Building an empirical mass spectra library for screening of organic pollutants by ultra-high-pressure liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

Hybrid quadrupole time-of-flight mass spectrometry (QTOF MS) has gained wide acceptance in many fields of chemistry, for example, proteomics, metabolomics and small molecule analysis. This has been due to the numerous technological advances made to this mass analyser in recent years. In the environmental field, the instrument has proven to be one of the most powerful approaches for the screening of organic pollutants in different matrices due to its high sensitivity in full acquisition mode and mass accuracy measurements. In the work presented here, the optimum experimental conditions for the creation of an empirical TOF MS spectra library have been evaluated. For this model we have used a QTOF Premier mass spectrometer and investigated its functionalities to obtain the best MS data, mainly in terms of mass accuracy, dynamic range and sensitivity. Different parameters that can affect mass accuracy, such as lock mass, ion abundance, spectral resolution, instrument calibration or matrix effect, have also been carefully evaluated using test compounds (mainly pesticides and antibiotics). The role of ultra-high-pressure liquid chromatography (UHPLC), especially when dealing with complex matrices, has also been tested. In addition to the mass accuracy measurements, this analyser allows the simultaneous acquisition of low and high collision energy spectra. This acquisition mode greatly enhances the reliable identification of detected compounds due to the useful (de)protonated molecule and fragment ion accurate mass information obtained when working in this mode. An in-house empirical spectral library was built for approximately 230 organic pollutants making use of QTOF MS in MS(E) mode. All the information reported in this paper is made available to the readers to facilitate screening and identification of relevant organic pollutants by QTOF MS.     

Vibrationally state-selective spin-orbit transfer with strong nonresonant pulses

By dynamic Stark shift using strong nonresonant pulses, we show that it is in principle possible to prepare arbitrary superposition states of mixed multiplicity. By a proper choice of parameters, the transfer of population is shown to follow the Rabi formula, where the initial and target states are now vibrational states of two light-induced molecular potentials of different multiplicity. Starting from nonstationary wave packets, the spin transfer can proceed via parallel transfer using a single pulse or by sequential transfer using a pulse sequence. A simple model is proposed to analyze the properties of both schemes and the feasibility of their experimental implementation for spin-orbit transitions in Rb2.     

Determination of hexavalent plutonium using differential spectrophotometry

A differential spectrophotometric method has been developed for plutonium in hexavalent state using a double beam spectrophotometer. The absorbance measurements were made at 835 nm in 4M sulfuric acid using a 5 cm cell. In the method developed the absorbance of six Pu(VI) standards, taken in the sample cell, were recorded against a molybdenum blue solution of appropriate intensity in the reference cell. A least-squares fit of data on absorbance and concentration of plutonium standards gave slope F and intercept Co which were used to determine the unknown concentrations using the relationship, C=C₀+F·Ar where Ar is the absorbance of a plutonium solution of unknown concentration C mg/g. Various parameters like choice of acid and acidity, slit width, oxidant etc. were studied and the conditions optimized. Plutonium in the concentration range of 0.1–0.3 mg/g could be determined with a precision of ±0.5%. Uranium does not interfere. The method is useful for the analysis of a large number of samples on a routine basis.     

Monitoring Conformational Changes in an Enzyme Conversion Inhibitor Using Pure Shift Exchange NMR Spectroscopy

We report the acquisition of 2D NMR EXSY spectra with ultrahigh resolution, which allows for probing the slow conformational exchange process in a pharmaceutical compound. The resolution enhancement is achieved by implementing interferogram based PSYCHE homonuclear decoupling to generate a pure shift proton spectrum along the direct domain of the resulting data. The performance of this pure shift EXSY pulse sequence is compared to the standard experiment recorded under identical conditions. It is found that although being less sensitive and requiring a longer acquisition time, the quality of pure shift spectra allows for extracting exchange rates values that are coherent with the ones determined by standard approach, on a temperature range that demonstrates the robustness of the chosen homonuclear decoupling method. The resolution enhancement provided by the simplification of proton line shape allows for probing a higher number of proton sites whose analysis would have been biased using a standard method. These results open the way to a thorough and accurate study of chemical exchange processes based on a multi-site analysis of 2D pure shift EXSY spectra     

A secondary ion microprobe ion trap mass spectrometer

An ion trap mass analyzer has been attached to an organic secondary ion microprobe. A pressure differential >100 can be maintained between the ion trap and microprobe. The well-focused secondary ion beam can transit a small (2 mm) diameter tube, but gas flow from ion trap to microprobe is impeded. This pressure differential allows the microprobe to retain imaging capability. Ion trap and microprobe data systems are integrated by taking advantage of the highly reproducible periodicity of the ion trap operating in resonant ejection mode and asynchronous signal and data acquisition afforded by commercially available interface cards. Secondary ion mass spectra and images obtained indicate an approximately 10-fold improvement in sensitivity, although preliminary evidence indicates low (<1%) trapping efficiency. Image data acquisition using the ion trap for mass analysis requires at least 10 times as much time compared to using a quadrupole mass filter because the mass-selected instability mode is used for mass analysis, i.e., mass resolution in the ion trap is not continuous as it is in the quadrupole.     

A comparison of single and double pulse laser-induced breakdown spectroscopy of aluminum samples

Single and double pulse laser-induced breakdown spectroscopy (LIBS) was carried out on aluminum samples in air. In the case of double pulse excitation, experiments were conducted by using the same laser source operated at the same wavelength (1064 nm in most cases here presented). A lowering of the second pulse plasma threshold was observed, together with an overall enhancement in line emission for the investigated time delay between the two pulses (40–60 μs). The laser-induced plasma originated by a single and double pulse was investigated near ignition threshold with the aim to study possible dynamical mechanisms in different regimes. Currently available spectroscopic diagnostics of plasma, such as the line broadening and shift due Stark effects, have been used in the characterization in order to retrieve electron densities, while standard temperature measurements were based on Boltzmann plot. Plasma relevant parameters, such as temperature and electron density, have been measured in the plasma decay on a long time scale, and compared with crater shape (diameter and inferred volume). The comparison of double with single pulse laser excitation was carried out while keeping constant the energy per pulse; the influence of laser energy was investigated as well. Results here obtained suggest that use of the double pulse technique could significantly improve the analytical capabilities of LIBS technique in routine laboratory experiments.     

Divided-evolution-based pulse scheme for quantifying exchange processes in proteins: powerful complement to relaxation dispersion experiments

A method for quantifying millisecond time scale exchange in proteins is presented based on scaling the rate of chemical exchange using a 2D (15)N, (1)H(N) experiment in which (15)N dwell times are separated by short spin-echo pulse trains. Unlike the popular Carr-Purcell-Meiboom-Gill (CPMG) experiment where the effects of a radio frequency field on measured transverse relaxation rates are quantified, the new approach measures peak positions in spectra that shift as the effective exchange time regime is varied. The utility of the method is established through an analysis of data recorded on an exchanging protein-ligand system for which the exchange parameters have been accurately determined using alternative approaches. Computations establish that a combined analysis of CPMG and peak shift profiles extends the time scale that can be studied to include exchanging systems with highly skewed populations and exchange rates as slow as 20 s(-1).     

Pulsed voltammetric stripping at the thin-film mercury electrode

A computer-controlled data acquisition system was used to generate comparative data for thin-film anodic stripping voltammetry with staircase, differential pulse and squarewave waveforms. Each waveform was tested for its sensitivity and speed of analysis. The square-wave form is the most sensitive, whether square wave or differential pulse measurements are used. This waveform offers the advantages of fast analysis time and discrimination against charging currents.     

An inter-calibration campaign using various selected Pu spike isotopic reference materials

In nuclear safeguards, precise and accurate isotopic analyses are needed for two major elements from the nuclear fuel cycle: uranium and plutonium. This can be achieved by Isotope Dilution Mass Spectrometry (IDMS), which is one of the most reliable analytical techniques for the determination of plutonium amount content to a high level of accuracy. In order to achieve reliable isotope measurements isotopic reference materials with certified amount of plutonium and isotopic composition are required. At the Institute for Reference Materials and Measurements (IRMM) various plutonium spike reference materials for isotopes ²³⁹Pu, ²⁴⁰Pu, ²⁴²Pu and ²⁴⁴Pu are available. This enabled the setup of an inter-calibration campaign inter-linking selected plutonium spikes on a metrological basis applying state-of-the-art measurement procedures. The aim of this campaign is threefold: firstly to perform measurements on selected plutonium spike isotopic reference materials for quality control purposes, secondly to verify the amount content and the isotopic composition of the recently produced IRMM-1027m large sized dried (LSD) spikes and thirdly to demonstrate IRMM’s measurement capabilities for plutonium analysis via external quality tools. The obtained results using various spike isotopic reference materials will be presented and discussed in this paper. The measurement uncertainties of the IDMS results were calculated according to the guide to the expression of uncertainty in measurement (GUM).     

Pulse radiolysis system based on ELU-6E LINAC

Basic parameters and operating characteristics of the linear microwave accelerator ELU-6 and pulse radiolysis system are given. LINAC can operate in single pulse mode or with repetition of 10 to 200 pps. In nanosecond mode electron pulses of 7, 10 and 17 ns, beam currents10 A and an energy of 8 MeV are generated. In microsecond mode, duration of pulses can be varied from 0.3 to 4.5 s, beam current up to 1.2 A and energy not less than 5 MeV are available. Versatile pulse radiolysis system enables routine optical measurements in the range from 350 to 2500 nm on the time scale from 10 ns to a few seconds. Transient signals are recorded by a 400 MHz storage Tektronix oscilloscope or by an IWATSU TS8123 digital oscilloscope linked to an Apple II microcomputer incorporated into the Institute computer system. A set up for pulse conductivity measurements with s resolution time is also available.     

Pu-239/Pu-240 isotope ratios determined using high resolution emission spectroscopy in a laser-induced plasma

Laser-induced breakdown spectroscopy (LIBS) has been applied for the determination of plutonium isotope ratios through direct observation of atomic emission from laser-induced plasmas at high resolution. The Pu-239/Pu-240 isotope shift of −0.355 cm⁻¹ from the plutonium atomic line at 594.52202 nm (Blaise et al., The Atomic Spectrum of Plutonium, Argonne National Laboratory Report ANL-83-95, 1984) is clearly resolved in our plasma conditions. Atomic emission is dispersed through a 2-m spectrometer in double pass mode and collected on an electronically gated, intensified charge-coupled device (ICCD) camera. The integrated peak areas obtained from curve-fitting closely match the Pu-239/Pu-240 isotopic ratios obtained from standard methods of thermal ionization mass spectrometry and gamma spectrometry. The observed plutonium linewidths were 0.19 cm⁻¹ (0.0067 nm). These linewidths are within the experimental error of the ideal instrument-limited linewidth, which is calculated to be 0.15 cm⁻¹ (0.0052 nm) based upon the known modulation transfer function for the ICCD system. This linewidth should allow LIBS to be applicable for isotopic ratio measurements for all of the light actinides.     

A semi-automated approach for processing and extracting low-level plutonium from soil

A semi-automated approach for processing and extracting plutonium from large soil samples has been developed and implemented in our laboratory. The system approach was designed around basic plutonium techniques already published in the literature. The technique was carried out using modular work stations for the major analytical steps, namely; soil leaching, plutonium extraction and phase evaporation. This allowed the laboratory to operate in a batch mode which improved sample flow and significantly reduced analysis cost and time. The method has been validated and is routinely used in our laboratory. Chemical yield, accuracy and precision were comparable to those obtained from manual procedures. The system was designed with built-in flexibility to accommodate revised or similar procedures and to complement most existing laboratory equipment and resources.     

Determination of K-factors for isotope abundance measurements of uranium and plutonium by thermal ionisation mass spectrometry

K-factors (= certified isotope ratio/observed isotope ratio) are determined for the isotope abundance measurements of uranium and plutonium by thermal ionisation mass spectrometry. An mdf of 0.07% and 0.18% per mass unit differing by a factor of about 3, is obtained for uranium and plutonium, respectively, employing double rhenium filament assembly in the ion source and Faraday cup as the detector using the presently available isotopic reference materials of uranium and plutonium.     

Optimizing electron-capture detector performance for gas chromatographic analysis of polychlorinated biphenyls

Summary A modified simplex technique has been applied to optimize the performance of an electron-capture detector under constant-current and constant-frequency pulse modes for the gas chromatographic analysis of polychlorinated biphenyls. Optimal performance was obtained by adjusting the four experimental parameters of pulse voltage, detector temperature, make-up gas flow rate, and reference current (or pulse frequency) using the signal-to-noise ratio of the eluting peak as the criterion. The results show that constant-current pulse mode has better sensitivity and dynamic range whereas the constant-frequency pulse mode has the lower detection limit. Subsequent principal component factor analysis indicated that similar performance is achievable using only three parameters.     

Practical limits of high speed chromatography: Evaluation with a computer based theoretical model of several commercially available gas chromatographs

Several commercially available chromatographs have been evaluated with respect to their ability to support high speed chromatography. Both autoinjection and trap and thermal pulse injection are both found to be suitable procedures to deliver the required narrow sample injection peak widths. However, even with the appropriate injectors in place, the systems are burdened with relatively slow data acquisition rates and large amplifier time constants that further degrade system performance and make them unsuitable for the high speed chromatography discussed herein. The study was conducted using a computer implemented model of a gas chromatograph's output.     

Effect of mixed solvent media on the sorption and separation of uranium and plutonium on macroporous resins

Ion-exchange studies on uranium and plutonium using macroporous (MP) anion-exchange resins from an aqueous-organic solvent mixed media were carried out to develop a separation method. Out of the several water miscible organic solvents tried methanol and acetone were found to be best suited. Distribution data were obtained for U(VI) and Pu(IV) using three macroporous resins under various parameters. Based on these data, separation factors for Pu/U were calculated. Column experiments using Tulsion A-27(MP) were also carried out. The method has the advantage of loading plutonium from as low as 1M nitric acid in the presence of methanol or acetone and could be used satisfactorily for its recovery from solutions containing plutonium and uranium.     

HSQC-ADEQUATE: an investigation of data requirements

Utilizing ¹³C‐¹³C connectivity networks for the assembly of carbon skeletons from HSQC‐ADEQUATE spectra was recently reported. HSQC‐ADEQUATE data retain the resonance multiplicity information of the multiplicity‐edited GHSQC spectrum and afford a significant improvement in the signal‐to‐noise (s/n) ratio relative to the 1,1‐ADEQUATE data used in the calculation of the HSQC‐ADEQUATE spectrum by unsymmetrical indirect covariance (UIC) processing methods. The initial investigation into the computation of HSQC‐ADEQUATE correlation plots utilized overnight acquisition of the 1,1‐ADEQUATE data used for the calculation. In this communication, we report the results of an investigation of the reduction in acquisition time for the 1,1‐ADEQUATE data to take advantage of the s/n gain during the UIC processing to afford the final HSQC‐ADEQUATE correlation plot. Data acquisition times for the 1,1‐ADEQUATE spectrum can be reduced to as little as a few hours, while retaining excellent s/n ratios and all responses contained in spectra computed from overnight data acquisitions. Concatenation of multiplicity‐edited GHSQC and 1,1‐ADEQUATE data also allows the interrogation of submilligram samples with 1,1‐ADEQUATE data when using spectrometers equipped with 1.7‐mm Micro CryoProbes ?. Copyright © 2011 John Wiley & Sons, Ltd.     

Monitoring dynamic systems with multiparameter fluorescence imaging

A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.     

Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays

Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NFkappaB, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca(2+) sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.     

Simultaneous quantification and identification using <Superscript>18</Superscript>O labeling with an ion trap mass spectrometer and the analysis software application “ZoomQuant”

Stable isotope labeling with (18)O is a promising technique for obtaining both qualitative and quantitative information from a single differential protein expression experiment. The small 4 Da mass shift produced by incorporation of two molecules of (18)O, and the lack of available methods for automated quantification of large data sets has limited the use of this approach with electrospray ionization-ion trap (ESI-IT) mass spectrometers. In this paper, we describe a method of acquiring ESI-IT mass spectrometric data that provides accurate calculation of relative ratios of peptides that have been differentially labeled using(18)O. The method utilizes zoom scans to provide high resolution data. This allows for accurate calculation of (18)O/(16)O ratios for peptides even when as much as 50% of a (18)O labeled peptide is present as the singly labeled species. The use of zoom scan data also provides sufficient resolution for calculating accurate ratios for peptides of +3 and lower charge states. Sequence coverage is comparable to that obtained with data acquisition modes that use only MS and MS/MS scans. We have employed a newly developed analysis software tool, ZoomQuant, which allows for the automated analysis of large data sets. We show that the combination of zoom scan data acquisition and analysis using ZoomQuant provides calculation of isotopic ratios accurate to approximately 21%. This compares well with data produced from (18)O labeling experiments using time of flight (TOF) and Fourier transform-ion cyclotron resonance (FT-ICR) MS instruments.