Analytical and Bioanalytical Chemistry (v.384, #3)

Luminescent quantum dots in immunoassays by Ellen R. Goldman; Igor L. Medintz; Hedi Mattoussi (560-563).

Directing energy flow through quantum dots: towards nanoscale sensing by Dale M. Willard; Tina Mutschler; Ming Yu; Jaemyeong Jung; Alan Van Orden (564-571).
Nanoscale sensors can be created when an expected energetic pathway is created and then that pathway is either initiated or disrupted by a specific binding event. Constructing the sensor on the nanoscale could lead to greater sensitivity and lower limits of detection. To this end, quantum dots (QDs) can be considered prime candidates for the active components. Relative to organic chromophores, QDs have tunable spectral properties, show less susceptibility to photobleaching, have similar brightness, and have been shown to display electro-optical properties. In this review, we discuss recent articles that incorporate QDs into directed energy flow systems, some with the goal of building new and more powerful sensors and others that could lead to more powerful sensors. Figure
Keywords: Nanoparticles; Quantum dot cooperation; Switching; Biosensoring; Energy transfer; Quantum dots

Labeling strategies for bioassays by Christel Hempen; Uwe Karst (572-583).
Different labeling strategies for enzymatic assays and immunoassays are reviewed. Techniques which make use of direct detection of a label, e.g. radioimmunoassays, are discussed, as are techniques in which the label is associated with inherent signal amplification. Examples of the latter, e.g. enzyme-linked immunosorbent assays or nanoparticle-label based assays, are presented. Coupling of the bioassays to chromatographic separations adds selectivity but renders the assays more difficult to apply. The advantages and drawbacks of the different analytical principles, including future perspectives, are discussed and compared. Selected applications from clinical, pharmaceutical, and environmental analysis are provided as examples.
Keywords: Labeling; Immunoassay; Enzyme assay; Nanoparticle; Liquid chromatography

Nanomaterials in mass spectrometry ionization and prospects for biological application by Zhong Guo; Amel A. A. Ganawi; Qiang Liu; Lin He (584-592).
The rapid development of nanotechnology has revolutionized scientific developments in recent decades. Mass spectrometry (MS) measurements are no exception and have benefited greatly from integration of nanomaterials in every step of analysis. This brief review summarizes recent developments in the field with the focus on the use of nanomaterials as alternative media to facilitate analyte ionization in laser-desorption ionization–mass spectrometry (LDI–MS) and secondary ion mass spectrometry (SIMS). The biological applications of both techniques are also detailed. The use of nanomaterials in other aspects of MS analysis, for example in sample clean-up and indirect analyte quantification, is briefly discussed.
Keywords: Nanomaterials; Mass spectrometry; Bioanalysis

Synthesis of magnetic nanoparticles and their application to bioassays by Tetsuya Osaka; Tadashi Matsunaga; Takuya Nakanishi; Atsushi Arakaki; Daisuke Niwa; Hironori Iida (593-600).
Magnetic nanoparticles have been attracting much interest as a labeling material in the fields of advanced biological and medical applications such as drug delivery, magnetic resonance imaging, and array-based assaying. In this review, synthesis of iron oxide magnetic nanoparticles via a reverse micelle system and modification of their surface by an organosilane agent are discussed. Furthermore, as a practical biological assay system, the magnetic detection of biomolecular interactions is demonstrated by using the combination of a patterned substrate modified with a self-assembled monolayer and the magnetic nanoparticles.
Keywords: Magnetic nanoparticle; Iron oxide; Surface modification; Biomolecule detection; Patterned surface

The use of nanoparticles in electroanalysis: a review by Christine M. Welch; Richard G. Compton (601-619).
Nanoparticles can display four unique advantages over macroelectrodes when used for electroanalysis: enhancement of mass transport, catalysis, high effective surface area and control over electrode microenvironment. Therefore, much work has been carried out into their formation, characterisation and employment for the detection of many electroactive species. This paper aims to give an overview of the investigations carried out in this field. Particular attention is paid to examples of the advantages and disadvantages nanoparticles show when compared to macroelectrodes and the advantages of one nanoparticle modification over another. Most work has been carried out using gold, silver and platinum metals. However, iron, nickel and copper are also reviewed with some examples of other metals such as iridium, ruthenium, cobalt, chromium and palladium. Some bimetallic nanoparticle modifications are also mentioned because they can cause unique catalysis through the mixing of the properties of both metals.
Keywords: Nanoparticles; Electroanalysis; Electrocatalysis; Mass transport

Nano-oncology: drug delivery, imaging, and sensing by Nathaniel G. Portney; Mihrimah Ozkan (620-630).
Innovation in the last decade has endowed nanotechnology with an assortment of tools for delivery, imaging, and sensing in cancer research—stealthy nanoparticle vectors circulating in vivo, assembled with exquisite molecular control, capable of selective tumor targeting and potent delivery of therapeutics; intense and photostable quantum dot-based tumor imaging, enabling multicolor detection of cell receptors with a single optical excitation source; arrays of semiconducting nanowire and carbon nanotube sensor elements for selective multiplexed sensing of cancer markers without the need for probe labeling. These rapidly emerging tools are indicative of a burgeoning field ready to expand into medical applications. This review attempts to outline most of the current nanoparticle toolset for therapeutic release by liposomes, dendrimers, smart polymers, and virus-based systems. Advantages of nanoparticle-based imaging and targeting by use of nanoshells and quantum dots are also explored. Finally, emerging nanoelectronics-based sensing and a global discussion on the utility of each nanoparticle system addresses their fundamental advantages and shortcomings in cancer research.
Keywords: Nanotechnology; Sensing; Imaging; Detection; Cancer

Eu3+-doped Gd2O3 nanoparticles as reporters for optical detection and visualization of antibodies patterned by microcontact printing by Mikaela Nichkova; Dosi Dosev; Richard Perron; Shirley J. Gee; Bruce D. Hammock; Ian M. Kennedy (631-637).
Lanthanide oxide nanoparticles are promising luminescent probes in bioanalysis, because of their unique spectral properties, photostability, and low-cost synthesis. We report for the first time the application of europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles to the optical imaging of antibody micropatterns. The nanoparticles were synthesized by spray pyrolysis and coated with antibody (IgG) molecules by physical adsorption. Our experiments showed that the Eu:Gd2O3 is a good biocompatible solid support for antibody immobilization. The antibodies (anti-rabbit IgG) immobilized on the nanoparticles had excellent biological activity in the specific recognition reaction with rabbit IgG patterned in line strips (10 μm×10 μm) on a glass substrate by use of a micro-contact printing technique. The specific immunoreaction was confirmed by two independent microscopic techniques—fluorescence and scanning electron microscopy (SEM). Both microscopic images revealed that the nanoparticles were organized into designated structures as defined by the microcontact printing process with negligible non-specific binding. The nanoparticles can be used as fluorescent markers in a variety of immunosensing applications in a microscale format.
Keywords: Nanoparticles; Lanthanide oxide; Fluorescence; Immunoassay; Microarray; Microcontact printing

A highly sensitive fluorescent immunoassay based on avidin-labeled nanocrystals by King-Keung Sin; Cangel Pui-Yee Chan; Ting-Huen Pang; Matthias Seydack; Reinhard Renneberg (638-644).
Nanocrystals of the fluorogenic precursor fluorescein diacetate (FDA) were applied as labels in order to improve on the assay sensitivity achieved in our previous studies. Each FDA nanocrystal can be converted into ∼2.6×106 fluorescein molecules, which is useful for improving immunoassay sensitivity and limits of detection. NeutrAvidin was simply adsorbed onto the surface of the FDA nanocrystals, which were coated with distearoylglycerophosphoethanolamine (DSPE) modified with amino(poly(ethylene glycol))(PEG(2000)-Amine) as an interface for coupling biomolecules. This can be applied to detect different kinds of analytes that are captured by corresponding biotinylated biomolecules in different bioanalytical applications. The applicability of the NeutrAvidin-labeled nanocrystals was demonstrated in an immunoassay using the labeled avidin–biotin technique. Biotinylated antibody and FDA-labeled avidin were applied to the assay sequentially. The performance was compared with the traditional sandwich-type assay for mouse immunoglobulin G detection. Following the immunoreaction, the nanocrystals were released by hydrolysis and dissolution instigated by adding a large volume of organic solvent/sodium hydroxide mixture. The limit of detection was lower (by a factor of 2.5–21) and the sensitivity was (3.5–30-fold) higher than immunoassays using commercial labeling systems (FITC and peroxidase). This study shows that using fluorescent nanocrystals in combination with the avidin–biotin technique can enhance assay sensitivity and provide a lower limit of detection without requiring long incubation times as in enzyme-based labels.
Keywords: FDA; Avidin; NeutrAvidin; Biotin; Labeled avidin–biotin technique; Immunoassay; Fluorescent nanocrystals

The present paper describes a new, facile and fast technique that can be used to incorporate CdSe/ZnS core/shell nanocrystals into polystyrene beads. The nanocrystals were immobilized within the polymer beads by demixing two nonmiscible solvents. The resulting beads were bright, homogeneously coded, and did not show any surface texture or photobleaching under application-relevant conditions. The beads were subsequently used on a model streptavidin–biotin binding system in order to demonstrate the applicability of the technique to enzyme-linked assays and multiplexing applications.
Keywords: Luminescent nanocrystals; Quantum dots; High-throughput analysis; Fluorescence coding; Polymer beads

A rapid (5.5 min) one-step whole blood C–reactive protein (CRP) magnetic permeability immunoassay utilizing monoclonal antibody conjugated dextran iron oxide nanoparticles (70 nm) as superparamagnetic labels and mixed fractions (1:1 ratio of 15–40 and 60 μm) of polyclonal anti-CRP conjugated silica microparticles for enhanced sedimentation is described. In this one-step assay procedure, a whole blood sample (4 μl) is applied to an assay glass vial, containing both antibody conjugates, and mixed for 30 s. The target analyte, CRP, forms a sandwich complex between the conjugated nanoparticles and microparticles, and, subsequently, the complex sediments under normal gravitation within 5 min to the bottom of the vial. The magnetic permeability increase of the sediment due to the presence of the complexed superparamagnetic nanoparticles is determined using an inductance-based transducer. Assayed patient whole blood samples were compared with the Abbott Diagnostics Architect reference method. A strong linear correlation was observed for the CRP concentration range 0–260 mg/l in whole blood (y=1.001x+0.42, R 2=0.982, n=50). The CRP assay presented showed a limit of detection of 3 mg/l and a total imprecision (coefficient of variation) of 10.5%. On the basis of our observations, we propose a rapid, one-step, CRP assay for near-patient testing.
Keywords: Magnetic permeability; Superparamagnetic nanoparticles; Magneto immunoassay; C–reactive protein; Point-of-care

Multiplexed SNP genotyping using nanobarcode particle technology by Michael Y. Sha; Ian D. Walton; Scott M. Norton; Micah Taylor; Mark Yamanaka; Michael J. Natan; Chongjun Xu; Snezana Drmanac; Steve Huang; Adam Borcherding; Radoje Drmanac; Sharron G. Penn (658-666).
Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.
Keywords: SNP genotyping; Nanowires; Cytochrome P450; Encoded particles; Nanotechnology

Acoustic microsensors—the challenge behind microgravimetry by Ralf Lucklum; Peter Hauptmann (667-682).
Acoustic microsensors are commonly known as high-resolution mass-sensitive devices. This is a restricted view in many chemical and biosensor applications, especially in liquids. Sensitivity to non-gravimetric effects is a challenging feature of acoustic sensors. In this review we give an overview of recent developments in resonant sensors including micromachined devices and also list recent activity relating to the (bio)chemical interface of acoustic sensors. Major results from theoretical analysis of quartz crystal resonators, descriptive for all acoustic microsensors are summarized, and non-gravimetric contributions to the sensor signal from viscoelasticity and interfacial effects are discussed. We finally conclude with some future perspectives.
Keywords: Resonant microsensors; Acoustic sensors; Cantilever sensors

A novel method based on in-situ surface polymerization of methyl methacrylate (MMA) has been developed for rapid fabrication of poly(methyl methacrylate) (PMMA) electrophoresis microchips with sharp inlet tips. Prepolymerized MMA containing an ultraviolet (UV) initiator was directly sandwiched between a nickel template and a PMMA plate. The image of the relief on the nickel template was precisely replicated in the synthesized PMMA layer on the surface of the commercially available PMMA plate during UV-initiated polymerization at room temperature. The chips were subsequently assembled by thermal bonding of channel plates and cover sheets. The sample was directly introduced into the separation channel through a sharp inlet tip, which was placed in the sample vial, without use of an injection cross. The attractive performance of the novel PMMA microchips has been demonstrated by using contactless conductivity detection for determination of several inorganic ions. Such rapid and simple sample introduction leads to highly reproducible signals with relative standard deviations of less than 5% for peak responses. These new approaches significantly simplify the process of fabricating PMMA devices and show great promise for high-speed microchip analysis.
Keywords: Miniaturization; Microchip capillary electrophoresis; Contactless conductivity detection; Ions; Poly(methyl methacrylate)

Very often, the accuracy of quantitative analytical methods for the determination of mycotoxins by liquid chromatography (LC)-mass spectrometry (MS) and LC-MS/MS is limited by matrix effects during the ionization process in the MS source. Stable isotope labeled standards are best suited to correct for matrix effects and to improve both the trueness and the precision of analytical methods employing LC-MS and LC-MS/MS. This paper describes the successful use of fully 13C isotope labeled deoxynivalenol [(13C15)DON] as an internal standard (IS) for the accurate determination of DON in maize and wheat by LC electrospray ionization MS/MS. To show the full potential of (13C15)DON as IS, maize and wheat extracts were analyzed without further cleanup. Subsequent to calibration for the LC-MS end determination, DON was quantified in matrix reference materials (wheat and maize). Without consideration of the IS, apparent recoveries of DON were 29±6% (n=7) for wheat and 37±5% (n=7) for maize. However, the determination of DON in the reference materials yielded 95±3% (wheat) and 99±3% (maize) when (13C15)DON was used as an IS for data evaluation.
Keywords: Deoxynivalenol; Mass spectrometry; Internal standard; Stable isotope; Wheat; Maize

Separation and identification of selenotrisulfides in epithelial cell homogenates by LC–ICP–MS and LC–ESI-MS after incubation with selenite by Charlotte Gabel-Jensen; Bente Gammelgaard; Lars Bendahl; Stefan Stürup; Ole Jøns (697-702).
To elucidate how selenite is metabolised in the intestine after oral intake, it was incubated with homogenized epithelial cells from pigs. When the metabolites were analysed by LC–ICP–MS, two major selenium metabolites were separated in the supernatant from the homogenate. These metabolites were formed instantly but disappeared within 15 min. No other selenium-containing compounds appeared during this time. Hence, the secondary reaction products were either volatilised or precipitated. To verify the identity of the compounds, a larger amount of selenite was incubated with epithelial cells. The presence of Cys-Se-SG and GS-Se-SG was verified by LC–ESI-MS. Selenotrisulfides were synthesized by reaction of L-cysteine and L-glutathione with sodium selenite. The reaction mixture contained three main products: selenodicysteine (Cys-Se-Cys), selenocysteine glutathione (Cys-Se-SG), and selenodiglutathione (GS-Se-SG). The two transient selenium compounds in the epithelial cell incubation mixture co-eluted with the synthesized Cys-Se-SG and GS-Se-SG, respectively. The identities of these compounds were verified by LC–ESI-MS. Hence, these selenium metabolites have now been identified by ESI-MS after isolation from epithelial cells.
Keywords: Selenium metabolites; Selenotrisulfides; ICP–MS; Epithelial cells

We report on a new and convenient high-throughput fluorescence technique for determining antioxidant capacities of hydrophilic food samples. The new method is called αPROX (anti protein oxidation) and is based on an equimolar complex of diphenylhexatriene propionic acid (DPHPA) and bovine serum albumin (BSA) in aqueous buffer at pH 7.4. DPHPA is a reporter fluorophore that becomes nonfluorescent upon free radical-induced oxidation. In a typical assay, the DPHPA/BSA complex is challenged with peroxyl radicals and shows almost the same susceptibility to oxidation as unlabeled BSA. The progress of protein oxidation and its inhibition by antioxidants at physiological pH is determined from the time-dependent decrease in DPHPA fluorescence intensity. The αPROX method was compared to other techniques frequently used to measure antioxidant capacities. In this article, representative results are provided for the inhibitory effects of pure food components, fruit juices, wines, and various polar plant extracts on protein oxidation.
Keywords: Antioxidant capacity; Diphenylhexatriene propionic acid (DPHPA); Protein oxidation; Phenolics

An immunoassay for a urinary metabolite as a biomarker of human exposure to the pyrethroid insecticide permethrin by Ki Chang Ahn; Seung-Jin Ma; Hsing-Ju Tsai; Shirley J. Gee; Bruce D. Hammock (713-722).
Permethrin is the most popular synthetic pyrethroid insecticide used in agriculture and public health. For the assessment of human exposure to permethrin, a competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the glycine conjugate of a major metabolite, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was developed based on a polyclonal antibody. An assay based on an antibody with a high sensitivity was optimized and characterized. The IC50 value and the detection range for trans-DCCA–glycine, in the assay buffer were 1.2 and 0.2−7.0 μg/L, respectively. The antibody recognized trans-DCCA–glycine and the mixture of cis-/trans-DCCA–glycine with an isomer range from 30:70 to 50:50 nearly equally. Little or no cross-reactivity to permethrin and its other free metabolites or glycine conjugates was measured. The integration of the ELISA and solid-phase extraction which was used to reduce the matrix effect from human urine samples provided for analysis of total cis-/trans-DCCA–glycine at low parts per billion levels in the samples. The limit of quantitation of the target analyte was 1.0 μg/L in urine with a limit of detection of 0.1 μg/L in buffer. This assay might be a useful tool for monitoring human exposure to permethrin.
Keywords: ELISA; Permethrin; Human exposure; Glycine metabolite; Polyclonal antibodies

It is necessary to understand the changes that occur during the initial processing of lamb skins, because these will affect the final quality of the leather. The types of collagen, their macro and micro structures, the presence of proteins other than collagens, and the quantity and the type of proteoglycans, all have a profound effect on the quality of leather. Proteins isolated from untreated or raw sheep skin and from pickled skin (skins treated with sodium sulfide and lime followed by bating with enzymes, then preserved in sodium chloride and sulfuric acid) were significantly different when analysed by use of 2D gel electrophoresis and mass spectrometry. Agarose gel electrophoresis with a very sensitive sequential staining procedure has been used to identify the glycosaminoglycans present in raw and treated skin and their impact on quality of leather. Results showed that effective removal of proteoglycans acting as inter-fibrillar adhesives of collagen fibrils seemed to improve leather quality. Removal of these molecules not only opens up the fibre structure of the skin but may also be important in wool removal. The presence of elastin, which imparts elastic properties to skin, is of significant importance to tanners. The amino acids desmosine and isodesmosine, found exclusively in elastin, were quantitatively analysed to assess the role of elastin in leather quality.
Keywords: 2D gel electrophoresis; MALDI–TOF mass spectrometry; Collagens; Glycosaminoglycans; Elastins

Construction of glucose biosensor based on sorption of glucose oxidase onto multilayers of polyelectrolyte/nanoparticles by Suxia Zhang; Weiwei Yang; Yaming Niu; Yancai Li; Ming Zhang; Changqing Sun (736-741).
A new approach to constructing an enzyme-containing film on the surface of a gold electrode for use as a biosensor is described. A basic multilayer film (BMF) of (PDDA/GNPs) n /PDDA was first constructed on the gold electrode by electrostatic layer-by-layer self-assembly of poly(diallyldimethylammonium chloride) (PDDA) and gold nanoparticles (GNPs). Glucose oxidase (GOx) was then sorbed into this BMF by dipping the BMF-modified electrode into a GOx solution. The assembly of the BMF was monitored and tested via UV-vis spectroscopy and cyclic voltammetry (CV). The ferrocenemethanol-mediated cyclic voltammograms obtained from the gold electrode modified with the (PDDA/GNPs) n /PDDA/GOx indicated that the assembled GOx remained electrocatalytically active for the oxidation of glucose. Analysis of the voltammetric signals showed that the surface coverage of active enzyme was a linear function of the number of PDDA/GNPs bilayers. This result confirmed the penetration of GOx into the BMF and suggests that the BMF-based enzyme film forms in a uniform manner. Electrochemical impedance measurements revealed that the biosensor had a lower electron transfer resistance (R et) than that of a sensor prepared by layer-by-layer assembly of PDDA and GOx, due to the presence of gold nanoparticles. The sensitivity of the biosensor for the determination of glucose, which could be controlled by adjusting the number of PDDA/GNPs bilayers, was investigated.
Keywords: Glucose oxidase; Gold nanoparticles; Electrostatic self-assembly; Biosensors; Polyelectrolyte multilayers

Determination of human serum albumin using aurothiomalate as electroactive label by Alfredo de la Escosura-Muñiz; María Begoña González-García; Agustín Costa-García (742-750).
A new electroactive label has been used to monitor immunoassays in the determination of human serum albumin (HSA) using glassy-carbon electrodes as supports for the immunological reactions. The label was a gold(I) complex, sodium aurothiomalate, which was bound to rabbit IgG anti-human serum albumin (anti-HSA-Au). The HSA was adsorbed on the electrode surface and the immunological reaction with gold-labelled anti-HSA was then performed for one hour by non-competitive or competitive procedures. The gold(I) bound to the anti-HSA was electrodeposited in 0.1 mol L−1 HCl at −1.00 V for 5 min then oxidised in 0.1 mol L−1 H2SO4 solution at +1.40 V for 1 min. Silver electrodeposition at −0.14 V for 1 min followed by anodic stripping voltammetry were then performed in aqueous 1.0 mol L−1 NH3–2.0×10−4 mol L−1 AgNO3. For both non-competitive and competitive formats, calibration plots in the ranges 5.0×10−10 to 1.0×10−8 mol L−1 and 1.0×10−10 to 1.0×10−9 mol L−1 HSA, respectively, with estimated detection limits of 1.5×10−10 mol L−1 (10 ng mL−1) and 1.0×10−10 mol L−1 (7 ng mL−1), respectively, were obtained. Levels of HSA in two healthy volunteer urine samples were also evaluated, using both immunoassay formats.
Keywords: Electroactive label; Aurothiomalate; Immunosensor; Rabbit IgG; Human serum albumin; Glassy-carbon electrode

Capillary electrophoresis–electrospray mass spectrometry and HR–ICP–MS for the detection and quantification of 10B-boronophenylalanine (10B–BPA) used in boron neutron capture therapy by Aurélien Pitois; Laura Aldave de las Heras; Antonella Zampolli; Luca Menichetti; Ramon Carlos; Guido Lazzerini; Luca Cionini; Pietro Alberto Salvatori; Maria Betti (751-760).
Boron neutron capture therapy (BNCT) is a bimodal radiotherapeutic treatment based on the irradiation of neoplastic tissues with neutrons after the tissues have selectively accumulated molecules loaded with nuclides with large neutron capture cross-sections (such boron-10). Boron-10 carriers have been tested to a limited extent, and clinical trials have been conducted on sulfhydryl borane (10B-BSH) and boronophenylalanine (10B-BPA). However, precise and accurate measurements of boron-10 concentrations (0.1–100 μg/g) in specimens and samples of limited size (μg scale) are needed in order to be able to biologically characterise new compounds in predictive tissue dosimetry, toxicology and pharmacology studies as well as in clinical investigations. A new approach based on fast separation and detection of 10B-BPA performed by coupling capillary electrophoresis to electrospray mass spectrometry is reported. This method allows the quantitative analysis and characterisation of 10B-BPA in a short time with a high separation efficiency. Detection limits of 3 μM for 10B-BPA and 30 ng/mL for 10B were obtained with CE–ESI–MS. A quantification limit of 10 μM for 10B-BPA (100 ng/mL for 10B) was attained. The total boron-10 concentration was determined by high-resolution inductively coupled mass spectrometry in order to validate the method. Boron-10 isotope measurements were carried out by HR–ICP–MS at medium resolution (R=4000) due to the presence of an isobaric interference at mass 10. Good agreement was obtained between the values from CE–ESI–MS and those from HR–ICP–MS. The method has been successfully used to determine the 10B-BPA in two lines of cultured cells.
Keywords: Capillary electrophoresis; Electrospray mass spectrometry; High-resolution ICP-MS; Boronophenylalanine; Boron neutron capture therapy

Cotinine, the main metabolite of nicotine in human body, is widely used as a biomarker for assessment of direct or passive exposure to tobacco smoke. A method for molecularly imprinted solid-phase extraction (MISPE) of cotinine from human urine has been investigated. The molecularly imprinted polymer (MIP) with good selectivity and affinity for cotinine was synthesized using cotinine as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker. The imprinted polymer was evaluated for use as a SPE sorbent, in tests with aqueous standards, by comparing recovery data obtained using the imprinted form of the polymer and a non-imprinted form (NIP). Extraction from the aqueous solutions resulted in more than 80% recovery. A range of linearity for cotinine between 0.05 and 5 μg mL−1 was obtained by loading 1 mL blank urine samples spiked with cotinine at different concentrations in acetate buffer of pH 9.0, and by using double basic washing and acidic elution. The intra-day coefficient of variation (CV) was below 7% and inter-day CV was below 10%. This investigation has provided a reliable MISPE–HPLC method for determination of cotinine in human urine from both active smokers and passive smokers. Figure
Keywords: Molecularly imprinted polymer; Solid-phase extraction; Cotinine; Urine samples; MISPE–HPLC

A supramolecular catalytic kinetic spectrofluorimetric method was developed for the determination of platinum(IV) and the possible mechanism of catalytic reaction was discussed. The method was based on the fluorescence-enhancing reaction of salicylaldehyde furfuralhydrazone (SAFH) with potassium bromate, which was catalysed by platinum(IV) in a water–ethanol medium. β–Cyclodextrin (β-CD) obviously sensitized the determination at pH 5.20 and 25°C. Under optimum conditions, the β-CD–platinum–KBrO3–SAFH supramolecular kinetic catalytic reaction system had excitation and emission maxima at 372 and 461 nm, respectively. The linear range of this method was 0.60–180 ng ml−1 with a relative standard deviation of 1.2%, and the detection limit was 0.18 ng ml−1. Investigation of the mechanism and the effects of interferences is presented. The proposed method was applied successfully to determine trace platinum(IV) in the chemotherapeutic drug cisplatin and serum from patients with satisfactory results.
Keywords: Catalytic kinetic spectrofluorimetry; Platinum; Salicylaldehyde furfuralhydrazone; β-Cyclodextrin; Supramolecular complex

The determination of inorganic phosphorus in human urine is very important, since it has diagnostic value in some clinical cases. Here we apply a simple, sensitive and direct method to determine inorganic phosphorus in urine. This new ensemble is prepared by adding ytterbium chloride and pyrocatechol violet in a 2:1 molar ratio in an aqueous solution of 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid buffer at pH 7.0. The addition of the urine sample turned the blue ensemble yellow and altered the UV–vis absorption spectra. The ensemble exhibits excellent selectivity for inorganic phosphorus over other constituents of urine. We validate the accuracy of our method by the standard procedure (molybdenum blue assay for phosphate). The detection results are basically consistent with normal excretion of phosphate. Furthermore, we fabricated a new kind of inorganic phosphorus reagent kit, which enables us to inspect phosphate concentrations of urine with the naked eye. Fit for all kinds of various clinic uses, our reagent kit is a hopeful substitute for the molybdenum reagent kit.
Keywords: UV–vis spectra; Pyrocatechol violet–2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid–Yb; Inorganic phosphorus; Urine; Reagent kit

Simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma by rapid liquid chromatography/tandem mass spectrometry by Ramakrishna V. S. Nirogi; Vishwottam N. Kandikere; Manoj Shukla; Koteshwara Mudigonda; Wishu Shrivasthava; Praveen V. Datla; Anjaneyulu Yerramilli (780-790).
A simple, rapid, sensitive and selective liquid chromatography/electrospray tandem mass spectrometry method was developed and validated for the simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma using mosapride as an internal standard. The method involves a simple one-step liquid-liquid extraction with a diethyl ether and dichloromethane mixture (7:3). The analytes were chromatographed using an isocratic mobile phase on a reversed-phase C18 column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 370/288 for cilostazol, m/z 368/286 for 3,4-dehydrocilostazol and m/z 422/198 for the internal standard. The assay exhibited a linear dynamic range of 5–2,000 ng/mL for cilostazol and 5–400 ng/mL for 3,4-dehydrocilostazol in human plasma. The lower limit of quantitation was 5 ng/mL for both cilostazol and its metabolite. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetics, bioavailability or bioequivalence studies.
Keywords: Cilostazol; 3,4-Dehydrocilostazol; Human plasma; Liquid chromatography/tandem mass spectrometry; Pharmacokinetics study

Determination of polybrominated diphenyl ethers in indoor dust standard reference materials by Heather M. Stapleton; Tom Harner; Mahiba Shoeib; Jennifer M. Keller; Michele M. Schantz; Stefan D. Leigh; Stephen A. Wise (791-800).
Polybrominated diphenyl ethers (PBDEs) have been measured for the first time in three different indoor dust Standard Reference Materials (SRMs) prepared by the National Institute of Standards and Technology (NIST). Two of these, SRM 2583 (Trace Elements in Indoor Dust) and SRM 2584 (Trace Elements in Indoor Dust), have been certified previously for lead and other inorganic constituents. A third, SRM 2585 (Organics in Indoor Dust), is a new indoor dust reference material prepared by NIST which will be certified for various organic compounds (polycyclic aromatic hydrocarbons, pesticides and polychlorinated biphenyls) in 2005 including certified concentrations for 16 individual PBDE congeners and reference values for an additional three PBDE congeners. Dust SRMs were analyzed for 30 PBDE congeners using high-resolution gas chromatography combined with low-resolution mass spectrometry operated in both negative chemical ionization (GC/ECNI–MS) and electron impact ionization (GC/EI–MS) modes. Sensitivity was an order of magnitude higher using GC/ECNI–MS relative to GC/EI–MS. These SRMs have been characterized and compared to the three PBDE commercial products (pentaBDE, octaBDE and decaBDE). PentaBDE and DecaBDE were present in all three SRMs and were the dominant commercial products, making up approximately 33% and 58%, respectively. Recent studies suggest that house dust may be a leading source of human exposure to PBDEs. These SRMs are the first reference materials with certified concentrations for PBDEs, which will aid in validating future measurements of PBDEs in house dust and other similar matrices.
Keywords: PBDEs; Standard Reference Materials; House dust; Brominated flame retardants; NIST

A comparative study was carried out for the simultaneous determination of 11 pyrethroid insecticides in soil by gas chromatography (GC) - ion trap (IT)- mass spectrometry (MS), by means of two different ionization modes; electron impact and negative chemical ionization and three data acquisition procedures; full scan, selected ion monitoring and MS/MS. Pyrethroids investigated were tetramethrin, bifenthrin, phenothrin, λ-cyhalothrin, permethrin, cyfluthrin, cypermethrin, flucythrinate, esfenvalerate, fluvalinate and deltamethrin. Soil samples were treated with toluene/water by microwave-assisted extraction for 9 min at 700 W and a cleaning up with florisil was performed. Clean soil samples were spiked with pyrethroids at a spiking level of 10, 25 and 50 ng/g. The method employed provides a concentration factor of 10. The ionization gas employed in the negative chemical ionization mode was methane. The use of MS/MS acquisition, in electron impact ionization, provided the best results, due to its high selectivity and sensitivity, giving very low limits of detection from 0.08 to 0.54 ng/g. In negative chemical ionization full scan and selected ion monitoring methods detection limits from 0.12 to 1.40 ng/g were found. The proposed methods were applied to several levels from 10 to 50 ng/g of spiked soils, being electron impact MS/MS method which minimizes matrix spectrum interferences and provided recovery average values from 84% to 120% with relative standard deviations which varied from 3.2 to 7.2%.
Keywords: Pyrethroid; Residues; Soil; Negative Chemical Ionization; MS/MS

A new procedure for determining eleven organochlorine pesticides in soils using microwave-assisted extraction (MAE) and headspace solid phase microextraction (HS-SPME) is described. The studied pesticides consisted of mirex, α- and γ-chlordane, p,p′-DDT, heptachlor, heptachlor epoxide isomer A, γ-hexachlorocyclohexane, dieldrin, endrin, aldrine and hexachlorobenzene. The HS-SPME was optimized for the most important parameters such as extraction time, sample volume and temperature. The present analytical procedure requires a reduced volume of organic solvents and avoids the need for extract clean-up steps. For optimized conditions the limits of detection for the method ranged from 0.02 to 3.6 ng/g, intermediate precision ranged from 14 to 36% (as CV%), and the recovery from 8 up to 51%. The proposed methodology can be used in the rapid screening of soil for the presence of the selected pesticides, and was applied to landfill soil samples.
Keywords: Microwave-assisted extraction; Solid phase microextraction; Chlorinated pesticides; Extraction methods; Landfill soil

This work is devoted to the EXAFS analysis of nanostructured iron(III) oxide synthesized inside the pore system of mesoporous carbon CMK-1. A detailed study of the recording, preparation and evaluation of data recorded in fluorescence mode at the iron K-edge with and without multiple scattering is shown. The results obtained show that the local structure of Fe3+ inside nanostructured iron(III) oxide is different to that of the bulk material. Due to the small particle size, data analysis is much more difficult and data preparation more complex. Incorporating multiple scattering paths in the Fourier transforms and back-transforms during data evaluation gives structural insights that cannot be obtained using other spectroscopic methods, and this technique was used to draw conclusions about the first four coordination spheres of the nanostructured iron(III) oxide.
Keywords: Fe-K XAFS; Nanostructured host/guest compounds; Mesoporous carbon; Nanostructured iron(III) oxide

Adsorption of mercury ions by mercapto-functionalized amorphous silica by Damián Pérez-Quintanilla; Isabel del Hierro; Fernando Carrillo-Hermosilla; Mariano Fajardo; Isabel Sierra (827-838).
Amorphous silicas have been functionalized by two different methods. In the heterogeneous route the silylating agent, 3-chloropropyltriethoxysilane, was initially immobilized onto the silica surface to give the chlorinated silica Cl-Sil. In a second reaction, multifunctionalized N,S donor compounds were incorporated to obtain the functionalized silicas, which are denoted as L-Sil-Het (where L=mercaptothiazoline, mercaptopyridine or mercaptobenzothiazole). In the homogeneous route, the functionalization was achieved through a one-step reaction between the silica and an organic ligand containing the chelating functions; this gave the modified silicas denoted as L-Sil-Hom. The functionalized silicas were characterized by elemental analysis, IR spectroscopy and thermogravimetry. These materials were employed as adsorbents for mercury cations from aqueous and acetone solutions at room temperature. The results indicate that, in all cases, mercury adsorption was higher in the modified silicas prepared by the homogeneous method. Figure
Keywords: Modified silica gel; Mercury adsorption; 2-Mercaptothiazoline; 2-Mercaptopyridine; 2-Mercaptobenzothiazole

An improvement of the peak parking technique is described for the serial determination of cations (Na+, $$NH^{ + }_{4} $$ , K+, Mg2+, and Ca2+) and anions (Cl, $$SO^{{2 - }}_{4} $$ , and $$NO^{ - }_{3} $$ ) using a single pump, a single eluent and a single detector. The present system used commercially-available unmodified cation exchange and anion exchange columns, which were attached to each switching valve. When 1.75 mM 5-sulfosalicylic acid was used as the eluent, serial separation of the above cations and anions was achieved in less than 20 min. The proposed ion chromatographic method was successfully applied to the serial determination of cations and anions in tap water and river water samples. The limits of detection at S/N=3 for an injection of 20 μl were 16–68 ppb (μg/l) for cations and 15–28 ppb for anions.
Keywords: Ion chromatography; Column switching; Peak parking; 5-Sulfosalicylic acid

In this study the possibility of derivatizing sugars using microwave irradiation was investigated. The amount of reagent, irradiation intensity, and derivatization time were optimized. In the derivatization of sugars with p-nitroaniline the reaction is complete within 5 min at 600 W when the p-nitroaniline-to-sugar and NaBH3CN-to-sugar mole ratios were above 1.4 and 3.1, respectively. A Doehlert design was used to optimize the mobile phase for separation of p-nitroaniline-labeled sugars; and the best separation was obtained by use of 0.01 mol L−1 acetate buffer at pH 4.40 containing 11.0% acetonitrile. Analysis using this method was highly sensitive and analysis time was short. Finally, a food sample was analyzed using the proposed method.
Keywords: Sugars; Microwave-assisted reaction; High-performance liquid chromatography; p-Nitroaniline; Doehlert design

A chemometrics approach has been used to optimize the separation of eight coumarin compounds by mixed micellar liquid chromatography. A utility function, a multi-criterion decision-making (MCDM) method, was tested for evaluation of two different measures of chromatographic performance (resolution and analysis time). The effect of six experimental parameters on a chromatographic response function (CRF) was investigated. The factors studied were the concentrations of SDS and Brij-35, alkyl-chain length of the alcohol used as organic modifier, organic modifier concentration, mobile phase pH, and temperature. The experiments were performed according to a face-centered cube response-surface experimental design. For evaluation of the chromatograms a simple linear response function was used which expressed as a summation of two optimization criteria, resolution and analysis time. Then calculated CRF values were fitted to a polynomial model to correlate the CRF values with the variables and their interactions. The regression model obtained was characterized by both its descriptive and predictive ability (R=0.963 and $${ ext{R}}^{{ ext{2}}} _{{{ ext{cv}}}}=0.861$$ ) and used, by means of a grid-search algorithm, to optimize the chromatographic conditions. Experiments performed under the optimum conditions predicted by the model produced a chromatogram of high quality. The model was also verified by the good agreement observed between predicted and experimental values of the chromatographic response function under the optimum conditions.
Keywords: Mixed micellar liquid chromatography; Coumarins; Utility function; Experimental design; Optimization