Analytical and Bioanalytical Chemistry (v.391, #2)

Food pathogen and toxin detection by Antje Baeumner (449-450).
is Assoiate Professor, Biological and Environmental Engineering, at Cornell University, Ithaca, NY. Her research focuses on the development of biosensors and lab-on-a-chip systems for detection of pathogens and toxins in food and environmental samples, and on clinically relevant analytes.

Trends and opportunities in food pathogen detection by S. R. Nugen; A. J. Baeumner (451-454).

The detection and identification of foodborne pathogens continue to rely on conventional culturing techniques. These are very elaborate, time-consuming, and have to be completed in a microbiology laboratory and are therefore not suitable for on-site monitoring. The need for a more rapid, reliable, specific, and sensitive method of detecting a target analyte, at low cost, is the focus of a great deal of research. Biosensor technology has the potential to speed up the detection, increase specificity and sensitivity, enable high-throughput analysis, and to be used for monitoring of critical control points in food production. This article reviews food pathogen detection methods based on electrochemical biosensors, specifically amperometric, potentiometric, and impedimetric biosensors. The underlying principles and application of these biosensors are discussed with special emphasis on new biorecognition elements, nanomaterials, and lab on a chip technology.
Keywords: Food pathogen; Electrochemical biosensor; Immunosensor; Genosensor; Lab on a chip

Microbiological contamination of foods continues to be a major concern in public health. Biological toxins are one class of important contaminants that can cause various human diseases. Outbreaks related to contamination by biological toxins or toxin-producing microorganisms have made it extremely important to develop rapid (approximately 20 min), sensitive and cost-effective analytical methods. This paper describes the development of a sensitive bioassay for the detection of cholera toxin (CT) in selected seafood samples, using ganglioside-incorporated liposomes. In this study, the assays were run with food samples spiked with various concentrations of CT. The limit of detection (LOD) increased by a factor of about 10–20 in most food samples, compared with the LOD in the buffer system previously reported. However, the LOD of toxins in food samples (8 × 10–3 × 103 fg/mL for CT) was still comparable to, or lower than, that previously reported for other assays. The results from this study demonstrate that the bioassays using ganglioside-liposomes can detect the toxin directly in the field screening of food samples rapidly, simply and reliably, without the need for complex instrumentation.
Keywords: Cholera toxin; Ganglioside-liposomes; Ganglioside-liposome immunoassay; Test strip bioassay; Lateral-flow assay

Liposome-based immunostrip for the rapid detection of Salmonella by Ja-an Annie Ho; Shi-Chin Zeng; Wei-Hsiang Tseng; Yong-Jen Lin; Chun-hsien Chen (479-485).
Salmonellae are ubiquitous human pathogens, which pose a danger to the elderly and children. Due to the increased number of outbreaks of human illness associated with the consumption of contaminated products in the USA and many other countries, there is an urgent need to develop rapid assays to detect common food-borne pathogens. This study demonstrates the feasibility of using a detectable label comprising methyl blue (MB), a visible dye, entrapped inside liposomes. Immunoliposomes tagged with anti-Salmonella common structural antigens (CSA) antibody encapsulating MB dye were prepared and used as the signal amplifier for the development of a field-portable colorimetric immunoassay to detect Salmonellae. Tapping mode atomic force microscopy (TMAFM), a scanning probe technique, was utilized to demonstrate the presence of anti-Salmonella antibody at the thus-prepared liposome. A plastic-backed nitrocellulose strip with two immobilized zones formed the basis of a sandwich assay. The first zone was the antigen capture zone (AC zone), used in a sandwich (noncompetitive) assay format; the other was the biotin capture zone (BC zone), used as a quality control index for the strip assay. During the capillary migration of the wicking reagent containing 80 μL of immunoliposomes and 40 μL of the test sample (heat-killed S. typhimurium), sample pathogens with surface-bound immunoliposomes were captured at the AC zone, while the unbound immunoliposomes continued to migrate and bind to the anti-biotin antibodies coated on the BC zone. The color density of the AC zone was directly proportional to the number of Salmonella typhimurium in the test sample. The detection limit of the current assay with heat-killed Salmonella typhimurium was 1,680 cells. The cross-reactivity of the proposed immunoassay was also investigated, and pathogens including E. coli O157:H7 and Listeria genus specific caused no interference with the detection of Salmonella typhimurium.
Keywords: Salmonella typhimurium ; Point-of-care diagnostics; Lateral flow immunoassay; Pathogen detection

Human pathogenic Cryptosporidium species bioanalytical detection method with single oocyst detection capability by John T. Connelly; Sam R. Nugen; Wlodek Borejsza-Wysocki; Richard A. Durst; Richard A. Montagna; Antje J. Baeumner (487-495).
A bioanalytical detection method for specific detection of viable human pathogenic Cryptosporidium species, C. parvum, C. hominis, and C. meleagridis is described. Oocysts were isolated from water samples via immunomagnetic separation, and mRNA was extracted with oligo-dT magnetic beads, amplified using nucleic acid sequence-based amplification (NASBA), and then detected in a nucleic acid hybridization lateral flow assay. The amplified target sequence employed was hsp70 mRNA, production of which is stimulated via a brief heat shock. The described method was capable of detecting one oocyst in 10 μL using flow-cytometer-counted samples. Only viable oocysts were detected, as confirmed using 4′,6-diamidino-2-phenylindole and propidium iodide (DAPI/PI) staining. The detection system was challenged by detecting oocysts in the presence of large numbers of common waterborne microorganisms and packed pellet material filtered from environmental water samples. When the method was compared with EPA Method 1622 for C. parvum detection, highly comparable results were obtained. Since the described detection system yields unambiguous results within 4.5 h, it is an ideal method for monitoring the safety of drinking water.
Keywords: Cryptosporidium ; mRNA; Detection; Liposome; Lateral flow; Human pathogen; Oligo-dT

An antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules by Andrew G. Gehring; David M. Albin; Sue A. Reed; Shu-I Tu; Jeffrey D. Brewster (497-506).
Intoxication and infection caused by foodborne pathogens are important problems worldwide, and screening tests for multiple pathogens are needed because foods may be contaminated with multiple pathogens and/or toxic metabolites. We developed a 96-well microplate, multiplex antibody microarray method to simultaneously capture and detect Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. typhimurium), as well as a biomolecule (chicken immunoglobulin G or IgG employed as a proteinaceous toxin analog) in a single sample. Microarrayed spots of capture antibodies against the targeted analytes were printed within individual wells of streptavidin-coated polystyrene 96-multiwell microtiter plates and a sandwich assay with fluorescein- or Cy3-labeled reporter antibodies was used for detection. (Printing was achieved with a conventional microarray printing robot that was operated with custom-developed microplate arraying software.) Detection of the IgG was realized from ca. 5 to 25 ng/mL, and detection of E. coli O157:H7 and S. typhimurium was realized from ca. 106 to 109 and ca. 107 to 109 cells/mL, respectively. Multiplex detection of the two bacteria and the IgG in buffer and in culture-enriched ground beef filtrate was established with a total assay (including detection) time of ca. 2.5 h. Detection of S. typhimurium was largely unaffected by high concentrations of the other bacteria and IgG as well as the ground beef filtrate, whereas a small decrease in response was observed for E. coli O157:H7. The multiwell plate, multiplex antibody microarray platform developed here demonstrates a powerful approach for high-throughput screening of large numbers of food samples for multiple pathogens and toxins.
Keywords: Antibody microarray; Bacteria; Fluorescence; Immunoassay; Multiwell, microtiter plate; Multiplex

Bacteriophage-amplified bioluminescent sensing of Escherichia coli O157:H7 by Steven Ripp; Patricia Jegier; Courtney M. Johnson; Jennifer R. Brigati; Gary S. Sayler (507-514).
Escherichia coli O157:H7 remains a continuous public health threat, appearing in meats, water, fruit juices, milk, cheese, and vegetables, where its ingestion at concentrations of perhaps as low as 10 to 100 organisms can result in potent toxin exposure and severe damage to the lining of the intestine. Abdominal pain and diarrhea develop, which in the very young or elderly can progress towards hemolytic uremic syndrome and kidney failure. To assist in the detection of E. coli O157:H7, a recombinant bacteriophage reporter was developed that uses quorum sensing (luxI/luxR) signaling and luxCDABE-based bioluminescent bioreporter sensing to specifically and autonomously respond to O157:H7 serotype E. coli. The bacteriophage reporter, derived from phage PP01, was tested in artificially contaminated foodstuffs including apple juice, tap water, ground beef, and spinach leaf rinsates. In apple juice, detection of E. coli O157:H7 at original inoculums of 1 CFU mL−1 occurred within approximately 16 h after a 6-h pre-incubation, detection of 1 CFU mL−1 in tap water occurred within approximately 6.5 h after a 6-h pre-incubation, and detection in spinach leaf rinsates using a real-time Xenogen IVIS imaging system resulted in detection of 1 CFU mL−1 within approximately 4 h after a 2-h pre-incubation. Detection in ground beef was not successful, however, presumably due to the natural occurrence of quorum sensing autoinducer (N-3-(oxohexanoyl)-l-homoserine lactone; OHHL), which generated false-positive bioreporter signals in the ground beef samples.
Keywords: Bacteriophage; Bioluminescence; Bioreporter; E. coli O157:H7; lux ; Quorum sensing

For most applications, 3–5 observations, or samplings (n), are utilized to estimate total aerobic plate count in an average population (μ) that is greater than about 50 cells, or colony forming units (CFU), per sampled volume. We have chosen to utilize a 6 × 6 drop plate method for bacterial colony selection because it offers the means to rapidly perform all requisite dilutions in a 96-well format and plate these dilutions on solid media using minimal materials. Besides traditional quantitative purposes, we also need to select colonies which are well-separated from each other for the purpose of bacterial identification. To achieve this goal using the drop plate format requires the utilization of very dilute solutions (μ < 10 CFUs per sampled drop). At such low CFU densities the sampling error becomes problematic. To address this issue we produced both observed and computer-generated colony count data and divided a large sample of individual counts randomly into N subsamples each with n = 2–24 observations (N × n = 360). From these data we calculated the average total mean-normalized ( $$overline{x} _{{{ ext{tot}}}} $$ , n = 360) deviation of the total standard deviation (s tot) from each jth subsample’s estimate (s j ), which we call Δ. When either observed or computer-generated Δ values were analyzed as a function of $$overline{x} _{{{ ext{tot}}}} $$ , a set of relationships ( $${ propto { oot { - 2} of {overline{x} _{{{ m{tot}}}} } }}$$ ) were generated which appeared to converge at an n of about 18 observations. This finding was verified analytically at even lower CFU concentrations ( $$overline{x} _{{{ ext{tot}}}} sim 1 - 10{ ext{ CFUs per observation}}$$ ). Additional experiments using the drop plate format and n = 18 samplings were performed on food samples along with most probable number (MPN) analyses and it was found that the two enumeration methods did not differ significantly.
Keywords: Sampling; Sampling error; E. coli ; Enumeration; 6 × 6 drop plate; MPN

We present herein the composition of bacterial communities occurring in ground chicken and the changes which arise in these populations based upon nonselective partitioning by commercially-available Dynal anti-Salmonella and anti-E. coli O157 immunomagnetic beads (IMB). Our enumeration and colony selection protocol was based upon a 6 × 6 drop plate method (n = 18 for each 25-g sub-sampling) using a dilution which resulted in ca. 4–8 colonies per drop. An average of 82 ± 13 colonies were selected from three 25-g ground chicken subsamplings per batch, each of which was repeated seasonally for one year. DNA was extracted from each colony and the composition of Eubacteria in each of these harvests was determined by sequence-based identification of 16S rDNA amplicons. The Gram-positive bacteria Brochothrix thermosphacta and Carnobacterium maltaromticum were the most commonly found organisms in both the total chicken wash (PBS) and in the IMB-bound (PBS-washed) fractions. The remaining background organisms which also adhered to varying degrees to commercial IMBs were: Pseudomonas oleovorans, Acinetobacter lwoffi, Serratia spp., and one Rahnella spp. A large number of the organisms were also cladistically evaluated based on rDNA basepair disparities: all Brochothrices were monophyletic; twelve different Pseudomonads were found along with eight Carnobacteria, seven Acinetobacteres, four Serratiae, and two Rahnellae. Carnobacterium alone showed an IMB-based concentration enhancement (ca. two to sixfold).
Keywords: Nonspecific adsorption; Immunomagnetic beads; Brochothrix ; Carnobacterium ; 16S rDNA

Absolute protein quantification has become an important challenge in modern bioanalytical chemistry. Among several approaches based on mass spectrometric techniques, inductively coupled plasma (ICP) as ionisation source provides element-selective and sensitive detection of heteroatoms, and thus, a potentially emerging tool in protein analysis. In this work we applied coupling of capillary liquid chromatography (μLC) and inductively coupled plasma-sector field mass spectrometry (ICP–SFMS) to the separation and determination of standard proteins. For quantification purposes, post-column isotope dilution of sulfur was applied and optimised for this type of hyphenated technique. Provided that the protein sequence is known (number of sulfur-containing amino acids, i.e. cysteines and methionines) the protein amount can then be directly calculated from the determined sulfur content in a certain protein fraction. In order to prove the reliability of the presented method, two different certified reference materials were analysed: CRM 393 (human apolipoprotein A-I) and CRM 486 (α-fetoprotein). For CRM 393 excellent agreement (37.0 ± 1.4 μmol L−1) was obtained with the certificate (37.7 ± 1.8 μmol L−1). However, the recovery rate for α-fetoprotein in CRM 486 was found to be about 60% indicating incomplete elution of the protein during the chromatographic separation.
Keywords: μLC; ICP–MS; Isotope dilution; Protein quantification; CRM 393; CRM 486

Protein films adsorbed on experimental dental materials: ToF-SIMS with multivariate data analysis by Falk Bernsmann; Nicole Lawrence; Matthias Hannig; Christiane Ziegler; Hubert Gnaser (545-554).
The proteins lysozyme, amylase, and bovine serum albumin (BSA) were adsorbed on two experimental dental materials, made of fluoroapatite particles embedded in polymer matrices, and on silicon wafers. The protein films were prepared as single-component layers, as binary mixtures, and as double layers. These systems were investigated by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the multivariate data analysis technique of discriminant principal-component analysis (DPCA). During adsorption of a single protein film on to the solid surfaces, the three proteins could be clearly distinguished by the scores of their mass spectra after selection of amino acid-related peaks and DPCA. Furthermore, very similar results were obtained on the two different fluoroapatite substrates. For samples coated with binary layers of two proteins adsorbed simultaneously, it was found for both substrate types that BSA shows the strongest ability to adsorb followed by lysozyme, while amylase has the smallest ability. By contrast, the consecutive adsorption of two protein layers showed a strong influence of substrate type on the adsorption ability of the proteins.
Keywords: Protein adsorption; Fluoroapatite-containing materials; Time-of-flight secondary ion mass spectrometry; Discriminant principal-component analysis

Electrospray-ionization driven by dielectric polarization by Michael Schilling; Dirk Janasek; Joachim Franzke (555-561).
A non-conductive piezo ceramic plate has been used to induce an electric field to generate an electrospray as ionization method for mass spectrometric determination. This technique decreases the risk of undesired discharges, induced by high electric currents. The applicability of the technique is demonstrated and compared with a commercial electrospray for mass spectrometric determination of reserpine and myoglobin.
Keywords: ESI; Dielectric polarization; MS

This paper describes a new and rapid method for accurate quantification of the six ergot alkaloids, ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine, by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The six ergot alkaloids studied have been defined by the European Food Safety Authority (EFSA) as among the most common and physiologically active ones. In addition, the method enables the quantification of the corresponding six epimers (ergo-inines) of these ergot alkaloids. This is of considerable importance in terms of the differences in toxicity of the isomeric forms. The method involves extraction under alkaline conditions using a mixture of acetonitrile and ammonium carbonate buffer followed by a rapid clean-up using dispersive solid-phase extraction with PSA (primary secondary amine) and a short chromatographic LC-run (21 min) with subsequent MS–MS detection. The method was developed and validated using ten different cereal and food samples. The major strength of the new method compared with previously published techniques is the simplicity of the clean-up procedure and the short analysis time. The limits of quantification were 0.17 to 2.78 μg kg−1 depending on the analyte and matrix. Recovery values for the 12 ergot alkaloids spiked into ten different matrices at levels of 5, 50, and 100 μg kg−1 were between 69 and 105% for 85 of 90 recovery measurements made over six days. Measurement uncertainty values were highly satisfactory. At a concentration level of 5 μg kg−1 the expanded measurement uncertainty ranged from ±0.56 to ±1.49 μg kg−1, at a concentration level of 100 μg kg−1 the expanded measurement uncertainty ranged from ±8.9 to ±20 μg kg−1. Both LOQs and measurement uncertainties were dependent on the analyte but almost independent of the matrix. The method performance was satisfactory when tested in a mini-intercomparison study between three laboratories from three different countries.
Keywords: Cereals; HPLC; Ergot alkaloids; Foods; Mass spectrometry

We present a highly sensitive method for the determination of platinum (Pt) in DNA extracts of peripheral blood mononuclear cells (PBMCs) and tissue samples from patients treated with cisplatin. The method is based on the measurement of Pt by inductively coupled plasma mass spectrometry (ICP-MS) and allows quantification of Pt-DNA adducts in PBMCs isolated from 10 mL blood and 1 mg tissue. The lower limit of quantification is 0.75 pg Pt or 7.5 fg Pt μg−1 DNA when using 100 μg DNA. The method proved to be accurate and precise. The results obtained using the ICP-MS method were in good agreement with results from the alternative 32P-postlabelling assay. The ICP-MS method was, however, more sensitive and proved to be less laborious. The advantages of the presented ICP-MS technique were demonstrated by the analysis of PBMCs, normal gastric tissue, and gastric tumour tissue of patients treated with cisplatin.
Keywords: ICP-MS; Platinum; DNA adducts; Peripheral blood mononuclear cells; Tissue

Development of analytical procedures for the determination of hexavalent chromium in corrosion prevention coatings used in the automotive industry by F. Séby; A. Castetbon; R. Ortega; C. Guimon; F. Niveau; N. Barrois-Oudin; H. Garraud; O. F. X. Donard (587-597).
The European directive 2000/53/EC limits the use of Cr(VI) in vehicle manufacturing. Although a maximum of 2 g of Cr(VI) was authorised per vehicle for corrosion prevention coatings of key components, since July 2007 its use has been prohibited except for some particular applications. Therefore, the objective of this work was to develop direct analytical procedures for Cr(VI) determination in the different steel coatings used for screws. Instead of working directly with screws, the optimisation of the procedures was carried out with metallic plates homogeneously coated to improve the data comparability. Extraction of Cr(VI) from the metallic parts was performed by sonication. Two extraction solutions were tested: a direct water extraction solution used in standard protocols and an ammonium/ammonia buffer solution at pH 8.9. The extracts were further analysed for Cr speciation by high-performance liquid chromatography (HPLC) inductively coupled plasma (ICP) atomic emission spectrometry or HPLC ICP mass spectrometry depending on the concentration level. When possible, the coatings were also directly analysed by solid speciation techniques (X-ray photoelectron spectroscopy, XPS, and X-ray absorption near-edge structure, XANES) for validation of the results. Very good results between the different analytical approaches were obtained for the sample of coating made up of a heated paint containing Zn, Al and Cr when using the extracting buffer solution at pH 8.9. After a repeated four-step extraction procedure on the same portion test, taking into account the depth of the surface layer reached, good agreement with XPS and XANES results was obtained. In contrast, for the coatings composed of an alkaline Zn layer where Cr(VI) and Cr(III) are deposited, only the extraction procedure using water allowed the detection of Cr(VI). To elucidate the Cr(VI) reduction during extraction at pH 8.9, the reactivity of Cr(VI) towards different species of Zn generally present in the coatings (metallic Zn and zinc oxide) was studied. The results showed that metallic Zn rapidly reduces Cr(VI), whereas this reaction is less evident in the presence of zinc oxide. Water was then retained for coatings containing metallic Zn.
Keywords: Hexavalent chromium determination; Corrosion prevention coating; Automotive industry; Hexavalent chromium extraction procedures; Zinc and hexavalent chromium reactivity

Headspace mass spectrometry methodology: application to oil spill identification in soils by J. L. Pérez Pavón; C. García Pinto; A. Guerrero Peña; B. Moreno Cordero (599-607).
In the present work we report the results obtained with a methodology based on direct coupling of a headspace generator to a mass spectrometer for the identification of different types of petroleum crudes in polluted soils. With no prior treatment, the samples are subjected to the headspace generation process and the volatiles generated are introduced directly into the mass spectrometer, thereby obtaining a fingerprint of volatiles in the sample analysed. The mass spectrum corresponding to the mass/charge ratios (m/z) contains the information related to the composition of the headspace and is used as the analytical signal for the characterization of the samples. The signals obtained for the different samples were treated by chemometric techniques to obtain the desired information. The main advantage of the proposed methodology is that no prior chromatographic separation and no sample manipulation are required. The method is rapid, simple and, in view of the results, highly promising for the implementation of a new approach for oil spill identification in soils. Figure PCA score plots illustrate clear discrimination of types of crude oil in polluted soil samples (e.g. results are shown for vertisol)
Keywords: Crude oils; Polluted soil samples; Headspace mass spectrometry; Chemometrics

Sugar alcohols are widely used as food additives and drug excipients. Erythritol (INS 968) is an important four-carbon sugar alcohol in the food industry. Erythritol occurs naturally in certain fruits, vegetables, and fermented foods. Currently, HPLC and GC methods are in use for the quantification of erythritol in natural/processed foods. However, an immunoassay for erythritol has not been developed so far. We have utilized affinity-purified erythritol-specific antibodies generated earlier [9] to develop an indirect competitive ELISA. With erythritol–BSA conjugate (54 mol/mol; 100 ng/well) as the coating antigen, a calibration curve was prepared using known amounts of standard meso-erythritol (0.1–100,000 ng) in the immunoassay. Watermelon (Citrullus lanatus) and red wine were selected as the food sources containing meso-erythritol. The amount of meso-erythritol was calculated as 2.36 mg/100 g fresh weight of watermelon and 206.7 mg/L of red wine. The results obtained from the immunoassay are in close agreement with the reported values analyzed by HPLC and GC (22–24 mg/kg in watermelon and 130–300 mg/L in red wine). The recovery analyses showed that added amounts of meso-erythritol were recovered fairly accurately with recoveries of 86–105% (watermelon) and 85–93.3% (red wine). The method described here for erythritol is the first report of an immunoassay for a sugar alcohol. Figure Indirect competitive ELISA for quantitation of erythritol
Keywords: meso-Erythritol; Food additive; Immunoassay; Indirect competitive ELISA; INS 968; Red wine; Watermelon

Studies on toxic oil syndrome: development of an enzyme-linked immunosorbent assay for 3-(N-phenylamino)propane-1,2-diol in human urine by Anna Martínez-Cabot; Begoña Varela; Maia Lloveras; Rafael Campos; M.-Pilar Marco; Angel Messeguer (617-624).
The fatty acid esters of 3-(N-phenylamino)propane-1,2-diol (PAP) are biomarkers of toxic oil batches that caused toxic oil syndrome (TOS), an intoxication that caused over 400 deaths and affected 20,000 people in Spain in 1981. PAP esters are converted into PAP by human pancreatic lipase. The in vivo biotransformation of PAP in two mouse strains generated potentially toxic metabolites. Here we report an enzyme-linked immunosorbent assay (ELISA) for PAP detection incorporating antibodies generated using PAP-hapten derivatives 1 and 2. The immunizing haptens were designed to recognize the phenylamino and hydroxymethylene moieties of the PAP structure. The antisera raised against 1-HCH showed greater affinity for free PAP, as demonstrated in competitive experiments using either 1-BSA or 2-BSA as coating antigens. The developed ELISA detects PAP at a threshold of 130 μg L−1 and can be used over a wide range of pH and ionic strength values. The assay can be applied to human urine samples, after a simple treatment method, with good recovery according to the correlation obtained when analyzing blind spiked urine samples. Figure Development of an ELISA for PAP in human urine
Keywords: Toxic oil syndrome; 3-(N-Phenylamino)propane-1,2-diol; Hapten design; Immunoassay

Characterization of interactions between polyphenolic compounds and human serum proteins by capillary electrophoresis by Andréa Diniz; Laura Escuder-Gilabert; Norberto P. Lopes; Rosa María Villanueva-Camañas; Salvador Sagrado; María José Medina-Hernández (625-632).
The interaction of ten natural polyphenolic compounds (chlorogenic acid, apigenin, catechin, epicatechin, flavanone, flavone, quercetin, rutin, vicenin-2 and vitexin) with human serum albumin and mixtures of human serum albumin and α1-acid glycoprotein under near physiological conditions is studied by capillary electrophoresis–frontal analysis. Furthermore, the binding of these polyphenolic compounds to total plasmatic proteins is evaluated using ultrafiltration and capillary electrophoresis. In spite of the relatively small differences in the chemical structures of the compounds studied, large differences were observed in their binding behaviours to plasmatic proteins. The hydrophobicity, the presence/absence of some functional groups, steric hindrance and spatial arrangement seem to be key factors in the affinity of natural polyphenols towards plasmatic proteins.
Keywords: Natural polyphenolic compounds; Polyphenol–protein interactions; Human serum albumin; α1-Acid glycoprotein; Whole plasma; Frontal analysis; Capillary electrophoresis

Natural wax constituents of a supercritical fluid CO2 extract from quince (Cydonia oblonga Mill.) pomace by Peter Lorenz; Melanie Berger; Julia Bertrams; Kristian Wende; Kristin Wenzel; Ulrike Lindequist; Ulrich Meyer; Florian C. Stintzing (633-646).
The chemical constituents of a lipophilic extract from quince (Cydonia oblonga Mill.), obtained by supercritical fluid CO2 extraction of the dried fruit pomace were investigated. Solvent partition of quince wax with n-hexane or acetone yielded an insoluble (crystalline) and a soluble (oily) fraction. Both fractions were analyzed separately using gas chromatography/mass spectrometry (GC/MS). The insoluble fraction consisted of saturated n-aldehydes, n-alcohols and free n-alkanoic acids of carbon chain lengths between 22 and 32, with carbon chain lengths of 26 and 28 dominating. Also odd-numbered unbranched hydrocarbons, mainly C27, C29 and C31, were detected particularly in the acetone-insoluble fraction (total, 15.8%). By means of vacuum liquid chromatography, triterpenoic acids were separated from the hexane-insoluble matter and identified as a mixture of ursolic, oleanolic and betulinic acids. The major constituents of the hexane-soluble fraction were glycerides of linoleic [Δ9,12, 18:2] and oleic [Δ9, 18:1] acids, accompanied by free linoleic, oleic and palmitic acids (C16). Moreover β-sitosterol, Δ5-avenasterol as well as trace amounts of other sterols were assigned. Finally the carotenoids phytoene and phytofluene were identified and quantified by UV/vis and high-performance liquid chromatography/MS techniques, yielding 1.0 and 0.3% of the quince wax, respectively. It is anticipated that the complex of lipid constituents from quince wax may exert interesting biological activities, the elucidation of which awaits further studies. Figure Quince fruits and some of their fruit wax constituents. Clockwise (starting at one o’clock): structure formulas of Δ5-avenasterol, linoleic acid, oleanolic acid, and phytofluen.
Keywords: Aldehydes; Long-chain alkanes; Fatty alcohols; Fatty acids; Carotenoids; Sterols; Triterpenoic acids

CE–LIF determination of salivary cadaverine and lysine concentration ratio as an indicator of lysine decarboxylase enzyme activity by Tamás Tábi; Zsolt Lohinai; Melinda Pálfi; Martin Levine; Éva Szökő (647-651).
Salivary bacteria produce the enzyme lysine decarboxylase which converts lysine to cadaverine. In the absence of appropriate oral hygiene, overgrowth of these bacteria depletes lysine. This may contribute to gingival inflammation, while cadaverine contributes to oral malodor. A selective and sensitive capillary electrophoresis method with laser-induced fluorescence detection has been developed for the determination of cadaverine and lysine in saliva, as an indicator of lysine decarboxylase enzyme activity. The diamino compounds were separated in acidic background electrolyte in their mono-labeled form after derivatization with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F). Linearity and reproducibility of the method in the range 1–50 μmol L−1 have been demonstrated using saliva samples. The method was applied for the measurement of cadaverine and lysine in the saliva of healthy volunteers with or without proper oral hygiene. In the absence of oral hygiene, the mol fraction of cadaverine to cadaverine plus lysine in saliva increased significantly (0.65 ± 0.13 vs. 0.39 ± 0.18, P < 0.001), indicating the presence of higher amount of bacterial lysine decarboxylase, that may contribute to periodontal diseases.
Keywords: Cadaverine; 4-Fluoro-7-nitrobenz-2-oxa-1, 3-diazole; Lysine; Lysine decarboxylase; Periodontal disease; Saliva

The simultaneous determination of three isomers of phenylenediamines (o, m, and p-phenylenediamine) and two isomers of dihydroxybenzenes (catechol and resorcinol) in hair dyes was performed by capillary zone electrophoresis coupled with amperometric detection (CZE–AD). The effects of working electrode potential, pH and concentration of running buffer, separation voltage, and injection time on CZE–AD were investigated. Under the optimum conditions the five analytes could be perfectly separated in 0.30 mol L−1 borate–0.40 mol L−1 phosphate buffer (pH 5.8) within 15 min. A 300 μm diameter platinum electrode had good responses at +0.85 V (versus SCE) for the five analytes. Their linear ranges were from 1.0 × 10−6 to 1.0 × 10−4 mol L−1 and the detection limits were as low as 10−7 mol L−1 (S/N = 3). This working electrode was successfully used to analyze eight kinds of hair dye sample with recoveries in the range 91.0–108.0% and RSDs less than 5.0%. These results demonstrated that capillary zone electrophoresis coupled with electrochemical detection using a platinum working electrode as detector was convenient, highly sensitive, highly repeatable and could be used in the rapid determination of practical samples. Figure Electropherograms obtained from 10 mg mL−1 hair dye sample solutions at a platinum working electrode under optimum CZE–AD conditions: (a) natural black (I), (b) golden: (1) p-phenylenediamine, (2) m-phenylenediamine, (3) o-phenylenediamine, (4) resorcinol, and (5) catechol
Keywords: Isomers; Hair dyes; Capillary zone electrophoresis; Amperometric detection; Platinum working electrode

A method based on anion exchange (AE) and affinity (AF)-HPLC (AE-AF-HPLC) hyphenated to inductively coupled plasma-(quadrupole) mass spectrometry (ICP-QMS) was developed for the speciation analysis of selenoprotein P (SelP), glutathione peroxidase (GPx) and selenoalbumin (SeAlb) in human serum. AE-HPLC is proposed here for the on-line alleviation of Cl and Br spectral interferences on 77Se (40Ar37Cl) and 82Se (81Br1H). Separation of GPx, SelP and SeAlb by AE-AF-HPLC was obtained within a total chromatographic runtime of <20 min. On-line (post-column) isotope dilution (ON-ID) and on-line external calibration (ON-EC)-ICP-QMS were used for the quantification of Se in GPx, SelP and SeAlb. ON-EC using a Se-L-cystine standard was shown to be a suitable approach for the routine simultaneous speciation analysis of serum GPx, SelP and SeAlb. The method validation was carried out by direct ICP-sector field MS determination of Se in GPx, SelP and SeAlb fractions collected after AE-AF-HPLC separation. In addition, the method accuracy for the determination of total protein-bound Se was assessed by analyzing a human serum reference material (BCR-637) certified for total Se content. Figure A methodology for the alleviation of Cl and Br interferences in the accurate simultaneous speciation analysis of glutathione peroxidase, selenoprotein P and selenoalbumin in human serum by affinity HPLC coupled on-line with ICP-quadrupole MS is proposed. This approach may be particularly useful for clinical laboratories that only have an ICP-quadrupole MS without a collision cell, or that lack an expensive ICP-SFMS (high-resolution) instrument
Keywords: Speciation analysis; Human serum selenoproteins; Anion exchange; Affinity chromatography; Inductively coupled plasma quadrupole mass spectrometry; On-line isotope dilution

Interdisciplinary study for the evaluation of biochemical alterations on mussel Mytilus galloprovincialis exposed to a tributyltin-polluted area by Emanuele Magi; Camilla Liscio; Erika Pistarino; Barbara Santamaria; Marina Di Carro; Micaela Tiso; Andrea Scaloni; Giovanni Renzone; M. Elisabetta Cosulich (671-678).
An interdisciplinary approach was employed to monitor the concentration and the effects of butyltin compounds in mussels (Mytilus galloprovincialis). Tissues from animals exposed to a marine area (Vado Ligure harbour) with a high concentration of tributyltin (TBT) were analysed and compared with control samples. TBT concentrations were measured by gas chromatography–mass spectrometry and the protein pattern in gill tissues was studied by proteomic analysis. Several proteomic signatures associated with contaminant exposure were observed; spots that were significantly increased in all contaminated samples were identified by mass spectrometry as fragments of β-tubulin. The degradation of β-tubulin was then confirmed by western blot analysis with specific anti-β-tubulin antibody. The effects observed on mussel gills after exposure in the TBT-polluted area are discussed.
Keywords: Tributyltin; Mytilus galloprovincialis ; Proteomic analysis; Mass spectrometry; Immunodetection

Catechol determination in compost bioremediation using a laccase sensor and artificial neural networks by Lin Tang; Guangming Zeng; Jianxiao Liu; Xiangmin Xu; Yi Zhang; Guoli Shen; Yuanping Li; Can Liu (679-685).
An electrochemical biosensor based on the immobilization of laccase on magnetic core-shell (Fe3O4–SiO2) nanoparticles was combined with artificial neural networks (ANNs) for the determination of catechol concentration in compost bioremediation of municipal solid waste. The immobilization matrix provided a good microenvironment for retaining laccase bioactivity, and the combination with ANNs offered a good chemometric tool for data analysis in respect to the dynamic, nonlinear, and uncertain characteristics of the complex composting system. Catechol concentrations in compost samples were determined by using both the laccase sensor and HPLC for calibration. The detection range varied from 7.5 × 10–7 to 4.4 × 10–4 M, and the amperometric response current reached 95% of the steady-state current within about 70 s. The performance of the ANN model was compared with the linear regression model in respect to simulation accuracy, adaptability to uncertainty, etc. All the results showed that the combination of amperometric enzyme sensor and artificial neural networks was a rapid, sensitive, and robust method in the quantitative study of the composting system. Figure Structure of the magnetic carbon paste electrode used in the electrochemical biosensor
Keywords: Catechol; Compost bioremediation; Laccase sensor; Artificial neural networks; Electrochemical determination

Determination of trace elements in bone by two-jet plasma atomic emission spectrometry by Natalia P. Zaksas; Toktobubu T. Sultangazieva; Vladimir A. Gerasimov (687-693).
This paper describes an analytical method for trace element determination in bone tissues. The study of the influence of the bone matrix showed that the addition of 25% ground bone to graphite powder with introduced impurities did not affect the analytical signal of elements in the spectral excitation in a two-jet plasma. On basis of these investigations a method for direct multielement analysis of bone tissues was suggested. The sample preparation procedure consisted in mixing powdered bone (particle size 30 μm or less) with a spectroscopic buffer (graphite powder plus NaCl) in ratio 1:3 or to a greater extent depending on the analyte concentration. Reference samples based on graphite powder were used for construction of calibration curves. The NaCl concentration in analyzed and calibration samples was 15 wt%. The effect of particle size was revealed from the determination of Ba, Sr, and Mg. To eliminate this effect, treatment of the samples with nitric acid was proposed. The validation of the technique was confirmed by comparison of the analysis results of a bone sample with those obtained by inductively coupled plasma atomic emission spectrometry after wet acid digestion. The limits of detection estimated for 20 elements were the following (μg g-1): 0.1 (Ag), 1.0 (Al), 1.0 (Ba), 0.1 (Be), 1.2 (Bi), 0.4 (Cd), 1.0 (Co), 0.2 (Cu), 0.6 (Cr), 1.9 (In), 2 (Fe), 0.3 (Ga), 0.4 (Mn), 0.4 (Mo), 0.7 (Ni), 1.0 (Pb), 0.7 (Sn), 0.8 (Tl), 5 (Sr), 1.0 (Zn).
Keywords: Bone; Two-jet plasma; Hydroxyapatite; Matrix effect; Graphite powder

A novel method employing high-performance cation chromatography in combination with inductively coupled plasma dynamic reaction cell mass spectrometry (ICP–DRC–MS) for the simultaneous determination of the herbicide glyphosate (N-phosphonomethylglycine) and its main metabolite aminomethyl phosphonic acid (AMPA) is presented. P was measured as 31P16O+ using oxygen as reaction gas. For monitoring the stringent target value of 0.1 μg L−1 for glyphosate, applicable for drinking and surface water within the EU, a two-step enrichment procedure employing Chelex 100 and AG1-X8 resins was applied prior to HPIC–ICP–MS analysis. The presented approach was validated for surface water, revealing concentrations of 0.67 μg L−1 glyphosate and 2.8 μg L−1 AMPA in selected Austrian river water samples. Moreover, investigations at three waste water-treatment plants showed that elimination of the compounds at the present concentration levels was not straightforward. On the contrary, all investigated plant effluents showed significant amounts of both compounds. Concentration levels ranged from 0.5–2 μg L−1 and 4–14 μg L−1 for glyphosate and AMPA, respectively.
Keywords: Glyphosate; AMPA; Waste water treatment; ICP–MS; Dynamic reaction cell

Optically detected magnetophoretic acceleration mass analysis of an individual micro-particle in an atmosphere has been remarkably improved in sensitivity by using a reflective microscope objective, by which forward scattered light from a particle could be effectively collected. From the light-scattering simulation, the detection limit for the radius of a micro-particle was estimated to be smaller than 0.4 μm, and about 60 times intensity enhancement was observed for a polystyrene particle with a radius of 2.8 μm. For both paramagnetic and diamagnetic micro-particles, the mass magnetic susceptibility and the relaxation time could be determined without knowing any parameters of the particles. From the relaxation time, the mass of a particle was obtained if the radius or the density of the particle was known. For a test sample silica particles were used to adsorb paramagnetic dysprosium(III), the surface concentration of dysprosium(III) on a single particle could be successfully determined by use of this method.
Keywords: Magnetophoresis; Microparticle analysis; Mass spectrometry; Magnetic susceptibility; Light-scattering detection

Validation of a screening method for rapid control of macrocyclic lactone mycotoxins in maize flour samples by Mohammed Zougagh; Helena Téllez; Alberto Sánchez; Manuel Chicharro; Angel Ríos (709-714).
A procedure for the analytical validation of a rapid supercritical fluid extraction amperometric screening method for controlling macrocyclic lactone mycotoxins in maize flour samples has been developed. The limit established by European legislation (0.2 mg kg−1), in reference to zearalenone (ZON) mycotoxin, was taken as the reference threshold to validate the proposed method. Natural ZON metabolites were also included in this study to characterize the final screening method. The objective was the reliable classification of samples as positive or negative samples. The cut-off level was fixed at a global concentration of mycotoxins of 0.17 mg kg−1. An expanded unreliability zone between 0.16 and 0.23 mg kg−1 characterized the screening method for classifying the samples. A set of 30 samples was used for the final demonstration of the reliability and usefulness of the method.
Keywords: Sample screening method; Sample classification; Validation; Macrocyclic lactone mycotoxins; Maize flour samples

A single optosensing device based on lanthanide-sensitized luminescence was developed for determination of p-aminobenzoic acid (PABA). The method is based on the formation of a complex between PABA and Tb(III) immobilized on the solid phase (QAE A-25 resin) placed inside the flow cell. NaCl (1 M) was used as carrier solution and HCl (0.05 M) as eluent. The sample solutions of PABA (100 μL) containing Tb(III) and buffered at pH = 6.0 were injected into the carrier stream and the luminescence was measured at λ ex = 290 nm and λ em = 546 nm. The method shows a linear range from 0.2 to 6.0 μg mL−1 with an RSD of 1.2% (n = 10) and a sampling frequency of 22 h−1. A remarkable characteristic of the method is its high selectivity which allows it to be satisfactorily applied to the analysis of PABA in pharmaceutical samples without prior treatment. Figure Typical emission bands of Tb(III) in a solid-phase PABA–Tb(III) luminescence spectrum
Keywords: Flow injection; Lanthanide-sensitized luminescence; Flow-through sensor; p-Aminobenzoic acid