Analytical and Bioanalytical Chemistry (v.401, #3)
The emergence of multimodal imaging methods for real-time nanoscopy by Cyril Petibois (783-784).
is Assistant Professor of Biochemistry at the University of Bordeaux, France, and is involved in the development of imaging methods for analysis of biosamples (CNRS UMR 5248 research unit; “Spectro-imaging of Biosystems” group). His research interest is to develop multimodal imaging methods for full characterization of biosamples, from synthetic models to individual cells and small animals, combining elemental, morphological, molecular, and chemical techniques. The laboratory is also engaged in the development of high-performance imaging methods using synchrotron radiation, notably using IR and X-ray beamlines at major European and Asian synchrotron radiation facilities.
A review of external microbeams for ion beam analyses by L. Giuntini (785-793).
Ion microbeam facilities are analytical tools with high spatial resolution exploiting MeV ion beams. The interactions of beam particles with atoms and nuclei of the target induce the emission of characteristic radiation, the energy of which provides signatures of the compositional and/or structural properties of the target; Ion-Beam Analysis (IBA) techniques, based on the detection of such radiation, enable characterisation of samples of interest, e.g. in material and earth sciences, cultural heritage, biology, medicine, and environmental studies. External beams, obtained by extracting the particles into the atmosphere through a thin window, have many attractive features, e.g. non-destructive/non-invasive analysis and ease of working, so many laboratories have dedicated beam-lines to ex-vacuo IBA analyses. External microprobes have made it possible to obtain probes in the micron range by adopting strong focusing lenses, ultra-thin windows for beam extraction, and short/ultra-short external path of beam particles in light gases; they have also made possible the use of new external IBA techniques, e.g. BS, ERDA, STIM, and IBIC. By adopting systems to raster scan the beam over the sample, imaging capabilities have also become available for ex-vacuo analysis. External scanning microprobes + IBA techniques have enabled the characterisation of samples with high spatial resolution, comparable with that achievable in-vacuum for thick samples, avoiding sample damage. Figure IBA analysis of a precious lapis lazuli artefact at the Florence external microbeam
Keywords: External microbeam; Ion-Beam Analysis; Ionoluminescence; PIGE; Backscattering Spectrometry; IBIC
Functional histology of glioma vasculature by FTIR imaging by Razia Noreen; Raphael Pineau; Chia-Chi Chien; Mariangela Cestelli-Guidi; Yeukuang Hwu; Augusto Marcelli; Michel Moenner; Cyril Petibois (795-801).
Fourier-transform infrared (FTIR) imaging has been used to investigate brain tumor angiogenesis using a mice solid tumor model and bare-gold (∅ 25 nm) or BaSO4 (∅ 500 nm) nanoparticles (NP) injected into blood vasculature. FTIR images of 20-μm-thick tissue sections were used for chemical histology of healthy and tumor areas. Distribution of BaSO4-NP (using the 1,218–1,159 cm−1 spectral interval) revealed clearly all details of blood vasculature with morphological abnormalities of tumor capillaries, while Au-NP (using the 1,046–1,002 cm−1 spectral interval) revealed also diffusion properties of leaky blood vessels. Diffusion of Au-NP out of vascular space reached 64 ± 29 μm, showing the fenestration of “leaky” tumor blood vessels, which should allow small NP (<100 nm, as for Au-NP) to diffuse almost freely, while large NP should not (as for BaSO4-NP in this study). Therefore, we propose to develop FTIR imaging as a convenient tool for functional molecular histology imaging of brain tumor vasculature, both for identifying blood capillaries and for determining the extravascular diffusion space offered by vessel fenestration.
Keywords: Bioanalytical methods; IR spectroscopy/Raman spectroscopy; Nanoparticles/Nanotechnology; Spectroscopy/Instrumentation
Synchrotron radiation phase-contrast X-ray CT imaging of acupuncture points by Dongming Zhang; Xiaohui Yan; Xinyi Zhang; Chenglin Liu; Ruishan Dang; Tiqiao Xiao; Peiping Zhu (803-808).
Three-dimensional (3D) topographic structures of acupuncture points were investigated by using synchrotron radiation in-line X-ray phase contrast computerized tomography. Two acupuncture points, named Zhongji (RN3) and Zusanli (ST36), were studied. We found an accumulation of microvessels at each acupuncture point region. Images of the tissues surrounding the acupuncture points do not show such kinds of structure. This is the first time that 3D images have revealed the specific structures of acupuncture points.
Keywords: Acupuncture point; Synchrotron radiation; X-ray; Phase contrast; Microvessel
Imaging the cellular uptake of tiopronin-modified gold nanoparticles by Xiaoqing Cai; Hsiang-Hsin Chen; Cheng-Liang Wang; Shin-Tai Chen; Sheng-Feng Lai; Chia-Chi Chien; Yi-Yun Chen; Ivan M. Kempson; Yeukuang Hwu; C. S. Yang; G. Margaritondo (809-816).
Well-dispersed gold nanoparticles (NP) coated with tiopronin were synthesized by X-ray irradiation without reducing agents. High-resolution transmission electron microscopy shows that the average core diameters of the NPs can be systematically controlled by adjusting the tiopronin to Au mole ratio in the reaction. Three methods were used to study the NP uptake by cells: quantitative measurements by inductively coupled plasma mass spectrometry, direct imaging with high lateral resolution transmission electron microscopy and transmission X-ray microscopy. The results confirmed that the NP internalization mostly occurred via endocytosis and concerned the cytoplasm. The particles, in spite of their small sizes, were not found to arrive inside the cell nuclei. The synthesis without reducing agents and solvents increased the biocompatibility as required for potential applications in analysis and biomedicine in general. Figure A high resolution Transmission X-ray microscope image (A) captured the internalization and aggregation of tiopronin-coated Au nanoparticles in the vicinity of cell nucleus, the light dark area, of an EMG-6 cell. (B) One of the corresponding pictures produced by three-dimensional tomography reconstruction. The complete movie sequence of such pictures provides three-dimensional visual confirmation of the internalization and location of tiopronin-coated Au nanoparticles.
Keywords: Gold nanoparticles; X-ray synthesis; Cellular uptake; Transmission X-ray microscopy
3D configuration of mandibles and controlling muscles in rove beetles based on micro-CT technique by Dee Li; Kai Zhang; Peiping Zhu; Ziyu Wu; Hongzhang Zhou (817-825).
X-ray micro-CT is a powerful tool to visualize without damage details of the inner structures of beetles, the largest order of insects with a hard external skeleton. This contribution shows the three-dimensional (3D) reconstruction of the head morphology of three rove beetle species (Insecta, Coleoptera, Staphylinidae)—Noddia sp., Creophilus maxillosus, and Hesperosoma sp.—using X-ray microtomography at a spatial resolution of 6 μm. The details of skeletal muscle fiber insertions are described, giving a comprehensive overview of mandible mobility and organization. With the support of 3D rendering, we discuss the relationship among the mandible forms, the development of the muscles controlling the movement, and the head morphology. The well-developed posterior part of the head capsule is always accompanied by a well-developed mandible, a large adductor muscle, and a large apodeme for the wide areas of the muscle fiber attachment. In Noddia sp., muscles connected to the posterolateral angle of the head capsule are mainly short muscles, whereas in Creophilus maxillosus, the latter are mainly long muscles, and in Hesperosoma sp. no mandible adductor muscle fibers are present on the posterolateral angle of the head capsule. These results offer new invaluable information regarding the biting functions of beetle mandibles and the trend of their morphological change during their long-term evolution.
Keywords: Mandible; Mandible muscles; Three-dimensional reconstruction; Head capsule; Staphylininae
Detecting small lung tumors in mouse models by refractive-index microradiology by Chia-Chi Chien; Guilin Zhang; Y. Hwu; Ping Liu; Weisheng Yue; Jianqi Sun; Yan Li; Hongjie Xue; Lisa X. Xu; Chang Hai Wang; Nanyow Chen; Chien Hung Lu; Ting-Kuo Lee; Yuh-Cheng Yang; Yen-Ta Lu; Yu-Tai Ching; T. F. Shih; P. C. Yang; J. H. Je; G. Margaritondo (827-835).
Refractive-index (phase-contrast) radiology was able to detect lung tumors less than 1 mm in live mice. Significant micromorphology differences were observed in the microradiographs between normal, inflamed, and lung cancer tissues. This was made possible by the high phase contrast and by the fast image taking that reduces the motion blur. The detection of cancer and inflammation areas by phase contrast microradiology and microtomography was validated by bioluminescence and histopathological analysis. The smallest tumor detected is less than 1 mm3 with accuracy better than 1 × 10−3 mm3. This level of performance is currently suitable for animal studies, while further developments are required for clinical application. Figure Refractive-index microradiology detects small lung cancer tumors (<1 mm) in vivo, with precise size measurements, and yields tomographically reconstructed pictures of tumors on the same scale
Keywords: Lung cancer; Synchrotron X-ray imaging; Real-time imaging
Study of OSEM with different subsets in grating-based X-ray differential phase-contrast imaging by Kai Zhang; Youli Hong; Peiping Zhu; Qingxi Yuan; Wanxia Huang; Zhili Wang; Shengqi Chu; Samuel A. McDonald; Federica Marone; Marco Stampanoni; Ziyu Wu (837-844).
Impressive developments in X-ray imaging are associated with X-ray phase contrast computed tomography based on grating interferometry, a technique that provides increased contrast compared with conventional absorption-based imaging. A new “single-step” method capable of separating phase information from other contributions has been recently proposed. This approach not only simplifies data-acquisition procedures, but, compared with the existing phase step approach, significantly reduces the dose delivered to a sample. However, the image reconstruction procedure is more demanding than for traditional methods and new algorithms have to be developed to take advantage of the “single-step” method. In the work discussed in this paper, a fast iterative image reconstruction method named OSEM (ordered subsets expectation maximization) was applied to experimental data to evaluate its performance and range of applicability. The OSEM algorithm with different subsets was also characterized by comparison of reconstruction image quality and convergence speed. Computer simulations and experimental results confirm the reliability of this new algorithm for phase-contrast computed tomography applications. Compared with the traditional filtered back projection algorithm, in particular in the presence of a noisy acquisition, it furnishes better images at a higher spatial resolution and with lower noise. We emphasize that the method is highly compatible with future X-ray phase contrast imaging clinical applications.
Keywords: X-ray phase-contrast imaging; Computed tomography; Phase retrieval; OSEM
Detection of collagens in brain tumors based on FTIR imaging and chemometrics by Razia Noreen; Chia-Chi Chien; Maylis Delugin; Seydou Yao; Raphael Pineau; Yeukuang Hwu; Michel Moenner; Cyril Petibois (845-852).
Fourier transform infrared (FTIR) imaging has been used as a molecular histopathology tool on brain tissue sections after intracranial implantation and development of glioma tumors. Healthy brain tissue (contralateral lobe) as well as solid and diffuse tumor tissues were compared for their collagen contents. IR spectra were extracted from IR images for determining the secondary structure of protein contents and compared to pure product spectra of collagens (types I, III, IV, V, and VI). Multivariate statistical analyses of variance and correspondence factorial analysis were performed to differentiate healthy and tumor brain tissues as well as their classification according to their secondary structure profiles. Secondary structure profiles revealed that no collagen was present in healthy tissues; they are also significantly different from solid and diffuse tumors (p < 0.05). Solid and diffuse tumors could be discriminated with respect to the secondary structure profile of fibrillar and non-fibrillar collagens, respectively. We can thus propose to develop FTIR imaging for histopathology examination of tumors on the basis of collagen contents.
Keywords: Bioanalytical methods; Chemometrics; Statistics; IR spectroscopy; Collagen types
Quantitative comparison of preparation methodologies for x-ray fluorescence microscopy of brain tissue by Simon A. James; Damian E. Myers; Martin D. de Jonge; Stefan Vogt; Chris G. Ryan; Brett A. Sexton; Pamela Hoobin; David Paterson; Daryl L. Howard; Sheridan C. Mayo; Matteo Altissimo; Gareth F. Moorhead; Stephen W. Wilkins (853-864).
X-ray fluorescence microscopy (XFM) facilitates high-sensitivity quantitative imaging of trace metals at high spatial resolution over large sample areas and can be applied to a diverse range of biological samples. Accurate determination of elemental content from recorded spectra requires proper calibration of the XFM instrument under the relevant operating conditions. Here, we describe the manufacture, characterization, and utilization of multi-element thin-film reference foils for use in calibration of XFM measurements of biological and other specimens. We have used these internal standards to assess the two-dimensional distribution of trace metals in a thin tissue section of a rat hippocampus. The data used in this study was acquired at the XFM beamline of the Australian Synchrotron using a new 384-element array detector (Maia) and at beamline 2-ID-E at the Advanced Photon Source. Post-processing of samples by different fixation techniques was investigated, with the conclusion that differences in solvent type and sample handling can significantly alter elemental content. The present study highlights the quantitative capability, high statistical power, and versatility of the XFM technique for mapping trace metals in biological samples, e.g., brain tissue samples in order to help understand neurological processes, especially when implemented in conjunction with a high-performance detector such as Maia.
Keywords: X-ray fluorescence microscopy; Bioinorganic chemistry; Elemental mapping; Maia detector system; Sample preparation
Investigation of the partially coherent effects in a 2D Talbot interferometer by Xin Ge; Zhili Wang; Kun Gao; Kai Zhang; Youli Hong; Dajiang Wang; Zhongzhu Zhu; Peiping Zhu; Ziyu Wu (865-870).
The recent use of a one-dimensional (1D) X-ray Talbot interferometer has triggered great interest in X-ray differential phase contrast imaging. As an improved version of a 1D interferometer, the development of two-dimensional (2D) grating interferometry strongly stimulated applications of grating-based imaging. In the framework of Fresnel diffraction theory, we investigated the self-image of 2D-phase gratings under partially coherent illumination. The fringe visibility of the self-image has been analyzed as a function of the spatial coherence length. From the viewpoint of self-image visibility, it is possible to find the optimal 2D grid for 2D X-ray grating interferometer imaging. Numerical simulations have been also carried out for quantitative evaluation. Results, in good agreement with theoretical analysis, indicate the spatial coherence requirements of the radiation illuminating a 2D grating interferometer. Moreover, our results can be used to optimize performances of a 2D grating interferometer and for further theoretical and experimental research on grating-based imaging systems.
Keywords: X-ray phase contrast; 2D Talbot interferometer; Partially coherent illumination; Visibility
ANAKON 2011 – German thoroughness meets Swiss precision by Oliver Bleher; Melanie Ewald; Felix Kolarov; A. Katrin Krieg; Alexander F. Le Blanc; Sabrina Rau (871-872).
Recombinant cell bioassays for the detection of (gluco)corticosteroids and endocrine-disrupting potencies of several environmental PCB contaminants by Toine F. H. Bovee; Richard J. R. Helsdingen; Astrid R. M. Hamers; Bram A. Brouwer; Michel W. F. Nielen (873-882).
Sensitive and robust bioassays for glucocorticoids are very useful for the pharmaceutical industry, environmental scientists and veterinary control. Here, a recombinant yeast cell was constructed that expresses the human glucocorticoid receptor alpha and a green fluorescent reporter protein in response to glucocorticoids. Both the receptor construct and the reporter construct were stably integrated into the yeast genome. The correct and specific functioning of this yeast glucocorticoid bioassay was studied by exposures to cortisol and other related compounds and critically compared to a GR-CALUX bioassay based on a human bone cell. Although less sensitive, the new yeast glucocorticoid bioassay showed sensitivity towards all (gluco)corticoids tested, with the following order in relative potencies: budesonide >> corticosterone > dexamethasone > cortisol = betamethasone > prednisolone > aldosterone. Hormone representatives for other hormone nuclear receptors, like 17β-estradiol for the oestrogen receptor, 5α-dihydrotestosterone for the androgen receptor and progesterone for the progesterone receptor, showed no clear agonistic responses, whilst some polychlorinated biphenyls were clearly able to interfere with the GR activity.
Keywords: Activity screening; Bioassay; Contaminants; Detection; Glucocorticoid receptor
Carbon nanotubes/pentacyaneferrate-modified chitosan nanocomposites platforms for reagentless glucose biosensing by A. M. Parra-Alfambra; E. Casero; M. A. Ruiz; L. Vázquez; F. Pariente; E. Lorenzo (883-889).
The design, characterization and applicability of a nanostructured biosensor platform are described. The biosensor is developed through the immobilization of three components: a polymeric chitosan network previously modified with a redox mediator (denoted as PCF-Pyr-Ch), an enzyme (glucose oxidase, chosen as a model) and carbon nanotubes onto a solid glassy carbon electrode (C). In order to assess the influence of the nanomaterial in the performance of the resulting analytical device, a second biosensor, free of carbon nanotubes, is developed. The characterization of both biosensing platforms was performed in aqueous phosphate buffer solutions using atomic force microscopy technique. In the presence of glucose, both systems exhibit a clear electrocatalytic activity, and glucose could be amperometrically determined at +0.35 V versus Ag/AgCl. The performance of both biosensors was evaluated in terms of sensitivity, detection limit and linear response range. Finally, the enhancement of the analytical response induced by the presence of carbon nanotubes was evaluated.
Keywords: Carbon nanotubes; Chitosan; Glucose oxidase; Reagentless electrochemical biosensor; AFM
Quantification of plasma phospholipids by ultra performance liquid chromatography tandem mass spectrometry by Yannick Rabagny; Wolfgang Herrmann; Jürgen Geisel; Susanne H. Kirsch; Rima Obeid (891-899).
We describe a fast and robust ultra performance liquid chromatography tandem mass spectrometry method for the quantification of phospholipid (PL) species in EDTA-plasma samples. We quantified total phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), and sphingomyelin (SM) and several species within these classes using one or two external calibrators and one internal standard for each class. Inter-assay coefficients of variation were <10% for the most abundant species and <20% for all quantified PC, LPC, and SM species and the three most abundant PE species. Coefficients of linear regression were R 2 > 0.98. Mean recoveries were between 83% and 123%. The limits of detection were 0.37 μmol/L for PC, 4.02 μmol/L for LPC, 3.75 μmol/L for PE, and 0.86 μmol/L for SM. Quantification was linear over the physiological ranges for PE, LPC, and SM and up to 500 μmol/L for PC. The concentrations of PLs in the plasma of healthy donors yielded results that were comparable with those of previous works.
Keywords: Phospholipid; Phosphatidylcholine; Phosphatidylethanolamine; Mass spectrometry
Distribution profiles of nitroxide spin probes in human skin—a combined study using spatially resolved electron spin resonance spectroscopy and mass spectrometry by U. Hochkirch; W. Herrmann; R. Stößer; M. Linscheid; H.-H. Borchert (901-907).
Electron spin resonance spectroscopy and mass spectrometry are two analytical methods that are very rarely used in combination. In this paper, we will show that the methods complement one another in the example of the distribution of stable nitroxide radicals in human skin, including the spatial resolution of these distribution processes. There are many ESR investigations dealing with this subject, but unfortunately, they are all limited to the detection of paramagnetic species. The combination with MS allows the successful examination of the distribution profile of the main biotransformation product of the nitroxide radicals, the respective “ESR-silent” hydroxylamines. In order to maintain the biological state of the sample material as far as possible, atmospheric pressure matrix-assisted laser desorption/ionization with ion trap detection has been used for the mass spectrometric investigations. The results validate the former findings of the strong reduction of stable free radicals by biological material; moreover, the diamagnetic species formed during these processes have been identified. Figure Comparison of the ESR and MS results concerning the distribution of the nitroxide radical CAT-1 and CAT-1-H in a human skin biopsy
Keywords: Nitroxide radicals; Hydroxylamine; AP-MALDI mass spectrometry; Electron spin resonance spectroscopy; Spatial resolution; Human skin
Using laser ablation/inductively coupled plasma mass spectrometry to bioimage multiple elements in mouse tumors after hyperthermia by Yi-Kong Hsieh; Pei-Shin Jiang; Bing-Shen Yang; Tian-Ye Sun; Hsu-Hsia Peng; Chu-Fang Wang (909-915).
In this study, we employed laser ablation/inductively coupled plasma mass spectrometry (LA-ICP-MS) to map the spatial distribution of Gd-doped iron oxide nanoparticles (IONPs) in one tumor slice that had been subjected to magnetic fluid hyperthermia (MFH). The mapping results revealed the high resolution of the elemental analysis, with the distribution of Gd atoms highly correlated with that of the Fe atoms. The spatial distributions of C, P, S, and Zn atoms revealed that the effect of MFH treatment was significantly dependent on the diffusion of the magnetic fluid in the tissue. An observed enrichment of Cu atoms after MFH treatment was probably due to inflammation in the tumor. The abnormal distribution of Ni atoms suggests a probable biochemical reaction in the tumor. Therefore, this LA-ICP-MS mapping technique can provide novel information regarding the spatial distribution of elements in tumors after cancer therapy. Figure Mapping and ion intensities of a 56Fe and b 158Gd atoms. The red line indicates the path taken during the time-resolved analyses of Fe and Gd atoms
Keywords: Laser ablation ICP-MS; Elemental imaging; Magnetic fluid hyperthermia
A piezoelectric immunosensor for Leishmania chagasi antibodies in canine serum by Joilson Ramos-Jesus; Kellyanne A. Carvalho; Rosana A. S. Fonseca; Geraldo G. S. Oliveira; Stella M. Barrouin Melo; Neuza M. Alcântara-Neves; Rosa F. Dutra (917-925).
The American visceral leishmaniasis is an important cause of morbidity and mortality in Brazil for both humans and dogs. Attempts to make a diagnosis of this disease need to be improved, especially in endemic areas, and in the tracking and screening of asymptomatic dogs, which are their main host in urban areas. A quartz crystal microbalance immunosensor for the diagnosis of the canine visceral leishmaniasis using a recombinant antigen of Leishmania chagasi (rLci2B-NH6) was developed. The rLci2B-NH6 was tightly immobilized on a quartz crystal gold electrode by self-assembled monolayer based on short-chain length thiol. The strategy was the use of the antigen-histidine tail covalently linked to glutaraldehyde performing a Schift base which permits a major exposure of epitopes and a reduced steric hindrance. The immunosensor showed good results regarding sensitivity and reproducibility, being able to distinguish positive and negative canine serum for L. chagasi. Furthermore, the immunosensor can be reused through exposure to sodium dodecyl sulfate solution, which promotes the dissociation of antigen–antibody binding, restoring the sensor surface with immobilized biologically active antigens for further analysis.
Keywords: Leishmania chagasi ; Immunosensor; Self-assembled monolayer; Piezoelectric
Determination of pyrethroid metabolites in human urine using liquid phase microextraction coupled in-syringe derivatization followed by gas chromatography/electron capture detection by Chiu-Hwa Lin; Cheing-Tong Yan; Ponnusamy Vinoth Kumar; Hong-Ping Li; Jen-Fon Jen (927-937).
Metabolites of synthetic pyrethroids such as cis-3-(2,2-dibromovinyl)-2,2-di-methylcyclo-propane-1-carboxylic acid, cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid), 3-phenoxybenzoic acid (3-PBA), and 4-fluoro-3-PBA are biomarkers for exposure to phenothrin, tetramethrin, cyfluthrin, cypermethrin, deltamethrin, and permethrin. In this study, the pyrethroid metabolites in workers’ urine samples were monitored for the first time with a novel sample pretreatment process combining hollow fiber liquid phase microextraction (HF-LPME) and in-syringe derivatization (ISD) followed by gas chromatography–electron capture detector (GC-ECD) analysis. A micro-syringe pre-filled with derivatizing agents and syringe needle connected to an extracting solvent impregnated hollow fiber segment was used as the LPME probe. Pyrethroid metabolites were extracted and enriched simultaneously from urine samples by HF-LPME sampling and acid hydrolysis at 70 °C for 10 min. After sampling, the ISD was performed by mixing the extracting solution and derivatizing agents through plunger movements, followed by GC-ECD analysis. Parameters influencing the HF-LPME efficiency and ISD were investigated and optimized. Under optimum conditions, the method provided enrichment factors of 69.8–154.6, repeatability from 5.0 to 12% (n = 5), and good linearity (R 2 = 0.9980–0.9998) for interested analytes spiked in urine samples. The method detection limits ranged from 1.6 to 17 ng/mL. A comparison was performed between the proposed method and conventional methods. The proposed method was applied to analyze pyrethroid metabolites in the urine samples collected from workers of pesticide formulation plants. The results suggested that the proposed HF-LPME coupled ISD method was a rapid, simple, efficient, and eco-friendly technique in the biomonitoring of metabolites of pyrethroids in workers’ urine.
Keywords: Pyrethroids; Metabolites; Urine samples; Hollow fiber; Liquid phase microextraction; In-syringe derivatization
Identification of heparin samples that contain impurities or contaminants by chemometric pattern recognition analysis of proton NMR spectral data by Qingda Zang; David A. Keire; Lucinda F. Buhse; Richard D. Wood; Dinesh P. Mital; Syed Haque; Shankar Srinivasan; Christine M. V. Moore; Moheb Nasr; Ali Al-Hakim; Michael L. Trehy; William J. Welsh (939-955).
Chemometric analysis of a set of one-dimensional (1D) 1H nuclear magnetic resonance (NMR) spectral data for heparin sodium active pharmaceutical ingredient (API) samples was employed to distinguish USP-grade heparin samples from those containing oversulfated chondroitin sulfate (OSCS) contaminant and/or unacceptable levels of dermatan sulfate (DS) impurity. Three chemometric pattern recognition approaches were implemented: classification and regression tree (CART), artificial neural network (ANN), and support vector machine (SVM). Heparin sodium samples from various manufacturers were analyzed in 2008 and 2009 by 1D 1H NMR, strong anion-exchange high-performance liquid chromatography, and percent galactosamine in total hexosamine tests. Based on these data, the samples were divided into three groups: Heparin, DS ≤ 1.0% and OSCS = 0%; DS, DS > 1.0% and OSCS = 0%; and OSCS, OSCS > 0% with any content of DS. Three data sets corresponding to different chemical shift regions (1.95–2.20, 3.10–5.70, and 1.95–5.70 ppm) were evaluated. While all three chemometric approaches were able to effectively model the data in the 1.95–2.20 ppm region, SVM was found to substantially outperform CART and ANN for data in the 3.10–5.70 ppm region in terms of classification success rate. A 100% prediction rate was frequently achieved for discrimination between heparin and OSCS samples. The majority of classification errors between heparin and DS involved cases where the DS content was close to the 1.0% DS borderline between the two classes. When these borderline samples were removed, nearly perfect classification results were attained. Satisfactory results were achieved when the resulting models were challenged by test samples containing blends of heparin APIs spiked with non-, partially, or fully oversulfated chondroitin sulfate A, heparan sulfate, or DS at the 1.0%, 5.0%, and 10.0% (w/w) levels. This study demonstrated that the combination of 1D 1H NMR spectroscopy with multivariate chemometric methods is a nonsubjective, statistics-based approach for heparin quality control and purity assessment that, once standardized, minimizes the need for expert analysts. Figure Contour plot from grid search of the optimal values of γ and C for the SVM model
Keywords: Heparin; Proton nuclear magnetic resonance (1H NMR); Pattern recognition; Classification and regression tree (CART); Artificial neural network (ANN); Support vector machine (SVM)
A biospectroscopic interrogation of fine needle aspirates points towards segregation between graded categories: an initial study towards diagnostic screening by Jemma G. Kelly; Abdullah A. Ahmadzai; Paul Hermansen; Mark A. Pitt; Zuhair Saidan; Pierre L. Martin-Hirsch; Francis L. Martin (957-967).
Fine needle aspirates (FNAs) of suspicious breast lesions are often used to aid the diagnosis of female breast cancer. Biospectroscopy tools facilitate the acquisition of a biochemical cell fingerprint representative of chemical bonds present in a biological sample. The mid-infrared (IR; 4,000–400 cm−1) is absorbed by the chemical bonds present, allowing one to derive an absorbance spectrum. Complementary to IR spectroscopy, Raman spectroscopy measures the scattering by chemical bonds following excitation by a laser to generate an intensity spectrum. Our objective was to apply these methods to determine whether a biospectroscopy approach could objectively segregate different categories of FNAs. FNAs of breast tissue were collected (n = 48) in a preservative solution and graded into categories by a cytologist as C1 (non-diagnostic), C2 (benign), C3 (suspicious, probably benign) or C5 (malignant) [or C4 (suspicious, probably malignant); no samples falling within this category were identified during the collection period of the study]. Following washing, the cellular material was transferred onto BaF2 (IR-transparent) slides for interrogation by Raman or Fourier-transform IR (FTIR) microspectroscopy. In some cases where sufficient material was obtained, this was transferred to low-E (IR-reflective) glass slides for attenuated total reflection–FTIR spectroscopy. The spectral datasets produced from these techniques required multivariate analysis for data handling. Principal component analysis followed by linear discriminant analysis was performed independently on each of the spectral datasets for only C2, C3 and C5. The resulting scores plots revealed a marked overlap of C2 with C3 and C5, although the latter pair were both significantly segregated (P < 0.001) in the Raman spectra. Good separation was observed between C3 and C5 in all three spectral datasets. Analysis performed on the average spectra showed the presence of three distinct cytological groups. Our findings suggest that biospectroscopy tools coupled with multivariate analysis may support the current FNA tests whilst increasing the sensitivity and associated reliability for improved diagnostics. Figure Average IR spectra derived from different categories of FNA specimens
Keywords: Breast cancer; Fine needle aspirate; IR spectroscopy; Principal component analysis; Linear discriminant analysis; Raman spectroscopy
Segregation of human prostate tissues classified high-risk (UK) versus low-risk (India) for adenocarcinoma using Fourier-transform infrared or Raman microspectroscopy coupled with discriminant analysis by Imran I. Patel; Júlio Trevisan; Paras B. Singh; Caroline M. Nicholson; R. K. Gopala Krishnan; Shyam S. Matanhelia; Francis L. Martin (969-982).
Vibrational spectroscopy techniques can be applied to identify a susceptibility-to-adenocarcinoma biochemical signature. A sevenfold difference in incidence of prostate adenocarcinoma (CaP) remains apparent amongst populations of low- (e.g. India) compared with high-risk (e.g. UK) regions, with migrant studies implicating environmental and/or lifestyle/dietary causative factors. This study set out to determine the biospectroscopy-derived spectral differences between risk-associated cohorts to CaP. Benign prostate tissues were obtained using transurethral resection from high-risk (n = 11, UK) and low-risk (n = 14, India) cohorts. Samples were analysed using attenuated total reflection Fourier-transform infrared (FTIR) spectroscopy, FTIR microspectroscopy and Raman microspectroscopy. Spectra were subsequently processed within the biochemical cell region (1,800−1–500 cm–1) employing principal component analysis (PCA) and linear discriminant analysis (LDA) to determine whether wavenumber–absorbance/intensity relationships might reveal biochemical differences associated with region-specific susceptibility to CaP. PCA-LDA scores and corresponding cluster vector plots identified pivotal segregating biomarkers as 1,582 cm−1 (Amide I/II trough); 1,551 cm−1 (Amide II); 1,667 cm−1 (Amide I); 1,080 cm−1 (DNA/RNA); 1,541 cm−1 (Amide II); 1,468 cm−1 (protein); 1,232 cm−1 (DNA); 1,003 cm−1 (phenylalanine); 1,632 cm−1 [right-hand side (RHS) Amide I] for glandular epithelium (P < 0.0001) and 1,663 cm−1 (Amide I); 1,624 cm−1 (RHS Amide I); 1,126 cm−1 (RNA); 1,761, 1,782, 1,497 cm−1 (RHS Amide II); 1,003 cm−1 (phenylalanine); and 1,624 cm−1 (RHS Amide I) for adjacent stroma (P < 0.0001). Primarily protein secondary structure variations were biomolecular markers responsible for cohort segregation with DNA alterations exclusively located in the glandular epithelial layers. These biochemical differences may lend vital insights into the aetiology of CaP. Figure The first study to apply biospectroscopy techniques to identify the underlying differences in the aetiology of prostate cancer between low- (India) compared to high-risk (UK) cohorts
Keywords: Aetiology; Biomarkers; FTIR microspectroscopy; PCA-LDA; Prostate cancer; Raman
Screening nucleotide binding to amino acid-coated supports by surface plasmon resonance and nuclear magnetic resonance by Carla Cruz; Eurico J. Cabrita; João A. Queiroz (983-993).
Here, we describe a rapid and efficient screening method using surface plasmon resonance (SPR) and saturation transfer difference–nuclear magnetic resonance (STD-NMR) spectroscopy to yield information regarding the residues involved in nucleotide binding to amino acid-coated supports. The aim of this work was to explore the use of these spectroscopic techniques to study amino acid–nucleotide interactions in order to improve the binding specificity of the amino acid ligands used to purify plasmid DNA. For SPR, we present a strategy that immobilizes arginine and lysine on a surface as model supports, and we analyze binding responses when synthetic homo-deoxyoligonucleotides are injected over the amino acid surface. The binding responses are detectable and reproducible despite the small size of the immobilized amino acids. Using STD-NMR, we performed epitope mapping of homo-deoxyoligonucleotides bound to l-arginine–bisoxyran–Sepharose and l-lysine–Sepharose supports. Polynucleotide binding preferences differed; for example, polyC interacted preferentially through its backbone with the two supports, whereas polyT bound the supports through its thymine moiety. STD-NMR combined with SPR measurements was successfully used to screen amino acid–nucleotide interactions and determine the binding affinities of the complexes.
Keywords: Saturation transfer difference–nuclear magnetic resonance; Surface plasmon resonance; Amino acid supports; Nucleotides; Binding affinities; Amino acid–nucleotide interactions
Measurement of bisphenol A, bisphenol A ß-d-glucuronide, genistein, and genistein 4′-ß-d-glucuronide via SPE and HPLC-MS/MS by Janis L. Coughlin; Bozena Winnik; Brian Buckley (995-1002).
Bisphenol A (BPA) is a synthetic industrial reactant used in the production of polycarbonate plastics, and genistein is a natural phytoestrogen abundant in the soybean. Current studies investigating the endocrine-disrupting effects of concomitant exposures to BPA and genistein have warranted the development of an analytical method for the simultaneous measurement of BPA and genistein, as well as their primary metabolites, bisphenol A ß-d-glucuronide (BPA gluc) and genistein 4′-ß-d-glucuronide (genistein gluc), respectively. All four analytes were extracted from rat plasma via solid phase extraction (SPE). Three SPE cartridges and four elution schemes were tested. Plasma extraction using Bond Elut Plexa cartridges with sequential addition of ethyl acetate, methanol, and acetonitrile yielded optimal average recoveries of 98.1 ± 1.8% BPA, 94.9 ± 8.0% genistein, 91.4 ± 6.1% BPA gluc, and 103 ± 6.1% genistein gluc. Identification and quantification of the four analytes were performed by a validated HPLC-MS/MS method using electrospray ionization and selective reaction monitoring. This novel analytical method should be applicable to the measurement of BPA, genistein, BPA gluc, and genistein gluc in urine, cultures, and tissue following in vivo exposures. While reports of the determination of BPA and genistein independently exist, the simultaneous optimized extraction and detection of BPA, genistein, BPA gluc, and genistein gluc have not previously been reported. Figure BPA and genistein co-exposure scenario. BPA-laden polycarbonate plastic baby bottle filled with soymilk, a rich source of genistein, provides a classic exposure scenario to young children—a population that is particularly vulnerable to the effects of endocrine-disrupting compounds
Keywords: Bisphenol A (BPA); Genistein; Bisphenol A ß-d-glucuronide; Genistein 4′-ß-d-glucuronide; HPLC; Mass spectrometry; Electrospray ionization; Chromatographic techniques
Bioaccumulation assessment of the sunscreen agent 2-ethylhexyl 4-(N,N-dimethylamino)benzoate in human semen by automated online SPE-LC-MS/MS by Zacarías León-González; Carlos Ferreiro-Vera; Feliciano Priego-Capote; María Dolores Luque de Castro (1003-1011).
The proven endocrine disruption nature of the sunscreen ingredient 2-ethylhexyl 4-(N,N-dimethylamino)benzoate (EDP) calls for research to understand its distribution and bioaccumulation in the human body. A sensitive analytical method to determine EDP and its metabolites in human semen based on online SPE-LC-MS/MS is described. The method has been fully validated and a standard addition calibration has been used for quantification to correct the observed matrix effects. The on-column detection limits of the analytes are between 0.2 and 0.6 ng, depending on the analyte and the sample. The repeatability of the method, expressed as relative standard deviation, was in the range 4.6–9.4%. The method was satisfactorily applied to semen samples from male volunteers who were subjected to single and repeated whole-body applications of an EDP-containing sunscreen product. EDP metabolites were found at different concentrations in semen samples from the repeated application study, thus showing evidences of bioaccumulation in humans.
Keywords: 2-Ethylhexyl 4-(N,N-dimethylamino)benzoate; Sunscreen; Semen; Bioaccumulation; Online solid-phase extraction; LC-MS/MS
Development and validation of an HPLC–UV detection assay for the determination of rufinamide in human plasma and saliva by Iolanda Mazzucchelli; Manuela Rapetti; Cinzia Fattore; Valentina Franco; Giuliana Gatti; Emilio Perucca (1013-1021).
The development of a simple and rapid high-performance liquid chromatography (HPLC) method for the determination of the new antiepileptic drug rufinamide (RFN) in human plasma and saliva is reported. Samples (250 μl) are alkalinized with ammonium hydroxide (pH 9.25) and extracted with dichloromethane using metoclopramide as internal standard. Separation is achieved with a Spherisorb silica column (250 × 4.6 mm i.d., 5 μm) at 30 °C using as mobile phase a solution of methanol/dichloromethane/n-hexane 10/25/65 (vol/vol/vol) mixed with 6 ml ammonium hydroxide. The instrument used was a Shimadzu LC-10Av chromatograph and flow rate was 1.5 ml min-1, with a LaChrom L-7400 UV detector set at 230 nm. Calibration curves are linear [r 2 = 0.998 ± 0.002 for plasma (n = 10) and r 2 = 0.999 ± 0.001 for saliva (n = 9)] over the range of 0.25–20.0 μg ml-1, with a limit of quantification at 0.25 μg ml-1. Precision and accuracy are within current acceptability standards. The assay is suitable for pharmacokinetic studies in humans and for therapeutic drug monitoring.
Keywords: Plasma; Saliva; HPLC; UV detection; Rufinamide
Development and validation of an HPLC–MS/MS method to quantify clopidogrel acyl glucuronide, clopidogrel acid metabolite, and clopidogrel in plasma samples avoiding analyte back-conversion by Luigi Silvestro; Mihaela Gheorghe; Adriana Iordachescu; Valentin Ciuca; Ariana Tudoroniu; Simona Rizea Savu; Isabela Tarcomnicu (1023-1034).
A new sensitive and fast quantitative analytical method for the simultaneous determination of clopidogrel, its main metabolite clopidogrel carboxylic acid, and the newly described acyl glucuronide metabolite, in human plasma samples, is presented. The analytical procedures (plasma storage, handling, and extract storage in the autosampler) were optimized in order to avoid back-conversion; a known drawback in measurements of clopidogrel. Clopidogrel acyl glucuronide was confirmed as a major source of back-conversion to the parent drug in the presence of methanol, and thorough stability experiments were carried out to find the most appropriate conditions for an accurate analysis of clopidogrel and the two metabolites. The method was validated by assessing selectivity, sensitivity, linearity, accuracy, and precision for all three analytes, in accordance to Food and Drug Administration guidelines. Spiked quality controls in plasma as well as incurred samples were used to verify back-conversion in the selected conditions, with results meeting European Medicines Agency acceptance criteria (concentrations within 80–120% of the first reading). The method was then applied to a pharmacokinetic study, and for the first time, a pharmacokinetic curve of clopidogrel acyl glucuronide in human plasma is presented. The concentrations ranged up to 1,048.684 ng/mL, with a mean of 470.268 ng/mL, while clopidogrel had a mean C max of 1.348 ng/mL; these orders of magnitude show how much the back-conversion of this metabolite may influence clopidogrel quantification if it is not properly controlled.
Keywords: Clopidogrel; Clopidogrel acyl glucuronide; Liquid chromatography; Tandem mass spectrometry; Pharmacokinetics; Back-conversion
Folate analysis in foods by UPLC-MS/MS: development and validation of a novel, high throughput quantitative assay; folate levels determined in Australian fortified breads by Maria V. Chandra-Hioe; Martin P. Bucknall; Jayashree Arcot (1035-1042).
An ultra-performance liquid chromatography-tandem mass spectrometry method was developed, optimised and validated for the quantification of synthetic folic acid (FA), also called pteroyl-l-glutamic acid or vitamin B9 and naturally occurring 5-methyltetrahydrofolate (5-MTHF) found in folate-fortified breads. Optimised sample preparation prior to analysis involved addition of 13C5 labelled internal standards, treatments with α-amylase and rat serum, solid-phase extraction using aromatic-selective cartridges and ultra-filtration. Analytes were separated on a Waters ACQUITY HSS T3 column during a 6-min run and analysed by positive ion electrospray selected reaction monitoring MS/MS. Standard calibration curves for the two analytes were linear over the range of 0.018–14 μg FA/g of fresh bread (r 2 = 0.997) and 9.3–900 ng 5-MTHF/g of fresh bread (r 2 = 0.999). The absolute recoveries were 90% and 76% for FA and 5-MTHF, respectively. Intra-day coefficients of variation were 3% for FA and 18% for 5-MTHF. The limit of detection was 9.0 ng/g for FA and 4.3 ng/g for 5-MTHF, determined using pre-extracted tapioca starch as the blank matrix. The assay is rugged, fast, accurate and sensitive, applicable to a variety of food matrices and is capable of the detection and quantification of the naturally occurring low levels of 5-MTHF in wheat breads. The findings of this study revealed that the FA range in Australian fortified breads was 79–110 μg/100 g of fresh bread and suggest that the flour may not have the mandated FA fortification level (200–300 μg/100 g of flour), though this cannot be determined conclusively from experimental bread data alone, as variable baking losses have been documented by other authors. Figure Chromatogram of labelled folic acid using UPLC-MS/MS
Keywords: Folic acid; 5-MTHF; UPLC; MS/MS; HPLC-MS; Fortification; Bread; Quantitation
Palytoxin in seafood by liquid chromatography tandem mass spectrometry: investigation of extraction efficiency and matrix effect by Patrizia Ciminiello; Carmela Dell’Aversano; Emma Dello Iacovo; Ernesto Fattorusso; Martino Forino; Luciana Tartaglione; Rachele Rossi; Vittorio Soprano; Daniela Capozzo; Luigi Serpe (1043-1050).
Blooms of Ostreopsis spp. have been recently reported along the Mediterranean coasts of Spain, France, Italy, and Greece posing serious risks to human health. Occurrence of Ostreopsis spp. may result in palytoxin contamination of seafood and, in order to prevent sanitary risks, the need exists to develop efficient extraction procedures to be coupled to rapid and sensitive monitoring methods of palytoxin-like compounds in seafood. In the present study, the best conditions for both extraction of palytoxin from seafood and palytoxin quantification by using liquid chromatography tandem mass spectrometry (LC-MS/MS) were investigated. Three seafood matrices (mussels, sea-urchins, and anchovies) were selected and five different extraction systems were tested, namely: the official protocol for extraction of lipophilic toxins and various aqueous methanol or acetonitrile solutions (MeOH/H2O 1:1, MeOH/H2O 8:2, MeCN/H2O 8:2 and MeOH 100%). Extraction with MeOH/H2O 8:2 provided the best results in terms of accuracy and matrix interference on LC-MS/MS detection of palytoxin. Accuracy and intra-day reproducibility (n = 3) were evaluated for all the selected matrices but only for mussels at three spiking concentration levels, including the provisional limit proposed by the Community Reference Laboratory for marine biotoxins (250 μg kg−1). Limits of quantitation of palytoxin in mussels, sea-urchins and anchovies tissues were calculated using matrix-matched standards; taking into account extraction efficiency of MeOH/H2O 8:2, they resulted to be 228, 343, and 500 μg kg−1, respectively. Figure Palytoxin in mussels, sea-urchins, and anchovies: extraction efficiency and matrix effect in LC-MS/MS determination
Keywords: Palytoxin; LC-MS/MS; Mussels; Seafood; Matrix effect; Accuracy
Residue analysis of four diacylhydrazine insecticides in fruits and vegetables by Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method using ultra-performance liquid chromatography coupled to tandem mass spectrometry by Xingang Liu; Jun Xu; Fengshou Dong; Yuanbo Li; Wenchen Song; Yongquan Zheng (1051-1058).
The new analytical method using Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) procedure for simultaneous determination of diacylhydrazine insecticide residues in fruits and vegetables was developed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The four insecticides (tebufenozide, methoxfenozide, chromafenozide, and halofenozide) were extracted from six fruit and vegetable matrices using acetonitrile and subsequently cleaned up using primary secondary amine (PSA) or octadecylsilane (C18) as sorbent prior to UPLC-MS/MS analysis. The determination of the target compounds was achieved in less than 3.0 min using an electrospray ionization source in positive mode (ESI+) for tebufenozide, methoxfenozide, and halofenozide and in negative mode (ESI−) for chromafenozide. The limits of detection were below 0.6 μg kg−1, while the limit of quantification did not exceed 2 μg kg−1 in different matrices. The QuEChERS procedure by using two sorbents (PSA and C18) and the matrix-matched standards gave satisfactory recoveries and relative standard deviation (RSD) values in different matrices at four spiked levels (0.01, 0.05, 0.1, and 1 mg kg−1). The overall average recoveries for this method in apple, grape, cucumber, tomato, cabbage, and spinach at four levels ranged from 74.2% to 112.5% with RSDs in the range of 1.4–13.8% (n = 5) for all analytes. This study provides a theoretical basis for China to draw up maximum residue limits and analytical method for diacylhydrazine insecticide in vegetables and fruits.
Keywords: Residue analysis; Ultra-performance liquid chromatography-mass spectrometry; Diacylhydrazine insecticide; Fruits; Vegetables
Sample preparation procedure for the determination of polycyclic aromatic hydrocarbons in petroleum vacuum residue and bitumen by Ewelina Gilgenast; Grzegorz Boczkaj; Andrzej Przyjazny; Marian Kamiński (1059-1069).
This paper describes a novel method of sample preparation for the determination of trace concentrations of polycyclic aromatic hydrocarbons (PAHs) in high-boiling petroleum products. Limits of quantitation of the investigated PAHs in materials of this type range from tens of nanograms per kilogram to <20 μg/kg. The studies revealed that in order to separate most of interferences from the analytes without a significant loss of PAHs, it is necessary to use size exclusion chromatography as the first step of sample preparation, followed by adsorption using normal-phase liquid chromatography. The use of orthogonal separation procedure described in the paper allows the isolation of only a group of unsubstituted and substituted aromatic hydrocarbons with a specific range of molar mass. The lower the required limit of quantitation of PAHs, the larger is the scale of preparative liquid chromatography in both steps of sample preparation needed. The use of internal standard allows quantitative results to be corrected for the degree of recovery of PAHs during the sample preparation step. Final determination can be carried out using HPLC-FLD, GC-MS, or HPLC-UV–VIS/DAD. The last technique provides a degree of identification through the acquired UV–VIS spectra. Figure Chromatograms obtained using UV-DAD detection with wavelength programming (A) and fluorimetric detection (B) for the separation of 18 PAH standards ((A) and (B)) and the fraction containing PAHs from road asphalt 50/70 prepared according to the procedure described in this work (C). Peak designation: 1 naphthalene, 2 acenaphthylene, 3 acenaphthene, 4 fluorene, 5 phenanthrene, 6 anthracene, 7 fluoranthene, 8 pyrene, 9 benzo[a]anthracene, 10 chrysene, 11 benzo[b]fluoranthene, 12 benzo[k]fluoranthene, 13 benzo[a]pyrene, 14 dibenzo[a,h]anthracene, 15 indeno[1,2,3-cd]pyrene, 16 benzo[ghi]perylene, 17 benzo[j]fluoranthene, 18 benzo[e]pyrene,19 highly polar components of road asphalt 50/70 eluted during backflush. BF backflush point
Keywords: Sample preparation techniques; Multidimensional liquid chromatography; Group separation; Size exclusion chromatography; Normal-phase adsorption chromatography; High-boiling petroleum products; Polycyclic aromatic hydrocarbons (PAHs); Trace analysis
Determination of some sulfonylurea herbicides in soil by a novel liquid-phase microextraction combined with sweeping micellar electrokinetic chromatography by Shuaihua Zhang; Xiaofang Yin; Qian Yang; Chun Wang; Zhi Wang (1071-1081).
A novel method for the determination of five sulfonylurea herbicides in soil was developed by a dispersive solid-phase extraction (DSPE) clean-up followed by dispersive liquid–liquid microextraction (DLLME), prior to sweeping micellar electrokinetic chromatography (MEKC). In the DSPE-DLLME, 10 g of soil sample was first extracted with 10 mL of acetonitrile containing 5% formic acid (pH 3.0). The extract was then cleaned-up by a DSPE with C18 as sorbent. A 1 mL aliquot of the resulting extract was then added into a centrifuge tube containing 5 mL of water adjusted to pH 2.0 and 60.0 μL chlorobenzene (as extraction solvent) for DLLME procedure. Then, the organic sample extraction solution was evaporated to dryness, and reconstituted with 20.0 μL of 1.0 mmol L−1 Na2HPO4 (pH 10.0) for sweeping-MEKC analysis after DLLME. Under optimized conditions, the method provided as high as 3,000- to 5,000-fold enrichments factors. The linearity of the method was in the range of 3.3–200 ng g−1 for chlorimuron ethyl and bensulfuron methyl, and in the range of 1.7–200 ng g−1 for tribenuron methyl, chlorsulfuron and metsulfuron methyl, with the correlation coefficients (r) ranging from 0.9965 to 0.9983, respectively. The limits of detection (LODs) ranged from 0.5 to 1.0 ng g−1. The intraday relative standard deviations (RSDs, n = 5) were below 5.3% and interday RSDs (n = 15) within 6.8%. The recoveries of the method for the five sulfonylureas from soil samples at spiking levels of 5.0, 20.0, and 100.0 ng g−1 were 76.0–93.5%, respectively. The developed method has been successfully applied to the analysis of the target sulfonylurea herbicide residues in soil samples with a satisfactory result.
Keywords: Dispersive solid-phase extraction clean-up; Dispersive liquid–liquid microextraction; Sulfonylurea herbicides; Soil samples; Sweeping; Micellar electrokinetic chromatography
Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium exchange to characterize allosteric regulation by Charulata B. Prasannan; Antonio Artigues; Aron W. Fenton (1083-1086).
There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g., active and allosteric sites). Both nuclear magnetic resonance (NMR), by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both H2O and D2O. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in D2O. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in D2O, but the effector continues to elicit an allosteric response. Figure Determining allosteric coupling in H2O vs D2O. Kapp-Phe as a function of the concentration of PEP
Keywords: Hydrogen/deuterium exchange; Mass spectrometry; Allosteric regulation; Allostery; Pyruvate kinase
Erratum to: Detecting small lung tumors in mouse models by refractive-index microradiology by Chia-Chi Chien; Guilin Zhang; Yeukuang Hwu; Ping Liu; Weisheng Yue; Jianqi Sun; Yan Li; Hongjie Xu; Lisa X. Xu; Chang Hai Wang; Nanyow Chen; Chien Hung Lu; Ting-Kuo Lee; Yuh-Cheng Yang; Yen-Ta Lu; Yu-Tai Ching; T. F. Shih; P. C. Yang; J. H. Je; Giorgio Margaritondo (1087-1087).