Analytical and Bioanalytical Chemistry (v.396, #3)

Chemical characterization of engineered nanoparticles by Michael R. Winchester; Ralph E. Sturgeon; José Manuel Costa-Fernández (951-952).
is a research chemist in the Analytical Chemistry Division of the National Institute of Standards and Technology (NIST) in Gaithersburg, Maryland, USA. His current research interests include the development of atomic spectroscopic and chromatographic methods of exceptionally high accuracy and precision and analytical methods for chemical analysis of nanoparticles. He is responsible for approximately 75 NIST Standard Reference Materials, including the 3100 series of single-element standard solutions and the 3180 series of anion standard solutions. is currently a principal research officer and group leader for Chemical Metrology at the Institute for National Measurement Standards, National Research Council Canada. His interests lie in inorganic analytical chemistry, comprising trace element analysis, vapor generation, instrument development, organometallic speciation, and production of certified reference materials with a focus on atomic and mass-spectrometric detection. is an associate professor of analytical chemistry in the Department of Physical and Analytical Chemistry at the University of Oviedo (Spain), and is Vice-Dean of the Chemistry Faculty. His scientific interests include development of functionalized photoluminescent nanomaterials (e.g., quantum dots) for biolabeling and sensing applications and the use of plasma-based elemental mass spectrometry for direct analysis of solids and chemical speciation studies.

earned a B.Sc. in Toxicology from the University of Guelph and a Ph.D. in Biochemistry/Toxicology from Michigan State University. Prior to his graduate work, he was employed as a Scientific Evaluator with Health Canada where he was involved in the evaluation of chemicals on the Canadian DSL. In 2006, he joined The Dow Chemical Company as a toxicology specialist where he is responsible for leading research programs in the areas of nanotoxicology, respiratory allergy and sensitization, and toxicogenomics and serves as a representative on the American Chemistry Council Nanotechnology Panel. is Manager of Toxicology for Industrial Chemicals in BASF Corporation. He received his Ph.D. in Pharmacology from the University of Louisville, after which he was a Postdoctoral Fellow at the Chemical Institute of Toxicology in Research Triangle Park. Dr. David worked for 8 years at Microbiological Associates in Bethesda, Maryland where he managed the Inhalation and Mammalian Toxicology Departments. He also spent 14 years at Eastman Kodak in Rochester New York as Senior Toxicologist before joining BASF in 2006. Dr. David has experience conducting inhalation, pulmonary, reproductive, and systemictoxicity studies. He was responsible for EH&S issues for nanotechnology at Eastman Kodak Company, and is currently responsible for nanotechnology issues in BASF Corporation. Nanotechnology is a rapidly emerging field of great interest and promise. As new materials are developed and commercialized, hazard information also needs to be generated to reassure regulators, workers, and consumers that these materials can be used safely. The biological properties of nanomaterials are closely tied to the physical characteristics, including size, shape, dissolution rate, agglomeration state, and surface chemistry, to name a few. Furthermore, these properties can be altered by the medium used to suspend or disperse these water-insoluble particles. However, the current toxicology literature lacks much of the characterization information that allows toxicologists and regulators to develop “rules of thumb” that could be used to assess potential hazards. To effectively develop these rules, toxicologists need to know the characteristics of the particle that interacts with the biological system. This void leaves the scientific community with no options other than to evaluate all materials for all potential hazards. Lack of characterization could also lead to different laboratories reporting discordant results on seemingly the same test material because of subtle differences in the particle or differences in the dispersion medium used that resulted in altered properties and toxicity of the particle. For these reasons, good characterization using a minimal characterization data set should accompany and be required of all scientific publications on nanomaterials.
Keywords: Nanotoxicology; Nanomaterial characterization; Nanomaterials; Nanotoxicity

The current standardization activities of ISO (International Organization for Standardization) TC229 on “Nanotechnology” are introduced with focus on the work of WG2 (Working Group 2) for “measurement and characterization”. Seven project groups of WG2 are actively preparing standard protocols (technical specifications) for characterization of single-wall carbon nanotubes (SWCNTs) by measurement methods such as TEM (transmission electron microscopy), SEM (scanning electron microscopy), EDX (energy-dispersive X-ray analysis), UV–Vis–NIR (ultraviolet-visible-near infrared) absorption spectroscopy, NIR-photoluminescence spectroscopy, EGA (evolved gas analysis)–GC–MS (gas chromatography–mass spectrometry), TGA (thermogravimetric analysis), and Raman spectroscopy; this work is described. The features of purity evaluation of SWCNTs by these methods are also briefly described and compared. Also described are two project groups of WG2 that are preparing standard protocols for characterization of multiwall CNTs (MWCNTs), aiming at the purity control by measurement of moisture content, ash content, metallic constituents, volatile content, polyaromatic hydrocarbon content, and carbon materials excluding MWCNTs. Other important properties for characterization of MWCNT, for example disorder, burning property, stacking nature, length, morphology, and inner/outer diameter, etc., are also mentioned. Finally, the importance and urgency of standardization for potential risk assessment of CNTs is briefly described, and current joint activity of ISO TC229 WG2 and WG3 for physicochemical characterization of engineered nanoscale materials for toxicological assessment is introduced.
Keywords: Nanotechnology; Standardization; Measurement and characterization; Carbon nanotubes; ISO (International Organization for Standardization)

Chemical modifications of nanoparticle (NP) surfaces are likely to regulate their activities, remove their toxic effects, and enable them to perform desired functions. It is urgent to develop analytical strategies for acquiring structural and quantitative information about small molecules linked to the surface of NP. Recent progress in characterizing the surface chemistry of NPs using nuclear magnetic resonance (NMR) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, liquid chromatography–mass spectroscopy (LC–MS), X-ray photoelectron spectroscopy (XPS), and combustion elemental analysis are reviewed.
Keywords: Nanoparticle; NMR; FTIR; MS; XPS; Combustion elemental analysis

Application of surface chemical analysis tools for characterization of nanoparticles by D. R. Baer; D. J. Gaspar; P. Nachimuthu; S. D. Techane; D. G. Castner (983-1002).
The important role that surface chemical analysis methods can and should play in the characterization of nanoparticles is described. The types of information that can be obtained from analysis of nanoparticles using Auger electron spectroscopy (AES), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (TOF-SIMS), low-energy ion scattering (LEIS), and scanning-probe microscopy (SPM), including scanning tunneling microscopy (STM) and atomic force microscopy (AFM), are briefly summarized. Examples describing the characterization of engineered nanoparticles are provided. Specific analysis considerations and issues associated with using surface-analysis methods for the characterization of nanoparticles are discussed and summarized, with the impact that shape instability, environmentally induced changes, deliberate and accidental coating, etc., have on nanoparticle properties. Figure Atomic force microscopy image of Cu2O nanodots formed on a SrTiO3 substrate.
Keywords: Nanoparticles; Nanotechnology; X-ray spectroscopy (XPS XRF EDX); Catalysts

Chemical and structural characterization of carbon nanotube surfaces by Kevin A. Wepasnick; Billy A. Smith; Julie L. Bitter; D. Howard Fairbrother (1003-1014).
To utilize carbon nanotubes (CNTs) in various commercial and scientific applications, the graphene sheets that comprise CNT surfaces are often modified to tailor properties, such as dispersion. In this article, we provide a critical review of the techniques used to explore the chemical and structural characteristics of CNTs modified by covalent surface modification strategies that involve the direct incorporation of specific elements and inorganic or organic functional groups into the graphene sidewalls. Using examples from the literature, we discuss not only the popular techniques such as TEM, XPS, IR, and Raman spectroscopy but also more specialized techniques such as chemical derivatization, Boehm titrations, EELS, NEXAFS, TPD, and TGA. The chemical or structural information provided by each technique discussed, as well as their strengths and limitations. Particular emphasis is placed on XPS and the application of chemical derivatization in conjunction with XPS to quantify functional groups on CNT surfaces in situations where spectral deconvolution of XPS lineshapes is ambiguous. Figure Of the more common techniques, Raman, TEM, & XPS are often used in the analysis of surface modified carbon nanotubes
Keywords: X-ray spectroscopy (XPS | XRF | EDX); Nanoparticles/nanotechnology; Spectroscopy/instrumentation; Potentiometry/titrations; IR spectroscopy/Raman spectroscopy; Interface/surface analysis

Single-walled carbon nanotubes (SWCNTs) are a family of structurally related artificial nanomaterials with unusual properties and many potential applications. Most SWCNTs can emit spectrally narrow near-IR fluorescence at wavelengths that are characteristic of their precise diameter and chiral angle. Near-IR fluorimetry therefore offers a powerful approach for identifying the structural species present in SWCNT samples. Such characterization is increasingly important for nanotube production, study, separation, and applications. General-purpose and specialized instruments suitable for SWCNT fluorimetric analysis are described, and methods for interpreting fluorimetric data to deduce the presence and relative abundances of different SWCNT species are presented. Fluorescence methods are highly effective for detecting SWCNTs in challenging samples such as complex environmental or biological specimens because of the methods’ high sensitivity and selectivity and the near absence of interfering background emission at near-IR wavelengths. Current limitations and future prospects for fluorimetric characterization of SWCNTs are discussed.
Keywords: Single-walled carbon nanotube; Near-IR fluorescence; Fluorimetric analysis; Fluorimetry

Laser desorption/ionization mass spectrometry analysis of monolayer-protected gold nanoparticles by Bo Yan; Zheng-Jiang Zhu; Oscar R. Miranda; Apiwat Chompoosor; Vincent M. Rotello; Richard W. Vachet (1025-1035).
Monolayer-protected gold nanoparticles (AuNPs) feature unique surface properties that enable numerous applications. Thus, there is a need for simple, rapid, and accurate methods to confirm the surface structures of these materials. Here, we describe how laser desorption/ionization mass spectrometry (LDI-MS) can be used to characterize AuNPs with neutral, positively, and negatively charged surface functional groups. LDI readily desorbs and ionizes the gold-bound ligands to produce both free thiols and disulfide ions in pure and complex samples. We also find that LDI-MS can provide a semi-quantitative measure of the ligand composition of mixed-monolayer AuNPs by monitoring mixed disulfide ions that are formed. Overall, the LDI-MS approach requires very little sample, provides an accurate measure of the surface ligands, and can be used to monitor AuNPs in complex mixtures. Figure LDI-MS analysis of ligand percentage on mixed monolayer AuNPs.
Keywords: Nanoparticles; Laser desorption/ionization; Mass spectrometry; Surface ligands

Coupled thermogravimetry, mass spectrometry, and infrared spectroscopy for quantification of surface functionality on single-walled carbon nanotubes by Christopher T. Kingston; Yadienka Martínez-Rubí; Jingwen Guan; Michael Barnes; Christine Scriver; Ralph E. Sturgeon; Benoit Simard (1037-1044).
We have successfully applied coupled thermogravimetry, mass spectrometry, and infrared spectroscopy to the quantification of surface functional groups on single-walled carbon nanotubes. A high-purity single-walled carbon nanotube sample was subjected to a rapid functionalization reaction that attached butyric acid moieties to the nanotube sidewalls. This sample was then subjected to thermal analysis under inert desorption conditions. Resultant infrared and mass spectrometric data were easily utilized to identify the desorption of the butyric acid groups across a narrow temperature range and we were able to calculate the degree of substitution of the attached acid groups within the nanotube backbone as 1.7 carbon atoms per hundred, in very good agreement with independent analytical measurements made by inductively coupled plasma optical emission spectrometry (ICP-OES). The thermal analysis technique was also able to discern the presence of secondary functional moieties on the nanotube samples that were not accessible by ICP-OES. This work demonstrates the potential of this technique for assessing the presence of multiple and diverse functional addends on the nanotube sidewalls, beyond just the principal groups targeted by the specific functionalization reaction. Figure 3D contour map of the FTIR spectra of the species desorbed from the GAP-functionalized SWCNT sample as a function of temperature.
Keywords: Single-walled carbon nanotubes; Functionalization; Thermogravimetric analysis; ICP-OES; TG-MS-FTIR

In this work, we have aimed to merge the advantages of nanotechnology and biophotonics in conjunction with vibrational spectroscopic techniques in order to understand the various aspects of new kinds of synthetic bionanoparticles originating from self-forming synthetic biopolymers known as polyethylene glycol (PEG)ylated lipids. In particular, two complementary molecular spectroscopic techniques based on thin-layered Fourier transform infrared and confocal laser tweezers. Raman spectroscopy has been employed for the investigations of newly developed artificial PEGylated lipids trademarked as QuSomes. These novel types of synthetic lipids are composed of 1,2-dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12), 1,2-dioleoyl-rac-glycerol-3-dodecaethylene glycol (GDO-12), and 1,2-distearoyl-rac-glycerol-3-triicosaethylene glycol (GDS-23). The lipid labeled GDM-12 has saturated 14 acyl chains whereas GDO-12 is characterized by monounsaturated 18 acyl chains, and GDS-23 is composed of saturated 18 acyl chains in their hydrophobic chain. Similarly, GDM-12 and GDO-12 contain 12 units, and GDS-23 contains 23 units of hydrophilic PEG head groups. In contrast to conventional phospholipids, this novel kind of lipid can form liposomes spontaneously upon hydration, without the input of external activation energy. In addition, fluorescence correlation spectroscopy has been utilized to measure the size distribution of such nanoparticles in suspension as well as scanning electron microscopy has been applied for the imaging purposes. Although such PEGylated lipids show a common spectral pattern, important differences in the spectra have been observed, enabling us to distinguish these different lipids on the basis of characteristic features calculated from the spectroscopic band component analysis. Finally, in this study, detailed spectroscopic results due to the vibrational band assignments and band component analysis corresponding to various functional groups for individual nanoparticles have been analyzed and discussed.
Keywords: PEGylated lipids; QuSomes; Spectroscopic analysis; Vibrational bands; FT-IR; LTRS

A colloidal synthesis method was developed to produce face centered cubic (fcc) Cu nanoparticles in the presence of surfactants in an organic solvent under an Ar environment. Various synthetic conditions were explored to control the size of the as-prepared nanoparticles by changing the precursor, varying the amount of surfactants, and tuning the reaction temperature. Transmission electron microscopy (TEM), selected-area electron diffraction, and high-resolution TEM were used as the main characterization tools. Upon exposure to air, these nanoparticles are oxidized at different levels depending on their sizes: (1) an inhomogeneous layer of fcc Cu2O forms at the surface of Cu nanoparticles (about 30 nm); (2) Cu nanoparticles (about 5 nm) are immediately oxidized into fcc Cu2O nanoparticles (about 6 nm). The occurrence of these different levels of oxidization demonstrates the reactive nature of Cu nanoparticles and the effect of size on their reactivity. Furthermore, utilization of their chemical reactivity and conversion of spherical Cu nanoparticles into CuS nanoplates through the nanoscale Kirkendall effect were demonstrated. The oxidization and sulfidation of Cu nanoparticles were compared. Different diffusion and growth behaviors were involved in these two chemical transformations, resulting in the formation of isotropic Cu2O nanoparticles during oxidization and anisotropic CuS nanoplates during sulfidation. Figure Transmission electron microscopy images of Cu nanoparticles (left), Cu2O nanoparticles (middle), and CuS nanoplates (right)
Keywords: Copper nanoparticles; Copper oxide nanoparticles; Copper sulfide nanoplates; Kirkendall effect; Nanoplates; Transmission electron microscopy

Applications of TGA in quality control of SWCNTs by Elisabeth Mansfield; Aparna Kar; Stephanie A. Hooker (1071-1077).
Carbon nanotubes exhibit a range of chemistries, including mixtures of different nanotube diameters, lengths, and chiralities coupled with various concentrations of metallic and non-nanotube-carbon impurities. The performance of a given material for a specific application depends on the chemistry, which is dictated in large part by the manufacturing process. Here, thermogravimetric analysis is utilized as a bulk characterization method for determining nanotube quality after manufacturing. The application of thermogravimetric analysis for quantifying basic nanotube chemistry is described (e.g., carbon-to-metal content, homogeneity). In addition, extension of the method to analyze specific nanotube properties (i.e., length and diameter) is reported. Results indicate that thermogravimetric analysis is sufficiently sensitive to enable quality control at both the macro-scale (carbon-to-metal ratio) and nano-scale (single-walled to multi-walled) and can detect subtle modifications in manufacturing processes.
Keywords: Thermogravimetric analysis; Quality control; Carbon nanotubes

Pristine single-walled carbon nanotube purity evaluation by using 1H NMR spectroscopy by Donna J. Nelson; Christopher N. Brammer (1079-1086).
Proper purity characterization of single-walled carbon nanotubes (SWCNTs) is an increasingly hot topic in the area of carbon nanotechnology. There are inconsistencies in purity characterization of SWCNT from manufacturers and in the literature. Purity of “as received,” oven dried, and NaHCO3-washed SWCNTs of three commercially available brands (NanoLab, SWeNT, and HiPco) is explored by using a consistent methodology via proton nuclear magnetic resonance (1H NMR) spectroscopy comparison, across three NMR solvents: DMSO-d 6, CDCl3, and D2O. Important insights into the purity of commercially available SWCNT and the importance of washing (cleaning) samples before use are offered.
Keywords: NMR; Nanoparticles; Quality control

Electrophoretic properties of BSA-coated quantum dots by Wendelin Bücking; Salam Massadeh; Alexei Merkulov; Shu Xu; Thomas Nann (1087-1094).
Low toxic InP/ZnS quantum dots (QDs), ZnS:Mn2+/ZnS nanocrystals and CdSe/ZnS nanoparticles were rendered water-dispersible by different ligand-exchange methods. Eventually, they were coated with bovine serum albumin (BSA) as a model protein. All particles were characterised by isotachophoresis (ITP), laser Doppler velocimetry (LDV) and agarose gel electrophoresis. It was found that the electrophoretic mobility and colloidal stability of ZnS:Mn2+/ZnS and CdSe/ZnS nanoparticles, which bore short-chain surface ligands, was primarily governed by charges on the nanoparticles, whereas InP/ZnS nanocrystals were not charged per se. BSA-coated nanoparticles showed lower electrophoretic mobility, which was attributed to their larger size and smaller overall charge. However, these particles were colloidally stable. This stability was probably caused by steric stabilisation of the BSA coating.
Keywords: Quantum dots; BSA; Gel electrophoresis; Dynamic light scattering; Laser Doppler electrophoresis; Isotachophoresis

Evaluation of quantum dots applied as switchable layer in a light-controlled electrochemical sensor by Zhao Yue; Waqas Khalid; Marco Zanella; Azhar Zahoor Abbasi; Andrea Pfreundt; Pilar Rivera Gil; Kirsten Schubert; Fred Lisdat; Wolfgang J. Parak (1095-1103).
Gold electrodes with switchable conductance are created by coating the gold surface with different colloidal quantum dots. For the quantum dot immobilization, a dithiol compound was used. By polarizing the electrode and applying a light pointer, local photocurrents were generated. The performance of this setup was characterized for a variety of different nanoparticle materials regarding drift and signal-to-noise ratio. We varied the following parameters: quantum dot materials and immobilization protocol. The results indicate that the performance of the sensor strongly depends on how the quantum dots are bound to the gold electrode. The best results were obtained by inclusion of an additional polyelectrolyte film, which had been fabricated using layer-by-layer assembly. Figure Gold electrode with switchable conductance created by the coating the gold surface with different colloidal quantum dots
Keywords: Light-addressable potentiometric sensor; Quantum dots; Photocurrent

Characterization of gold nanorods in vivo by integrated analytical techniques: their uptake, retention, and chemical forms by Liming Wang; Yu-Feng Li; Liangjun Zhou; Ying Liu; Li Meng; Ke Zhang; Xiaochun Wu; Lili Zhang; Bai Li; Chunying Chen (1105-1114).
Integrated analytical techniques were used to study the tissue distribution and structural information of gold nanorods (Au NRs) in Sprague-Dawley rats through tail intravenous injection. Before in vivo experiments were conducted, careful characterization of Au NRs was performed. The zeta potential proved that adsorption of bovine serum albumin on Au NRs turned the surface charges from positive to negative as in an in vitro simulation. The biodistribution of Au NRs was investigated quantitatively by inductively coupled plasma mass spectrometry at different time points after injection. As target tissues, both liver and spleen were chosen to further demonstrate the intracellular localization of Au NRs by the combination of transmission electron microscopy and energy-dispersive X-ray spectroscopy. Moreover, synchrotron-radiation-based X-ray absorption spectroscopy was employed and it was observed that long-term retention of Au NRs in liver and spleen did not induce obvious changes in the oxidation states of gold. Therefore, the present systematic method can provide important information about the fates of Au NRs in vivo and can also be extended to study the biological effects of other metallic nanomaterials in the future. Figure Systematic method to study biodistribution and characterization of metallic nanomaterials in vivo
Keywords: Gold nanorods; Biodistribution; Characterization in vivo; Transmission electron microscopy; Inductively coupled plasma mass spectrometry; X-ray absorption spectroscopy

Multifunctional ZrO2 nanoparticles (NPs) and ZrO2-SiO2 nanorods (NRs) have been successfully applied as the matrices for cyclodextrins and as affinity probes for enrichment of peptides (leucine-enkephalin, methionine-enkephalin and thiopeptide), phosphopeptides (from tryptic digestion products of β-casein) and phosphoproteins from complex samples (urine and milk) in atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and MALDI time-of-flight (TOF) MS. The results show that the ZrO2 NPs and ZrO2-SiO2 NRs can interact with target molecules (cyclodextrins, peptides, and proteins), and the signal intensities of the analytes were significantly improved in MALDI-MS. The maximum signal intensities of the peptides were obtained at pH 4.5 using ZrO2 NPs and ZrO2-SiO2 NRs as affinity probes. The limits of detection of the peptides were found to be 75-105 fmol for atmospheric pressure MALDI-MS and those of the cyclodextrins and β-casein were found to be 7.5-20 and 115-125 fmol, respectively, for MALDI-TOF-MS. In addition, these nanomaterials can be applied as the matrices for the analysis of cyclodextrins in urine samples by MALDI-TOF-MS. ZrO2 NPs and ZrO2-SiO2 NRs efficiently served as electrostatic probes for peptide mixtures and milk proteins because 2–11 times signal enhancement can be achieved compared with use of conventional organic matrices. Moreover, we have successfully demonstrated that the ZrO2 NPs can be effectively applied for enrichment of phosphopeptides from tryptic digestion of β-casein. Comparing ZrO2 NPs with ZrO2-SiO2 NRs, we found that ZrO2 NPs exhibited better affinity towards phosphopeptides than ZrO2-SiO2 NRs. Furthermore, the ZrO2 and ZrO2-SiO2 nanomaterials could be used to concentrate trace amounts of peptides/proteins from aqueous solutions without tedious washing procedures. This approach is a simple, straightforward, separation-and washing-free approach for MALDI-MS analysis of cyclodextrins, peptides, proteins, and tryptic digestion products of phosphoproteins. Figure Multifunctional ZrO2 nanoparticles and ZrO2-SiO2 nanorods for improved MALDI-MS
Keywords: Nanoparticles; Peptides; Phosphoproteins; Phosphopeptides; Cyclodextrins; Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Enhancing the analytical performance of immunoassays that employ metal-enhanced fluorescence by Robert Nooney; Andrew Clifford; Xavier LeGuevel; Ondrej Stranik; Colette McDonagh; Brian D. MacCraith (1127-1134).
In this work, we used a model assay system (polyclonal human IgG–goat antihuman IgG) to elucidate some of the key factors that influence the analytical performance of bioassays that employ metal-enhanced fluorescence (MEF) using silver nanoparticles (NPs). Cy5 dye was used as the fluorescent label, and results were compared with a standard assay performed in the absence of NPs. Two sizes of silver NPs were prepared with respective diameters of 60 ± 10 and 149 ± 16 nm. The absorption spectra of the NPs in solution were fitted accurately using Mie theory, and the dipole resonance of the 149-nm NPs in solution was found to match well with the absorption spectrum of Cy5. Such spectral matching is a key factor in optimizing MEF. NPs were deposited uniformly and reproducibly on polyelectrolyte-coated polystyrene substrates. Compared to the standard assay performed without the aid of NPs, significant improvements in sensitivity and in limit of detection (LOD) were obtained for the assay with the 149-nm NPs. An important observation was that the relative enhancement of fluorescence increased as the concentration of antigen increased. The metal-assisted assay data were analyzed using standard statistical methods and yielded a LOD of 0.086 ng/mL for the spectrally matched NPs compared to a value of 5.67 ng/mL obtained for the same assay in the absence of NPs. This improvement of ∼66× in LOD demonstrates the potential of metal-enhanced fluorescence for improving the analytical performance of bioassays when care is taken to optimize the key determining parameters. Metal nanoparticle-enhanced fluorescence immunoassay using a sandwich assay format
Keywords: Nanoparticles; Plasmonic enhancement; Fluorescence immunoassay

Highly sensitive rare cell detection based on quantum dot probe fluorescence analysis by Yi-Heui Hsieh; Shih-Jen Liu; Hsin-Wei Chen; Yao-Kwang Lin; Keng S. Liang; Lee-Jene Lai (1135-1141).
This study presents an efficient and sensitive method for detecting rare cells without cell culture, in which cells are analyzed quantitatively using quantum dots (QDs) as a fluorescent probe. By the conjugation of QDs with cells, the biotin–streptavidin reaction functions as a bridge to connect QDs and cells. The cells can be quantified based on the correlation of the QD fluorescence intensity with the cell population. Non-specific adsorption and cross-reaction of QD625–streptavidin on T cell membrane are neglected by reacting with biotin anti-human CD3 and mixing with red blood cell, respectively. Additionally, the photo-activation period and pH can be controlled to enhance the fluorescence of cell populations, which increases linearly with the number of T cells from 40 to 100,000, not only in a single T cell line but also in mixing with a total of 106 red blood cells. Moreover, the specific T cells can be detected in less than 15 min, even though rare specific cells may number only 40 cells. Among the advantages, the proposed system for detecting rare cells include simplicity of preparation, low cost, rapid detection, and high sensitivity, all of which can facilitate the detection of circulating tumor cells in early stages of diagnosis or prognosis. Figure Quantum dot is indirectly conjugated with the membrane of T cell through the antigen-antibody reaction. The fluorescent intensity at an emission wavelength (λem) of 625 nm was recorded by quantitative analysis of the T cell populations using an excitation wavelength (λex) of 365 nm.
Keywords: Quantum dot; pH; Fluorescence; T lymphocyte cells; Red blood cell

Use of the quartz crystal microbalance to monitor ligand-induced conformational rearrangements in HIV-1 envelope protein gp120 by Hyun-Su Lee; Mark Contarino; M. Umashankara; Arne Schön; Ernesto Freire; Amos B. Smith III; Irwin M. Chaiken; Lynn S. Penn (1143-1152).
We evaluated the potential of a quartz crystal microbalance with dissipation monitoring (QCM-D) to provide a sensitive, label-free method for detecting the conformational rearrangement of glycoprotein gp120 upon binding to different ligands. This glycoprotein is normally found on the envelope of the HIV-1 virus and is involved in viral entry into host cells. It was immobilized on the surface of the sensing element of the QCM-D and was exposed to individual solutions of several different small-molecule inhibitors as well as to a solution of a soluble form of the host cell receptor to which gp120 binds. Instrument responses to ligand-triggered changes were in qualitative agreement with conformational changes as suggested by other biophysical methods. Figure Graphic to accompany the on-line abstract for "Use of the quartz crystal microbalance to monitor ligand-induced conformational rearrangements in HIV-1 envelope protein gp120," by Hyun-Su Lee, Mark Contarino, M. Umashankara, Arne Schön, Ernesto Freire, Amos B. Smith, III, Irwin M. Chaiken, and Lynn S. Penn
Keywords: Quartz crystal microbalance (QCM-D); Human immunodeficiency virus 1 (HIV-1); Ligand–gp120 complex; s-CD4

Engineered synapse model cell: genetic construction and chemical evaluation for reproducible high-throughput analysis by Satoshi Migita; Akito Tateishi; Kari Keinänen; Tetsuya Haruyama (1153-1157).
Bioassay models of neural functions must lend themselves to high-throughput analysis in neural drug discovery. However, smart analysis methods for these functions have not yet been fully established. Here, we describe the development of a synapse model for cell-based biosensing. The engineered synapse model cell expresses ionotropic glutamate receptor on its surface, like the neural postsynaptic membrane. The advantages of the model cell are the ease of handling and reproducibility as compared with the cultured neural cell, and it can be employed to evaluate receptor function through ion flux analysis. The agonist-induced sodium influx was monitored as an agonist concentration-dependent increase in the observed fluorescence signal. Furthermore, we found that our model cell enables the correction of uneven cellular signal levels using a reporter system. Our engineered synapse model cell can be employed as a powerful tool for the screening of lead substances in pharmaceutical high-throughput analysis.
Keywords: Qualified analysis; Cellular biosensing; Cell-based biosensor; High-throughput analysis; Ligand-gated ion channel

Positionally controlled growth of cells using a cytophobic fluorinated polymer by Sarvesh Varma; Jessica McLachlan; Amanda M. Leclair; Betty C. Galarreta; Peter R. Norton; François Lagugné-Labarthet (1159-1165).
This paper presents a novel method for cell positioning on a substrate which combines the optical quality of glass and the cell-repelling property of fluoropolymers. The process employs plasma lithography, which utilizes the high-resolution patterning of photolithography along with the versatility of the plasma polymerization. When mammalian cells were grown over these substrates, they avoided the fluoropolymer regions and grew almost exclusively within the exposed glass areas (windows). The patterned surface reproduces the initial design of the mask, offering the possibility to control cell distances and interactions with a versatile arrangement whilst keeping the optical quality of glass for microscopy observation, in particular, when a pristine substrate in needed. This approach opens up possibilities for analysis of biological processes, such as studying cell interactions, with the integration of optical or electrical sensors.
Keywords: Fluoropolymer; Plasma lithography; Cell patterning; Fluorescence microscopy; Cytophobic surface

1H NMR-based metabolomics approach for exploring urinary metabolome modifications after acute and chronic physical exercise by C. Enea; F. Seguin; J. Petitpas-Mulliez; N. Boildieu; N. Boisseau; N. Delpech; V. Diaz; M. Eugène; B. Dugué (1167-1176).
Metabolomics is a comprehensive method for metabolite assessment that involves measuring the overall metabolic signature of biological samples. We used this approach to investigate biochemical changes due to acute and chronic physical exercise. Twenty-two women using identical oral contraceptives were segregated into an untrained (n = 10) or trained (n = 12) group depending on their physical training background. The subjects performed two exercises in a randomized order: a prolonged exercise test (75% of their $$ mathop Vlimits^cdot {{ ext{O}}_{2,;max }} $$ until exhaustion) and a short-term, intensive exercise test (short-term, intensive exercise anaerobic test). Urine specimens were collected before and 30 min after each test. The samples were analyzed by 1H NMR spectroscopy, and multivariate statistical techniques were utilized to process the data. Distinguishing characteristics were observed only in the urine profiles of specimens collected before vs. 30 min after the short-term, intensive exercise test. The metabolites responsible for such changes were creatinine, lactate, pyruvate, alanine, β-hydroxybutyrate, acetate, and hypoxanthine. In both groups, the excretion of lactate, pyruvate, alanine, β-hydroxybutyrate, and hypoxanthine increased similarly after the completion of the short-term, intensive exercise test (p < 0.03). However, acetate excretion increased to a lesser extent in trained than in untrained subjects (p < 0.05). In conclusion, metabolomics is a promising tool in order to gain insight into physiological status and to clarify the changes induced by short-term, intense physical exercise.
Keywords: Biological variation; Metabolomics; Physical exercise; Pre-analytical factor; Urine analysis; Young women

Early detection of apoptosis in living cells by fluorescence correlation spectroscopy by Michelle M. Martinez; Randall D. Reif; Dimitri Pappas (1177-1185).
Early detection of apoptotic cells via caspase activity is demonstrated with fast response time. Fluorescence correlation spectroscopy (FCS) is used to identify the presence of a cleaved fluorogenic probe based on the fluorescence of rhodamine 110 in Jurkat cells. FCS curves are shown to be markedly different for autofluorescent (non-apoptotic) cells, whereas cells with cleaved probe showed diffusion and molecular brightness characteristic of rhodamine 110. Using FCS measurements, cells were identified as apoptotic on the basis of the presence of autocorrelated fluorescence, average molecular brightness (η), and molecular dwell time (τ D). Apoptotic cells identified in this manner were detected as early as 45 min after induction. Unlike other methods with similar identification times, such as western blotting and electron microscopy, cells remain viable for further analysis. This multi-parameter approach is rapid, flexible, and does not require transfection of the cells prior to analysis, enabling apoptosis to be identified early in a wide variety of cell types. Figure Concept of fluorescence correlation spectroscopy in living cells. In the absence of cleaved fluorogenic probe, the cell autofluorescence is largely uncorrelated. Apoptosis (and caspase activity) produces cleaved rhodamine 110 molecules in the cell, leading to correlated fluorescence.
Keywords: Fluorescence correlation spectroscopy; Apoptosis; Caspases; Single-cell analysis; Cancer

Dual-cardiac marker capillary waveguide fluoroimmunosensor based on tyramide signal amplification by Aristeidis E. Niotis; Christos Mastichiadis; Panagiota S. Petrou; Ion Christofidis; Sotirios E. Kakabakos; Athanasia Siafaka-Kapadai; Konstantinos Misiakos (1187-1196).
The early diagnosis of acute myocardial infarction requires the determination of several markers in serum shortly after its incidence. The markers most widely employed are the isoenzyme MB of creatine kinase (CK-MB) and the cardiac troponin I (cTnI). In the present work, a capillary waveguide fluoroimmunosensor for fast and highly sensitive simultaneous determination of these markers in serum samples is demonstrated. The dual-analyte immunosensor was realized using glass capillaries internally modified with an ultrathin poly(dimethylsiloxane) film by creating discrete bands of analyte-specific antibodies. The capillary was then filled with a mixture of sample and biotinylated detection antibodies followed by reaction with streptavidin–horseradish peroxidase and incubation with a fluorescently labeled tyramide derivative to accumulate fluorescent labels onto immunoreaction bands. Upon scanning the capillary with a laser beam, part of the emitted fluorescence is trapped and waveguided through the capillary wall to a photomultiplier placed on one of its ends. The employment of tyramide signal amplification provided detection limits of 0.2 and 0.5 ng/mL for cTnI and CK-MB, respectively, in a total assay time of 30 min compared to 0.8 and 0.6 ng/mL obtained for the corresponding assays when the conventional fluorescent label R-phycoerythrin was used in a 65-min assay. In addition, the proposed immunosensor provided accurate and repeatable measurements (intra-assay and interassay coefficients of variation lower than 10%), and the values determined in serum samples were in good agreement with those obtained with commercially available enzyme immunoassays. Thus, the proposed capillary waveguide fluoroimmunosensor has all the required characteristics for fast and reliable diagnosis of acute myocardial infarction. Figure Assay formats employed for the determination of CK-MB and cTnI using as label either R-phycoerythrin or tyramide signal amplification system (left) and CK-MB and cTnI calibration curves obtained using the capillary immunosensor and AlexaFluor-488-labeled tyramide conjugate as label (right)
Keywords: Capillary waveguide immunosensor; Cardiac troponin I; Creatine kinase MB; Tyramide signal amplification; Ultrathin PDMS film

An “off–on” rhodamine-based fluorescence probe for the selective signaling of Cr(III) has been designed by exploiting the guest-induced structure transform mechanism. This system shows a sharp Cr(III)-selective fluorescence enhancement response in 100% aqueous system under physiological pH value and possesses high selectivity against the background of environmentally and biologically relevant metal ions including Cr(VI), Al(III), Fe(III), Cd(II), Co(II), Cu(II), Ni(II), Zn(II), Mg(II), Ba(II), Pb(II), Na(I), and K(I). Under optimum conditions, the fluorescence intensity enhancement of this system is linearly proportional to Cr(III) concentration from 5.0 × 10−8 to 7.0 × 10−6 mol L−1 with a detection limit of 1.6 × 10−8 mol L−1.
Keywords: Chromium; Probe; Fluorescence; Rhodamine

Filter paper saturated by urine sample in metabolic disorders detection by proton magnetic resonance spectroscopy by Hélène Blasco; Marie-Ange Garrigue; Aymeric De Vos; Catherine Antar; François Labarthe; François Maillot; Christian R Andres; Lydie Nadal-Desbarats (1205-1211).
NMR spectroscopy of urine samples is able to diagnose many inborn errors of metabolism (IEM). However, urinary metabolites have a poor stability, requiring special care for routine analysis (storage of urine at −20 or −80 °C, fast transport). The aim of our study was to investigate the reliability of dried urine filter paper for urine storage and transport and to evaluate the ability of NMR to detect several IEM using this method. Urine samples from five healthy subjects were analyzed by 1H NMR following different storage conditions (−20 vs 4 °C vs dried on filter paper) and at different time points (24 h, 48 h, 96 h, and 7 days). Urine pattern of fresh urine was considered as a reference. We analyzed the conservation of some amino acids and organic acids using Bland and Altman plot with intraclass correlation coefficient determination. Then, we evaluated the use of filter paper to detect four different IEM (methylmalonic and isovaleric acidurias, ornithine transcarbamylase deficiency, and cystinuria). Analysis of urine samples from healthy subjects revealed a high stability of studied molecules (ICC > 0.8) even after 7 days of storage on filter paper. Moreover, an excellent preservation of metabolites specifically accumulated in IEM was observed when analysis of dried urine filter paper was compared to fresh urine (coefficient of variation < 15%). This preliminary study demonstrates that storage of dried urine on filter paper is reliable for 1H NMR spectroscopy analysis. Preservation of urine molecules over time using that method is convenient for routine clinical practice. Graphical Abstract Urine spectra of a patient with isovaleric aciduria from urine sample (a) and dried urine filter paper (b). IVG : isovaleryl glycine. Crn : creatinine, TMAO : trimethylamine oxyde
Keywords: Proton magnetic resonance spectroscopy; Inborn errors of metabolism; Urinary metabolite; Filter paper

The enzyme diisopropyl fluorophosphatase (DFPase, EC 3.1.8.2) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Until now, determination of kinetic data has been achieved by use of techniques such as pH-stat titration, ion-selective electrodes, and a recently introduced method based on in situ Fourier-transform infrared (FTIR) spectroscopy. We report the use of 1D 1H–31P HSQC NMR spectroscopy as a new method for real-time quantification of the hydrolysis of toxic organophosphonates by DFPase. The method is demonstrated for the agents sarin (GB), soman (GD), and cyclosarin (GD) but can also be used for V-type nerve agents, for example VX. Besides buffered aqueous solutions the method was used to determine enzymatic activities in a biodiesel-based bicontinuous microemulsion that serves as an example of complex decontamination media, for which other established techniques often fail. The method is non-invasive and requires only limited manual handling of small volumes of liquid (700 μL), which adds to work safety when handling highly toxic organophosphorus compounds. Limits of detection are slightly below 100 μmol L−1 on a 400 MHz spectrometer with 16 FIDs added for a single time frame. The method is not restricted to DFPase but can be used with other phosphotriesterases, for example paraxonase (PON), and even reactive chemicals, for example oximes and other nucleophiles, as long as the reaction components are compatible with the NMR experiment. Figure Staggered view of consecutive 1D 1H–31P HSQC NMR spectra recorded during the hydrolysis of GF catalyzed by DFPase in aqueous buffer solution. During the course of the enzyme-catalyzed reaction the doublet of doublets (dd) signal of the nerve agent is decreasing and at the same time the doublet (d) signal of the phosphonate is increasing. The phosphonate (d) signal is used for quantification because of a better signal-to-noise ratio compared with the (dd) of the agent
Keywords: Phosphotriesterases; Enzymes; Nerve agents; NMR; DFPase; Hydrolases

Precipitation and selective extraction of human serum endogenous peptides with analysis by quadrupole time-of-flight mass spectrometry reveals posttranslational modifications and low-abundance peptides by Declan Williams; Suzanne Ackloo; Peihong Zhu; Peter Bowden; Kenneth R. Evans; Christina L. Addison; Chris Lock; John G. Marshall (1223-1247).
The endogenous peptides of human serum may have regulatory functions, have been associated with physiological states, and their modifications may reveal some mechanisms of disease. In order to correlate levels of specific peptides with disease alongside internal standards, the polypeptides must first be reliably extracted and identified. Endogenous blood peptides can be effectively enriched by precipitation of the serum with organic solvents followed by selective extraction of peptides using aqueous solutions modified with organic solvents. Polypeptides on filter paper were assayed with Coomasie brilliant blue binding. The polypeptides were resolved by detergent tricine polyacrylamide electrophoresis and visualized by diamine silver staining. Peptides in the extracts were collected by C18 and analyzed by matrix-assisted laser desorption/ionization and liquid chromatography–electrospray ionization–tandem mass spectrometry (MS/MS) quadrupole time-of-flight MS/MS. Peptides were resolved as multiple isotopic peaks in MS mode with mass deviation of 0.1 Da or less and similar accuracy for fragments. The sensitivity of MS and MS/MS analysis was estimated to be in the picomolar range or less. The peptide composition of the extracts was dependent on solvent formulation. Multiple peptides from apolipoproteins, complement proteins, coagulation factors, and many others were identified by X!Tandem with high mass accuracy of peptide ions and fragments from collision-induced dissociation. Many previously unreported posttranslational modifications of peptides including phosphorylations, oxidations, glycosylations, and others were detected with high mass accuracy and may be of clinical importance. About 4,630 redundant peptides were identified with 99% confidence separately, and together some 1,251 distinct proteins were identified with 99% confidence or greater using the Paragon algorithm.
Keywords: Bioanalytical methods; Mass spectrometry/ICP–MS; Amino acids/peptides

Determination of naltrexone and 6β-naltrexol in human blood: comparison of high-performance liquid chromatography with spectrophotometric and tandem-mass-spectrometric detection by Sonja Brünen; Ralf Krüger; Susann Finger; Felix Korf; Falk Kiefer; Klaus Wiedemann; Karl J. Lackner; Christoph Hiemke (1249-1257).
We present data for a comparison of a liquid-chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) and a high-performance liquid-chromatographic method with column switching and UV spectrophotometric detection. The two methods were developed for determination of naltrexone and 6β-naltrexol in blood serum or plasma aiming to be used for therapeutic drug monitoring to guide the treatment of patients with naltrexone. For the high-performance liquid chromatography (HPLC)/UV detection, online sample cleanup was conducted on Perfect Bond C18 material with 2% (vol/vol) acetonitrile in deionized water. Drugs were separated on a C18 column using 11.5% (vol/vol) acetonitrile and 0.4% (vol/vol) N,N,N,N-tetramethylethylenediamine within 20 min. LC-MS/MS used naltrexone-d 3 and 6β-naltrexol-d 4 as internal standards. After protein precipitation, the chromatographic separation was performed on a C18 column by applying a methanol gradient (5–100%, vol/vol) with 0.1% formic acid over 9.5 min. The HPLC/UV method was found to be linear for concentrations ranging from 2 to 100 ng/ml, with a regression correlation coefficient of r 2 > 0.998 for naltrexone and 6β-naltrexol. The limit of quantification was 2 ng/ml for naltrexone and 6β-naltrexol. For the LC-MS/MS method the calibration curves were linear (r² > 0.999) from 0.5 to 200 ng/ml for both substances, and the limit of quantification was 0.5 ng/ml. The concentrations measured by the two methods correlated significantly for both substances (r² > 0.967; p < 0.001). Both methods could be used for therapeutic drug monitoring. The HPLC/UV method was advantageous regarding automatization and costs, whereas LC-MS/MS was superior with regard to sensitivity.
Keywords: Naltrexone; 6ß-Naltrexol; Therapeutic drug monitoring; High-performance liquid chromatography/UV detection; Liquid chromatography/tandem mass spectrometry

Application of electrospray ionization hybrid ion trap/time-of-flight mass spectrometry in the rapid characterization of quinocetone metabolites formed in vitro by Zhao-Ying Liu; Ling-Li Huang; Dong-Mei Chen; Meng-Hong Dai; Yan-Fei Tao; Yu-Lian Wang; Zong-Hui Yuan (1259-1271).
The application of electrospray ionization hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography (LC/MS–IT–TOF) in the rapid characterization of in vitro metabolites of quinocetone was developed. Metabolites formed in rat liver microsomes were separated using a VP-ODS column with gradient elution. Multiple scans of metabolites in MS and MS2 modes and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only a 30-min analysis. Most measured mass errors were less than 10 ppm for both protonated molecules and fragment ions using external mass calibration. The elemental compositions of all fragment ions of quinocetone and its metabolites could be rapidly assigned based upon the known compositional elements of protonated molecules. The structure of metabolites were elucidated based on the combination of three techniques: agreement between their proposed structure, the accurate masses, and the elemental composition of ions in their mass spectra; comparison of their changes in accurate molecular masses and fragment ions with those of parent drug or metabolite; and the elemental compositions of lost mass numbers in proposed fragmentation pathways. Twenty-seven phase I metabolites were identified as 11 reduction metabolites, three direct hydroxylation metabolites, and 13 metabolites with a combination of reduction and hydroxylation. All metabolites except the N-oxide reduction metabolite M6 are new metabolites of quinocetone, which were not previously reported. The ability to conduct expected biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurement, all in a single experimental run, is one of the most attractive features of this methodology. The results demonstrate the use of LC/MS–IT–TOF approach appears to be rapid, efficient, and reliable in structural characterization of drug metabolites. Figure The accurate extracted mass chromatograms (EIC) of quinocetone metabolites in rat liver microsomes incubated with quinocetone for 2h. M0 quinocetone, M1–M27 metabolites of quinocetone.
Keywords: Ion trap/time-of-flight; Accurate mass measurements; Mass spectrometry; Elemental composition; Quinocetone; Metabolites

Analysis was performed on four different categories of phospholipids (phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA)) from urine in patients with breast cancer. This quantitative analysis was conducted using nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry (nLC-ESI-MS-MS). This study shows the profiling of the phospholipids (PLs) that can be identified by the negative ion mode of MS. A previous study (Kim et al. Anal. Bioanal. Chem. 393:1649, 21) focused on only two PL classes: phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) and were identified by positive ion mode. PLs were extracted by lyophilization of 1 mL of urine from both healthy normal females and breast cancer patients before and after surgery. Separation of PLs was performed by nLC followed by structural identification of PLs using data-dependent collision-induced dissociation. A total of 34 urinary PL molecules (12 PSs, 12 PIs, four PGs, and six PAs) were quantitatively examined. Among the four PL categories examined in this study, most PL classes showed an increase in the total amounts in the cancer patients, yet PIs exhibited some decreases. The present study suggests that the lipid composition found in the urine of breast cancer patients can be utilized for the possible development of disease markers, when the analysis is performed with negative ion mode of nLC-ESI-MS-MS. Figure Calibration curves of the concentration dependency (solid line) of each acyl chain length of PS standard deduced from the relationship (dotted lines) between relative peak area (vs. IS) and acyl chain length
Keywords: Phospholipids; Quantitative analysis; nLC-ESI-MS-MS; Urine; Breast cancer

Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis by Carmen Calinescu; Rodolfo Federico; Bruno Mondovi; Mircea Alexandru Mateescu (1281-1290).
A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 °C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 °C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin. Figure Figure Zymographic assay of DAO with peroxidase included in polyacrylamide gels
Keywords: Diamine oxidase; Zymographic assay; Entrapped peroxidase polyacrylamide gel; Proteolytic stability; Simulated gastro-intestinal conditions

The analysis of potential genotoxic impurities (PGIs) in active pharmaceutical ingredients (APIs) is a challenging task. The target limit of detection for a PGI in an API is typically 1 ppm (1 µg/g API). This is about 500 times lower than for classical impurity analysis. Consequently, analytical methods for trace analysis, mostly in combination with MS detection, need to be applied for the qualitative and quantitative determination of these impurities. A two-dimensional capillary GC method is presented that can be used for the determination of some target PGIs. A concentrated solution of the API sample is directly introduced in the GC-MS system, using an apolar column for first-dimension separation. The fraction (heart-cut) containing the PGIs is transferred to a second capillary column, installed in a low-thermal-mass oven (LTM). The LTM focuses the heart-cut(s) and allows independent temperature-programmed analysis with a polar second-dimension column. The API, solvent, and derivatization agents are not introduced in the second column or in the MS detector, avoiding contamination, column degradation, and target analyte peak detection/integration issues. The performance of this set-up is illustrated by the analysis of some Michael-reactive acceptor PGIs and haloalcohols in carbamazepine as test matrix. Excellent reproducibility (<10% RSD) at the low parts per million level and low detection limits (<1 ppm) were obtained.
Keywords: Two-dimensional capillary GC; Capillary-flow technology; Low-thermal-mass oven; Pharmaceuticals; Potential genotoxic impurities

Analysis and occurrence of alkylphenolic compounds and estrogens in a European river basin and an evaluation of their importance as priority pollutants by Rikke Brix; Cristina Postigo; Susana González; Marta Villagrasa; Asuncion Navarro; Marina Kuster; Maria J. Lopez de Alda; Damià Barceló (1301-1309).
As a part of a project aiming to assess the potential toxicological effects of contaminants in aquatic ecosystems, the objective of this work was to determine the occurrence of several selected endocrine-disrupting compounds in water and sediment and to estimate the estrogenicity of the water. The study consisted of four sampling campaigns at seven sampling points in the lower Llobregat catchment area (NE Spain). Water and sediment samples underwent chemical target analysis for 19 steroid estrogens and alkylphenols, which are known to be endocrine-disrupting compounds. In this study, the only estrogens detected in the water samples were estrone and estrone-sulfate, which were found at low levels (2–5 ng l−1). The alkylphenolic compound showing the highest concentrations was nonylphenol di-ether carboxylate (NP2EC), which was found at levels up to 30.62 µg l−1 in water samples and 535 ng g−1 in sediment samples. K d was determined for several alkylphenolic compounds and showed the expected trend of decreasing K d with increasing polarity. The concentrations of nonylphenol and octylphenol only exceeded the annual average of the European Union’s environmental quality standards (EQS) in one sampling point. However, the calculated estrogenic potential surpassed the expected effect concentration in several sampling points, indicating a potential risk. Therefore, we recommend that future EQS include short-chain alkylphenol ethoxylates and carboxylates.
Keywords: Endocrine-disrupting compounds; Estrogenicity; European quality standards

Development of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry for analysis of halogenated flame retardants in wastewater by Simon Ningsun Zhou; Eric J. Reiner; Chris Marvin; Paul Helm; Nicole Riddell; Frank Dorman; Michelle Misselwitz; Li Shen; Patrick Crozier; Karen MacPherson; Ian D. Brindle (1311-1320).
Until recently, atmospheric pressure photoionization (APPI) has typically been used for the determination of non-polar halogenated flame retardants (HFRs) by liquid chromatography (LC) tandem mass spectrometry. In this study, we demonstrated the feasibility of utilizing liquid chromatography atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-APCI-MS/MS) for analysis of 38 HFRs. This developed method offered three advantages: simplicity, rapidity, and high sensitivity. Compared with APPI, APCI does not require a UV lamp and a dopant reagent to assist atmospheric pressure ionization. All the isomers and the isobaric compounds were well resolved within 14-min LC separation time. Excellent instrument detection limits (6.1 pg on average with 2.0 μL injection) were observed. The APCI mechanism was also investigated. The method developed has been applied to the screening of wastewater samples for screening purpose, with concentrations determined by LC-APCI-MS/MS agreeing with data obtained via gas chromatography high resolution mass spectrometry. Figure LC-APCI-MS/MS for analysis of halogenated flame reterdants
Keywords: Atmospheric pressure chemical ionization (APCI); Liquid chromatography tandem mass spectrometry (LC-MS/MS); Halogenated flame retardants (HFRs); Wastewater

Determination of 43 polycyclic aromatic hydrocarbons in air particulate matter by use of direct elution and isotope dilution gas chromatography/mass spectrometry by Zheng Li; Erin N. Pittman; Debra A. Trinidad; Lovisa C. Romanoff; James Mulholland; Andreas Sjödin (1321-1330).
We are reporting a method for measuring 43 polycyclic aromatic hydrocarbons (PAH) and their methylated derivatives (Me-PAHs) in air particulate matter (PM) samples using isotope dilution gas chromatography/high-resolution mass spectrometry (GC/HRMS). In this method, PM samples were spiked with internal standards, loaded into solid phase extraction cartridges, and eluted by dichloromethane. The extracts were concentrated, spiked with a recovery standard, and analyzed by GC/HRMS at 10,000 resolution. Sixteen 13C-labeled PAHs and two deuterated Me-PAHs were used as internal standards to account for instrument variability and losses during sample preparation. Recovery of labeled internal standards was in the range of 86–115%. The proposed method is less time-consuming than commonly used extraction methods, such as sonication and accelerated solvent extraction (ASE), and it eliminates the need for a filtration step required after the sonication extraction method. Limits of detection ranged from 41 to 332 pg/sample for the 43 analytes. This method was used to analyze reference materials from the National Institute of Standards and Technology. The results were consistent with those from ASE and sonication extraction, and these results were also in good agreement with the certified or reference concentrations. The proposed method was then used to measure PAHs on PM2.5 samples collected at three sites (urban, suburban, and rural) in Atlanta, GA. The results showed distinct seasonal and spatial variation and were consistent with an earlier study measuring PM2.5 samples using an ASE method, further demonstrating the compatibility of this method and the commonly used ASE method. Figure A schematic illustration of the direct elution set up
Keywords: Polycyclic aromatic hydrocarbon; PAH; PM; Method; GC/MS

This article presents the different modes and configurations of liquid-phase microextraction (LPME) through comparison with headspace solid-phase microextraction (HS-SPME) for the simultaneous extraction/methylation of the nine haloacetic acids (HAAs) found in water. This is the first analytical case reported of solvent bar extraction–preconcentration–derivatisation assisted by an ion-pairing transfer for HAAs. In this method, 5 μL of the organic extractant, decane, was confined within a hollow-fibre membrane that was placed in a stirred aqueous sample containing the derivatising reagents (dimethylsulphate with a tetrabutylammonium salt). With heating at 45 °C in the HS-SPME method, some organic solvents (extractant, excess of derivatising reagent) are also volatilised and compete with the esters on the fibre (the fibre is damaged and it can be reused only 50−60 times). In addition, the HS-SPME method provides inadequate sensitivity (limits of detections between 0.3 and 5 µg/L) to quantify HAAs at the level usually found in drinking waters. Alternative headspace LPME methods for HAAs require heating (45 °C, 25 min) to derivatise and volatilise the esters but, by using solvent bar microextraction (SBME), the extraction/methylation takes place at room temperature without degradation of HAAs to trihalomethanes. Adequate precision (relative standard deviation of approximately 8%), linearity (0.1–500 µg/L) and sensitivity (10 times higher than the HS-SPME alternative) indicate that the SBME method can be a candidate for routine determination of HAAs in tap water. Finally, the SBME method was applied for the analysis of HAAs in tap and swimming pool water and the results were compared with those of a previous validated headspace gas chromatography–mass spectrometry method. Figure The solvent bar microextraction technique is used for haloacetic acid (the major class of nonvolatile chlorination by-products) extraction/methylation of tap water before gas chromatography–mass spectrometry determination
Keywords: Water analysis; Haloacetic acids; Solvent bar microextraction; Headspace solid-phase microextraction; Gas chromatography–mass spectrometry

Compatibility of quantum dots with immunobuffers, and its effect on signal/background of quantum dot-based immunoassay by Xiaoshan Zhu; Dayue Duan; Steen Madsen; Nelson G. Publicover (1345-1353).
In this work, the compatibility of quantum dots (QDs) with immunobuffers was studied by investigating the fluorescence stability of QDs in immunobuffers (in this research immunobuffers were defined as buffers for immunoaffinity binding or separation). Experimentally, the fluorescence signals of QDs with different surface chemistries (amine-terminated, streptavidin-coated, or antibody-conjugated) in commonly used immunobuffers were monitored versus time. The effect of some buffer composition on the compatibility of QDs with these buffers was also explored. Based on experimental data, the QD compatibility with these buffers is summarized, and it is found that a trace amount of bovine serum albumin added to most of these buffers helps QDs to achieve compatibility with them. Moreover, with QD as fluorescence label and C-reactive protein as a model analyte, a magnetic bead-based assay was performed using compatible and incompatible QD–immunobuffer systems. It is shown that compatible QD–immunobuffer systems can be used to achieve a higher assay signal/background ratio. Figure Fluorescence signals of streptavidin-coated QDs in 8 mol L−1 urea in PBS of pH 7.4 containing 1% SDS and 0.1% BSA vs. the measurement time (QD concentrations were 1 pmol L−1, 10 pmol L−1, 0.1 nmol L−1, and 1 nmol L−1, and there was 30 min vortex mixing between measurements)
Keywords: Nanoparticles/nanotechnology; Fluorescence/luminescence; Immunoassays/ELISA

Sequential extraction (water, Driselase, protease XIV) and extraction with simulated gastric and intestinal fluids were proposed to characterize the binding and the bioaccessibility of chromium in two commercial food supplements obtained by incorporation of this element into yeast. Chromium in Cr-enriched yeast was found to be hardly extractable with water, Driselase, or simulated gastric fluid (recoveries of approximately 10–20%), but proteolysis or gastrointestinal fluid digestion released more than half of the chromium present. Fractionation with size-exclusion chromatography with Cr-specific detection by inductively coupled plasma mass spectrometry (ICP MS) allowed the distinction of two fractions: one below approximately 1 kDa and one 1–5 kDa; they contained the entirety of the released Cr with proportions varying as a function of the extracting solution and the origin of sample. When collected and investigated by reversed-phase high-performance liquid chromatography–ICP MS, the low molecular mass fraction was found to release Cr(III), whereas the heavier one showed most of Cr bound in fairly stable hydrophobic complexes. However, an attempt of their identification by electrospray ionization MS/MS and matrix-assisted laser desorption ionization MS was not successful. Figure
Keywords: Speciation; Chromium; Yeast

Stripping analysis of heavy metals in tap water using the bismuth film electrode by Núria Serrano; José Manuel Díaz-Cruz; Cristina Ariño; Miquel Esteban (1365-1369).
A commercially available screen-printed carbon electrode coated with an ex situ deposited bismuth film (BiSPCE) has been applied to the determination of Pb(II) and Zn(II) ions in tap water (Barcelona water distribution network) by means of stripping voltammetry (SV) and stripping chronopotentiometry (SCP). A good reproducibility of the measurements and a satisfactory agreement between SV and SCP data were observed for both heavy metal ions. Although, in principle, the procedure could be also suited to the determination of Cd(II), this species was not detected. The results were also consistent with the routine ICP-OES measurements of the water distribution company, thus confirming the potential usefulness of such BiSPCE disposable devices for the analysis of heavy metals in natural waters. Figure DPASV measurements of Pb(II)-Cd(II)-Zn(II) system in 0.01 mol L-1 KNO3 on bismuth film coated screen-printed carbon electrode (BiSPCE
Keywords: Bismuth film screen-printed carbon electrode; Ex situ deposition; Constant-current stripping chronopotentiometry; Stripping voltammetry; Heavy metals in tap water