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Analytical and Bioanalytical Chemistry (v.404, #10)

Thanks to reviewers in 2012 (pp. 2775-2786).

New strategies for teaching maths to chemistry students by Paul C. Yates (pp. 2787-2792).

is the Discipline Lead for the Physical Sciences at the Higher Education Academy. In this capacity he supports the teaching of university-level chemistry in the UK through links with departments and other appropriate bodies. His background is in physical chemistry, and he has a long-standing interest in developing the mathematical skills of chemists.

New strategies for teaching maths to chemistry students by Paul C. Yates (pp. 2787-2792).

Davy Guillarme and Jean-Luc Veuthey (Eds.): UHPLC in life sciences by Francois Lestremau (pp. 2793-2794).

Optical biochemical and chemical sensors by Laura M. Lechuga (pp. 2795-2796).

is Head of the Nanobiosensors and Bioanalytical Applications Group at the Center on Nanoscience and Nanotechnology (CIN2, CSIC) in Barcelona (Spain). Her main research interests are the development of biosensor devices based on plasmonics, magnetoplasmonics, silicon photonics, and nanomechanics principles, including surface biofunctionalization, microfluidics, and lab-on-a-chip integration and their application in diagnostics. She has published over 150 original articles and has eight families of patents, and has participated in the establishment of two spin-offs.

Optical biochemical and chemical sensors by Laura M. Lechuga (pp. 2795-2796).

New luminescent oxygen-sensing and temperature-sensing materials based on gadolinium(III) and europium(III) complexes embedded in an acridone–polystyrene conjugate by Sergey M. Borisov; I. Klimant (pp. 2797-2806).

New sensing materials have been developed which rely on the use of luminescent europium(III) and gadolinium(III) complexes with thenoylacetylacetonate embedded in an acridone–polystyrene conjugate. Acridone acts as an antenna which efficiently absorbs violet light. Covalent coupling to the polystyrene backbone prevents aggregation and enables very high antenna loading (16 % w/w). Energy transfer from the antenna to the lanthanide complexes results in efficient red luminescence from the Eu(III) complex or green phosphorescence originating from the Gd(III) chelate. The luminescence of the material based on the Eu(III) complex is only slightly affected by oxygen but is highly sensitive to temperature under physiological conditions (20–40 °C). The Gd(III) complex has long phosphorescence decay times of approximately 1 ms and high sensitivity to oxygen. Ultra-thin (250 nm) sensing layers with sufficient absorption at the excitation wavelength enable monitoring of rapid oxygen changes virtually in real time. Immobilization of both complexes in a single matrix results in a dual-luminescence material with emissions almost ideally matching the red and green channels of a digital camera. Thus, oxygen imaging using a very simple and inexpensive set-up can be realized. Additionally, the material can be used for simultaneous sensing of oxygen and temperature. Figure Phosphorescent oxygen-sensing material based on a gadolinium(III) complex

Keywords: Europium; Gadolinium; Luminescence; Acridone; Oxygen sensor; Temperature sensor

Optimization of size, morphology and colloidal stability of fluorescein dye-doped silica NPs for application in immunoassays by Robert I. Nooney; Eoin McCormack; Colette McDonagh (pp. 2807-2818).

Fluorescent nanoparticle (NP) labels are of great interest for point-of-care medical diagnostics where high fluorescence signals combined with low limits of detection are required. In this work, hydrophilic and hydrophobic fluorescein dye derivatives were covalently doped into silica NPs. The NPs were prepared in a range of sizes from 16 to 80 nm using both ternary and quaternary microemulsion methods where the diameter varied linearly with changes in the water to surfactant ratio. The morphology and colloidal stability of the NPs were characterised using transmission electron microscopy and photon correlation spectroscopy; NPs doped with hydrophobic fluorescein dye were significantly smaller and more polydispersed. Optical properties including absorption, fluorescence and quantum efficiency were also determined. Representative NPs from each microemulsion method (ternary, Ø = 25 nm and quaternary, Ø = 80 nm) were tested as labels in a fluorescence based immunoassay for the detection of human IgG and human chorionic gonadotropin. Both sets of nanoparticle assays showed lower limits of detection and better coefficients of variance than a free dye label with good day to day reproducibility. The optimal surface coverage of detection antibody was also found to depend on the size of the nanoparticle. Figure Synthesis of fluorescein dye doped NPs and their application in a standard immunoassay for the detection of hIgG.

Keywords: Fluorescence; Nanoparticles; Colloidal stability; Immunoassay

Optimization of size, morphology and colloidal stability of fluorescein dye-doped silica NPs for application in immunoassays by Robert I. Nooney; Eoin McCormack; Colette McDonagh (pp. 2807-2818).

Keywords: Fluorescence; Nanoparticles; Colloidal stability; Immunoassay

Spectroscopic detection and quantification of heme and heme degradation products by U. Neugebauer; A. März; T. Henkel; M. Schmitt; J. Popp (pp. 2819-2829).

Heme and heme degradation products play critical roles in numerous biological phenomena which until now have only been partially understood. One reason for this is the very low concentrations at which free heme, its complexes and the partly unstable degradation products occur in living cells. Therefore, powerful and specific detection methods are needed. In this contribution, the potential of nondestructive Raman spectroscopy for the detection, quantification and discrimination of heme and heme degradation products is investigated. Resonance Raman spectroscopy using different excitation wavelengths (413, 476, 532, and 752 nm) is employed to estimate the limit of detection for hemin, myoglobin, biliverdin, and bilirubin. Concentrations in the low micromolar range (down to 3 μmol/L) could be reliably detected when utilizing the resonance enhancement effect. Furthermore, a systematic study on the surface-enhanced Raman spectroscopy (SERS) detection of hemin in the presence of other cellular components, such as the highly similar cytochrome c, DNA, and the important antioxidant glutathione, is presented. A microfluidic device was used to reproducibly create a segmented flow of aqueous droplets and oil compartments. Those aqueous droplets acted as model chambers where the analytes have to compete for the colloid. With the help of statistical analysis, it was possible to detect and differentiate the pure substances as well as the binary mixtures and gain insights into their interaction. Figure Resonance Raman spectrum of met-myoglobin and Raman intensity - concentration - calibration plot.

Keywords: Limit of detection; Resonance and surface-enhanced Raman spectroscopy; Biliverdin; Bilirubin; Microfluidic

Spectroscopic detection and quantification of heme and heme degradation products by U. Neugebauer; A. März; T. Henkel; M. Schmitt; J. Popp (pp. 2819-2829).

Keywords: Limit of detection; Resonance and surface-enhanced Raman spectroscopy; Biliverdin; Bilirubin; Microfluidic

Chemical silicon surface modification and bioreceptor attachment to develop competitive integrated photonic biosensors by Jorge Escorihuela; María José Bañuls; Javier García Castelló; Veronica Toccafondo; Jaime García-Rupérez; Rosa Puchades; Ángel Maquieira (pp. 2831-2840).

Methodology for the functionalization of silicon-based materials employed for the development of photonic label-free nanobiosensors is reported. The studied functionalization based on organosilane chemistry allowed the direct attachment of biomolecules in a single step, maintaining their bioavailability. Using this immobilization approach in probe microarrays, successful specific detection of bacterial DNA is achieved, reaching hybridization sensitivities of 10 pM. The utility of the immobilization approach for the functionalization of label-free nanobiosensors based on photonic crystals and ring resonators was demonstrated using bovine serum albumin (BSA)/anti-BSA as a model system.

Keywords: Photonic nanobiosensor; Silicon-based materials; Label free; DNA microarray

Filter-free integrated sensor array based on luminescence and absorbance measurements using ring-shaped organic photodiodes by Tobias Abel; Martin Sagmeister; Bernhard Lamprecht; Elke Kraker; Stefan Köstler; Birgit Ungerböck; Torsten Mayr (pp. 2841-2849).

An optical waveguiding sensor array featuring monolithically integrated organic photodiodes as integrated photo-detector, which simplifies the readout system by minimizing the required parts, is presented. The necessity of any optical filters becomes redundant due to the proposed platform geometry, which discriminates between excitation light and sensing signal. The sensor array is capable of measuring luminescence or absorption, and both sensing geometries are based on the identical substrate. It is demonstrated that background light is virtually non-existent. All sensing and waveguide layers, as well as in- and out-coupling elements are assembled by conventional screen-printing techniques. Organic photodiodes are integrated by layer-by-layer vacuum deposition onto glass or common polymer foils. The universal and simple applicability of this sensor chip is demonstrated by sensing schemes for four different analytes. Relative humidity, oxygen, and carbon dioxide are measured in gas phase using luminescence-based sensor schemes; the latter two analytes are also measured by absorbance-based sensor schemes. Furthermore, oxygen and pH in aqueous media were enabled. The consistency of calibration characteristics extending over different sensor chips is verified. Figure Integrated fluorescence (left) and absorbance (right) based sensor waveguide

Keywords: Optical sensor; Organic photodiode; Sensor array; Integrated sensor; Optoelectronic; Screen-printing

LED–LED portable oxygen gas sensor by I. M. Perez de Vargas-Sansalvador; C. Fay; M. D. Fernandez-Ramos; D. Diamond; F. Benito-Lopez; L. F. Capitan-Vallvey (pp. 2851-2858).

A portable instrument for oxygen determination, based on the quenching of phosphorescent octaethylporphyrin by gaseous O₂, has been developed using the fluorimetric paired emitter–detector diode technique (FPEDD). The instrument configuration consists of two light-emitting diodes (LEDs) facing each other, with an interchangeable support containing a phosphorescent membrane in between, in which one of the LEDs is used as the light source (emitter LED) and the other, working in reverse bias mode, as the light detector. The feasibility of using a LED as a luminescence detector is studied. Its small size enables integration of the instrument into a portable measurement system. A systematic study of the system capabilities as a portable instrument was performed to optimize range, sensitivity, short term and long term stability, dynamic behaviour, effect of temperature and humidity, and temporal drift.

Keywords: Oxygen sensor; Gas sensor; Optical sensor; Paired emitter detector-diode sensor; Portable instrumentation

LED–LED portable oxygen gas sensor by I. M. Perez de Vargas-Sansalvador; C. Fay; M. D. Fernandez-Ramos; D. Diamond; F. Benito-Lopez; L. F. Capitan-Vallvey (pp. 2851-2858).

Keywords: Oxygen sensor; Gas sensor; Optical sensor; Paired emitter detector-diode sensor; Portable instrumentation

Angle-dependent resonance of localized and propagating surface plasmons in microhole arrays for enhanced biosensing by Ludovic S. Live; Anuj Dhawan; Kirsty F. Gibson; Hugo-Pierre Poirier-Richard; Duncan Graham; Michael Canva; Tuan Vo-Dinh; Jean-François Masson (pp. 2859-2868).

The presence of microhole arrays in thin Au films is suited for the excitation of localized and propagating surface plasmon (SP) modes. Conditions can be established to excite a resonance between the localized and propagating SP modes, which further enhanced the local electromagnetic (EM) field. The co-excitation of localized and propagating SP modes depends on the angle of incidence (θ _exc) and refractive index of the solution interrogated. As a consequence of the enhanced EM field, enhanced sensitivity and an improved response for binding events by about a factor of 3 to 5 was observed with SPR sensors in the Kretschmann configuration for a set of experimental conditions (λ _SPR, θ _exc, and η). Thus, microhole arrays can improve sensing applications of SPR based on classical prism-based instrumentation and are suited for SP-coupled spectroscopic techniques. Fig Co-excitation of localized and propagating SP enhances sensitivity of SPR

Keywords: Localized and propagating surface plasmons; Enhanced sensitivity; Angle-dependent amplification; Surface-enhanced Raman scattering; Biosensing; Microhole arrays

Biofunctionalized gold nanoparticles for SPR-biosensor-based detection of CEA in blood plasma by Tomáš Špringer; Jiří Homola (pp. 2869-2875).

We report on the use of new biofunctionalized gold nanoparticles (bio-AuNPs) that enable a surface plasmon resonance (SPR) biosensor to detect low levels of carcinoembryonic antigen (CEA) in human blood plasma. Bio-AuNPs consist of gold nanoparticles functionalized both with (1) streptavidin, to provide high affinity for the biotinylated secondary antibody used in the second step of the CEA sandwich assay, and with (2) bovine serum albumin, to minimize the nonspecific interaction of the bio-AuNPs with complex samples (blood plasma). We demonstrate that this approach makes it possible for the SPR biosensor to detect CEA in blood plasma at concentrations as low as 0.1 ng/mL, well below normal physiological levels (approximately nanograms per milliliter). Moreover, the limit of detection achieved using this approach is better by a factor of more than 1,000 than limits of detection reported so far for CEA in blood plasma using SPR biosensors.

Keywords: Surface plasmon resonance; Carcinoembryonic antigen; Biosensor; Nanoparticles

Biofunctionalized gold nanoparticles for SPR-biosensor-based detection of CEA in blood plasma by Tomáš Špringer; Jiří Homola (pp. 2869-2875).

Keywords: Surface plasmon resonance; Carcinoembryonic antigen; Biosensor; Nanoparticles

Direct surface plasmon resonance immunosensing of pyraclostrobin residues in untreated fruit juices by E. Mauriz; C. García-Fernández; J. V. Mercader; A. Abad-Fuentes; A. M. Escuela; L. M. Lechuga (pp. 2877-2886).

A surface plasmon resonance (SPR) immunoassay for on-line detection of the strobilurin fungicide pyraclostrobin in untreated fruit juices is presented. The analysis of pyraclostrobin residues is accomplished in apple, grape, and cranberry samples by monitoring the recognition events occurring separately in a two-channel home-made SPR biosensor. Covalent coupling of the analyte derivative results in a reversible method, enabling more than 80 measurements on the same sensor surface. Optimization of the immunoassay conditions provides limits of detection as low as 0.16 μg L⁻¹. The selectivity and reproducibility of the analysis is ensured by studying both non-specific interactions with unrelated compounds and inter-assay coefficients of variation. Excellent recovery ranging from 98 to 103 % was achieved by a simple 1:5 dilution of fruit juice with assay buffer before the analysis. The lack of previous cleaning and homogenization procedures reduces the analysis time of a single food sample to only 25 min, including the regeneration cycle. Figure Schematic representation of the SPR platform

Keywords: SPR; Biosensor; Immunoassay; Pyraclostrobin; Food analysis

Silicon photonic sensors incorporated in a digital microfluidic system by Cristina Lerma Arce; Daan Witters; Robert Puers; Jeroen Lammertyn; Peter Bienstman (pp. 2887-2894).

Label-free biosensing with silicon nanophotonic microring resonator sensors has proven to be an excellent sensing technique for achieving high-throughput and high sensitivity, comparing favorably with other labeled and label-free sensing techniques. However, as in any biosensing platform, silicon nanophotonic microring resonator sensors require a fluidic component which allows the continuous delivery of the sample to the sensor surface. This component is typically based on microchannels in polydimethylsiloxane or other materials, which add cost and complexity to the system. The use of microdroplets in a digital microfluidic system, instead of continuous flows, is one of the recent trends in the field, where microliter- to picoliter-sized droplets are generated, transported, mixed, and split, thereby creating miniaturized reaction chambers which can be controlled individually in time and space. This avoids cross talk between samples or reagents and allows fluid plugs to be manipulated on reconfigurable paths, which cannot be achieved using the more established and more complex technology of microfluidic channels where droplets are controlled in series. It has great potential for high-throughput liquid handling, while avoiding on-chip cross-contamination. We present the integration of two miniaturized technologies: label-free silicon nanophotonic microring resonator sensors and digital microfluidics, providing an alternative to the typical microfluidic system based on microchannels. The performance of this combined system is demonstrated by performing proof-of-principle measurements of glucose, sodium chloride, and ethanol concentrations. These results show that multiplexed real-time detection and analysis, great flexibility, and portability make the combination of these technologies an ideal platform for easy and fast use in any laboratory. Online Abstract Figure Droplet moving on the surface a photonic chip with a digital microfluidic system

Keywords: Silicon nanophotonic ring resonator sensors; Silicon-on-insulator; Digital microfluidics; Electrowetting-on-dielectric; Microdroplets

Silicon photonic sensors incorporated in a digital microfluidic system by Cristina Lerma Arce; Daan Witters; Robert Puers; Jeroen Lammertyn; Peter Bienstman (pp. 2887-2894).

Keywords: Silicon nanophotonic ring resonator sensors; Silicon-on-insulator; Digital microfluidics; Electrowetting-on-dielectric; Microdroplets

Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS by Rawi Ramautar; Reinald Shyti; Bart Schoenmaker; Lotte de Groote; Rico J. E. Derks; Michel D. Ferrari; Arn M. J. M. van den Maagdenberg; André M. Deelder; Oleg A. Mayboroda (pp. 2895-2900).

The need for sensitive analytical technologies applicable to metabolic profiling of volume-restricted biological samples is high. Here, we demonstrate feasibility of capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (MS) with sheathless nano-electrospray interface for non-targeted profiling of ionogenic metabolites in body fluids of experimental animals. A representative mixture of the metabolites and body fluids of mice such as cerebrospinal fluid (CSF), urine and plasma were used as examples of low-volume biological samples for method evaluation. An injection volume of only 9 nL resulted in limits of detection between 0.7 and 12 nM for the metabolite mixture. The method allowed the detection of ∼350 molecular features in mouse CSF (an injection volume of ca. 45 nL), while ∼400 features were observed in mouse plasma and ∼3,500 features in mouse urine (an injection volume of ca. 9 nL). The low-volume body fluid samples were analyzed directly after only 1:1 dilution with water, thereby fully retaining sample integrity, which is of crucial importance for non-targeted metabolic profiling. As little is known about the metabolic composition of mouse CSF, we identified a fraction of the molecular features in mouse CSF using accurate mass information, migration times, MS/MS data, and comparison with authentic standards. We conclude that sheathless CE-MS can be used for sensitive metabolic profiling of volume-restricted biological samples.

Keywords: Mass spectrometry; Sheathless interface; Metabolic profiling; Cerebrospinal fluid; Urine; Low-volume samples

High-capacity NO₂ denuder systems operated at various temperatures (298–473 K) by Jan-Christoph Wolf; Reinhard Niessner (pp. 2901-2907).

In this study, we investigated several coatings for high-temperature, high-capacity, and high-efficiency denuder-based NO₂ removal, with the scope to face the harsh conditions and requirements of automotive exhaust gas sampling. As first coating, we propose a potassium iodide (KI)/polyethylene glycol coating with a high removal efficiency (ε > 98 %) for about 2 h and 50 ppm NO₂ at room temperature (298 K). At elevated temperatures (423 K), the initial capacity (100 ppmh) is decreased to 15 ppmh. Furthermore, this is the first proposal of the ionic liquid methyl-butyl-imidazolium iodide ([BMIm⁺][I⁻]) as denuder coating material. At room temperature, this ionic liquid exhibits far greater capacity (300 ppmh) and NO₂ removal efficiency (ε > 99.9 %) than KI. Nevertheless, KI exhibits a slightly (~10 %) higher capacity at elevated temperatures than [BMIm⁺][I⁻]. Both coatings presented are suitable for applications requiring selective denuding of NO₂ at temperatures up to 423 K.

Keywords: Nitrogen dioxide; Denuder; Potassium iodide; Ionic liquid; Elevated temperature

High-capacity NO₂ denuder systems operated at various temperatures (298–473 K) by Jan-Christoph Wolf; Reinhard Niessner (pp. 2901-2907).

Keywords: Nitrogen dioxide; Denuder; Potassium iodide; Ionic liquid; Elevated temperature

A fast and reliable method for the quantitative determination of benzimidazoles and metabolites in milk by LC-MS/MS with on-line sample treatment by Diego García-Gómez; Marta García-Hernández; Encarnación Rodríguez-Gonzalo; Rita Carabias-Martínez (pp. 2909-2914).

This work reports the development of a simple, reliable and automated method based on LC-MS/MS for the quantitative determination of benzimidazole residues and some of their metabolites in milk. The method involves the use of an extraction cartridge coupled on-line to the chromatographic system for the clean-up of the milk samples, efficiently eliminating matrix macromolecules and providing appropriate selectivity for the determination of such compounds. In the online method developed here, only a reduced manual sample manipulation was required (protein precipitation and filtration) prior to injection into the chromatographic system. The limits of detection of the target anthelmintics ranged from 0.1 to 0.8 ng mL⁻¹ in milk samples, these values being below the maximum residue limit established for these compounds. The whole method developed was validated in real samples according to the requirements set by the Commission Decision 2002/657/EC. The optimized method was successfully applied to commercial and raw milk samples of different origin demonstrating that the proposed method may find application in routine laboratory analyses of food control safety. Figure On-line instrumental setup (switching valve in loading position) and total ion chromatogram (TIC) for a milk sample spike at 10 ng ml^-1.

Keywords: Benzimidazole drugs; Milk; Restricted access material; On-line sample treatment; Liquid chromatography–tandem mass spectrometry; Validation according to Commission Decision 2002/657/EC

Developmental phases of individual mouse preimplantation embryos characterized by lipid signatures using desorption electrospray ionization mass spectrometry by Christina R. Ferreira; Valentina Pirro; Livia S. Eberlin; Judy E. Hallett; R. Graham Cooks (pp. 2915-2926).

Knowledge of the lipids present in individual preimplantation embryos is of interest in fundamental studies of embryology, in attempts to understand cellular pluripotency and in optimization of in vitro culture conditions necessary for the application and development of biotechnologies such as in vitro fertilization and transgenesis. In this work, the profiles of fatty acids and phospholipids (PL) in individual mouse preimplantation embryos and oocytes were acquired using an analytical strategy based on desorption electrospray ionization mass spectrometry (DESI-MS). The methodology avoids sample preparation and provides information on the lipids present in these microscopic structures. Differences in the lipid profiles observed for unfertilized oocytes, two- and four-cell embryos, and blastocysts were characterized. For a representative set of embryos (N = 114) using multivariate analysis (specifically principal component analysis) unfertilized oocytes showed a narrower range of PL species than did blastocysts. Two- and four-cell embryos showed a wide range of PLs compared with unfertilized oocytes and high abundances of fatty acids, indicating pronounced synthetic activity. The data suggest that the lipid changes observed in mouse preimplantation development reflect acquisition of a degree of cellular membrane functional and structural specialization by the blastocyst stage. It is also noteworthy that embryos cultured in vitro from the two-cell through the blastocyst stage have a more homogeneous lipid profile as compared with their in vivo-derived counterparts, which is ascribed to the restricted diversity of nutrients present in synthetic culture media. The DESI-MS data are interpreted from lipid biochemistry and previous reports on gene expression of diverse lipids known to be vital to early embryonic development.

Keywords: Desorption electrospray ionization; Lipid analysis; Embryology; Preimplantation development

Three-dimensional molecular reconstruction of rat heart with mass spectrometry imaging by Lara Fornai; Annalisa Angelini; Ivo Klinkert; Frans Giskes; Andras Kiss; Gert Eijkel; Erika A. Amstalden-van Hove; Leendert A. Klerk; Marny Fedrigo; Giuseppe Pieraccini; Gloriano Moneti; Marialuisa Valente; Gaetano Thiene; Ron M. A. Heeren (pp. 2927-2938).

Cardiovascular diseases are the world’s number one cause of death, accounting for 17.1 million deaths a year. New high-resolution molecular and structural imaging strategies are needed to understand underlying pathophysiological mechanism. The aim of our study is (1) to provide a molecular basis of the heart animal model through the local identification of biomolecules by mass spectrometry imaging (MSI) (three-dimensional (3D) molecular reconstruction), (2) to perform a cross-species validation of secondary ion mass spectrometry (SIMS)-based cardiovascular molecular imaging, and (3) to demonstrate potential clinical relevance by the application of this innovative methodology to human heart specimens. We investigated a MSI approach using SIMS on the major areas of a rat and mouse heart: the pericardium, the myocardium, the endocardium, valves, and the great vessels. While several structures of the heart can be observed in individual two-dimensional sections analyzed by metal-assisted SIMS imaging, a full view of these structures in the total heart volume can be achieved only through the construction of the 3D heart model. The images of 3D reconstruction of the rat heart show a highly complementary localization between Na⁺, K⁺, and two ions at m/z 145 and 667. Principal component analysis of the MSI data clearly identified different morphology of the heart by their distinct correlated molecular signatures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging. Figure Workflow of the 3D reconstruction. A Tissue section, B gold deposition is done by sputter coating, C, C1 SIMS-ToF mass analyzer, C, C2 mass spectral peaks, C, C3 datacube images; D, E Reconstruction of the heart showing 3D-spatial distributions of three different ions 145 m/z (red), 23 m/z (green), and 39 m/z (blue); F coregistration of 40 individual MS imaging

Keywords: 3D molecular reconstruction; Secondary ion mass spectrometry; Heart; Imaging

Speciation of antimony in injectable drugs used for leishmaniasis treatment (Glucantime^®) by HPLC-ICP-MS and DPP by F. Séby; C. Gleyzes; O. Grosso; B. Plau; O. F. X. Donard (pp. 2939-2948).

Meglumine antimonate is the active of Glucantime® used for the treatment of leishmaniasis, a tropical disease caused by parasitic protozoa, and it is estimated that 12 million people worldwide are affected. This drug mainly contains Sb(V) under the form of an organic complex with N-methylglucamine (NMG). During the synthesis of this molecule, traces of Sb(III) may be present, also probably complexed. Due to the fact that Sb(III) is considered more toxic than Sb(V), it is important to evaluate the Sb(III) concentration in the drug samples. In the literature, very different concentrations for residual concentrations of Sb(III) in the drug ampoules are found. Therefore, to have a true insight of antimony speciation, two independent analytical methods were developed in this work. We used an anion exchange method coupled with inductively coupled plasma mass spectrometry (ICP-MS) which was cross-referenced with an electrochemistry method (differential pulse polarography (DPP)) that could be used for routine analysis on the production site. To obtain Sb species in detectable forms, the complexes between Sb species and NMG need to be broken. This was obtained by diluting samples in hydrochloric acid in deaerated conditions to avoid Sb redox reactions. For the two analytical methods, the HCl concentration was optimized to obtain simultaneously a complete destruction of the complexes as well as limited redox reactions for Sb(V) and Sb(III) released species. For high-performance liquid chromatography (HPLC)-ICP-MS, a dilution with 5 M HCl gives the better results. The side reaction is an oxidation of Sb(III) which can be limited by the removal of oxygen. When DPP is used, the major problem is the reduction of Sb(V) which is present in high amount in the samples. Working with 0.6 M HCl allows this problem to be minimized. When applied to different lots of Glucantime®, Sb(III) concentration values are in good agreement for the two analytical methods, with, for HPLC-ICP-MS, the advantage of the simultaneous detection of both Sb redox species.

Keywords: Antimony; Speciation; DPP; HPLC-ICP-MS; Drug; Leishmaniasis

Speciation of antimony in injectable drugs used for leishmaniasis treatment (Glucantime^®) by HPLC-ICP-MS and DPP by F. Séby; C. Gleyzes; O. Grosso; B. Plau; O. F. X. Donard (pp. 2939-2948).

Keywords: Antimony; Speciation; DPP; HPLC-ICP-MS; Drug; Leishmaniasis

Phosphorylcholine-containing lipid molecular species profiling in biological tissue using a fast HPLC/QqQ-MS method by Chuan-Ho Tang; Po-Nien Tsao; Ching-Yu Lin; Lee-Shing Fang; Shu-Hui Lee; Wei-Hsien Wang (pp. 2949-2961).

A fast reversed-phase liquid chromatography-electrospray ionization triple quadrupole mass spectrometry method was developed for the molecular species profiling of glycerophosphocholine (GPC) and sphingomyelin (SM) in total lipid extracts. A two-stage mass spectrometry strategy was adopted to analyze in detail the composition of lipid molecular species. Precursor ion analysis was first conducted to obtain the preliminary composition profile of the phosphorylcholine-containing lipid. The product ion spectra were sequentially acquired for each recorded signal to determine the molecular structure of the lipid. A total of 150 GPCs and 12 SMs were identified in the fetal mouse lung with relative amounts ranging from 13.7 % to less than 0.002 % (normalizing by the total signal response). A column packed with core–shell particles was used to obtain excellent chromatographic separation with a shorter time demand in a conventional high-performance liquid chromatography system. Considering the compromise between the chromatographic efficiency and the electrospray signal response, the optimization of the mobile phase improves the chromatographic plate number to approximately 40,000 and the detection limits to less than 0.001 mg/L. The applicability of the method was validated through a study of chemically induced early lung maturation. The metabolic alteration in the fetal mouse lung was clearly reflected in the GPC and SM composition with several characteristics of the molecular structure that related to the character of the phospholipid layer upon the epithelial lining of alveoli and the relevant cell function. The results indicated that this analytical strategy is reliable for comprehensive molecular species profiling of GPC and SM and might be extended to the analysis of other phospholipids. Figure Glycerophosphocholine and sphingomyelin molecular species profiling using a fast HPLC/QqQ-MS method adopting a two-stage mass spectrometry strategy composed of preliminary phosphorylcholine-containing lipid profiling and sequential lipid molecular structure determination

Keywords: Glycerophosphocholine; Lipidomics; Mass spectrometer; Molecular species profiling; Sphingomyelin

New “hyphenated” CPC-HPLC-DAD-MS strategy for simultaneous isolation, analysis and identification of phytochemicals: application to xanthones from Garcinia mangostana by Thomas Michel; Emilie Destandau; Laëtitia Fougère; Claire Elfakir (pp. 2963-2972).

Centrifugal partition chromatography (CPC) coupled online with high-performance liquid chromatography (HPLC) with diode-array detection (DAD) and mass spectrometry (MS) is presented in this work. This strategy offers the possibility to obtain simultaneously CPC fractionation of natural extracts, the HPLC fingerprint of separated fractions and structural information on molecules contained in each fraction. This new approach was applied to the fractionation and purification of xanthones from Garcinia mangostana (Clusiaceae) pericarp. A biphasic solvent system of heptane/ethyl acetate/methanol/water (2:1:2:1, v/v) was used for the CPC separation of 175 mg crude ethanolic extract. The HPLC analysis was conducted with a reversed-phase monolithic column allowing fast and repeatable separation. This combined CPC-HPLC-DAD-MS method led to isolation of 33 mg α-mangostin and 6 mg γ-mangostin at 98 % and 98.5 % purity, respectively, in 140 min. Furthermore, in the same time a total of 16 other xanthones were detected in the extract, and ten of them were identified on the basis of their UV and MS spectra.

Keywords: Centrifugal partition chromatography; Centrifugal partition chromatography–high-performance liquid chromatography–diode-array detection–mass spectrometry; Hyphenation; Garcinia mangostana ; Xanthones

Measurement of the elution strength and peak shape enhancement at increasing modifier concentration and temperature in RPLC by J. J. Baeza-Baeza; Y. Dávila; J. J. Fernández-Navarro; M. C. García-Álvarez-Coque (pp. 2973-2984).

Two approaches are proposed to measure the effect of different experimental factors (such as the modifier concentration and temperature) on the elution strength and peak shape in reversed-phase liquid chromatography, which quantify the percentage change in the retention factor and peak width (referred to the weakest conditions) per unit change in the experimental factor. The approaches were applied to the separation of a set of flavonoids with aqueous micellar mobile phases of the surfactant Brij-35 (polyoxyethylene(23)dodecanol), in comparison with acetonitrile–water mixtures, using an Eclipse XDB-C18 column. The particular interaction of each flavonoid with the oxyethylene chains of Brij-35 molecules (adsorbed on the stationary phase or forming micelles) changed the elution window, distribution of chromatographic peaks and partitioning kinetics, depending on the hydroxyl substitution in the aromatic rings of flavonoids. At 25 °C, peak shape with Brij-35 mobile phases was significantly poorer with regard to acetonitrile–water mixtures. At increasing temperature, the efficiency of Brij-35 increased, approaching at 80 °C the values obtained at equilibrium conditions, already reached with acetonitrile at 25 °C.

Keywords: Reversed-phase liquid chromatography; Elution strength; Peak shape enhancement; Flavonoids; Brij-35; Temperature

Comparison of classical chromatographic tests with a chromatographic test applied to stationary phases prepared by thermal immobilization of poly(methyloctylsiloxane) onto silica by Endler M. Borges; Melvin R. Euerby; Carol H. Collins (pp. 2985-3002).

Stationary-phase evaluation in reversed-phase liquid chromatography (RP-LC) is not a straightforward process. A number of tests to characterize and classify stationary phases have been suggested. The results of these various tests, however, do not always describe the real properties of the stationary phase. This study critically compares several tests for RP-LC stationary phases, including the Engelhardt, Tanaka, and SRM 870 tests, as well as an in-house test, with emphasis on the stationary-phase descriptors of hydrophobicity and silanol activity. The stationary phases were prepared by thermal immobilization of poly(methyloctylsiloxane) onto silica. Hydrophobicity data from the tests were generally good and interchangeable between the several tests. In contrast, the silanol activity results of the various tests differ significantly. As a consequence, stationary phase classification with respect to silanol activity depends considerably on the test method applied. A new classification method for silanol activity is proposed.

Keywords: Reversed-phase liquid chromatography; Basic solutes; Chemometrics; Chromatographic tests; Silanol activity; Stationary phase classification

The relative brightness of PEG lipid-conjugated polymer nanoparticles as fluid-phase markers in live cells by Lawrence P. Fernando; Prakash K. Kandel; P. Christine Ackroyd; Kenneth A. Christensen (pp. 3003-3014).

While conjugated polymer nanoparticles (CPNs) have been widely touted as ultra-bright labels for biological imaging, no direct comparative measurements of their intracellular brightness have been reported. Simple in vitro comparisons are not definitive since fluorophore brightness in vitro may not correspond with intracellular brightness. We have compared the fluorescence brightness of J774A.1 cells loaded with 24 nm methoxy-capped 2,000 M _r polyethylene glycol lipid PFBT nanoparticles (PEG lipid-PFBT CPNs) to cells loaded with carboxy-functionalized quantum dots (Qdots) or a dextran-linked small molecule organic dye, Alexa Fluor 488 dextran (AF488-dex). Under conditions likely to be used for biological imaging or flow cytometry, these CPNs are 175× brighter than Qdots and 1,400× brighter than AF488-dex in cells. Evaluation of the minimum incubation concentration required for detection of nanoparticle fluorescence with a commercial flow cytometer indicated that the limit of detection for PEG lipid-PFBT CPNs was 19 pM (86 ppb), substantially lower than values obtained for Qdots (980 pM) or AF488-dex (11.2 nM). Investigation of the mechanism of cellular uptake of the three fluid-phase labels indicates that these particles are passively taken into macrophage cells via macropinocytosis without interaction with cell surface receptors, and ultimately localize in lysosomes. In addition, no cytotoxicity could be observed at any of the CPN concentrations tested. Together, these data suggest that these CPNs are appropriate and attractive candidates as fluid-phase markers with significantly greater fluorescence brightness than existing dyes or nanoparticles. We expect that these CPNs will find application in both imaging and flow cytometry.

Keywords: Alexa dextran; Quantum dots; Conjugated polymer nanoparticles; Semiconducting polymer nanoparticles; LAMP-1; Cellular toxicity

Quantifying dithiothreitol displacement of functional ligands from gold nanoparticles by De-Hao Tsai; Melanie P. Shelton; Frank W. DelRio; Sherrie Elzey; Suvajyoti Guha; Michael R. Zachariah; Vincent A. Hackley (pp. 3015-3023).

Dithiothreitol (DTT)-based displacement is widely utilized for separating ligands from their gold nanoparticle (AuNP) conjugates, a critical step for differentiating and quantifying surface-bound functional ligands and therefore the effective surface density of these species on nanoparticle-based therapeutics and other functional constructs. The underlying assumption is that DTT is smaller and much more reactive toward gold compared with most ligands of interest, and as a result will reactively displace the ligands from surface sites thereby enabling their quantification. In this study, we use complementary dimensional and spectroscopic methods to characterize the efficiency of DTT displacement. Thiolated methoxypolyethylene glycol (SH-PEG) and bovine serum albumin (BSA) were chosen as representative ligands. Results clearly show that (1) DTT does not completely displace bound SH-PEG or BSA from AuNPs, and (2) the displacement efficiency is dependent on the binding affinity between the ligands and the AuNP surface. Additionally, the displacement efficiency for conjugated SH-PEG is moderately dependent on the molecular mass (yielding efficiencies ranging from 60 to 80 % measured by ATR-FTIR and ≈90 % by ES-DMA), indicating that the displacement efficiency for SH-PEG is predominantly determined by the S–Au bond. BSA is particularly difficult to displace with DTT (i.e., the displacement efficiency is nearly zero) when it is in the so-called normal form. The displacement efficiency for BSA improves to 80 % when it undergoes a conformational change to the expanded form through a process of pH change or treatment with a surfactant. An analysis of the three-component system (SH-PEG + BSA + AuNP) indicates that the presence of SH-PEG decreases the displacement efficiency for BSA, whereas the displacement efficiency for SH-PEG is less impacted by the presence of BSA. Figure Schematic displacement of ligands from a AuNP by DTT

Keywords: Gold; Nanoparticle; Dithiothreitol; Bovine serum albumin; Polyethylene glycol; Ligand displacement; Thiol

The in vivo biodistribution and fate of CdSe quantum dots in the murine model: a laser ablation inductively coupled plasma mass spectrometry study by TsingHai Wang; HuiAn Hsieh; YiKong Hsieh; ChiShiun Chiang; YuhChang Sun; ChuFang Wang (pp. 3025-3036).

Understanding the cytotoxicity of quantum dots strongly relies upon the development of new analytical techniques to gather information about various aspects of the system. In this study, we demonstrate the in vivo biodistribution and fate of CdSe quantum dots in the murine model by means of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). By comparing the hot zones of each element acquired from LA-ICP-MS with those in fluorescence images, together with hematoxylin and eosin-stained images, we are able to perceive the fate and in vivo interactions between quantum dots and rat tissues. One hour after intravenous injection, we found that all of the quantum dots had been concentrated inside the spleen, liver and kidneys, while no quantum dots were found in other tissues (i.e., muscle, brain, lung, etc.). In the spleen, cadmium-114 signals always appeared in conjunction with iron signals, indicating that the quantum dots had been filtered from main vessels and then accumulated inside splenic red pulp. In the liver, the overlapped hot zones of quantum dots and those of phosphorus, copper, and zinc showed that these quantum dots have been retained inside hepatic cells. Importantly, it was noted that in the kidneys, quantum dots went into the cortical areas of adrenal glands. At the same time, hot zones of copper appeared in proximal tubules of the cortex. This could be a sign that the uptake of quantum dots initiates certain immune responses. Interestingly, the intensity of the selenium signals was not proportional to that of cadmium in all tissues. This could be the result of the decomposition of the quantum dots or matrix interference. In conclusion, the advantage in spatial resolution of LA-ICP-MS is one of the most powerful tools to probe the fate, interactions and biodistribution of quantum dots in vivo.

Keywords: Laser ablation inductively coupled plasma mass spectrometry; Quantum dots; Biodistribution; Fate of quantum dots; In vivo

Evaluation of dried blood spot (DBS) technology versus plasma analysis for the determination of MK-1775 by HILIC-MS/MS in support of clinical studies by Yang Xu; Wei Fang; Wei Zeng; Suzanne Leijen; Eric J. Woolf (pp. 3037-3048).

The collection of human blood samples as dried blood spots (DBS) for the pharmacokinetic assessment of investigational drugs in clinical trials offers a number of advantages over conventional plasma sampling, namely, small sample volume, simplified sample handling, and cost-effective shipping and storage. The use of DBS coupled with liquid chromatography–tandem mass spectrometry analysis was evaluated for the quantification of MK-1775, a Wee-1 inhibitor under development as a chemo/radio-sensitizer for the treatment of cancer. The DBS method exhibited an assay performance comparable to that of the existing plasma assay, which is currently used in support of clinical studies. Both assays used the same linear dynamic range of 2–1,000 ng/mL, with a lower limit of quantification of 2 ng/mL. Based on the intra-day assay validation results, the accuracy of the DBS method ranged from 94.0 to 105.0 %, with a coefficient of variation of <4.8 %. The blood-to-plasma ratio calculated from the DBS data (blood concentrations) and the plasma data (plasma concentrations) was in good agreement with the one obtained from the in vitro assessment using conventional methodology. No significant hematocrit impact on the assay was observed as hematocrit ranged from 16 to 85 %. The correlation between the measured MK-1775 concentrations in plasma and that determined in dried blood spots from oncology patients during the ongoing clinical study was discussed.

Keywords: MK-1775; Dried blood spot (DBS); Hematocrit impact; Hydrophilic interaction liquid chromatography (HILIC); Tandem mass spectrometry (MS/MS); Blood-to-plasma (B/P) ratio

Development of a novel method for quantification of sterols and oxysterols by UPLC-ESI-HRMS: application to a neuroinflammation rat model by Sophie Ayciriex; Anne Regazzetti; Mathieu Gaudin; Elise Prost; Delphine Dargère; France Massicot; Nicolas Auzeil; Olivier Laprévote (pp. 3049-3059).

Cholesterol and oxysterols are involved as key compounds in the development of severe neurodegenerative diseases and in neuroinflammation processes. Monitoring their concentration changes under pathological conditions is of interest to get insights into the role of lipids in diseases. For numerous years, liquid chromatography coupled to mass spectrometry has been the method of choice for metabolites identification and quantification in biological samples. However, sterols and oxysterols are relatively apolar molecules poorly adapted to electrospray ionization (ESI). To circumvent this drawback, we developed a novel and robust analytical method involving derivatization of these analytes in cholesteryl N-4-(N,N-dimethylamino)phenyl carbamates under alkaline conditions followed by ultra-performance liquid chromatography–high resolution mass spectrometry analysis (UPLC-HRMS). Optimized UPLC conditions led to the separation of a mixture of 11 derivatized sterols and oxysterols especially side chain substituted derivatives after 6 min of chromatographic run. High sensitivity time-of-flight mass analysis allowed analytes to be detected in the nanomolar range. The method was validated for the analysis of oxysterols and sterols in mice brain in respect of linearity, limits of quantification, accuracy, precision, analyte stability, and recovery according to the Food and Drug Administration (FDA) guidelines. The developed method was successfully applied to investigate the impact of lipopolysaccharide (LPS) treatment on the rat cerebral steroidome.

Keywords: UPLC/ESI/HRMS; Cholesterol; Oxysterols; Derivatization; Quantification

Microalgal motility measurement microfluidic chip for toxicity assessment of heavy metals by Guoxia Zheng; Yunhua Wang; Jianhua Qin (pp. 3061-3069).

A polydimethylsiloxane microfluidic chip has been developed for the estimation of toxic heavy metals based on measurement of mobility of marine microalgae. The chip is mainly composed of an upstream concentration gradient generator and a downstream perfusion-based chemotatic module. The processes of toxic liquid dilution and diffusion, microalgal culturing, cell stimulation, and online screening can be integrated in this chip, which makes it an attractive approach to simplify toxicity testing procedures. The microalgal motility was adopted as a microfluidic bioassay signal and was evaluated as the percentage of motile cells, curvilinear velocity, average path velocity, and straight line velocity. Two mobile marine microalgae, Platymonas subcordiformis and Platymonas helgolandica var. tsingtaoensis, were confined in the chemotatic module and stimulated by the eight concentration gradients of Cu and Cd generated by the concentration gradient generator. In all cases, a toxic response was detected (i.e., a dose-related inhibition of motility was observed). Only 1.5 h was needed to predict EC₅₀ values. Thus, the microfluidic chip developed was proved to be useful as a simple and rapid approach in heavy metal detection and might be expanded as a conventional test method in environmental toxicity assessment.

Keywords: Microfluidic chip; Marine microalgae; Motility; Toxicity assessment; Chemostatic module

Microalgal motility measurement microfluidic chip for toxicity assessment of heavy metals by Guoxia Zheng; Yunhua Wang; Jianhua Qin (pp. 3061-3069).

Keywords: Microfluidic chip; Marine microalgae; Motility; Toxicity assessment; Chemostatic module

Progress of elemental anomalies of hippocampal formation in the pilocarpine model of temporal lobe epilepsy—an X-ray fluorescence microscopy study by J. Chwiej; J. Kutorasinska; K. Janeczko; K. Gzielo-Jurek; L. Uram; K. Appel; R. Simon; Z. Setkowicz (pp. 3071-3080).

In the present paper, X-ray fluorescence microscopy was applied to follow the processes occurring in rat hippocampal formation during the post-seizure period. In the study, one of the status epilepticus animal models of epilepsy was used, namely the model of temporal lobe epilepsy with pilocarpine-induced seizures. In order to analyze the dynamics of seizure-induced elemental changes, the samples taken from seizure-experiencing animals 3 h and 1, 4, and 7 days after proconvulsive agent administration were analyzed. The obtained results confirmed the utility of X-ray fluorescence microscopy in the research of mechanisms involved in the pathogenesis and progress of epilepsy. The topographic and quantitative elemental analysis of hippocampal formations from different periods of epileptogenesis showed that excitotoxicity, mossy fibers sprouting, and iron-induced oxidative stress may be the processes responsible for seizure-induced neurodegenerative changes and spontaneous recurrent seizures occurring in the chronic phase of the pilocarpine model. The analysis of correlations between the recorded elemental anomalies and quantitative parameters describing animal behavior in the acute period of pilocarpine-induced status epilepticus showed that the areal densities of selected elements measured in the latent period strongly depend on the progress of the acute phase. Especially important seem to be the observations done for Ca and Zn levels which suggest that the intensity of the pathological processes such as excitotoxicity and mossy fibers sprouting depend on the total time of seizure activity. These results as well as dependencies found between the levels of S, K, and Cu and the intensity of maximal seizures clearly confirm how important it is to control the duration and intensity of seizures in clinical practice.

Keywords: Pilocarpine model of epilepsy; Topographic and quantitative elemental analysis; X-ray fluorescence microscopy; Synchrotron radiation

Evaluation of different extraction approaches for the determination of phenolic compounds and their metabolites in plasma by nanoLC-ESI-TOF-MS by R. Quirantes-Piné; V. Verardo; D. Arráez-Román; S. Fernández-Arroyo; V. Micol; M. F. Caboni; A. Segura-Carretero; A. Fernández-Gutiérrez (pp. 3081-3090).

Sample preparation is an important step for the determination of phenolic compounds in biological samples. Different extraction methods have been tested to determine phenolic compounds and their metabolites in plasma by nano-liquid chromatography coupled to electrospray ionisation-time-of-flight mass spectrometry (nanoLC-ESI-TOF-MS). The sample treatment optimisation was performed using commercial foetal bovine serum spiked with representative phenolic standards, namely naringenin, luteolin, verbascoside, apigenin, rutin, syringic acid and catechin. Different protein-precipitation conditions were evaluated as well as enzymatic digestion with trypsin and solid-phase extraction using different phases such as C-18, ABN and ENV+, working at different pH values. The optimum extraction procedure consisted of a previous protein-precipitation step using HCl 200 mmol/L in methanol for 2.5 h at 50 °C followed by a solid-phase extraction using C-18 cartridges at pH 2.5. This procedure was finally applied to the plasma of rats overfed with a phenolic-rich Lippia citriodora extract. These samples were analysed by nanoLC-ESI-TOF-MS, enabling the identification of five compounds previously found in the administered L. citriodora extract and one metabolite.

Keywords: Phenolic compounds; Extraction procedure; Plasma; NanoLC-ESI-TOF-MS

Selective sampling using confocal Raman spectroscopy provides enhanced specificity for urinary bladder cancer diagnosis by Ishan Barman; Narahara Chari Dingari; Gajendra Pratap Singh; Rajesh Kumar; Stephen Lang; Ghulam Nabi (pp. 3091-3099).

In recent years, Raman spectroscopy has shown substantive promise in diagnosing bladder cancer, especially due to its exquisite molecular specificity. The ability to reduce false detection rates in comparison to existing diagnostic tools such as photodynamic diagnosis makes Raman spectroscopy particularly attractive as a complementary diagnostic tool for real-time guidance of transurethral resection of bladder tumor (TURBT). Nevertheless, the state-of-the-art high-volume Raman spectroscopic probes have not reached the expected levels of specificity thereby impeding their clinical translation. To address this issue, we propose the use of a confocal Raman probe for bladder cancer diagnosis that can boost the specificity of the diagnostic algorithm based on its suppression of the out-of-focus non-analyte-specific signals emanating from the neighboring normal tissue. In this article, we engineer and apply such a probe, having depth of field of approximately 280 μm, for Raman spectral acquisition from ex vivo normal and cancerous TURBT samples. Using this clinical dataset, a diagnostic algorithm based on principal component analysis and logistic regression is developed. We demonstrate that this approach results in comparable sensitivity but significantly higher specificity in relation to high-volume Raman spectral data. The application of only two principal components is sufficient for the discrimination of the samples underlining the robustness of the algorithm. Further, no discordance between replicate spectra is observed emphasizing the reproducible nature of the current diagnostic assessment. The high levels of sensitivity and specificity achieved in this proof-of-concept study opens substantive avenues for application of a confocal Raman probe during endoscopic procedures related to diagnosis and treatment of bladder cancer. Figure Artistic depiction of the working principle of the confocal Raman spectroscopic sensor for urinary bladder cancer diagnosis

Keywords: Confocal Raman spectroscopy; Urinary bladder cancer; Optical diagnosis; Raman probe; Transurethal resection of bladder tumor

Metabolic profiling reveals new serum biomarkers for differentiating diabetic nephropathy by Akiyoshi Hirayama; Eitaro Nakashima; Masahiro Sugimoto; Shin-ichi Akiyama; Waichi Sato; Shoichi Maruyama; Seiichi Matsuo; Masaru Tomita; Yukio Yuzawa; Tomoyoshi Soga (pp. 3101-3109).

Capillary electrophoresis coupled with time-of-flight mass spectrometry was used to explore new serum biomarkers with high sensitivity and specificity for diabetic nephropathy (DN) diagnosis, through comprehensive analysis of serum metabolites with 78 diabetic patients. Multivariate analyses were used for identification of marker candidates and development of discriminative models. Of the 289 profiled metabolites, orthogonal partial least-squares discriminant analysis identified 19 metabolites that could distinguish between DN with macroalbuminuria and diabetic patients without albuminuria. These identified metabolites included creatinine, aspartic acid, γ-butyrobetaine, citrulline, symmetric dimethylarginine (SDMA), kynurenine, azelaic acid, and galactaric acid. Significant correlations between all these metabolites and urinary albumin-to-creatinine ratios (p < 0.009, Spearman’s rank test) were observed. When five metabolites (including γ-butyrobetaine, SDMA, azelaic acid and two unknowns) were selected from 19 metabolites and applied for multiple logistic regression model, AUC value for diagnosing DN was 0.927 using the whole dataset, and 0.880 in a cross-validation test. In addition, when four known metabolites (aspartic acid, SDMA, azelaic acid and galactaric acid) were applied, the resulting AUC was still high at 0.844 with the whole dataset and 0.792 with cross-validation. Combination of serum metabolomics with multivariate analyses enabled accurate discrimination of DN patients. The results suggest that capillary electrophoresis-mass spectrometry based metabolome analysis could be used for DN diagnosis.

Keywords: Diabetic nephropathy; Capillary electrophoresis-mass spectrometry; Metabolome; Biomarker; Multiple logistic regression; Orthogonal partial least-squares discriminant analysis

Dynorphin A 1–17 biotransformation in inflamed tissue, serum and trypsin solution analysed by liquid chromatography–tandem mass spectrometry by M. Morgan; H. M. D. R. Herath; P. J. Cabot; P. N. Shaw; A. K. Hewavitharana (pp. 3111-3121).

Dynorphin A 1–17 (DYN A) is an endogenous neuropeptide that is of interest due to its diverse roles in analgesia, inflammation and addiction. Upon release, DYN A is subject to metabolism by a range of enzymes and its biotransformation is dependent on the site and environment into which it is released. In this study, we investigated the biotransformation of DYN A in rat inflamed tissue at pH 7.4 and 5.5, in rat serum and in trypsin solution. DYN A-porcine was incubated at 37 °C in each matrix over a range of incubation periods. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Incubation of DYN A in trypsin solution and in rat serum resulted in 6 and 14 fragments, respectively. Incubation in inflamed rat paw tissue occasioned 21 fragments at pH 7.4 and 31 fragments at pH 5.5. Secondary breakdown of some larger primary fragments was also observed in this study.

Keywords: Dynorphin; Metabolism; Fragments; Inflamed tissue; LC-MS-MS

Metabonomic classification and detection of small molecule biomarkers of malignant pleural effusions by Xian-Mei Zhou; Cui-Cui He; Yu-Mei Liu; Yang Zhao; Dan Zhao; Yun Du; Wei-Yi Zheng; Jian-Xin Li (pp. 3123-3133).

To date, most research has been focused on the benign molecules in pleural effusions, and diagnosis of malignant ones still remains challenging. In the present study, targeting the small molecules as potential biomarkers to predict the malignancy of the effusions, the metabolic profiles of 81 clinical pleural effusions (41 malignant effusions from lung cancer and 40 benign ones) were investigated through a NMR-based metabonomic approach. In ¹H NMR analysis, a total of ten small molecules in the effusions were simultaneously determined. Significantly higher mean values of valine, lactate, and alanine and markedly lower signal intensities of acetoacetate, trimethylamine-N-oxide, and α- and β-glucose were observed in malignant pleural effusions compared with those in benign ones. DFA modeling of NMR spectra subjected to a validation allowed the malignant effusions to be discriminated from benign ones in both training and validation groups. Currently, the conventional clinical analyses on chemical constituents in effusions could not provide a reliable prediction of malignancy of the effusions; the present results revealed that the small molecules might serve as useful biomarkers for diagnosis of the effusions, and the present NMR-based metabonomic approach provided a valuable potential to rapidly and sensitively predict the malignancy of the pleural effusions. Figure NMR based metabonomic analysis of pleural effusions and diagnostic results with discriminant function analysis

Keywords: Pleural effusions; Lung cancer; Small molecules; Biomarkers; ¹H NMR; Discriminant function analysis (DFA)

Transformation of cocaine during water chlorination by Iria González-Mariño; José Benito Quintana; Isaac Rodríguez; Noemí Sánchez-Méndez; Rafael Cela (pp. 3135-3144).

The stability of cocaine and its two main human metabolites, benzoylecgonine and ecgonine methyl ester, in chlorine-containing waters has been investigated by direct injection of different reaction time aliquots in a liquid chromatograph (LC) coupled to a quadrupole-time-of-flight mass spectrometer (QTOF-MS). Factors potentially affecting cocaine degradation (the only compound showing a significant decrease in the preliminary study) were evaluated in detail by means of a Box–Behnken experimental design. Sample pH resulted to be the most important variable, increasing both the rate of chlorination-mediated reactions and the ester hydrolysis process. From these reactions, and due to the high mass accuracy measurements obtained with the QTOF system, four by-products could be positively identified: benzoylecgonine, norcocaine, norbenzoylecgonine and N-formylnorcocaine. Finally, their formation and cocaine degradation yields were assessed under chlorination experiments with two real surface water samples. In one of them, showing a low anthropogenic impact, benzoylecgonine and norcocaine were notably generated even after only 1 h of reaction, whereas at higher contact times also norbenzoylecgonine and N-formylnorcocaine could be determined with a lower yield. On the other hand, the second sample, with a higher organic matter content, consumed rapidly the chlorine, so that only benzoylecgonine was produced. These findings point out the convenience of monitoring the described transformation products, in addition to the precursor illicit drug, during drinking water production, taking into account that cocaine traces might be present in water catchments and particularly in areas with high population densities.

Keywords: Cocaine; Drugs of abuse; Chlorination; By-products; Environmental fate; Liquid chromatography-mass spectrometry (LC-MS); Time-of-flight (TOF)

Transformation of cocaine during water chlorination by Iria González-Mariño; José Benito Quintana; Isaac Rodríguez; Noemí Sánchez-Méndez; Rafael Cela (pp. 3135-3144).

Keywords: Cocaine; Drugs of abuse; Chlorination; By-products; Environmental fate; Liquid chromatography-mass spectrometry (LC-MS); Time-of-flight (TOF)

UPLC-UV-MS^E analysis for quantification and identification of major carotenoid and chlorophyll species in algae by Weiqi Fu; Manuela Magnúsdóttir; Sigurður Brynjólfson; Bernhard Ø. Palsson; Giuseppe Paglia (pp. 3145-3154).

A fast method for quantification and identification of carotenoid and chlorophyll species utilizing liquid chromatography coupled with UV detection and mass spectrometry has been demonstrated and validated for the analysis of algae samples. This method allows quantification of targeted pigments and identification of unexpected compounds, providing isomers separation, UV detection, accurate mass measurements, and study of fragment ions for structural elucidation in a single run. This is possible using parallel alternating low- and high-energy collision spectral acquisition modes, which provide accurate mass full scan chromatograms and accurate mass high-energy chromatograms. Here, it is shown how this approach can be used to confirm carotenoid and chlorophyll species by identification of key diagnostic fragmentations during high-energy mode. The developed method was successfully applied for the analysis of Dunaliella salina samples during defined red LED lighting growth conditions, identifying 37 pigments including 19 carotenoid species and 18 chlorophyll species, and providing quantification of 7 targeted compounds. Limit of detections for targeted pigments ranged from 0.01 ng/mL for lutein to 0.24 ng/mL for chlorophyll a. Inter-run precision ranged for of 3 to 24 (RSD%) while inter-run inaccuracy ranged from −17 to 11. Figure Identification of carotenoids and chlorophylls combining UPLC separation, UV detection, accurate mass measurements and study of fragment ions in a single run

Keywords: Carotenoids; Chlorophylls; UPLC; Mass spectrometry; MS^E ; Dunaliella salina

Dynamic processes and chemical composition of Lepidium sativum seeds determined by means of field-cycling NMR relaxometry and NMR spectroscopy by A. Rachocki; L. Latanowicz; J. Tritt-Goc (pp. 3155-3164).

Proton nuclear magnetic resonance (NMR) techniques, such as field-cycling relaxometry, wide-line NMR spectroscopy, and magic angle spinning NMR spectroscopy, were applied to study the seeds of cress, Lepidium sativum. Field-cycling NMR relaxometry was used for the first time to investigate the properties of the whole molecular system of dry cress seeds. This method not only allowed the dynamics to be studied, but was also successful in the differentiation among the solid (i.e., carbohydrates, proteins, or fats forming a solid form of lipids) and liquid-like (oil compounds) components of the seeds. The ¹H NMR relaxation dispersion of oils was interpreted as a superposition of intramolecular and intermolecular contributions. The intramolecular part was described in terms of a Lorentzian spectral density function, whereas a log–Gaussian distribution of correlation times was applied for the intermolecular dipole–dipole contribution. The models applied led to very good agreement with the experimental data and demonstrate that the contribution of the intermolecular relaxation to the overall relaxation should not be disregarded, especially at low frequencies. A power-law frequency dependence of the proton relaxation dispersion was used for the interpretation of the solid components. From the analysis of the ¹H wide-line NMR spectra of the liquid-like component of hydrated cress seeds, we can conclude that the contribution of oil protons should always be taken into account when evaluating the spin–lattice relaxation times values or measuring the moisture and oil content. The application of ¹H magic angle spinning NMR significantly improves resolution in the liquid-like spectrum of seeds and allows the determination of the chemical composition of cress seeds. Figure Proton wide-line and magic angle spining NMR spectra of dry cress seeds

Keywords: Cress; Lepidium sativum ; Seeds; ¹H field-cycling NMR relaxometry; Molecular dynamics; ¹H wide-line NMR spectroscopy; ¹H magic angle spinning NMR spectroscopy

Enzymeless determination of total sugar by luminol–tetrachloroaurate chemiluminescence on chip to analyze food samples by Al-Mahmnur Alam; Mohammad Kamruzzaman; Trung-Dung Dang; Sang Hak Lee; Young Ho Kim; Gyu-Man Kim (pp. 3165-3173).

Chemiluminescence (CL) emission from luminol–tetrachloroaurate ([AuCl₄]⁻) system studied in presence of monosaccharide sugars such as glucose and fructose was investigated on a microfluidic chip fabricated by the soft lithography technique. CL emission from the luminol–[AuCl₄]⁻ system at 430 nm was intensified remarkably by the catalytic activity of glucose and fructose at room temperature. Under optimized conditions, the CL emission intensity of the system was found to be linearly related to the concentration of the sugars. Based on this observation, nonenzymatic determination of total sugar (glucose, fructose, or hydrolyzable sucrose) was performed in a rapid and sensitive analytical method. The results revealed that the linearity ranged from 9 to 1,750 μM for glucose and 80 to 1,750 μM for fructose, with a limit of detection of 0.65 and 0.69 μM, respectively. The relative standard deviations determined at 250 μM based on six repetitive injections were 1.13 and 1.15 % for glucose and fructose, respectively. The developed method was successfully applied for determination of the total sugar concentration in food and beverages. Figure Schematic diagram and plausible chemical reaction scheme of microfluidic chip based enzymless determination of total sugar concentration. (a) CL emission for reaction between luminol and [AuCl₄]- in absence of sugar; (b) Enhanced CL emission when reaction mixture of reducing sugar and [AuCl₄]- merge with the luminol stream in the chip. SP-1, SP-2, and SP-3 represent the syringe pumps that deliver H₂O/Sugar sample, [AuCl₄]- and luminol solution, respectively, to the chip. M first mixing zone; D mixing and detection zone, W waste out

Keywords: Chemiluminescence; Microfluidic chip; Luminol; Tetrachloroaurate; Sugar; Nonenzymatic

Determination of biocides in different environmental matrices by use of ultra-high-performance liquid chromatography–tandem mass spectrometry by Zhi-Feng Chen; Guang-Guo Ying; Hua-Jie Lai; Feng Chen; Hao-Chang Su; You-Sheng Liu; Fu-Qiang Peng; Jian-Liang Zhao (pp. 3175-3188).

A sensitive and robust method using solid-phase extraction and ultrasonic extraction for preconcentration followed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS–MS) has been developed for determination of 19 biocides: eight azole fungicides (climbazole, clotrimazole, ketoconazole, miconazole, fluconazole, itraconazole, thiabendazole, and carbendazim), two insect repellents (N,N-diethyl-3-methylbenzamide (DEET), and icaridin (also known as picaridin)), three isothiazolinone antifouling agents (1,2-benzisothiazolinone (BIT), 2-n-octyl-4-isothiazolinone (OIT), and 4,5-dichloro-2-n-octyl-isothiazolinone (DCOIT)), four paraben preservatives (methylparaben, ethylparaben, propylparaben, and butylparaben), and two disinfectants (triclosan and triclocarban) in surface water, wastewater, sediment, sludge, and soil. Recovery of the target compounds from surface water, influent, effluent, sediment, sludge, and soil was mostly in the range 70–120 %, with corresponding method quantification limits ranging from 0.01 to 0.31 ng L⁻¹, 0.07 to 7.48 ng L⁻¹, 0.01 to 3.90 ng L⁻¹, 0.01 to 0.45 ng g⁻¹, 0.01 to 6.37 ng g⁻¹, and 0.01 to 0.73 ng g⁻¹, respectively. Carbendazim, climbazole, clotrimazole, methylparaben, miconazole, triclocarban, and triclosan were detected at low ng L⁻¹ (or ng g⁻¹) levels in surface water, sediment, and sludge-amended soil. Fifteen target compounds were found in influent samples, at concentrations ranging between 0.4 (thiabendazole) and 372 ng L⁻¹ (methylparaben). Fifteen target compounds were found in effluent samples, at concentrations ranging between 0.4 (thiabendazole) and 114 ng L⁻¹ (carbendazim). Ten target compounds were found in dewatered sludge samples, at concentrations ranging between 1.1 (DEET) and 887 ng g⁻¹ (triclocarban).

Keywords: Biocides; Water; Sludge; Solid-phase extraction; Ultrasonic extraction; Liquid chromatography; Tandem mass spectrometry

Fast determination of trace dimethyl fumarate in milk with near infrared spectroscopy following fluidized bed enrichment by Ya-Jie Xie; Zhuan Wang; Wan-Peng Hu; Song Xu (pp. 3189-3194).

Near infrared spectroscopy (NIRS) has been proved to be a powerful analytical tool in different fields. However, because of the low sensitivity in near infrared region, it is a significant challenge to detect trace analytes with normal NIRS technique. A novel enrichment technique called fluidized bed enrichment has been developed recently to improve sensitivity of NIRS which allows a large volume solution to pass through within a short time. In this paper, fluidized bed enrichment method was applied in the determination of trace dimethyl fumarate in milk. Macroporous styrene resin HZ-816 was used as adsorbent material, and 1 L solution of dimethyl fumarate was run to pass through the material for concentration. The milk sample was pretreated to remove interference matters such as protein, fat, and then passed through the material for enrichment; after that, diffuse reflection NIR spectra were measured for the analyte concentrated on the material directly without any elution process. The enrichment and spectral measurement procedures were easy to operate. NIR spectra in 900–1,700 nm were collected for dimethyl fumarate solutions in the concentration range of 0.506–5.060 μg/mL and then used for multivariate calibration with partial least squares (PLS) regression. Spectral pretreatment methods such as multiplicative scatter correction, first derivative, second derivative, and their combinations were carried out to select the optimal PLS model. Root mean square error of cross-validation calculated by leave-one-out cross-validation is 0.430 μg/mL with ten PLS factors. Ten samples in an independent test set were predicted by the model with the mean relative error of 5.33 %. From the results shown in this work, it can be concluded that the NIR technique coupled with on-line enrichment method can be expanded for the determination of trace analytes, and its applications in real liquid samples like milk and juice may also be feasible.

Keywords: Near infrared spectroscopy (NIRS); Enrichment; Fluidized bed; Dimethyl fumarate; Milk; PLS

Fast determination of trace dimethyl fumarate in milk with near infrared spectroscopy following fluidized bed enrichment by Ya-Jie Xie; Zhuan Wang; Wan-Peng Hu; Song Xu (pp. 3189-3194).

Keywords: Near infrared spectroscopy (NIRS); Enrichment; Fluidized bed; Dimethyl fumarate; Milk; PLS

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Analytical and Bioanalytical Chemistry (v.404, #10)

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Analytical and Bioanalytical Chemistry (v.404, #10)