluations of synthesized columns, an imprinted monolith with good recognition capacity (the imprinting factors for YPLG and oxytocin were 4.499 and 4.013, respectively) and high column efficiency (theoretical plates for YPLG and oxytocin were 22,995 plates/m and 16,952 plates/m, respectively) was achieved. In addition, the effects of various experimental parameters on the recognition of oxytocin, including the organic modifier content, the buffer concentration, and the pH value, were studied systematically. Furthermore, a mixture of oxytocin and other proteins was analyzed using this monolithic CEC column, and oxytocin was eluted much more slowly than other large biomolecules, which demonstrated the high selective recognition ability of such an imprinted monolith for oxytocin with PLG (Pro-Leu-Gly) as the epitope. Figure Separation of a mixture of oxytocin, BSA, bovine hemoglobin, ovalbumin, and lysozyme on the open column, the blank monolithic column, and the monolithic YPLG-imprinted column

Keywords: Molecularly imprinted polymer; Capillary electrochromatography; Monolithic column; Epitope approach; Oxytocin

Determination of drug–serum protein interactions via fluorescence polarization measurements by Ulf Mathias; Manfred Jung (pp. 1147-1156).

New fast methods for the determination of pharmacokinetic behaviour of potential drug candidates are receiving increasing interest. We present a new homogeneous method for the determination of drug binding and drug competition for human serum albumin and α₁-acid glycoprotein that is amenable to high-throughput-screening. It is based on selective fluorescent probes and the measurement of fluorescence polarization. This leads to decreased interference with fluorescent drugs as compared with previously published methods based on similar probes and the measurement of fluorescence intensity. The binding of highly fluorescent drugs that still interfere with the probes can be measured by simply titrating the drugs in a two-component system with the serum protein. The assay may also be used to discover strongly binding protein ligands that are interesting for drug-targeting strategies. Additionally, binding data could be obtained from larger libraries of compounds for in silico predictive pharmacokinetics. Figure Fluorescence polarization displacement titration of dansylsarcosine (3D-structure as insert) bound to human serum albumin (HSA) by naproxene

Keywords: Human serum albumin; α₁-acid glycoprotein; High-throughput-screening; Early-ADME tests; Fluorescence polarization

ESI–MS method for in vitro investigation of skin penetration by copper–amino acid complexes: from an emulsion through a model membrane by Lena Mazurowska; Kinga Nowak-Buciak; Mirosław Mojski (pp. 1157-1163).

Copper can be found in many cosmetic formulations, mainly as complexes with peptides, hydroxyacids or amino acids. The main reason that the usage of this element in this context is still increasing is its beneficial biochemical activity, although the mechanism that enables its complexes to permeate through skin barriers is largely unknown. The ability of copper complexes with amino acids to penetrate through the stratum corneum and participate in copper ion transport processes is key to their cosmetic and pharmaceutical activities. The penetration process was studied in vitro in a model system, a Franz diffusion cell with a liposome membrane, where a liquid crystalline system with physicochemical properties similar to those of the intercellular cement of stratum corneum was used to model the skin barrier. The influences of various ligands on the model membrane migration rate of copper ions was studied, and the results highlighted the crucial roles of metal ion complex structure and stability in this process.

Keywords: Copper; Amino acids; Diffusion cell; Model membrane permeation; ESI–MS

Analysis of mismatched DNA by mismatch binding ligand (MBL)–Sepharose affinity chromatography by Yuki Goto; Hitoshi Suda; Akio Kobori; Kazuhiko Nakatani (pp. 1165-1173).

Mismatch binding molecules (MBLs), strongly and selectively bound to the mismatched base pair in duplex DNA, were immobilized on Sepharose. Three MBL–Sepharose columns were prepared with three MBLs, naphthyridine dimer (ND), naphthyridine–azaquinolone (NA), and aminonaphthyridine dimer (amND), which exhibited different binding profiles to the mismatched base pairs. These three MBL–Sepharose columns showed characteristic elution profiles for DNA duplexes containing mismatched base pairs. The ND–Sepharose column separated the G–G and G–A mismatched DNA from fully matched duplexes. The NA–Sepharose column separated the A–A and G–A mismatched DNA from other DNA duplexes. The amND–Sepharose column separated the C–C mismatched DNA. These chromatographic profiles were very consistent with the binding preference of each MBL. By changing the elution conditions from sodium hydroxide to sodium chloride, MBL–Sepharose columns were also able to separate the mismatched DNA that weakly bound to the MBL from fully matched DNA duplex. Figure MBL-Sepharose affinity chromatography successfully separates the mismatched duplex DNA from fully matched duplex.

Keywords: Mismatch binding ligand; Affinity chromatography; Mutation detection; Melting temperature; Single nucleotide polymorphism

Multiwell fluorometric and colorimetric microassays for the evaluation of beta-secretase (BACE-1) inhibitors by Francesca Mancini; Marina Naldi; Vanni Cavrini; Vincenza Andrisano (pp. 1175-1183).

The amyloid beta (Abeta) peptide is responsible for toxic amyloid plaque formation and is central to the aetiology of Alzheimer’s disease (AD). It is generated by proteolytic processing of the amyloid precursor protein (APP) by beta-secretase (BACE-1) and gamma-secretase. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach to the treatment of Alzheimer’s disease. This paper reports on improved microtiter plate-based fluorescence and colorimetric assays for the high-throughput screening (HTS) of BACE-1 inhibitors achieved by employing, for the first time, casein fluorescein isothiocyanate (casein-FITC) and N-α-benzoyl-D,L-arginine p-nitroanilide (BAPNA) as substrates, since they are known to be readily available and convenient substrates for proteases. The methods are based on the fluorescence enhancement following casein-FITC proteolysis and the visible absorbance of the p-nitroaniline (pNA) produced by BAPNA hydrolysis, with both reactions catalysed by BACE-1. Casein-FITC is a high-affinity substrate (K _m = 110 nM) for BACE-1, more so than the Swedish (SW) type peptide (a peptide containing the Swedish mutant of APP, a familiar mutation that enhances Abeta production). BACE-1 catalysis of casein-FITC proteolysis exhibited Michaelis–Menten kinetic. Therefore, it was found that BACE-1 was saturable with casein-FITC that was processed in a time- and pH-dependent manner with greater catalytic efficiency than observed for the SW peptide. The enantioselective hydrolysis of L-BAPNA by BACE-1 was observed. l-BAPNA was hydrolysed ten times more efficiently by BACE-1 than the WT (wild-type peptide). The novel methods were validated using a FRET assay as an independent reference method. Therefore, in order to select new leads endowed with multifunctional activities, drugs for Alzheimer’s disease (AD)—potent acetylcholinesterase (AChE) inhibitors—were tested for BACE-1 inhibition using the proposed validated assays. Among these, donepezil, besides being an acetylcholinesterase inhibitor, was also found to be a BACE-1 inhibitor that displayed submicromolar potency (170 nM).

Keywords: Beta-secretase; Multiwell assay; Casein-FITC; BAPNA; Alzheimer’s disease drugs

Gold nanoparticle aggregation-based highly sensitive DNA detection using atomic force microscopy by Minh-Phuong Ngoc Bui; Taek Jin Baek; Gi Hun Seong (pp. 1185-1190).

The potential ability of atomic force microscopy (AFM) as a quantitative bioanalysis tool is demonstrated by using gold nanoparticles as a size enhancer in a DNA hybridization reaction. Two sets of probe DNA were functionalized on gold nanoparticles and sandwich hybridization occurred between two probe DNAs and target DNA, resulting in aggregation of the nanoparticles. At high concentrations of target DNA in the range from 100 nM to 10 μM, the aggregation of gold nanoparticles was determined by monitoring the color change with UV-vis spectroscopy. The absorption spectra broadened after the exposure of DNA–gold nanoparticles to target DNA and a new absorption band at wavelengths >600 nm was observed. However, no differences were observed in the absorption spectra of the gold nanoparticles at low concentrations of target DNA (10 pM to 10 nM) due to insufficient aggregation. AFM was used as a biosensing tool over this range of target DNA concentrations in order to monitor the aggregation of gold nanoparticles and to quantify the concentration of target DNA. Based on the AFM images, we successfully evaluated particle number and size at low concentrations of target DNA. The calibration curve obtained when mean particle aggregate diameter was plotted against concentration of target DNA showed good linearity over the range 10 pM to 10 nM, the working range for quantitative target DNA analysis. This AFM-based DNA detection technique was three orders of magnitude more sensitive than a DNA detection method based on UV-vis spectroscopy.

Keywords: AFM; Gold nanoparticles; Sandwich DNA hybridization; DNA detection

Fluorescence-detecting cationic surfactants using luminescent CdTe quantum dots as probes by Xue-Lian Diao; Yun-Sheng Xia; Tian-Long Zhang; Yan Li; Chang-Qing Zhu (pp. 1191-1197).

A novel fluorescence quenching method for the determination of cationic surfactants (CS), specifically cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), and cetylpyridinium chloride (CPC), has been developed using water-soluble luminescent CdTe quantum dots (QDs) modified with thioglycolic acid (TGA). The possible interference from heavy and transition metals (HTM) has been efficiently eliminated through simple sample treatment with mercapto cotton made in-house. Under optimum conditions, the extent of fluorescence quenching of CdTe QDs is linearly proportional to the concentration of CS from 2.0 × 10⁻⁷ to 7.0 × 10⁻⁶ mol L⁻¹ with a detection limit of 5.0 × 10⁻⁸ mol L⁻¹. The relative standard deviation for 1.0 × 10⁻⁶ mol L⁻¹ CTAB is 2.5% (n = 6). The proposed method exhibits high sensitivity and selectivity and furthermore avoided the use of toxic organic solvents and tedious solvent extraction procedures. It has been applied to the determination of trace CS in natural river water and commodity samples with satisfactory results. Potential interference from heavy and transition metals is eliminated during photoluminescence detection of CS through simple sample pre-treatment with mercapto cotton

Keywords: Cationic surfactants; CdTe Quantum dots; Fluorescence quenching; Mercapto cotton

Detection of flavonoids and assay for their antioxidant activity based on enlargement of gold nanoparticles by Jing Wang; Nandi Zhou; Zhiqiang Zhu; Junyi Huang; Genxi Li (pp. 1199-1205).

We report our findings that natural flavonoids such as quercetin, daizeol and puerarin can act as reductants for the enlargement of gold nanoparticles (Au-NPs). Consequently, the UV–vis spectra of a solution containing Au-NPs will be gradually changed, and the molecules of the natural herbs can be detected by making use of changes in the UV–visible spectra. Furthermore, we have prepared a self-assembled monolayer modified electrode by modifying cysteamine on a gold substrate electrode, which is further modified by some Au-NP seeds. When the modified electrode is immersed in a solution containing flavonoids and tetrachloroauric acid as a gold source for the growth of the Au-NP seeds, with the increase of the concentration of flavonoids, the Au-NP seeds on the surface of the modified electrode can be enlarged to varying degrees. As a result, the peak currents in the corresponding cyclic voltammograms are inversely decreased, and simultaneously the peak separation is increased. Therefore, an electrochemical method to detect flavonoids is also proposed. Compared with the optical detection method, the electrochemical method has an extraordinarily lower detection limit and a significantly extended detection range. Moreover, the optical and electrochemical experimental results can be also used to assay and compare the relative antioxidant activities of the flavonoids. Figure Enlargement of Au nanoparticles by flavonoids at cysteamine modified electrode

Keywords: Gold nanoparticles; Flavonoid; Antioxidant; Detection

Fourier-transform infrared (FTIR) spectroscopy for monitoring and determining the degree of crystallisation of polyhydroxyalkanoates (PHAs) by Mustafa Kansiz; Ana Domínguez-Vidal; Don McNaughton; Bernhard Lendl (pp. 1207-1213).

FTIR spectroscopy has been used to monitor and determine the degree of crystallisation in a sample of polyhydroxybutyrate-co-14%valerate (PHB–co-14%HV). Time series spectra of solution-cast films of the polymer revealed spectral changes attributed to the onset of crystallisation. Curve fitting was used to obtain an absolute measure of crystallinity. Mean centred principal-component analysis (PCA) revealed that 99.9% of the spectral variance could be attributed to factor 1. The loadings plot for factor 1 contained features attributable to crystalline and amorphous phases. These features were opposite in sign, indicating that changes in the spectra with the onset of crystallisation are simultaneous and opposite in direction, i.e. as the crystalline band increases the amorphous band decreases. Cross-peaks in asynchronous 2D correlation maps indicate there are likely to be very minor components that are changing out of phase. The presence of these minor components is supported by examination of the loadings of higher factors in the PCA model. PCA has been shown to be suitable for determining the number of dynamic spectral features and has enabled relative and objective monitoring of crystallisation kinetics.

Keywords: FTIR; Polyhydroxyalkanoates (PHA); Crystallinity; Principal-component analysis (PCA); Curve fitting; 2D correlation analysis

Determination of molar absorptivity coefficients for major type-B trichothecenes and certification of calibrators for deoxynivalenol and nivalenol by Rudolf Krska; Patricia Schubert-Ullrich; Ralf D. Josephs; Håkan Emteborg; Gerhard Buttinger; Hans Pettersson; Hans P. van Egmond; Ronald C. Schothorst; Susan MacDonald; Danny Chan (pp. 1215-1226).

This paper presents results from the European Commission-funded project Doncalibrant, the objective of which was to produce calibrators with certified mass fractions of the Fusarium toxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-Ac-DON), 15-acetyldeoxynivalenol (15-Ac-DON), and nivalenol (NIV), in acetonitrile. The calibrators, available in ampoules, were sufficiently homogeneous, with between-bottle variations (s _bb) of less than 2%. Long-term stability studies performed at four different temperatures between −18 and 40 °C revealed no significant negative trends (at a confidence level of 95%). Molar absorptivity coefficients (in L mol⁻¹ cm⁻¹) were determined for all four toxins (DON: 6805 ± 126, NIV: 6955 ± 205, 3-Ac-DON: 6983 ± 141, 15-Ac-DON: 6935 ± 142) on the basis of a mini-interlaboratory exercise. The overall uncertainty of the calibrators’ target values for DON and NIV were evaluated on the basis of gravimetric preparation data and include uncertainty contributions from possible heterogeneity, storage, and transport. The Doncalibrant project resulted in the production of calibrators for DON (IRMM-315) and NIV (IRMM-316) in acetonitrile with certified mass fractions of 25.1 ± 1.2 μg g⁻¹ and 24.0 ± 1.1 μg g⁻¹, respectively. Both CRMs became commercially available from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) at the beginning of 2007.

Keywords: Mycotoxin; Certified reference material; Calibrator

Determination of two phototransformation products of bentazone using quadrupole time-of-flight mass spectrometry by Manuela Peschka; Mira Petrovic; Thomas P. Knepper; Damià Barceló (pp. 1227-1234).

The transformation products 2-(isopropylcarbamoyl)phenylsulfamic acid and 2-(1-hydroxypropane-2-yl)-1,2-dihydroindazol-3-one could be determined during the photolysis of the herbicide bentazone. Degradation experiments were carried out with different types of water in a natural sunlight simulating system. Besides the anticipated hydroxylated bentazone, the second transformation product was identified by means of exact mass measurement using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QqToF MS). Both phototransformation products occurred in all water types tested. The required irradiation time was matrix dependent. 2-(Isopropylcarbamoyl)phenylsulfamic acid was detected in a drainage channel in the Ebro river delta (Catalonia, Spain).

Keywords: Bentazone; Photolysis; Structure elucidation; Quadrupole time-of-flight mass spectrometry

The photochemical behaviour of five household pyrethroid insecticides and a synergist as studied by photo-solid-phase microextraction by M. Fernández-Álvarez; M. Lores; M. Llompart; C. García-Jares; R. Cela (pp. 1235-1247).

In the present study, solid-phase microextraction in photochemical studies was used to investigate UV light induced photodegradation of five pyrethroids (empenthrin, transfluthrin, allethrin, phenothrin and cyphenothrin) and a synergist (piperonyl butoxide), which are common ingredients of household insecticides. Gas chromatography coupled with mass spectrometry was used to separate and tentatively identify the parent compounds and their corresponding photoproducts generated in the same polydimethylsiloxane fibre. Kinetics curves were obtained and apparent first-order rate constants and half-lives were estimated. Twenty-six photoproducts were tentatively identified and photodegradation pathways for the compounds investigated were proposed. It is a matter of some concern that three of the photoproducts identified [(3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzaldehyde and (3-phenoxyphenyl)methanol] have been reported to be endocrine disruptors. There is no record of previous studies of cyphenothrin and empenthrin photodegradation, and therefore the present study represents the first attempt to elucidate the photochemical behaviour of these compounds. Figure Photo-SPME for Pyrethroids

Keywords: Synthetic pyrethroids; Piperonyl butoxide; UV photodegradation; Photo-solid-phase microextraction; Gas chromatography–mass spectrometry

Monitoring of the surface of paper samples exposed to UV light by ATR-FT-IR spectroscopy and use of multivariate control charts by Elisa Robotti; Marco Bobba; Andrea Panepinto; Emilio Marengo (pp. 1249-1263).

The effect of exposure of paper samples to UV light was monitored by use of ATR-FT-IR spectroscopy and multivariate statistical tools. Three types of paper were tested: common laser-printer paper, newsprint, and thermal fax paper. The samples were first characterised by ATR-FT-IR spectroscopy to determine natural experimental variability. They were then exposed to UV light for 30 h and the effects of the exposure were monitored by use of the same spectroscopic technique. Finally, multivariate statistical tools were applied to the final dataset, coupled with construction of multivariate control charts, to identify the effects of UV light on the sample surfaces.

Keywords: Paper; ATR-FT-IR spectroscopy; Multivariate control charts; UV light; Accelerated ageing; Industrial assessment of paper stability

Trace analysis of sulfonylurea herbicides and their metabolites in water using a combination of off-line or on-line solid-phase extraction and liquid chromatography–tandem mass spectrometry by François Perreau; Philippe Bados; Lucien Kerhoas; Sylvie Nélieu; Jacques Einhorn (pp. 1265-1273).

Two alternatives for the rapid simultaneous quantification of six sulfonylurea herbicides and five of their main degradation products in natural water are proposed. For concentration, the compounds were extracted on a polystyrene–divinylbenzene solid phase under pH and elution conditions that suppressed any hydrolysis. The eluates were analysed by liquid chromatography coupled to electrospray tandem mass spectrometry within 20 min. The whole method was validated and shown to give no hydrolysis artefacts. The application of off-line and on-line SPE of sulfonylureas enabled the 0.1 μg L⁻¹ and 1 ng L⁻¹ LOQ levels to be reached, respectively. The on-line SPE–LC–MS–MS method allowed the accurate quantitation of all sulfonylureas and three degradation products at 0.1 μg L⁻¹ or below in natural water, with an average repeatability of 8%.

Keywords: Herbicide; Sulfonylurea; Degradation product; On-line solid-phase extraction; LC–MS–MS analysis

Determination of acetone in seawater using derivatization solid-phase microextraction by Edward D. Hudson; Kadek Okuda; Parisa A. Ariya (pp. 1275-1282).

Acetone plays an important role in the chemistry of both the atmosphere and the ocean, due to its potential effect on the tropospheric HO_x (= HO + HO₂) budget, as well as its environmental and health effects. We discuss the development of a mobile, sensitive, selective, economical and facile method for the determination of acetone in seawater. The method consists of derivatizing acetone to its pentafluorobenzyl oxime using 1,2,3,4,5-pentafluorobenzylhydroxylamine (PFBHA), followed by solid-phase microextraction (SPME) and analysis by gas chromatography/mass spectrometry (GC/MS). A detection limit of 3.0 nM was achieved. The buffering capacity of seawater imposes challenges in using the method’s optimum pH (3.7) on seawater samples, requiring calibration standards to be made in buffered salt water and the acidification of seawater samples and standards prior to extraction. We employed the technique for analysis of selected surface seawater samples taken on the Nordic seas during the ARK-XX/1 cruise (R.V. Polarstern). An upper limit of 5.5–9.6 nM was observed for acetone in these waters, the first acetone measurements reported for far North Atlantic and Arctic waters. Simplified schematic of transformations of organic compounds at the atmosphere–ocean interface

Keywords: Ketones; Volatile organic compounds; Gas chromatography/mass spectrometry; Dissolved organic carbon

Optimisation of a selective method for the determination of organophosphorous triesters in outdoor particulate samples by pressurised liquid extraction and large-volume injection gas chromatography–positive chemical ionisation–tandem mass spectrometry by José Benito Quintana; Rosario Rodil; Purificación López-Mahía; Soledad Muniategui-Lorenzo; Darío Prada-Rodríguez (pp. 1283-1293).

A selective analytical method for the determination of nine organophosphate triesters and triphenylphosphine oxide (TPPO) in outdoor particulate matter is presented. It involves a fully automated pressurised liquid extraction (PLE) step, integrating an alumina clean-up process, and subsequent determination by large-volume injection gas chromatography–positive chemical ionisation–tandem mass spectrometry (LVI-GC–PCI–MS/MS). The extraction variables (solvent, amount of adsorbent, temperature, time and number of cycles) were optimised using a multicriteria strategy which implements a desirability function that maximises both extraction and clean-up efficiencies while searching for the best-compromise PLE conditions. The final method affords quantification limits of between 0.01 and 0.3 μg g⁻¹ and recoveries of >80%, with the exceptions of the most polar analytes, TCEP and TPPO (~65%) for both urban dust and PM10 samples. Moreover, the method permitted the levels of these compounds in dust deposited outdoors (between LOD and 0.5 μg g⁻¹ for TEHP) and PM10 samples (between LOD and 2.4 μg m⁻³ for TiBP) to be measured and reported for the first time.

Keywords: Organophosphorous triesters; Flame retardants; Plasticizers; Urban dust; Particulate matter; PM10; Pressurised liquid extraction; Accelerated solvent extraction; GC–MS; Experimental design; Multicriteria optimisation; Desirability function

Combined use of total metal content and size fractionation of metal biomolecules to determine the provenance of pine nuts (Pinus pinea) by J. L. Gómez-Ariza; A. Arias-Borrego; T. García-Barrera (pp. 1295-1302).

Four essential elements (Mn, Ni, Zn, and Cu) and their molecular-size distribution patterns have been determined, for twenty four samples of pine nuts from eight areas in Spain and Portugal (Huelva, Cádiz, Badajoz, Cataluña, Castilla, Madrid, Faro, and Coimbra), by size-exclusion liquid chromatography (SEC) coupled on-line to UV and inductively coupled plasma mass spectrometric (ICP–MS) detection. The variability observed in total element content and the size-exclusion profiles of elements in samples from distant areas were considered as chemical descriptors for characterization of geographic origin. A pattern-recognition technique, the display method principal component analysis, was used as visualization technique to determine the provenance of the pine nuts collected. The results obtained confirmed that size fractionation profiles give more information for assessing the provenance of pine nuts than the total elements composition traditionally used for this purpose. Combination of these chemical descriptors was the most suitable choice for the samples studied. Figure This paper shows the application of an analytical approach based on total elements concentrations and the relative abundance of metal-biomolecules, estimated by the size-exclusion fractions, as chemical descriptors to determine the provenance of pine nuts. Principal component analysis (PCA) has been used as a visualization technique.

Keywords: Pine nuts; Metals; Multielement molecular-size fractionation; Size-exclusion chromatography; Inductively coupled plasma–mass spectrometry (ICP–MS); Food provenance

A critical comparison of analytical flow systems exploiting streamlined and pulsed flows by Ana C. B. Dias; Joâo L. M. Santos; José L. F. C. Lima; Cristina M. Quintella; Angelo M. V. Lima; Elias A. G. Zagatto (pp. 1303-1310).

Multipumping (MPFS) and multicommuted (MCFS) flow systems relying on pulsed and laminar flows were critically compared. The mixing conditions and dispersion associated with both systems were evaluated by simulating the sample with bromocresol green. The molybdenum blue method for phosphate determination in soil extracts was also implemented in both flow systems. Furthermore, laser-induced fluorescence (LIF) was applied to visualize the dispersing sample; rhodamine B was used as the fluorescent species. The pulsed flow enhanced the mixing of the solutions involved, thus reducing reagent consumption (48 and 96 μl for MPFS and MCFS), and improving sampling rate (67 and 144 h⁻¹ for MCFS and MPFS). For phosphate determination, results obtained with both systems were precise (r.s.d. < 0.5%; n = 10) and accurate. Analyses of the absorbance vs time/space LIF plots revealed that exploitation of pulsed flow led to a pronounced radial dispersion and to a limited axial dispersion, typical aspects of turbulent flows.

Keywords: Flow analysis; Pulsed flow; Multipumping; Multicommutation

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Analytical and Bioanalytical Chemistry (v.388, #5-6)