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The recent technological and methodological developments in NMR spectroscopy have dramatically decreased its detection limit, hence expanding its application areas. Herein, we report the first experimental detection of 2H-13C isotopomeric isomers (isotopomers) in natural abundance (1.7 X 10-4%) both in liquids and oriented systems by combining cryoprobe, high magnetic field, and carefully chosen 2D heteronuclear correlation techniques. These experunental results require the detection of one molecule among 600 0011 and demonstrate the sensivity and the selectivity of the employed NMR techniques. Besides, we show that 2H-13C enantio-isotopomers can be distinguished using 2D NMR in chiral polypeptide alignment media, thus providing a new tool for the chiral analysis. This method opens fruitful propects in various analytical fields such as molecular source authentication, metabolism studies, or the fight against counterfeiting. [ABSTRACT FROM AUTHOR]

Monolayer stability of core-shell nanoparticles is a key determinant of their utility in biological studies such as imaging and drug delivery. Intracellular thiols (e.g., cysteine, cysteansine, and glutathione) can trigger the release of thiolate- bound monolayers from nanoparticles, a favorable outcome for controllable drug release applications but an unfavorable outcome for imaging agents. Here, we describe a method to quantify the monolayer release of gold nanoparticles (AuNPs) in living cells using parallel measurements by laser desorption/ ionization (LDI) and inductively coupled plasma (ICP) mass spectrometry. This combination of methods is tested using AuNPs with structural features known to influence monolayer stability and on cetis types with varying concentrations of gtutathione. On the basis of our results, we predict that this approach should help efforts to engineer nanopartide suthce monolayers with tunable stability, providing stable platforms for imaging agents and controlled release of therapeutic monolayer payloads. [ABSTRACT FROM AUTHOR]

Total internal reflection (TIR) Raman and reflectivity spectra were collected for nonresonant analytes as a function of incident angle at sapphire or sapphire/smooth 50 rim gold interfaces using 785 nm excitation. For both interfaces, the Raman signal as a function of incident angle is well-modeled by the calculated interfacial mean square electric field (MSEF) relative to the incident field times the thickness of the layer being probed in the Ransan measurement (Das). The Raman scatter was reproducibly enhanced at the interface containing a gold film relative to the sapphire interface by a factor of 4.3-4.6 for aqueous pyridine or 2.2-3.7 for neat nitrobenzene, depending on the analyzed vibrational mode. The mechanism for the increased Raman signal is the enhanced MSEF at incident angles where propagating surface plasmons are excited in the metal film. The background from the TIR prism was reduced by 89-95% with the addition of the gold film, and the percent relative uncertainty in peak area was reduced from 15 to 1.7% for the 1347 cm-1 mode of nitrobenzene. Single monolayers of benzenethiol (S/N = 6.8) and 4-mercaptopyridine (S/N = 16.5) on gold films were measured by TW Raman spectroscopy with 785 rim excitation (210 mW) without resonant enhancement in 1 mm. [ABSTRACT FROM AUTHOR]

We present a method for the qualitative and quantitative study of transient metabolic flux of phage infection at the molecular level. The method is based on statistical total correlation spectroscopy (STOCSY) and partial least squares discriminant analysis (PLS-DA) applied to nuclear magnetic resonance (NMR) metabonomic data sets. An algorithm for this type of study is developed and demonstrated. The method has been implemented on 1H NMR data sets of growth media in planktonic cultures of Pseudomonas aeruginosa infected with bacteriophage pf1. Transient metabolic flux of various important metabolites, identified by STOCSY and PLS-DA analysis applied to the NMR data set, are estimated at various stages of growth. The opportunistic and nosocomial pathogen P. aeruginosa is one of the best-studied model organism for bacterial biofilms. Complete information regarding metabolic connectivity of this system is not possible by conventional spectroscopic approach. Our study presents temporal comparative 1H NMR metabonomic analyses of filamentous phage pf1 infection in planktonic cultures of P. aeruginosa K strain (PAK). We exemplify here the potential of STOCSY and PLS-DA tools to gain mechanistic insight into subtle changes and to determine the transient flux associated with metabolites following metabolic perturbations resulting from phage infection. Our study has given new avenues in correlating existing postgenomic data with current metabonomic results in P. aeruginosa biofilms research. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is a post-translational modification that is essential for the regulation of many important cellular activities, including proliferation and differentiation. Current techniques for detecting protein phosphorylation in single cells often involve the use of fluorescence markers, such as antibodies or genetically expressed proteins. In contrast, infrared spectroscopy is a label-free and noninvasive analytical technique that can monitor the intrinsic vibrational signatures of chemical bonds. Here, we provide direct evidence that protein phosphorylation in individual living mammalian cells can be measured with synchrotron radiation-based Fourier transform-infrared (SR-FT-IR) spectromicroscopy. We show that PC12 cells stimulated with nerve growth factor (NGF) exhibit statistically significant temporal variations in specific spectral features, correlating with changes in protein phosphorylation levels and the subsequent development of neuron-like phenotypes in the cells. The spectral phosphorylation markers were confirmed by bimodal (FT-IR/fluorescence) imaging of fluorescently marked PC12 cells with sustained protein phosphorylation activity. Our results open up new possibilities for the label-free real-time monitoring of protein phosphorylation inside cells. Furthermore, the multimolecule sensitivity of this technique will be useful for unraveling the associated molecular changes during cellular signaling and response processes. [ABSTRACT FROM AUTHOR]

Fluorescence correlation spectroscopy (FCS) is a powerful approach to characterizing the binding and transport dynamics of macromolecules. The unbiased interpretation of FCS data relies on the evaluation of multiple competing hypotheses to describe an underlying physical process under study, which is typically unknown a priori. Bayesian inference provides a convenient framework for this evaluation based on the temporal autocorrelation function (TACF), as previously shown theoretically using model TACF curves (He, J., Guo, S., and Bathe, M. Anal. Chem. 2012, 84). Here, we apply this procedure to simulated and experimentally measured photon-count traces analyzed using a multitau correlator, which results in complex noise properties in TACF curves that cannot be modeled easily. As a critical component of our technique, we develop two means of estimating the noise in TACF curves based either on multiple independent TACF curves themselves or a single raw underlying intensity trace, including a general procedure to ensure that independent, uncorrelated samples are used in the latter approach. Using these noise definitions, we demonstrate that the Bayesian approach selects the simplest hypothesis that describes the FCS data based on sampling and signal limitations, naturally avoiding overfitting. Further, we show that model probabilities computed using the Bayesian approach provide a reliability test for the downstream interpretation of model parameter values estimated from FCS data. Our procedure is generally applicable to FCS and image correlation spectroscopy and therefore provides an important advance in the application of these methods to the quantitative biophysical investigation of complex analytical and biological systems. [ABSTRACT FROM AUTHOR]

High-resolution magic-angle sample spinning (HR-MAS) 1H NMR spectroscopy of tissue biopsies combined with chemometric techniques has emerged as a valuable methodology for disease diagnosis and environmental assessments. However, the tissue mass required for such experiments is of the order of 10 mg, and this can compromise the metabolic evaluation because of tissue heterogeneity. Tissue availability is often a limitation for clinical studies due to histopathological requirements, which are currently the gold standard for diagnosis, for example, in the case of tumors. Here, we introduce the use of a rotating micro-NMR detector that optimizes the coil filling factor such that mass-limited samples can be measured. We show the results for measuring nanoliter volume tissue biopsies using a commercial HR-MAS probe for the first time. The method has been tested with bovine muscle and human gastric mucosal tumor tissue samples. The gain in mass sensitivity is approximate up to 17-fold, and the adequate spectral resolution (3 Hz) allows the measurement of the metabolite profiles in nanoliter volume samples, thereby limiting the ambiguity resulting from heterogeneous tissues; thus, the approach presents diagnostic potential for studies by metabonomics of mass-limited biopsies. [ABSTRACT FROM AUTHOR]

The usefulness of infrared-reflection absorption spectroscopy (IR-RAS) for the rapid measurement of residual drug substances without sampling was evaluated. In order to realize the highly accurate rapid measurement, locally weighted partial least-squares (LW-PLS) with a new weighting technique was developed. LW-PLS is an adaptive method that builds a calibration model on demand by using a database whenever prediction is required. By adding more weight to samples closer to a query, LW-PLS can achieve higher prediction accuracy than PLS. In this study, a new weighting technique is proposed to further improve the prediction accuracy of LW-PLS. The root-mean-square error of prediction (RMSEP) of the IR-RAS spectra analyzed by LW-PLS with the new weighting technique was compared with that analyzed by PLS and locally weighted regression (LWR). The RMSEP of LW-PLS with the proposed weighting technique was about 36% and 14% smaller than that of PLS and LWR, respectively, when ibuprofen was a residual drug substance. Similarly, LW-PLS with the weighting technique was about 39% and 24% better than PLS and LWR in RMSEP, respectively, when magnesium stearate was a residual excipient. The combination of IR-RAS and LW-PLS with the proposed weighting technique is a very useful rapid measurement technique of the residual drug substances. [ABSTRACT FROM AUTHOR]

A novel functional imaging mass spectrometry technology is described that utilizes activity-based probes for imaging enzyme active sites in tissue sections. We demonstrate this technology using an activity-based probe (fluorophosphate) that is specific for serine hydrolases. A dendrimer containing multiple mass tags that is attached to the activity-based probe is used to analyze the binding sites of the probe through release and measurement of the mass tags on laser irradiation. A generation 8 poly(amido amine) dendrimer with 1024 amino groups was labeled with an azide group, and then, more than 900 mass tags were attached in order to achieve signal amplification of nearly 3 orders of magnitude. The experimental protocol first involves binding of the activity-based probe containing an alkyne group to serine hydrolases in the tissue section followed by attachment of the dendrimer labeled with mass tags to the bound probe by Click chemistry. On irradiation of the labeled tissue by the laser beam in a raster pattern, the mass tags are liberated and recorded by the mass analyzer; consequently, the ion image of the mass tag reveals the distribution of serine hydrolases in the tissue. This process was shown using rat brain and mouse embryo sections. Targeted imaging has the advantage of providing high spatial resolution and high sensitivity through the use of signal amplification chemistry with high target specificity through the use of an enzyme activity probe. [ABSTRACT FROM AUTHOR]

Inductively coupled planar resonators offer convenient integration of high-resolution NMR spectroscopy with microfluidic lab-on-a-chip devices. Planar spiral resonators are fabricated lithographically either by gold electroplating or by etching Cu laminated with polyimide. Their performance is characterized by NMR imaging as well as spectroscopy. A single-scan limit of detection LODt = 0.95 nmol s1/2 was obtained from sample volumes around 1 μL. The sensitivity of this approach is similar to that obtained by microstripline and microslot probes. [ABSTRACT FROM AUTHOR]

The space charge effect has an important role in instruments dealing with ion packets and charged particles in gas phase such as the mass spectrometer and ion mobility spectrometer (IMS). It has been shown that the space charge is partially responsible for peak broadening in IMS depending on the ion density. Here, we explore the effect of space charge on peak shifting in IMS. We show that the field created by a large peak influences the drift time of a neighboring small peak. An experimental method was introduced to accurately measure the effect of space charge between two peaks. In this method, a double pulse was applied to the shutter grid to create two closed ion packets with a given initial spacing. The final spacing was then measured at the collector through the separation of the two peaks. This study shows that space charge repulsion must be considered for accurate measurements of ion mobilities. The experiments were performed in both normal and inverse modes. A theoretical model was also proposed to describe the repulsion between two ion packets in IMS. [ABSTRACT FROM AUTHOR]

The ability to unequivocally identify a gunshot residue (GSR) when a firearm is discharged is a very important and crucial part of crime scene investigation. To date, the great majority of the analyses have focused on the inorganic components of GSR, but the introduction of "lead-free" or "nontoxic" ammunitions makes it difficult to prevent false negatives. This study introduces a fast methodology for the organic analysis of GSR using Raman spectroscopy. Six different types of ammunition were fired at short distances into cloth targets, and the Raman spectra produced by the GSR were measured and compared with the spectra from the unfired gunpowder ammunition. The GSR spectrum shows high similarity to the spectrum of the unfired ammunition, allowing the GSR to be traced to the ammunition used. Additionally, other substances that might be found on the victim's, shooter's, or suspect's clothes and might be confused with GSR, such as sand, dried blood, or black ink from a common ballpoint pen, were analyzed to test the screening capability of the Raman technique. The results obtained evidenced that Raman spectroscopy is a useful screening tool when fast analysis is desired and that little sample preparation is required for the analysis of GSR evidence. [ABSTRACT FROM AUTHOR]

In the paper, we present a novel single particle method, named spatially resolved scattering correlation spectroscopy (SRSCS), based on a total internal reflection (TIR) configuration and strong resonance light scattering (RLS) of silver nanoparticles (AgNPs). The principle of SRSCS is similar to fluorescence correlation spectroscopy (FCS), and it is based on measuring the RLS fluctuations in a small volume due to Brownian motion of single nanoparticles. We first established a highly sensitive SRSCS system. In the SRSCS system, a millimeter-scale hole is employed to efficiently separate nanoparticle scattering light from the background reflected beam, and an electron multiplying charge-coupled device (EMCCD) is used as an array detector. The SRSCS system was successfully used for detection and imaging of single AgNPs in solution. Furthermore, we developed the model of SRSCS according to the FCS method and systematically investigated the effects of certain factors such as particle concentration, viscosity of the solution, hardware and software binning and accumulation time on SRSCS measurements using AgNPs as a model sample. A series of calibration experiments were conducted, and the experimental data obtained were in good agreement with the SRSCS model. This new method is multiplexing, spatially resolved, and free of photobleaching and may become a useful method for study on heterogeneous systems, such as the motion of proteins on the cell membrane. [ABSTRACT FROM AUTHOR]

Accurate and reproducible quantification of glycans from protein drugs has become an important issue for quality control of therapeutic proteins in biopharmaceutical and biotechnology industries. Mass spectrometry is a promising tool for both qualitative and quantitative analysis of glycans owing to mass accuracy, dflciency, and reproducibility, but it has been of limited success in quantitative analysis for sialylated glycans in a high-throughput manner. Here, we present a solid-phase permethylation-based total N-glycan quantitative method that includes N-glycan releasing, purification, and derivatization on a 96-well plate platform. The solid-phase neutralization enabled us to perform reliable absolute quantification of the acidic Nglycans as well as neutral N-glycans from model glycoproteins (i.e., chicken ovalbumin and porcine thyroglobulin) by only using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, low-abundance sialylated N-glycans from human serum prostate specific antigen (PSA), an extremely valuable prostate cancer marker, were initially quantified, and their chemical compositions were proposed. Taken together, these results demonstrate that our allindusive glycan preparation method based on a 96-well plate platform may contribute to the precise and reliable qualitative and quantitative analysis of glycans. [ABSTRACT FROM AUTHOR]

Mass spectrometry allows sensitive, automated, and high-throughput analysis of small molecules. In principle, tandem mass specirometry allows us to identify ~unknown small molecules not in any database, but the automated interpretation of such data is in its infancy. Fragmentation trees have recently been introduced for the automated analysis of the fragmentation patterns of small molecules. We present a method for the automated comparison of such fragmentation patterns, based on aligning the compounds' fragmentation trees. We cluster compounds based solely on their fragmentation patterns and show a good agreement with known compound dasses. Fragmentation pattern similarities are strongly correlated with the chemical similarity of molecules. We present a tool for searching a database for compounds with fragmentation pattern similar to an unknown sample compound. We apply this tool to metabolites from Icelandic poppy. Our method allows fully automated computational identification of small molecules that cannot be found in any database. [ABSTRACT FROM AUTHOR]

Unique to ion mobility mass spectrometry (EM- MS) is the ability to provide collision cross section (CCS) data and the capacity to delineate any dissociation and/or unfolding of protein complexes. The strong correlation of the experimentally determined CCS with theory is indicative of the retention of native structure in the gas phase, which in turn, qualifies as a means in evaluating the tM-MS data The assessment of IM-MS data, however, is currently impeded due to the lack of appropriate stnactural coordinates to use as input in the in silico calculation of theory. To address this sane, this study invokes the use of rapid protein threading predictor (RAPTOR) to generate tertiary structures of dosely related monomeric chemokines (MCP-l, MCP- 3, MCP-4, and eotaxin) and, subsequently, utilize these models to estimate the theoretical values. Experimental CCS of both the model proteins and chemokines correlate well with theory generated by RAPTOR, All conformations for z = 5+ of chemokines fall within theoretical limits, Of the four chemokines, MCP-4 with z = 6+ appears to adopt an extended conformation, while eotaxin gradually unfolds, and the extended structures of MCP-1 and MCP-3 increase in abundance upon activation. Combining RAPTOR with lM-MS and collision-induced dissociation (CU)) enables us to interrogate the conformations of homologous proteins with very similar tertiary structures. [ABSTRACT FROM AUTHOR]

Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ∼700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (∼1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA. [ABSTRACT FROM AUTHOR]

A historical self-portrait painted by Sir Arthur Streeton (1867-1943) has been studied with fast-scanning X-ray fluorescence microscopy using synchrotron radiation. One of the technique's unique strengths is the ability to reveal metal distributions in the pigments of underlying brushstrokes, thus providing information critical to the interpretation of a painting. We have applied the nondestructive technique with the event-mode Maia X-ray detector, which has the capability to record elemental maps at megapixels per hour with the full X-ray fluorescence spectrum collected per pixeL The painting poses a difficult challenge to conventional - X-ray analysis, because - it was completely obscured with heavy brushstrokes of highly X-ray absorptive lead white paint (2PbCO3·Pb(OH)2) by the artist, making it an excellent candidate for the application of the synchrotron-based technique. The 25 megapixel elemental maps were successfully observed through the lead white paint across the 200 × 300 mm² scan area. The sweeping brushstrokes of the lead white overpaint contributed significant detrimental structure to the elemental maps. A corrective procedure was devised to enhance the visualization of the elemental maps by using the elastic X-ray scatter as a proxy for the lead white overpaint We foresee the technique applied to the most demanding of culturally significant artworks where conventional analytical methods are inadequate. [ABSTRACT FROM AUTHOR]

We present a micromachined silicon attenuated total reflection-infrared (ATR-IR) crystal with integrated nanofluidic glass channels which enables infrared spectroscopic studies with only 71 nL sample volume. Because of the short path length through silicon, the system allows IR spectroscopy down to 1200 cnf', which covers the typical fingerprint region of most organic compounds. To demonstrate proof-of- principle, the chip was used to study a Knoevenagel condensation reaction between malononitrile and p-anisaldehyde catalyzed by different concentrations of 1,8-diazabicyclo[5.4.0]undec-7-ene (OBU) in solvent acetonitrile. By in situ measurement, it was demonstrated for the first time that at certain concentrations of DBU, reaction intermediates become stabilized, an effect that slows down or even stops the reaction. This is thought to be caused by increased ionic character of the solvent, in which protonated DBU stabilizes the intermediates. This dearly demonstrates that infrared mechanistic studies of chemical reactions are feasible in volumes as little as 71 nL. [ABSTRACT FROM AUTHOR]

The article presents information on the use of mass spectrometry methods for mapping of glycoprotein glycosylation and determinations of detailed glycan structure. These methods are generally used in the pharmaceutical industry and academic biomedical field for the analysis of recombinant glycoproteins.

Plutonium (Pu) dioxide particles were produced from certified reference material (CRM) 136 solution (CRM 136-plutonium isotopic standard, New Brunswick Laboratory, Argonne, IL, U.S.A., 1987) using an atomizer system on December 3, 2009 after chemical separation of americium (Am) on October 27, 2009. The highest density of the size distribution of the particles obtained from 312 particles on a selected impactor stage was in the range of 0.7-0.8 μm. The flattening degree of 312 particles was also estimated. The isotopic composition of Pu and uranium (U) and the amount of Am were estimated by thermal ionization mass spectrometry (TIMS), inductively coupled plasma mass spectrometry (ICPMS), and α-spectrometry. Within uncertainties the isotopic composition of the produced particles is in agreement with the expected values, which were derived from the decay correction of the Pu isotopes in the CRM 136. The elemental ratio of Am to Pu in the produced particles was determined on the 317th and 674th day after Am separation, and the residual amount of Am in the solution was estimated. The analytical results of single particles by micro-Raman-scanning electron microscopy (SEM)-energy-dispersive X-ray spectrometry (EDX) indicate that the produced particles are Pu dioxide. Our initial attempts to measure the density of two single particles gave results with a spread value accompanied by a large uncertainty. [ABSTRACT FROM AUTHOR]

Evanescent wave cavity ringdown spectroscopy (EW-CRDS) is advocated as an approach for monitoring the formation of supported lipid bilayers (SLBs) on quartz substrates in situ and for the quantitative study of fast molecular adsorption kinetics at the resulting modified biomimetic surface. This approach is illustrated using SLBs of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Complementary atomic force microscopy (AFM) and quartz crystal microbalance with dissipation (QCM-D) measurements confirm the formation of bilayers on quartz. The subsequent interaction of the porphyrin, 5,10,15,20-tetraphenyl-21H,23H-porphine-p,p',p″,p‴-tetrasulfonic acid tetrasodium hydrate (TPPS) with the cationic bilayer-modified silica surface has been studied using EW-CRDS combined with an impinging-jet to deliver analyte to the surface in a well-defined manner. The adsorption of TPPS to the bilayer was kinetically controlled and the adsorption rate constant was found to be 1.7 (±0.6) x 10-4 cm s-1 from finite element modeling of the jet hydrodynamics and associated convective-diffusion equation, coupled to a first-order surface process describing adsorption. These proof-of-concept studies provide a platform for the investigation of molecular processes at biomembranes using EW-CRDS for chemical species showing optical absorbance in the visible and ultraviolet range. [ABSTRACT FROM AUTHOR]

The much anticipated overhaul of the International System of Units (SI) will result in new definitions of base units in terms of fundamental constants. However, redefinition of the kilogram in terms of the Planck constant (h) cannot proceed without consistency between the Avogadro and Planck constants, which are both related through the Rydberg constant. In this work, an independent assessment of the atomic weight of silicon in a highly enriched 28Si crystal supplied by the International Avogadro Coordination (IAC) was performed. This recent analytical approach, based on dissolution with NaOH and its isotopic characterization by multicollector inductively coupled plasma mass spectrometry, is critically evaluated. The resultant atomic weight Ar(Si) = 27.976?968?39(24)k=1 differs significantly from the most recent value of Ar(Si) = 27.976?970?27(23)k=1. Using the results generated herein for Ar(Si) along with other IAC measurement results for mass, volume, and the lattice spacing, the estimate of the Avogadro constant becomes NA = 6.022?140?40(19) x 1023 mol-1. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic sulfur heterocycles (PASH) are sulfur analogues of polycyclic aromatic hydrocarbons (PAH). Alkylated PAH attract much attention as carcinogens, mutagens, and as diagnostics for environmental forensics. PASH, in contrast, are mostly ignored in the same studies due to the conspicuous absence of gas chromatography/mass spectrometry (GC/MS) retention times and fragmentation patterns. To obtain these data, eight coal tar and crude oils were analyzed by automated sequential GC-GC. Sample components separated based on their interactions with two different stationary phases. Newly developed algorithms deconvolved combinatorially selected ions to identify and quantify PASH in these samples. Simultaneous detection by MS and pulsed flame photometric detectors (PFPD) provided additional selectivity to differentiate PASH from PAH when coelution occurred. A comprehensive library of spectra and retention indices is reported for the C1-C4 two-, three-, and four-ring PASH. Results demonstrate the importance of using multiple fragmentation patterns per homologue (MFPPH) compared to selected ion monitoring (SIM) or extraction (SIE) to identify isomers. Since SIM/SIE analyses dramatically overestimate homologue concentrations, MFPPH should be used to correctly quantify PASH for bioavailability, weathering, and liability studies. [ABSTRACT FROM AUTHOR]

Laser induced breakdown spectroscopy (LIBS) is shown to be capable of low volume (90 pL) quantitative elemental analysis of picogram amounts of dissolved metals in solutions. Single-pulse and collinear double-pulse LIBS were investigated using a 532 nm dual head laser coupled to a spectrometer with an intensified charge coupled device (CCD) detector. Aerosols were produced using a micronebulizer, conditioned inside a concentric spray chamber, and released through an injector tube with a diameter of 1 mm such that a LIBS plasma could be formed ∼2 mm from the exit of the tube. The emissions from both the aerosols and a single microdrop were then collected with a broadband high resolution spectrometer. Multielement calibration solutions were prepared, and continuing calibration verification (CCV) standards were analyzed for both aerosol and microdrop systems to calculate the precision, accuracy, and limits of detection for each system. The calibration curves produced correlation coefficients with R2 values > 0.99 for both systems. The precision, accuracy, and limit of detection (LOD) determined for aerosol LIBS were averaged and determined for the emission lines of Sr II (421.55 nm), Mg II (279.80 nm), Ba II (493.41 nm), and Ca II (396.84 nm) to be ∼3.8% RSD, 3.1% bias, 0.7 ∼g/mL, respectively. A microdrop dispenser was used to deliver single drops containing 90 pL into the space where a LIBS plasma was generated with a focused laser pulse. In the single drop microdrop LIBS experiment, the analysis of a single drop, containing a total mass of 45 pg, resulted in a precision of 13% RSD and a bias of 1% for the Al I (394.40 nm) emission line. The absolute limits of detection of single drop microdrop LIBS for the emission lines Al I (394.40 nm) and Sr II (421.5 nm) were approximately 1 pg, and Ba II (493.41 nm) produced an absolute detection limit of approximately 3 pg. Overall, the precision, accuracy, and absolute LOD determined for single microdrop LIBS resulted in a typical performance of ∼14% RSD, 6% bias, and 1 pg for the elements Sr II (421.55 nm), Al I(394.40 nm), Mg II (279.80), and Ba II(493.41 nm). [ABSTRACT FROM AUTHOR]

Chromatography combined with spectral analyzers provides great separation powers. However, the amount of data provided makes analysis difficult and laborious. The automated peak extraction and quantification (APEQ) method is a novel method for targeted and untargeted analysis. The method extracts retention times (tR), spectra, and signal intensity from data from chromatography with spectral detectors. It is a tiered approach consisting of five tiers: (1) the chromatograms are split into sections. (2) Peaks are detected and their elution profiles, spectra, and signal intensities are approximated. (3) The number of important peaks in each section is estimated. (4) For each section, the peaks and background are modeled using parallel factor analysis (PARAFAC). Several PARAFAC models with a different number of factors are made. (5) For each section, the optimal model is selected. The optimal model will in most cases be the model with lowest dimensionality (i.e., fewest factors) that has a factor describing the baseline. The APEQ method has been used to analyze gas chromatography/mass spectrometry (GC/MS) full-scan chromatograms of 12 samples, each containing 30 polycyclic aromatic hydrocarbons (PAHs). It detected all 30 PAHs and 14 compounds not intentionally added. It extracted the tRs, mass spectra, and relative concentrations. The quantification was compared to integration of extracted ion chromatograms. It had comparable quality for peaks with little or no overlap and significantly better for peaks with overlap. The total analysis time with default parameters was less than 10 min on an ordinary laptop. By optimizing the parameters, all 30 PAHs could be quantified in 142 s. [ABSTRACT FROM AUTHOR]

In this study, we combined surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) with HgTe nanostructures as matrix for the detection of several proteins (αl-antitrypsin, trypsin, IgG, protein G) and their complexes. We investigated the effects of several parameters (the concentration and nature of surfactants and metal ions, the pH, and concentration of the analytes in the sample matrixes) on the sensitivity of the detection of these proteins and their complexes. The presence of stabilizing Brij 76 surfactant and Zn(II) ions allowed the detection of weak protein complexes, such as αl-antitrypsin-trypsin and IgG-protein G complexes, at the picomole level. We observed multiply charged states at m/z 72 160 ([α1-antitrypsin + trypsin + H]+) and 86585 ([IgG + protein G + 2H}2+) for the α1-antitrypsin-trypsin and lgG-protein G complexes, respectively. To the best of our knowledge, detection of weak protein complexes and determination of their stoichiometry have not been demonstrated previously when a combination of SALDI-MS and nanostructures were used. This simple and reproducible SALDI-MS approach using HgTe nanostructures holds great potential for the detection of other proteins and their complexes. [ABSTRACT FROM AUTHOR]

Electron transfer dissociation (ETD) has improved the mass spectrometric analysis of proteins and peptides with labile post-translational modifications and larger intact masses. Here, the parameters governing the reaction rate of ETD are examined experimentally. Currently, due to reagent injection and isolation events as well as longer reaction times, ETD spectra require significantly more time to acquire than collision-induced dissociation (CID)) spectra (>100 ms), resulting in a trade-off in the dynamic range of tandem MS analyses when ETD-based methods are compared to CID-based methods. Through fine adjustment of reaction parameters and the selection of reagents with optimal characteristics, we demonstrate a drastic reduction in the time taken per ETD event. In fact, ETD can be performed with optimal efficiency in nearly the same time as CID at low precussor charge state (z = +3) and becomes faster at higher charge state (z > +3). [ABSTRACT FROM AUTHOR]

Nanospray desorption electrospray ionization (nano-DESI) combined with high-resolution mass spectrometry was used for the first time for the analysis of the polar constituents of liquid petroleum crude oil samples. The analysis was performed in both positive and negative ionization modes using three solvents, one of which (acetonitrile/toluene mixture) is commonly used in petroleomics studies while two other polar solvents (acetonitrile/water and methanol/water mixtures) are generally not compatible with petroleum characterization using mass spectrometry. The results demonstrate that nano-DESI analysis efficiently ionizes petroleum constituents soluble in a particular solvent. When acetonitrile/toluene is used as a solvent, nano-DESI generates electrospray-like spectra. In contrast, strikingly different spectra were obtained using acetonitrile/water and methanol/water. Comparison with the literature data indicates that these solvents selectively extract water-soluble constituents of the crude oil. Water-soluble compounds are predominantly observed as sodium adducts in nano-DESI spectra indicating that addition of sodium to the solvent may be a viable approach for efficient ionization of water-soluble crude oil constituents. Nano-DESI enables rapid screening of different classes of compounds in crude oil samples based on their solubility in solvents that are rarely used for petroleum characterization providing better coverage of the crude oil composition as compared to electrospray ionization (ESI). It also enables rapid characterization of water-soluble components of petroleum samples that is difficult to perform using traditional approaches. [ABSTRACT FROM AUTHOR]

Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents. [ABSTRACT FROM AUTHOR]

Electrically driven separations which contain nanoparticles offer detection and separation advantages but are often difficult to reproduce. To address possible sources of separation inconsistencies, anionic functionalized gold nanoparticles are thoroughly characterized and subsequently included in continuous full filling capillary electrophoresis separations of varying concentrations of three small molecules. Citrate stabilized gold nanospheres are functionalized with 11-mercaptoundecanoic acid, 6-mercaptohexanoic acid, or thioctic acid self-assembled monolayers (SAMs) and characterized using dynamic light scattering, extinction spectroscopy, zeta potential, and X-ray photoelectron spectroscopy prior to use in capillary electrophoresis. Several important trends are noted. First, the stability of these anionic nanoparticles in the capillary improves with increased ligand packing density as indicated by a ratio of absorbance collected at 520 to 600 nm. Second, increasing nanoparticle concentration from 0 to 2 nM (0-0.0025%, w/w) minimally impacts analyte migration times; however, when higher nanoparticle concentrations are included within the capillary, nanoparticle aggregation occurs which induces separation inconsistencies. Third, analyte peak areas are most significantly impacted as their concentration decreases. These trends are attributed to both sample enrichment and electrostatic interactions between the anionic carboxylic acid functionalized gold nanoparticles and sample. These important findings suggest that sample concentration-induced conductivity differences between the sample matrix and separation buffer as well as SAM packing density are important parameters to both characterize and consider when nanoparticles are used during continuous full filling separations and their subsequent use to enhance spectroscopic signals to improve in-capillary analyte detection limits. [ABSTRACT FROM AUTHOR]

In this paper, we describe experimental and modeling results that illucidate the nature of coupling between surface plasmon polaritons in a thin silver film with the molecular resonance of a zinc phthalocyanine dye film. This coupling leads to several phenomena not generally observed when plasmons are coupled to transparent materials. The increased absorption coefficient near a molecular resonance leads to a discontinuity in the refractive index, which causes branching of the plasmon resonance condition and the appearance of two peaks in the p-polarized reflectance spectrum. A gap exists between these peaks in the region of the spectrum associated with the molecular resonance and reflects quenching of the plasmon wave due to violation of the resonance condition. A second observation is the appearance of a peak in the s-polarized reflection spectra. The initial position of this peak corresponds to where the refractive index of the adsorbate achieves its largest value, which occurs at wavelengths just slightly larger than the maximum in the molecular resonance. Although this peak initially appears to be nondispersive, both experimental data and optical modeling indicate that increasing the film thickness shifts the peak position to longer wavelengths, which implies that this peak is not associated with the molecular resonance but, rather, is dispersive in nature. Indeed, modeling shows that this peak is due to a guided mode in the film, which appears in these conditions due to the abnormally high refractive index of the film near the absorbance maximum. Results also show that, with increasing film thickness, numerous additional guided modes appear and move throughout the visible spectrum for both s- and p-polarized light. Notably, these guided modes are also quenched near the location of the molecular resonance. The quenching of both the plasmon resonance and the guided modes can be explained by a large decrease in the in-plane wave propagation length that occurs near the molecular resonance, which is a direct result of the film's large absorption coefficient. [ABSTRACT FROM AUTHOR]

Several tablets of a formulation containing 1% w/w of the desired active pharmaceutical ingredient (API) form are spiked with minimal amounts of two different anhydrous polymorphs and an amorphous form. The amount of contaminant form was 2.5 to 10% of the total API concentration (0.025 to 0.1% w/w in the tablet), with five spiked tablets prepared. The presence of these contaminant particles are then identified using Raman microscopy/mapping. The entire surface of each of these tablets is Raman-probed through a grid based on our previous proposal (ŠaŠic, S.; Whitlock, M. Appl. Spectrosc.2008, 62, 916) about the minimal number of spectra to acquire that would guarantee identification of the targeted component (taking into account the limit of detection). All three forms have been clearly identified in the Raman mapping spectra of prepared "calibration" tablets; particularly of note is the 2.5% spike (0.025% w/w in the tablet) of the relatively weakly scattering amorphous form. The same method is then applied to packaged tablets on stability and demonstrates that none of the previously analyzed contaminant forms is detected, hence building confidence that the desired API form does not change during stability testing. [ABSTRACT FROM AUTHOR]

Because of its highly reproducible and quantitative nature and minimal requirements for sample preparation or separation, ¹H nuclear magnetic resonance (NMR) spectroscopy is widely used for profiling small-molecule metabolites in biofluids. However ¹H NMR spectra contain many overlapped peaks. In particular, blood serum/plasma and diabetic urine samples contain high concentrations of glucose, which produce strong peaks between 3.2 ppm and 4.0 ppm. Signals from most metabolites in this region are overwhelmed by the glucose background signals and become invisible. We propose a simple "Add to Subtract" background subtraction method and show that it can reduce the glucose signals by 98% to allow retrieval of the hidden information. This procedure includes adding a small drop of concentrated glucose solution to the sample in the NMR tube, mixing, waiting for an equilibration time, and acquisition of a second spectrum. The glucose-free spectra are then generated by spectral subtraction using Bruker Topspin software. Subsequent multivariate statistical analysis can then be used to identify biomarker candidate signals for distinguishing different types of biological samples. The principle of this approach is generally applicable for all quantitative spectral data and should find utility in a variety of NMR-based mixture analyses as well as in metabolite profiling. [ABSTRACT FROM AUTHOR]

We incorporated the specific recognition of adenine-rich singled-stranded DNA (ssDNA) into dual polarization interferometry (DPI) measurements for direct, selective, and sensitive detection of the small molecule coralyne, and we simultaneously employed the real-time and label-free technique for detailed investigation of the interaction between coralyne and adenine-rich ssDNA. Data from UV-visible spectroscopy, circular dichroism (CD) spectroscopy, and DNA melting firmly confirmed that 48-mer homoadenine ssDNA oligonucleotide (A48) had highly specific recognition for coralyne, whereas 48-mer homothymine ssDNA oligonucleotide (T48) as the control had no such recognition. The immobilization of ssDNA (A48 or T48) on a silicon oxynitride chip could be achieved through a preadsorbed poly(ethylenimine) (PEI) layer. Mass, thickness, and refractive index (RI) changes resolved by DPI during the whole process of ssDNA immobilization suggested that most ssDNA molecules were likely to lie on the PEI surface mainly in the form of a flat monolayer and insert themselves partly into the PEI layer. Qualitative and quantitative analysis of mass, thickness, and RI changes in A48/PEI layer upon addition of different concentrations of coralyne revealed that A48 most likely underwent a conformational change from single-stranded to double-stranded structure. By evaluation of the binding curves from changes in mass, the association rate constant (ka), dissociation rate constant (kd), and association constant (KA) between coralyne and A48 were determined to be 4.95 x 103 M-1 s-1, 0.031 s-1, and 1.6 x 105 M-1, respectively. Good linear correlations between coralyne concentrations ranging from 0.5 to 12 μM and three parameters (mass, thickness, and RI) resolved by the response to coralyne binding were obtained. The detection limits were 0.22 μM for mass calibration, 0.14 μM for thickness calibration, and 0.32 μM for RI calibration. The high selectivity of the biosensor to coralyne at the A48/PEI interface was successfully confirmed by using the other two interfaces (T48/PEI and PEI) and three typical intercalators (ethidium bromide, daunomycin, and methylene blue). It is expected that the biosensing platform may be extended to simultaneously detect and characterize the interactions of a variety of target molecules with functional DNA molecules with high sensitivity. [ABSTRACT FROM AUTHOR]

The article discusses techniques used to identify and describe phospholipid films located on solid surfaces. Emphasis is given to membrane research related to chemical composition, film formation, and lipids' molecular interactions. Instrumental methods discussed include force microscopy and spectroscopy, secondary ion mass spectrometry and imaging, and microfluidics.

The article presents an overview of research which investigated recent advances in instrumentation, techniques and applications of the highest resolution mass analyzers related to high resolution spectrometry between 2008 and 2012. A discussion of a need which exists for high mass resolution spectrometry, of several mass analyzer types and of their development and of the ways in which they can be used in bio-analysis and complex organic mixtures, is presented.

The article presents an overview of research which has been conducted on significant and essential progress which has been seen in X-ray spectrometry, was published between January of 2010 and August of 2011 and covered a variety of topics, including developments and improvements which were seen in X-ray optics, X-ray detectors and elemental imaging techniques. The applications that X-ray spectrometry can be used for are examined.

The article presents an overview of research which has investigated advances which have been seen in the use of nanoparticles for analytical measurements since 2002, and developments which have been seen in the field of nanotechnology since 2002 and which have resulted in new procedures for the synthesis of nanoscale materials. Advances in nanostructures which have been seen between January of 2010 and November of 2011 are discussed. The use of nanoparticles in spectroscopy is examined.