Analytical and Bioanalytical Chemistry (v.395, #4)

Antibiotics in food and environmental samples by María Cruz Moreno-Bondi (875-876).
is Professor of Analytical Chemistry at Complutense University in Madrid (Spain). Her main areas of research include the development of optical chemical sensors and biosensors based on fluorescence measurements and their application to environmental and food analysis, and the synthesis of biomimetic recognition elements for sensing and separation purposes. She received the Young Researcher’s Award from the Spanish Society of Analytical Chemistry in 1993. She is a member of the executive boards of the Spanish Society of Applied Spectroscopy, the Madrid Community Section of the Royal Spanish Society of Chemistry and also of the Advanced Study Course on Optical Sensors (ASCOS) series.

Antibiotics in food: Legislation and validation of analytical methodologies by R. Companyó; M. Granados; J. Guiteras; M. D. Prat (877-891).
In this paper, European food safety legislation is presented, and special attention is devoted to monitoring residues of veterinary drugs in foodstuffs of animal origin. After a short review of the state of the art of analytical methodology for antibiotic residue analysis, the paper focuses on validation of analytical methods, with Decision 657/2002/EC as reference document. Finally, the main issues of the quality control of the analytical data, i.e. analysis of reference materials and participation in proficiency tests, are briefly addressed.
Keywords: Residues of antibiotics; Food analysis; Method validation; Legislation; Quality control; Decision 2002/657/EC

Monitoring of food products from animal origin for the presence of antimicrobial residues is preferably done using microbial screening methods because of their high cost-effectiveness. Traditionally applied methods fail to detect the maximum residue limits which were established when EU Council Regulation 2377/90 came into effect. Consequently, during the last decade this has led to the development of improved microbial screening methods. This review provides an overview of the efforts expended to bring antibiotic screening methods into compliance with EU legislation. It can be concluded that the current situation is still far from satisfactory. Figure Microbial inhibition assay for muscle samples
Keywords: Drug monitoring/drug screening; Antibiotic residues; Microbial screening methods

Review on immunoanalytical determination of tetracycline and sulfonamide residues in edible products by Nuria Pastor-Navarro; Ángel Maquieira; Rosa Puchades (907-920).
The state of the art of immunoanalysis for the determination of tetracycline and sulfonamide residues in food products is reviewed. Special attention is paid to the design and synthesis of haptens, providing an overview of the efforts spent on developing antibiotic screening methods to determine residue levels in agreement with the legislation. The results and observations published, focused on tetracycline and sulfonamide enzyme-linked immunosorbent assays, are discussed.
Keywords: Tetracycline; Sulfonamide; Enzyme-linked immunosorbent assay; Edible products

An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples by María Cruz Moreno-Bondi; María Dolores Marazuela; Sonia Herranz; Erika Rodriguez (921-946).
Antibiotics are a class of pharmaceuticals that are of great interest due to the large volumes of these substances that are consumed in both human and veterinary medicine, and due to their status as the agents responsible for bacterial resistance. They can be present in foodstuffs and in environmental samples as multicomponent chemical mixtures that exhibit a wide range of mechanisms of action. Moreover, they can be transformed into different metabolites by the action of microorganisms, as well as by other physical or chemical means, resulting in mixtures with higher ecotoxicities and risks to human health than those of the individual compounds. Therefore, there is growing interest in the availability of multiclass methods for the analysis of antimicrobial mixtures in environmental and food samples at very low concentrations. Liquid chromatography (LC) has become the technique of choice for multiclass analysis, especially when coupled to mass spectrometry (LC-MS) and tandem MS (LC-MS2). However, due to the complexity of the matrix, in most cases an extraction step for sample clean-up and preconcentration is required before analysis in order to achieve the required sensitivities. This paper reviews the most recent developments and applications of multiclass antimicrobial determination in environmental and food matrices, emphasizing the practical aspects of sample preparation for the simultaneous extraction of antimicrobials from the selected samples. Future trends in the application of LC-MS-based techniques to multiclass antibiotic analysis are also presented.
Keywords: Antibiotic monitoring; Environmental analysis; Food analysis; Extraction; LC-MS

Residual antimicrobials in food constitute a risk to human health. Although epidemiological data on the real magnitude of their adverse effects are very scarce, they indicate that food could be an important vehicle for evolution and dissemination of antimicrobial-resistant bacteria. Public health agencies in many countries rely on detection by mass spectrometry (MS) for unambiguous identification of residues of antimicrobial agents in animal food products for human consumption. The introduction of relatively inexpensive and robust liquid chromatography (LC)–MS systems has given a strong impulse to the development of confirmatory methods for the above medicines in foodstuffs. The initial part of this review, after a brief introduction into the field of antimicrobials, is dedicated to the most important EU regulations and directives for control of residues of these substances in animal products. The main attention in this review is on the sample-treatment and MS detection systems in use today for analysing the most important classes of antimicrobials in various biological matrices (milk, animal tissues, eggs, and honey). As evidenced by this review, reversed-phase LC combined with tandem MS, usually triple-quadrupole MS (QqQMS), is currently the preferred technique in most residue analysis of a single-class of antimicrobials. A recently emerging analytical strategy is that of developing methods for detecting a large variety of veterinary drugs belonging to different classes, including pesticides (multi-class residue analysis). To do this, simple and generic extraction and separation techniques applicable to a broad range of compounds differing in physical and chemical properties have been adopted. Such methods are still based mainly on LC–QqQMS. Emerging alternative MS detection systems are time-of-flight MS, which provides accurate mass of the analyte(s), or Q–linear ion trap (IT) MS that eliminates some limitations of ITMS n .
Keywords: Drug monitoring/drug screening; Extraction (SFE, SPE, SPME); HPLC; Foods/beverages; Mass spectrometry, ICP–MS

Applications of capillary electrophoresis to the determination of antibiotics in food and environmental samples by Ana M. García-Campaña; Laura Gámiz-Gracia; Francisco J. Lara; Monsalud del Olmo Iruela; Carmen Cruces-Blanco (967-986).
In this paper we review applications of capillary electrophoresis (CE) to the determination of antibiotic residues in food derived from animals and in environmental samples. Although many CE methods have been used to determine antibiotics in the pharmaceutical field (drug quality control or therapeutic monitoring in biological samples), food and environmental applications have been increasing in recent years. Due to the maximum residue limits established by the EU, in Directive 2377/90/EEC, for foodstuffs of animal origin and considering the low levels that can be found in environmental or waste waters or soils, different strategies to increase sensitivity have been developed, including off-line preconcentration, on-line stacking modes to use higher sample volumes, or in-line solid-phase extraction. Also, several detection techniques, such as fluorescence, laser-induced fluorescence, electrochemical detection, or mass spectrometry have been used; the last of these also enables unequivocal identification of the residues, required by Commission Decision 2002/657/EC. All these aspects will be discussed in this paper, in relation to the main groups of antibiotics used in veterinary and human medicine, for which applications in food and environmental samples have been developed by using CE as an efficient alternative to liquid chromatography.
Keywords: Capillary electrophoresis; Antibiotics; Environmental analysis; Food analysis

Analysis of antibiotics in fish samples by F. Cañada-Cañada; A. Muñoz de la Peña; A. Espinosa-Mansilla (987-1008).
The use of antibiotics in food-producing animals has generated considerable interest because the widespread administration of these drugs may lead to the development of resistant human pathogens. A large increase in the demand for seafood products has occurred in the last century. This has led to a concomitant increase in high-intensity aquaculture methods, characterized by high stock density and volume, and the heavy use of formulated feeds containing antibiotics, among other substances. Therefore, accurate and sensitive determination of antibiotic residues is now a necessity. In order to protect human health, the European Union and other regulatory authorities worldwide have established maximum residue limits (MRL) for antibiotic residues in animal products entering the human food chain. This paper reviews the most recent methods for analysis of antibiotic residues in fish.
Keywords: Antibiotics; Fish; Analysis

Traceability of sulfonamide antibiotic treatment by immunochemical analysis of farm animal hair samples by Javier Adrian; Marta Gratacós-Cubarsí; Francisco Sánchez-Baeza; Jose-Antonio Garcia Regueiro; Massimo Castellari; M.-Pilar Marco (1009-1016).
The use of hair to trace use of unauthorized substances, therapeutic agents, or their misuse is becoming very attractive since residues can be detected for a long time after treatment. For this purpose, an indirect enzyme-linked immunosorbent assay (ELISA) has been evaluated for its capability to trace sulfonamide antibiotic treatment by analyzing cattle and pig hair samples. Pigmented and nonpigmented hair samples from control and sulfamethazine (SMZ)-treated pigs and calves were collected, extracted under different alkaline conditions, and analyzed by ELISA after just diluting the extracts with the assay buffer. Data analysis following the European recommendations for screening methods demonstrates that the ELISA can detect SMZ in hair samples with a limit of detection (90% of the zero dose (IC90)) between 30 and 75 ng g−1. The same samples have been analyzed by HPLC after a dual solid-phase extraction. The ELISA results matched very well those obtained by the chromatographic method, demonstrating that the immunochemical method can be used as a screening tool to trace animal treatments. Between the benefits of this method are the possibility to directly analyze hair extracts with sufficient detectability and its high-throughput capability. Preliminary validation data are reported using an experimental approach inspired on the Commission Decision 2002/657/EC criteria for screening methods.
Keywords: Sulfonamide antibiotics; Sulfamethazine; Hair analysis; ELISA; Immunoassay; HPLC-DAD; Treatment traceability

is Associate Professor in the Department of Chemistry at the Graduate School of Science, Hiroshima University, where he performs in situ microscopic investigations on reactions and studies phenomena occurring at liquid–liquid interfaces.

Spectroscopic studies of molecular interaction at the liquid–liquid interface by Jilska M. Perera; Geoffrey W. Stevens (1019-1032).
The development of techniques to study the liquid–liquid interface is a major challenge. Spectroscopy in all its forms provides a powerful method of investigation, especially when combined with other optical techniques. Over the last 30 years, there have been significant developments in the methods for studying heterogeneous interfaces. As technology progresses, the sensitivity of existing techniques has been improved but there are major challenges still to be met, such as the measurement of interfacial dielectric constant and viscosity. This paper aims to summarise the use of spectroscopy to study molecular interactions at the liquid–liquid interface.
Keywords: Liquid–liquid interface; Spectroscopy; Interfacial phenomena

Some new experimental methods for measuring the optical chirality of molecular aggregates formed at liquid–liquid interfaces have been reviewed. Chirality measurements of interfacial aggregates are highly important not only in analytical spectroscopy but also in biochemistry and surface nanochemistry. Among these methods, a centrifugal liquid membrane method was shown to be a highly versatile method for measuring the optical chirality of the liquid–liquid interface when used in combination with a commercially available circular dichroism (CD) spectropolarimeter, provided that the interfacial aggregate exhibited a large molar absorptivity. Therefore, porphyrin and phthalocyanine were used as chromophoric probes of the chirality of itself or guest molecules at the interface. A microscopic CD method was also demonstrated for the measurement of a small region of a film or a sheet sample. In addition, second-harmonic generation and Raman scattering methods were reviewed as promising methods for detecting interfacial optical molecules and measuring bond distortions of chiral molecules, respectively.
Keywords: Liquid–liquid interface; Interfacial aggregation; Optical chirality; Centrifugal liquid membrane method; Porphyrin and phthalocyanine

Rhombic-ordered microdomains of diprotonated 5,10,15,20-tetraphenylporphine aggregate, whose sizes were 10–200 µm, were formed at dodecane/aqueous H2SO4 interfaces. The light excitation of their two absorption bands (410 and 473 nm for H- and J-bands, respectively) led to one fluorescence band at longer wavelength (723 nm). The direction of the emission transition dipole moment (µ e) of individual rhombic microdomains, determined with an in situ optical microscope and a linear polarizer, was almost parallel to the major axis, which was also almost parallel to the direction of the absorption transition dipole moment of their J-bands. Their absorption and emission transition scheme was proposed.
Keywords: Interface/surface analysis; Spectroscopy/instrumentation; Fluorescence/luminescence

The spectrofluorometric behavior of a membrane potential-sensitive dye, 1-(3-sulfonatopropyl)-4-[β-{2-(di-N-butylamino)-6-naphthyl}vinyl]pyridinium betaine (di-4-ANEPPS), at the polarized 1,2-dichloroethane/water interface was studied by means of potential-modulated fluorescence (PMF) spectroscopy. The results, combined with those from cyclic and alternating current voltammetry, clearly suggested that the dye adsorbed at the interface underwent a reorientation with increasing the interfacial potential, giving a well-developed PMF response as well as a voltammetric response. In addition to the PMF response, another PMF response was observed by addition of dilauroyl phosphatidylcholine (DLPC). This additional response was well explained in terms of a reorientation of di-4-ANEPPS at the interface, which would be induced by the potential-dependent desorption of DLPC from the interface. Thus, the present study supported the reorientation/solvatochromic mechanism for the membrane potential-sensitive dye rather than the electrochromic mechanism. Figure Reaction model for the potential-induced reorientation of di-4-ANEPPS at the O/W interface in the presence of DLPC in the DCE phase.
Keywords: Membrane potential-sensitive dye; di-4-ANEPPS; Liquid/liquid interface; Potential-modulated fluorescence

Hydrogen-bonding molecular ruler surfactants as probes of specific solvation at liquid/liquid interfaces by A. Renee Siler; Michael R. Brindza; Robert A. Walker (1063-1073).
Resonance-enhanced, second harmonic generation (SHG) is used to measure the electronic structure of solutes sensitive to specific solvation adsorbed to liquid/liquid and liquid/solid interfaces. Here, specific solvation refers to solvent–solute interactions that are directional and localized. N-methyl-p-methoxyaniline (NMMA) is a solute whose first allowed electronic transition wavelength remains almost constant (∼315 nm) in non-hydrogen-bonding solvents regardless of solvent polarity. However, in hydrogen-bond-accepting solvents such as dimethylsulfoxide, NMMA’s absorbance shifts to longer wavelengths (320 nm), whereas in hydrogen-bond-donating solvents (e.g., water), the absorbance shifts to shorter wavelengths (∼300 nm). SHG experiments show that at alkane/silica interfaces, surface silanol groups serve as moderately strong hydrogen-bond donors as evidenced by NMMA’s absorbance of 307 nm. At the carbon tetrachloride/water interface, NMMA absorbance also shifts to slightly shorter wavelengths (298 nm) implying that water molecules at this liquid/liquid interface are donating strong hydrogen bonds to the adsorbed NMMA solutes. In contrast, experiments using newly developed molecular ruler surfactants with NMMA as a model hydrophobic solute and a hydrophilic, cationic headgroup imply that, as NMMA migrates across an aqueous/alkane interface, it carries with it water that functions as a hydrogen-bond-accepting partner. Figure This schematic diagram illustrates that specific solvation forces can change across a liquid/liquid interface. Data in this work show that neutral N-methylmethoxyaniline (NMMA) adsorbed to an alkane/aqueous interface accepts strong hydrogen bonds from the surrounding aqueous solvent. However, a NMMA analogue integrated into a molecular ruler surfactant appears to serve as a hydrogen bond donor to a strongly associated water molecule as the solute moves further into the alkane phase
Keywords: Liquid/liquid interface; Second harmonic generation; SHG; Hydrogen bonding; N-Methyl-p-methoxyaniline; Solvation

Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment by A. A. M. Stolker; M. J. Groot; J. J. P. Lasaroms; A. W. J. M. Nijrolder; M. H. Blokland; I. Riedmaier; C. Becker; H. H. D. Meyer; M. W. F. Nielen (1075-1087).
The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10–12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCβ levels (5–20 µg/kg) after 5–7 weeks. When treatment is repeated two times, the CCβ levels are reached after 9–11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but—in contrast with the pour-on application—after i.m. injection, significant increase of 17β-testosterone and 17β-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood.
Keywords: Liquid chromatography; Mass spectrometry; Hair; Testosterone ester; Estradiol benzoate; Pour-on treatment

Antibody-specific aptamer-based PCR analysis for sensitive protein detection by Yoshihito Yoshida; Katsunori Horii; Nobuya Sakai; Hiromi Masuda; Makio Furuichi; Iwao Waga (1089-1096).
Nucleic acid amplification techniques were applied to the enzyme linked immunosorbent assay (ELISA) with an antibody-specific aptamer, R18. This novel detection system is a modification of the original immuno-polymerase chain reaction (immuno-PCR), but oligonucleotide-labeled antibodies are not required in the assay. This method is performed with the usual ELISA protocol, using an RNA aptamer for rabbit IgG instead of the conventional secondary antibody. After the assay plate was washed, quantitative reverse transcription (RT)-PCR was performed. Ribonuclease (RNase) inhibitors are not needed for this method. The detection limit of the quantitative RT-PCR is over 100 times more sensitive than the original ELISA method, even with the same sandwich–antibody combination. Only 1 mg of aptamer is sufficient for more than 10 million assays. This aptamer-based quantitative PCR successfully detected 16 attomoles (16 × 10−18) of vascular endothelial growth factor (VEGF). This is a cost-effective and easy method to increase the sensitivity of the rabbit antibody-based ELISA systems. The new method is referred to as immuno-aptamer PCR (iaPCR), to distinguish it from the original immuno-PCR.
Keywords: Aptamer; Antibody; ELISA; Real-time immuno-PCR

UV cross-linking of unmodified DNA on glass surfaces by Thomas Schüler; Alla Nykytenko; Andrea Csaki; Robert Möller; Wolfgang Fritzsche; Jürgen Popp (1097-1105).
The performance of DNA microarrays strongly depends on their surface properties. Furthermore, the immobilization method of the capture molecules is of importance for the efficiency of the microarray in terms of sensitivity and specificity. This work describes the immobilization of single-stranded capture oligonucleotides by UV cross-linking on silanated (amino and epoxy) glass surfaces. Thereby we used amino (NH2) and poly thymine/poly cytosine modifications of the capture sequences as well as unmodified capture molecules. The results were compared to UV cross-linking of the same DNA oligonucleotides on unmodified glass surfaces. Immobilization and hybridization efficiency was demonstrated by fluorescence and enzyme-induced deposition of silver nanoparticles. We found out that single-stranded DNA molecules do not require a special modification to immobilize them by UV cross-linking on epoxy- or amino-modified glass surfaces. However, higher binding rates can be achieved when using amino-modified oligonucleotides on an epoxy surface. The limit of detection for the used settings was 5 pM.
Keywords: DNA microarray; Enzyme-induced silver deposition; UV cross-linking; Silanated glass surfaces

On-line ionic imprinted polymer selective solid-phase extraction of nickel and lead from seawater and their determination by inductively coupled plasma-optical emission spectrometry by Natalia García-Otero; Carmen Teijeiro-Valiño; Jacobo Otero-Romaní; Elena Peña-Vázquez; Antonio Moreda-Piñeiro; Pilar Bermejo-Barrera (1107-1115).
Nickel(II) and lead(II) ionic imprinted 8-hydroxyquinoline polymers were synthesized by a precipitation polymerization technique and were used as selective solid phase extraction supports for the determination of nickel and lead in seawater by flow injection solid phase extraction on-line inductively coupled plasma-optical emission spectrometry. An optimum loading flow rate of 2.25 mL min−1 for 2 min and an elution flow rate of 2.25 mL min−1 for 1 min gave an enrichment factor of 15 for nickel. However, a low dynamic capacity and/or rate for adsorption and desorption was found for lead ionic imprinted polymer and a flow rate of 3.00 mL min−1 for 4-min loading and a flow rate of 2.25 mL min−1 for 1-min elution gave a enrichment factor of 5. The limit of detection was 0.33 µg L−1 for nickel and 1.88 µg L−1 for lead, with a precision (n = 11) of 8% (2.37 µg Ni L−1) for nickel and 11% (8.38 µg Pb L−1) for lead. Accuracy was also assessed by analyzing SLEW-3 (estuarine water) and TM-24 (lake water) certified reference materials, and the values determined were in good agreement with the certified concentrations.
Keywords: On-line solid-phase extraction; Ionic imprinted polymer; Nickel; Lead; Seawater; Inductively coupled plasma-optical emission spectrometry

1H-nuclear magnetic resonance spectroscopy-based metabolic assessment in a rat model of obesity induced by a high-fat diet by So-Hyun Kim; Seung-Ok Yang; Hee-Su Kim; Yujin Kim; Taesun Park; Hyung-Kyoon Choi (1117-1124).
Obesity, whose prevalence is increasing rapidly worldwide, is recognized as a risk factor for diabetes, cardiovascular disease, liver disease, and renal disease. To investigate metabolic changes in the urine of a rat model of obesity induced by a high-fat diet (HFD), rats were divided into the following four groups based on the diet type and degree of weight gain: normal-diet (ND) low gainers, ND high gainers, HFD low gainers, and HFD high gainers. Biochemical analyses of visceral fat-pad weight, plasma, and liver tissues were performed. The 1H-nuclear magnetic resonance (1H-NMR) spectra of urine were analyzed using multivariate statistical analysis to identify the separation of the groups. It was observed that the metabolic profile of urine obtained by 1H-NMR-spectroscopy-based metabolomic analysis differed between ND low gainers and ND high gainers even though these animals consumed the same normal diet. Several key metabolites in urine, such as betaine, taurine, acetone/acetoacetate, phenylacetylglycine, pyruvate, lactate, and citrate contributed to the classification of these two groups. The metabolic profile of urine also differed between ND low gainers and HFD high gainers, which consumed the different diet and showed a different weight gain. This study has identified features of urine metabolites in various groups and demonstrated the reliability of an NMR-based metabolomics approach to investigate the effects of the diet and the physical constitution on obesity. Figure Intensity of the metabolites (normalized relative to the creatinine intensity) for ND low gainers vs. HFD high gainers. An independent t test was performed to assess the statistical significance between each group. The error bars are expressed as the SEM *P<0.025 vs. each group
Keywords: 1H-NMR; High-fat diet; Obesity; Metabolomics; Physical constitution

Preparation of magnetic molecularly imprinted polymer for rapid determination of bisphenol A in environmental water and milk samples by Yongsheng Ji; Juanjuan Yin; Zhigang Xu; Chuande Zhao; Huayu Huang; Haixia Zhang; Chunming Wang (1125-1133).
A magnetic molecularly imprinted polymer (M-MIP) of bisphenol A (BPA) was prepared by miniemulsion polymerization. The morphological and magnetic characteristics of the M-MIP were characterized by Fourier-transform infrared spectroscopy, transmission electron microscopy, and vibrating sample magnetometry. The adsorption capacities of the M-MIP and the nonimprinted polymer were investigated using static adsorption tests, and were found to be 390 and 270 mg g−1, respectively. Competitive recognition studies of the M-MIP were performed with BPA and the structurally similar compound DES, and the M-MIP displayed high selectivity for BPA. A method based on molecularly imprinted solid-phase extraction assisted by magnetic separation was developed to extract BPA from environmental water and milk samples. Various parameters such as the mass of sorbent, the pH of the sample, the extraction time, and desorption conditions were optimized. Under selected conditions, extraction was completed in 15 min. High-performance liquid chromatography with UV detection was employed to determine BPA after the extraction. For water samples, the developed method exhibited a limit of detection (LOD) of 14 ng L−1, a relative standard deviation of 2.7% (intraday), and spiked recoveries ranging from 89% to 106%. For milk samples, the LOD was 0.16 µg L−1, recoveries ranged from 95% to 101%, and BPA was found in four samples at levels of 0.45–0.94 µg L−1. The proposed method not only provides a rapid and reliable analysis but it also overcomes problems with conventional solid-phase extraction (SPE), such as the packing of the SPE column and the time-consuming nature of the process of loading large-volume samples.
Keywords: Magnetic molecularly imprinted polymer; Solid-phase extraction; Magnetic separation; High-performance liquid chromatography; Bisphenol A

Development of a voltammetric electronic tongue for discrimination of edible oils by Paolo Oliveri; M. Antonietta Baldo; Salvatore Daniele; Michele Forina (1135-1143).
In this paper, we propose a novel strategy to perform cyclic voltammetric measurements with a platinum microelectrode directly in edible oil samples. The microelectrode was employed as an electronic tongue that, along with the application of chemometrics to the current–potential responses, proved useful for discriminating oils on the basis of their quality and geographical origin. The method proposed here is based on the use of suitable room temperature ionic liquids, added to oils as supporting electrolytes to provide conductivity to the low-polarity samples. The entire voltammograms, recorded directly on the oil/RTIL mixtures, were processed via principal component analysis and a classification technique (K nearest neighbors), to extract information on samples characteristics. Data processing showed that oils having different nature (i.e. maize and olive) or geographical origin (i.e. olive oils coming from different regions) can be distinguished. Figure A novel strategy to perform voltammetric measurements with a platinum microelectrode directly in edible oil samples is presented. The microelectrode is employed as an electronic tongue that, along with the application of chemometrics to the voltammetric responses, allows oil discrimination according to their quality and geographical origin.
Keywords: Electronic tongue; Edible oils; Room temperature ionic liquids (RTILs); Voltammetry; Platinum microelectrode; Principal component analysis (PCA)

Interactions between rotenone and humic acids by means of FT-IR and fluorescence spectroscopies by Ivana Cavoski; Valeria D’Orazio; Teodoro Miano (1145-1158).
The aim of this work was to ascertain, on a comparative basis, the compositional, structural and functional differences occurring between three humic acids (HAs), HA S1 (isolated from a Mediterranean brown soil), HA S2 (isolated from a Bavarian brown soil), and HA SR (a Suwannee River standard aquatic HA, purchased from IHSS), and to investigate the influence of their intrinsic properties on the types of binding mechanisms toward the pesticide rotenone. Original HAs and their corresponding HA–rotenone products, obtained by two different interaction protocols, were analyzed for elemental and functional group composition, and spectroscopic techniques, such as Fourier-transform infrared (FT IR) with Fourier self-deconvolution (FSD) and fluorescence both in the single-scan and in three-dimensional modes. The HA S1 sample appeared to be characterized by a greater aromaticity degree and lower polarity with respect to the HA S2, featured by a mixed aromatic/aliphatic character, whereas mainly aliphatic and acidic resulted the HA SR. The data obtained suggested that the low water-soluble, non-polar pesticide rotenone resulted preferentially adsorbed onto HAs by hydrophobic interaction, that was the prevailing mechanism in the order HA S1 > HA S2 >>> HA SR, whereas hydrogen bonds resulted predominant in the opposite order.
Keywords: Rotenone; Humic acids; FT IR; Fluorescence; EEM fluorescence; Interaction mechanisms

Quantitative monitoring of an activated sludge reactor using on-line UV-visible and near-infrared spectroscopy by Mafalda C. Sarraguça; Ana Paulo; Madalena M. Alves; Ana M. A. Dias; João A. Lopes; Eugénio C. Ferreira (1159-1166).
The performance of an activated sludge reactor can be significantly enhanced through use of continuous and real-time process-state monitoring, which avoids the need to sample for off-line analysis and to use chemicals. Despite the complexity associated with wastewater treatment systems, spectroscopic methods coupled with chemometric tools have been shown to be powerful tools for bioprocess monitoring and control. Once implemented and optimized, these methods are fast, nondestructive, user friendly, and most importantly, they can be implemented in situ, permitting rapid inference of the process state at any moment. In this work, UV-visible and NIR spectroscopy were used to monitor an activated sludge reactor using in situ immersion probes connected to the respective analyzers by optical fibers. During the monitoring period, disturbances to the biological system were induced to test the ability of each spectroscopic method to detect the changes in the system. Calibration models based on partial least squares (PLS) regression were developed for three key process parameters, namely chemical oxygen demand (COD), nitrate concentration (N-NO 3 ), and total suspended solids (TSS). For NIR, the best results were achieved for TSS, with a relative error of 14.1% and a correlation coefficient of 0.91. The UV-visible technique gave similar results for the three parameters: an error of ~25% and correlation coefficients of ~0.82 for COD and TSS and 0.87 for N-NO 3 . The results obtained demonstrate that both techniques are suitable for consideration as alternative methods for monitoring and controlling wastewater treatment processes, presenting clear advantages when compared with the reference methods for wastewater treatment process qualification. Figure Raw spectra obtained in situ with the NIR analyzer between 900 and 1400 nm
Keywords: Activated sludge process; UV-visible; Near-infrared; Chemometrics; On-line monitoring; Wastewater treatment

Trialkyl esters of phosphoric acid are widely used as flame retardants. The corresponding dialkylphosphates are formed as the main metabolites in animal experiments. We extended a previously published method for the determination of four organophosphorus flame retardant metabolites [bis(2-chloroethyl) phosphate, diphenyl phosphate, di-m-cresyl phosphate and di-p-cresyl phosphate] to be able to determine di-n-butyl phosphate (DBP) and bis(2-chloropropyl) phosphate (BCPP) in human urine samples additionally in one run. After solid-phase extraction, derivatization with pentafluorobenzyl bromide and further solid-phase cleanup, the extracts were analysed by gas chromatography–tandem mass spectrometry. The limits of detection were 0.25 µg/l for both analytes. Interday imprecisions were 2–6%. To show the applicability of the method, the internal burden of 25 persons of the population was determined. Twelve percent of the urine samples analysed tested positive for BCPP at concentrations from below the limit of detection to 0.85 µg/l; one sample contained 0.26 µg/l DBP.
Keywords: Organophosphorus flame retardants; Dialkyl phosphate metabolites; Di-n-butyl phosphate; Bis(2-chloroethyl) phosphate; Di-2-chloroethyl phosphate

Detection of halogenated organic compounds using immobilized thermophilic dehalogenase by Philip G. Bachas-Daunert; Zachariah P. Sellers; Yinan Wei (1173-1178).
is a high school student at Paul Laurence Dunbar High School, 1600 Man O’ War Blvd, Lexington, KY 40513, USAEnvironmental pollutants containing halogenated organic compounds can cause a plethora of health problems. Detection, quantification, and eventual remediation of halogenated pollutants in the environment are important to human well-being. Toward this end, we previously identified a haloacid dehalogenase, L-HADST, from the thermophile Sulfolobus tokodaii. This thermophilic enzyme is extremely stable and catalyzes, stereospecifically, the dehalogenation of l-2-haloacids. In the current study, we covalently linked L-HADST to an N-hydroxysuccinimidyl Sepharose resin to construct a highly specific sensor with long shelf life for the detection of l-2-haloacids. The enzyme-modified resin was packed into disposable columns. Samples containing l-2-haloacids were first incubated in the column, and were then collected to quantify the chloride produced through the breakdown of the substrate. The optimum pH of the immobilized enzyme is around 9.5, similar to that of the soluble protein. Its catalytic activity increased with temperature up to the highest temperature measured (50 °C). The resin could be fully regenerated after multiple reaction cycles and retained 70% of the initial activity after being stored at 4 °C for 6 months. The L-HADST-modified resin could be used to breakdown and quantify l-2-haloacids spiked in the simulated environmental samples, indicating dehalogenases from extremophiles can potentially be employed in the detection and decontamination of l-2-haloacids. Figure Detection of halogenated organic compound using immobilized thermophilic dehalogenase
Keywords: Dehalogenase; Thermophilic; Enzyme immobilization; Electrochemistry

Dielectrophoresis for manipulation of micro/nano particles in microfluidic systems by C. Zhang; K. Khoshmanesh; A. Mitchell; K. Kalantar-zadeh (1179-1179).