Analysis Of Complex Mixtures Research Articles

Differentiation of oligosaccharide isomers by direct infusion multidimensional mass spectrometry.

Oligosaccharides demonstrate many bioactivities with applications in the pharmaceutical, cosmetic, and food industries. They also serve as biomarkers for various diseases including cancer and glycogen storage disorders. These make the structural characterization of oligosaccharides very important. Unfortunately, the structural diversity found in saccharides make their characterization challenging, necessitating the development of sophisticated instrumentation to enable isomer differentiation. Herein, we report the ability of halide (Cl- and Br-) adducts to enable direct differentiation of oligosaccharide isomers using conventional collision-induced dissociation (CID) tandem MS (MS/MS). The halide adducts were generated by direct infusion nano-electrospray ionization (nESI). For the first time, this traditional nESI CID MS/MS platform was used to differentiate stereoisomers of trisaccharides (cellotriose β(1 → 4) and maltotriose α(1 → 4), tetrasaccharides (cellotetraose and maltotetraose), and pentasaccharides (cellopentaose and maltopentaose)). In addition, the MS/MS of halide adducts enabled the differentiation of positional, structural, and linkage isomers from a total of 14 oligosaccharides. The isomer differentiation was realized by the generation of distinct diagnostic fragment ions in CID. We also performed principal component analysis using the entire range of MS/MS fragment ion profiles and found that negative-ion mode halide adduction provided more effective isomer differentiation compared with positive-ion mode sodium adduction. Finally, we demonstrated complex mixture analysis by spiking all 14 oligosaccharides into raw urine, of which we successfully distinguished species based on molecular weight (first dimension) and CID MS/MS fragmentation patterns as the second dimension separation. This work effectively showcases the potential to use direct infusion nESI-MS/MS to characterize synthetic oligosaccharide isomers in unpurified reaction mixture as well as from biofluids for diagnostic purposes.

Qualitative analysis of aromatic compounds via 1D TOCSY techniques

The aromatic compounds, including o-xylene, m-xylene, p-xylene, and ethylbenzene, primarily originate from the catalytic reforming of crude oil, and have a wide variety of applications. However, because of similar physical and chemical properties, these compounds are difficult to be identified by gas chromatography (GC) without standard samples. With the development of modern nuclear magnetic resonance (NMR) techniques, NMR has emerged as a powerful and efficient tool for the rapid analysis of complex and crude mixtures without purification. In this study, the parameters of one-dimensional (1D) total correlation spectroscopy (TOCSY) NMR techniques, including 1D selective gradient TOCSY and 1D chemical-shift-selective filtration (CSSF) with TOCSY, were optimized to obtain comprehensive molecular structure information. The results indicate that the overlapped signals in NMR spectra of nonpolar aromatic compounds (including o-xylene, m-xylene, p-xylene and ethylbenzene), polar aromatic compounds (benzyl alcohol, benzaldehyde, benzoic acid), and aromatic compounds with additional conjugated bonds (styrene) can be resolved in 1D TOCSY. More importantly, full molecular structures can be clearly distinguished by setting appropriate mixing time in 1D TOCSY. This approach simplifies the NMR spectra, provides structural information of entire molecules, and can be applied for the analysis of other structural isomers.

Online Bipolar Dual Spray for the Charge State Reduction and Characterization of Complex Synthetic Polymers.

Charge reduction mass spectrometry (CR/MS) hyphenated to liquid chromatography (LC) couples liquid-phase compound separation and mass spectral decompression to resolve and characterize multicomponent systems. LC/CR/MS has proven to be effective for complex mixture analysis, particularly synthetic polymers. A newer charge manipulation approach called bipolar dual spray has previously been demonstrated to reduce the observed charge state distribution of ammoniated polyethene glycol. In this approach, two electrospray emitters, in close proximity and of opposite polarity, fuse droplets from their electrospray plumes, which allows the subsequent chemistry. In this work, we investigate the ability of bipolar dual spray to reduce the charge of synthetic polyols, thereby simplifying complex mixture analysis and generating new compositional information only available through the coupling of charge reduction with LC/MS analysis. This work also represents the first demonstration of online charge reduction via dual spray. Polyethylene glycol (PEG) 7.2K subjected to LC/MS with dual spray reduced the average charge state from 8.2+ to 4.4+. LC/MS with dual spray was also applied to the characterization of an end-group-modified PEG 10K (i.e., aminated) containing several reaction impurities. This approach allowed for the identification of low-level starting material, tosylated PEG, and PEG mono(amine), where both LC/MS and direct infusion dual spray did not detect the impurities. Overall, the results demonstrated that bipolar dual spray can be incorporated into an LC/MS analysis and affords the ability to reduce the charge state distribution of PEG cations, decompress the m/z axis, lower spectra complexity, and enable/simplify data interpretation.

Single-Scan Ultraselective NMR Experiments with Preserved Sensitivity.

Selective NMR experiments provide rapid access to important structural information, and are essential to tackle the analysis of large molecules and complex mixtures. Single-scan ultraselective experiments are particularly useful, as they can rapidly select signals that overlap with other signals. Here, we describe a novel type of single-scan ultraselective NMR experiments that is robust against the effects of translational molecular diffusion, and thus make it possible to improve significantly the sensitivity of the experiment. This will largely broaden the applicability of this powerful class of experiments.

Single‐Scan Ultraselective NMR Experiments with Preserved Sensitivity

AbstractSelective NMR experiments provide rapid access to important structural information, and are essential to tackle the analysis of large molecules and complex mixtures. Single‐scan ultraselective experiments are particularly useful, as they can rapidly select signals that overlap with other signals. Here, we describe a novel type of single‐scan ultraselective NMR experiments that is robust against the effects of translational molecular diffusion, and thus make it possible to improve significantly the sensitivity of the experiment. This will largely broaden the applicability of this powerful class of experiments.

Hyperpolarized 1H and 13C NMR Spectroscopy in a Single Experiment for Metabolomics.

The application of NMR spectroscopy to complex mixture analysis and, in particular, to metabolomics is limited by the low sensitivity of NMR. We recently showed that dissolution dynamic nuclear polarization (d-DNP) could enhance the sensitivity of 13C NMR for complex metabolite mixtures, leading to the detection of highly sensitive 13C NMR fingerprints of complex samples such as plant extracts or urine. While such experiments provide heteronuclear spectra, which are complementary to conventional NMR, hyperpolarized 1H NMR spectra would also be highly useful, with improved limits of detection and compatibility with the existing metabolomics workflow and databases. In this technical note, we introduce an approach capable of recording both 1H and 13C hyperpolarized spectra of metabolite mixtures in a single experiment and on the same hyperpolarized sample. We investigate the analytical performance of this method in terms of sensitivity and repeatability, and then we show that it can be efficiently applied to a plant extract. Significant sensitivity enhancements in 1H NMR are reported with a repeatability suitable for metabolomics studies without compromising on the performance of hyperpolarized 13C NMR. This approach provides a way to perform both 1H and 13C hyperpolarized NMR metabolomics with unprecedented sensitivity and throughput.

Improved analysis of derivatized steroid hormone isomers using ion mobility-mass spectrometry (IM-MS).

Over the last decade, applications of ion mobility-mass spectrometry (IM-MS) have exploded due primarily to the widespread commercialization of robust instrumentation from several vendors. Unfortunately, the modest resolving power of many of these platforms (~40-60) has precluded routine separation of constitutional and stereochemical isomers. While instrumentation advances have pushed resolving power to >150 in some cases, chemical approaches offer an alternative for increasing resolution with existing IM-MS instrumentation. Herein we explore the utility of two reactions, derivatization by Girard's reagents and 1,1-carbonyldiimidazole (CDI), for improving IM separation of steroid hormone isomers. These reactions are fast (≤30min), simple (requiring only basic lab equipment/expertise), and low-cost. Notably, these reactions are structurally selective in that they target carbonyl and hydroxyl groups, respectively, which are found in all naturally occurring steroids. Many steroid hormone isomers differ only in the number, location, and/or stereochemistry of these functional groups, allowing these reactions to "amplify" subtle structural differences and improve IM resolution. Our results show that resolution was significantly improved amongst CDI-derivatized isomer groups of hydroxyprogesterone (two-peak resolution of Rpp = 1.10 between 21-OHP and 11B-OHP), deoxycortisone (Rpp = 1.47 between 11-DHC and 21-DOC), and desoximetasone (Rpp = 1.98 between desoximetasone and fluocortolone). Moreover, characteristic collision cross section (DTCCSN2) measurements can be used to increase confidence in the identification of these compounds in complex biological mixtures. To demonstrate the feasibility of analyzing the derivatized steroids in complex biological matrixes, the reactions were performed following steroid extraction from urine and yielded similar results. Additionally, we applied a software-based approach (high-resolution demultiplexing) that further improved theresolving power (>150). Overall, our results suggest that targeted derivatization reactions coupled with IM-MS can significantly improve the resolution of challenging isomer groups, allowing for more accurate and efficient analysis of complex mixtures.

Light-Induced Orthogonal Fragmentation of Crosslinked Peptides.

Crosslinking mass spectrometry provides pivotal information on the structure and interaction of proteins. MS-cleavable crosslinkers are regarded as a cornerstone for the analysis of complex mixtures. Yet they fragment under similar conditions as peptides, leading to mixed fragmentation spectra of the crosslinker and peptide. This hampers selecting individual peptides for their independent identification. Here, we introduce orthogonal cleavage using ultraviolet photodissociation (UVPD) to increase crosslinker over peptide fragmentation. We designed and synthesized a crosslinker that can be cleaved at 213 nm in a commercial mass spectrometer configuration. In an analysis of crosslinked Escherichia coli lysate, the crosslinker-to-peptide fragment intensity ratio increases from nearly 1 for a conventionally cleavable crosslinker to 5 for the UVPD-cleavable crosslinker. This largely increased the sensitivity of selecting the individual peptides for MS3, even more so with an improved doublet detection algorithm. Data are available via ProteomeXchange with identifier PXD040267.

Field-portable detection of fentanyl and its analogs: A review.

The need to detect fentanyl and its analogs in the field is an important capability to help prevent unintentional exposure or overdose on these substances, which may result in death. Many portable methods historically used in the field by first responders and other field users to detect and identify other chemical substances, such as hazardous materials, have been applied to the detection and identification of these synthetic opioids. This paper describes field portable spectroscopic methods used for the detection and identification of fentanyl and its analogs. The methods described are automated colorimetric tests including lateral flow assays; vibrational spectroscopy (mid-infrared and Raman); gas chromatography-mass spectrometry; ion mobility spectrometry, and high-pressure mass spectrometry. In each case the background and key details of these technologies are outlined, followed by a discussion of the application of the technology in the field. Attention is paid to the analysis of complex mixtures and limits of detection, including the required spectral databases and algorithms used to interrogate these types of samples. There is also an emphasis on providing actionable information to the (likely) non-scientist operators of these instruments in the field.

MultiplexMS: A Mass Spectrometry-Based Multiplexing Strategy for Ultra-High-Throughput Analysis of Complex Mixtures.

High-throughput chemical analysis of natural product mixtures lags behind developments in genome sequencing technologies and laboratory automation, leading to a disconnect between library-scale chemical and biological profiling that limits new molecule discovery. Here, we report a new orthogonal sample multiplexing strategy that can increase mass spectrometry-based profiling up to 30-fold over traditional methods. Profiled pooled samples undergo subsequent computational deconvolution to reconstruct peak lists for each sample in the set. We validated this approach using in silico experiments and demonstrated a high assignment precision (>97%) for large, pooled samples (r = 30), particularly for infrequently occurring metabolites of relevance in drug discovery applications. Requiring only 5% of the previously required MS acquisition time, this approach was repeated in a recent biological activity profiling study on 925 natural product extracts, leading to the rediscovery of all previously reported bioactive metabolites. This new method is compatible with MS data from any instrument vendor and is supported by an open-source software package: https://github.com/liningtonlab/MultiplexMS.

Ultrahigh performance LC/FT-MS non-targeted screening for biomass burning organic aerosol with MZmine2 and MFAssignR

In recent years, ultrahigh performance liquid chromatography Fourier transform mass spectrometry (LC/FT-MS) based non-targeted screening (NTS) methods have become increasingly popular for comprehensive analysis of complex organic mixtures. However, applying these methods for environmental complex mixture analysis is challenging due to the extreme complexity of natural samples and a lack of standard samples or surrogates for environmental complex mixtures. Furthermore, limited molecular markers in the databases and insufficient data processing software workflows make the application of these methods more challenging for environmental complex mixtures. In this work, we implement a new NTS data processing workflow to process data collected from ultrahigh performance liquid chromatography and Fourier transform Orbitrap Elite Mass Spectrometry (LC/FT-MS) by combining MZmine2 and MFAssignR, two opensource data processing tools and commercial Mesquite liquid smoke as a surrogate for biomass burning organic aerosol. MZmine2.53 data extraction followed MFAssignR molecular formula assignment offered noise free and highly accurate 1733 individual molecular formulas presented in liquid smoke with 4906 molecular species, including isomers. The results of this new approach were consistent with the results of direct infusion FT-MS analysis confirming its reliability. Over 90% of the molecular formulas presented in mesquite liquid smoke were matched with the molecular formulas of ambient biomass burning organic aerosol. This suggests the potential use of commercial liquid smoke is an acceptable surrogate for biomass burning organic aerosol research. The presented method significantly improves the identification of the molecular composition of biomass burning organic aerosol by successfully addressing some of the limitations related to the data analysis and giving a semi quantitative insight into the analysis.

An Optimized Two-Dimensional Quantitative Nuclear Magnetic Resonance Strategy for the Rapid Quantitation of Diester-Type C19-Diterpenoid Alkaloids from Aconitum carmichaelii.

With the development of nuclear magnetic resonance (NMR) spectrometers and probes, two-dimensional quantitative nuclear magnetic resonance (2D qNMR) technology with a high signal resolution and great application potential has become increasingly accessible for the quantitation of complex mixtures. However, the requirement that the relaxation recovery time be equal to at least five times T1 (longitudinal relaxation time) makes it difficult for 2D qNMR to simultaneously achieve high quantitative accuracy and high data acquisition efficiency. By comprehensively using relaxation optimization and nonuniform sampling, we successfully established an optimized 2D qNMR strategy for HSQC experiments at the half-hour level and then accurately quantified the diester-type C19-diterpenoid alkaloids in Aconitum carmichaelii. The optimized strategy had the advantages of high efficiency, high accuracy, good reproducibility, and low cost and thus could serve as a reference to optimize 2D qNMR experiments for quantitative analysis of natural products, metabolites, and other complex mixtures.

Hyperpolarized 13C NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization.

Hyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of NMR. Dissolution Dynamic Nuclear Polarization (d-DNP) provides a unique and general way to detect 13C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d-DNP now encompasses the analysis of complex mixtures at natural 13C abundance. However, it has in this area been limited to metabolite extracts. Here, we report the first d-DNP-enhanced 13C NMR analysis of a biofluid -urine- at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

Hyperpolarized 13C NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization

AbstractHyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of conventional NMR. Dissolution Dynamic Nuclear Polarization (d‐DNP) provides a unique and general way to detect 13C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d‐DNP now encompasses the analysis of complex mixtures at natural 13C abundance. However, the application of d‐DNP in this area has been limited to metabolite extracts. Here, we report the first d‐DNP‐enhanced 13C NMR analysis of a biofluid ‐urine‐ at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

High-Throughput Nanoliter Sampling and Direct Analysis of Biological Fluids Using Droplet Imbibition Mass Spectrometry.

A high-throughput droplet imbibition mass spectrometry (MS) experiment is reported for the first time that allows direct analysis of ultra-small volumes of complex mixtures. In this experiment, an array of optimized tips of glass capillaries containing the analyte solution is sampled by rapidly moving charged microdroplets, which picks up (i.e., imbibes) the analyte and transfers it to a proximal mass spectrometer. The advantages associated with this droplet imbibition experiment include (1) ultra-small sample consumption (1.3 nL/min), which reduces the matrix effect in complex mixture analysis, and (2) high surface activity, which eliminates ion suppression effects caused by competition for the space charge on the droplet surface. Collectively, the enhanced surface effect and small flow rates dramatically increase the sensitivity of the droplet imbibition MS approach. This was experimentally shown by constructing calibration curves for cocaine analysis in human raw urine and whole blood, achieving 2 and 7 pg/mL limits of detection, respectively. The high-throughput feature was demonstrated by analyzing five structurally different compounds in 20 s intervals. With the measured flow rate of 1.3 nL/min on a 5 μm glass tip size, the results described in the current study showcase droplet imbibition MS to be a powerful and high-throughput alternative for conventional nano-electrospray ionization (flow rate <100 nL/min), which is the most efficient method for transferring small sample volumes to mass spectrometers.

Environmentally sustainable computationally spectrophotometric resolution strategy for analysis single-tablet regimens of antihypertension with overlapped spectra

Single tablet regimens (STRs) for hypertension suppression improve patient satisfaction, quality of life, medication and adherence compared to multi-tablet regimens (MTRs). Development of simple cost effective and at the same time green an ecofriendly method for analysis and resolution of multicomponent and complex mixtures is an increasingly important issue in the field of pharmaceutical industry in general and analytical chemistry in specific. Thus, the presented research deals with two main goals, the first one is concerned with developing and applying a fully validated resolution plan using numerical data of signals on manipulated spectra for successive extraction of parent spectra of drugs in single-tablet Regimens (STRs) used in antihypertensive treatment. The STRs contains three drugs with deconvoluted spectra, Atenolol (ATL), Amiloride (AMD) and Chlortalidone (CLN). The resolution plan encompasses two simple and affordable spectrophotometric methods employing resolving spectra either normalized spectrum (NS) or factorized spectrum (FS) which acts as a master key for unlocking the overlapped spectra of ternary mixture with no need for any preliminary physical separation, sample pretreatment or pH adjustment. These two main methods namely, dual amplitude difference (DAD) and derivative ratio transformation (DDT). The presented approaches required a simple spectrophotometer with its built-in program using primitive mathematical operations to restore the parent spectrum for individually drug cited in the mixture understudy. The proposed methods validity was checked and evaluated through the ICH guidelines and displayed linearity within concentration range 4.0-40.0μg/mL for ATL, 3.0-20.0μg/mL for both AMD and CLN. While the specificity evaluation was performed via assaying the three drugs accurately and precisely in their synthetic mixtures and in their STRs. Generally, these methods could be recruited for the fast and effective analysis and determination of the purity index for AMD, CLN and ATL in bulk materials and available market formulation, Teklo ® tablets.The second main goal of the research was the evaluation of the methods greenness and whiteness where the applied UV-methods and the reported HPLC one had significantly different greenness scores when compared using three metrics: the Green Certificate Classification (GCC), the Complex Green Analytical Procedure Index (Complex-GAPI), and the Analytical Greenness metric approach (AGREE).Also, the whiteness scores, which reflected the degree of the achieved sustainability for the HPLC reported technique and the produced UV ones were done by using the ideologies of the white analytical chemistry (WAC) tool. All the differences between the four aforementioned metrics are discussed in this study.

Advances, Applications, and Challenges in RP HPLC Method Development

High-performance liquid chromatography (HPLC) has become an indispensable tool for modern analytical chemistry, and reversed-phase (RP) HPLC is one of the most widely used techniques for the separation and analysis of complex mixtures. In recent years, there have been numerous advances in RP HPLC method development, including the development of new stationary phases, improved column technology, and novel optimization strategies. These advances have led to increased speed, resolution, and sensitivity, as well as improved selectivity and reduced non-specific adsorption. Moreover, RP HPLC has found applications in a wide range of industries, including pharmaceuticals, food and beverage, environmental analysis, forensic science, and biotechnology. Despite its many benefits, RP HPLC method development is not without its challenges, including issues with column stability, sample preparation, and optimization. In this review article, we provide a comprehensive overview of the principles, optimization strategies, and recent advances in RP HPLC method development. We discuss the applications of RP HPLC in various industries and identify the common challenges and emerging trends in the field. Additionally, we evaluate the impact of RP HPLC on the accuracy, sensitivity, and selectivity of analysis and its potential to improve the quality and safety of products and the environment. Overall, this review highlights the importance of RP HPLC in modern analytical chemistry and provides insights and recommendations for researchers and practitioners who are interested in this technique. By exploring the latest advances, applications, and challenges in RP HPLC method development, we hope to inspire continued innovation and progress in this important field.

Improving annotation propagation on molecular networks through random walks: introducing ChemWalker.

Annotation of the mass signals is still the biggest bottleneck for the untargeted mass spectrometry analysis of complex mixtures. Molecular networks are being increasingly adopted by the mass spectrometry community as a tool to annotate large-scale experiments. We have previously shown that the process of propagating annotations from spectral library matches on molecular networks can be automated using Network Annotation Propagation (NAP). One of the limitations of NAP is that the information for the spectral matches is only propagated locally, to the first neighbor of a spectral match. Here, we show that annotation propagation can be expanded to nodes not directly connected to spectral matches using random walks on graphs, introducing the ChemWalker python library. Similarly to NAP, ChemWalker relies on combinatorial in silico fragmentation results, performed by MetFrag, searching biologically relevant databases. Departing from the combination of a spectral network and the structural similarity among candidate structures, we have used MetFusion Scoring function to create a weight function, producing a weighted graph. This graph was subsequently used by the random walk to calculate the probability of 'walking' through a set of candidates, departing from seed nodes (represented by spectral library matches). This approach allowed the information propagation to nodes not directly connected to the spectral library match. Compared with NAP, ChemWalker has a series of improvements, on running time, scalability and maintainability and is available as a standalone python package. ChemWalker is freely available at https://github.com/computational-chemical-biology/ChemWalker. ridasilva@usp.br. Supplementary data are available at Bioinformatics online.

In Silico Demonstration of Two-Dimensional Mass Spectrometry Using Spatially Dependent Fragmentation.

Two-dimensional mass spectrometry (2DMS) allows for the analysis of complex mixtures of all kinds at high speed and resolution without data loss from isolation or biased acquisition, effectively generating tandem mass spectrometry information for all ions at once. Currently, this technique is limited to instruments utilizing an ion trap such as the Fourier transform ion cyclotron resonance or linear ion traps. To overcome this limitation, new fragmentation waveforms were used in either a temporal or spatial configuration, allowing for the application of 2DMS on a much wider array of instruments. A simulated example of a time-of-flight-based instrument is shown with the new waveforms, which allowed for the correlation of fragment ions to their respective precursors through the processing of the modulation of fragmentation intensity with a Fourier transform. This application indicated that 2D modulation and Fourier precursor/fragment intensity correlation are possible in any case where separation, either temporally or spatially, can be achieved, allowing 2DMS to be applied to almost every type of mass spectrometry instrument.

19F-labeled molecular probes for NMR-based detection

Nuclear magnetic resonance (NMR) is a unique analytical technique because it is capable of providing structural information about molecules at the atomic level. However, the complicated spectral assignments and the inherently low sensitivity without resorting to hyperpolarization methods hamper the widespread applications of NMR techniques in the analysis of complex real-world samples. Through a combination of 19F NMR and ingeniously designed molecular probes, the dynamic recognitions of target analytes can be transduced into easily interpretable 19F NMR signals of characteristic chemical shifts. The method has demonstrated robust performance for the precise multi-component analysis of complex mixtures and the enantiodifferentiation of chiral analytes that are difficult to resolve by chiral chromatographic methods. Here, we highlight a few recently developed 19F-labeled probes and provide discussions on their recognition properties, analyte scopes, and applications under different scenarios.