MS and NMR Analysis of Isotopically Labeled Chloramination Disinfection Byproducts: Hyperlinks and Chemical Reactions.

FT-ICR MS and NMR analysis of an isotopically labeled complex mixture of water disinfection byproducts formed by chloramine disinfection of model phenolic acids is described. A new molecular formula assignment procedure using the CoreMS Python library able to assign isotopically enriched formulas is proposed. Statistical analysis of the assigned formulas showed that the number of compounds, the diversity of the mixture, and the chlorine count increase during the chloramination reaction. The complex reaction mixture was investigated as a network of reactions using PageRank and Reverse PageRank algorithms. Independent of the MS signal intensities, the PageRank algorithm calculates the formulas with the highest probability at convergence of the reaction; these were chlorinated and nitrated derivatives of the starting materials. The Reverse PageRank revealed that the most probable chemical transformations in the complex mixture were chlorination and decarboxylation. These agree with the data obtained from INADEQUATE NMR spectra and literature data, indicating that this approach could be applied to gain insight into reactions pathways taking place in complex mixtures without any prior knowledge.

Clean PDI-1 SQ: Suppression of HSQC artifacts in 2D proton-detected INADEQUATE spectra by pulse sequence redesign.

Proton-detected INADEQUATE NMR experiments are widely used for structure elucidation of small molecules, in particular the implementations that display 13C single-quantum rather than double-quantum frequencies in the indirect dimension of 2D spectra. But unfortunately, such spectra in addition to the desired 1H-13C two-bond correlations also contain HSQC artifacts of comparable magnitude. The redesigned versatile experiment presented in this paper requires no compromise based on different 13C multiplicities and suppresses the HSQC artifacts that are a source of possible spectral misinterpretation. Demonstration of the new method is shown by applications to typical small molecules of different complexity.

A gas-phase standard delivery system for direct breath analysis.

Applications for direct breath analysis by mass spectrometry (MS) are rapidly expanding. One of the more recent mass spectrometry-based approaches is secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS). Despite increasing usage, the SESI methodology still lacks standardization procedures for quality control and absolute quantification. In this study, we designed and evaluated a custom-built standard delivery system tailored for direct breath analysis. The system enables the simultaneous introduction of multiple gas-phase standard compounds into ambient MS setups in the lower parts-per-million (ppm) to parts-per-billion (ppb) range. To best mimic exhaled breath, the gas flow can be heated (37 °C-40 °C) and humidified (up to 98% relative humidity). Inter-laboratory comparison of the system included various SESI-HRMS setups, i.e. an Orbitrap and a quadrupole time-of-flight mass spectrometer (QTOF), and using both single- as well as multi-component standards. This revealed highly stable and reproducible performances with between-run variation <19% and within-run variation <20%. Independent calibration runs demonstrated high accuracy (96%-111%) and precision (>95%) for the single-compound standard acetone, while compound-specific performances were obtained for the multi-component standard. Similarly, the sensitivity varied for different compounds within the multi-component standard across all SESI-Orbitrap and -QTOF setups, yielding limits of detections from 3.1 ppb (forp-xylene) to 0.05 ppb (for 1,8-cineol). Routinely applying the standard system throughout several weeks, allowed us to monitor instrument stability and to identify technical outliers in exhaled breath measurements. Such routine deployment of standards would significantly improve data quality and comparability, which is especially important in longitudinal and multi-center studies. Furthermore, performance validation of the system demonstrated its suitability for reliable absolute quantification while it illustrated compound-dependent behavior for SESI.

More than ADEQUATE: doubling the sensitivity of 13CH-13CH correlations in double-quantum NMR experiments.

We present modifications of the ADEQUATE experiment which more than double the sensitivity of carbon-carbon correlations of 13CH-13CH moieties. Additionally, these improvements can be applied without a sensitivity penalty to obtain spectra with a 13C chemical shift axis in the indirectly detected dimension, instead of a double-quantum frequency, allowing simpler interpretation of spectra. The modified experiments, which use refocussing of 1JCH couplings and 1H decoupling during JCC evolution intervals, were tested on several molecules, including a pentasaccharide (20 mg, 19 mM), where on average a 2.6-fold signal-to-noise improvement was achieved and the number of observable correlations increased. Doubling sensitivity results in a 4-fold reduction of the experimental time, allowing ADEQUATE spectra to be recorded overnight instead of over multiple days.

Reduced dimensionality hyphenated NMR experiments for the structure determination of compounds in mixtures.

For the structure determination of molecules in mixtures using NMR spectroscopy, the dispersion of 13C chemical shifts provides much needed separation of resonances in the indirectly detected dimension of 2D heterocorrelated NMR experiments. This separation is crucial for establishing networks of coupled spins by hyphenated techniques that combine hetero- and homonuclear polarisation transfers. However, as the sample complexity increases, 13C chemical shifts stop being unique, hindering spectral interpretation. The resulting ambiguities can be removed by adding another dimension to these experiments. However, the spectra obtained from complex samples are riddled with overlapped signals, meaning that another dimension will only reduce the spectral resolution and prevent structure determination. A promising solution is to stay in two dimensions and use the combined 13C and 1H chemical shifts to separate signals. We have developed a suite of (3,2)D reduced dimensionality hyphenated NMR experiments that preserve the information content of 3D spectra but offer all of the advantages of 2D spectra - high resolution and ease of manipulation with only a mild sensitivity penalty. The proposed experiments complement the existing (3,2)D HSQC-TOCSY and include a (3,2)D HSQC-NOESY/ROESY, (3,2)D HSQC-CLIP-COSY and (3,2)D HSQC-HSQMBC. The new experiments represent a set of NMR techniques typically employed in the structure determination of complex compounds and have been adopted here for use on mixtures. The resolving power of these experiments is illustrated on the analysis of hot water extracts of green tea.