Elucidating the Gas-Phase Behavior of Nitazene Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry.

2-Benzylbenzimidazoles, or "nitazenes", are a class of novel synthetic opioids (NSOs) that are increasingly being detected alongside fentanyl analogs and other opioids in drug overdose cases. Nitazenes can be 20× more potent than fentanyl but are not routinely tested for during postmortem or clinical toxicology drug screens; thus, their prevalence in drug overdose cases may be under-reported. Traditional analytical workflows utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) often require additional confirmation with authentic reference standards to identify a novel nitazene. However, additional analytical measurements with ion mobility spectrometry (IMS) may provide a path toward reference-free identification, which would greatly accelerate NSO identification rates in toxicology laboratories. Presented here are the first IMS and collision cross section (CCS) measurements on a set of fourteen nitazene analogs using a structures for lossless ion manipulations (SLIM)-orbitrap MS. All nitazenes exhibited two high intensity baseline-separated IMS distributions, which fentanyls and other drug and druglike compounds also exhibit. Incorporating water into the electrospray ionization (ESI) solution caused the intensities of the higher mobility IMS distributions to increase and the intensities of the lower mobility IMS distributions to decrease. Nitazenes lacking a nitro group at the R1 position exhibited the greatest shifts in signal intensities due to water. Furthermore, IMS-MS/MS experiments showed that the higher mobility IMS distributions of all nitazenes possessing a triethylamine group produced fragment ions with m/z 72, 100, and other low intensity fragments while the lower mobility IMS distributions only produced fragment ions with m/z 72 and 100. The IMS, solvent, and fragmentation studies provide experimental evidence that nitazenes potentially exhibit three gas-phase protomers. The cyclic IMS capability of SLIM was also employed to partially resolve four sets of structurally similar nitazene isomers (e.g., protonitazene/isotonitazene, butonitazene/isobutonitazene/secbutonitazene), showcasing the potential of using high-resolution IMS separations in MS-based workflows for reference-free identification of emerging nitazenes and other NSOs.

[Establishment of Rapid Simultaneous Analysis Method for Plant Toxins by LC-TOF-MS].

Assuming food poisoning caused by toxic plants, an LC-TOF-MS-based method for the rapid and simultaneous analysis of 16 plant toxins was established. After adding water-methanol (1 : 9) and n-hexane, the samples were homogenized and extracted, and then subjected to centrifugal separation. Without any purification procedures, LC-TOF-MS measurements were performed, and qualitative and quantitative analyses using monoisotopic ion [M+H]+ (m/z) were conducted. The addition-recovery test using curry showed that qualitative analysis was possible under a setting with a retention time of ±0.2 minutes or less and mass accuracy of 5 ppm or lower and that quantitative analysis was possible with a recovery rate of 68-142% and a repeatability of 1.4-10.1%. Furthermore, measurements of the amount of plant toxins in the boiled plants and broths of cooked toxic plants demonstrated the transfer of plant toxins to broths. These suggest that in the event of food poisoning, broths may be used as an analysis sample, even when plants are not available.

Identification of Unique Fragmentation Patterns of Fentanyl Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry.

The opioid crisis in the United States is being fueled by the rapid emergence of new fentanyl analogs and precursors that can elude traditional library-based screening methods, which require data from known reference compounds. Since reference compounds are unavailable for new fentanyl analogs, we examined if fentanyls (fentanyl + fentanyl analogs) could be identified in a reference-free manner using a combination of electrospray ionization (ESI), high-resolution ion mobility (IM) spectrometry, high-resolution mass spectrometry (MS), and higher-energy collision-induced dissociation (MS/MS). We analyzed a mixture containing nine fentanyls and W-15 (a structurally similar molecule) and found that the protonated forms of all fentanyls exhibited two baseline-separated IM distributions that produced different MS/MS patterns. Upon fragmentation, both IM distributions of all fentanyls produced two high intensity fragments, resulting from amine site cleavages. The higher mobility distributions of all fentanyls also produced several low intensity fragments, but surprisingly, these same fragments exhibited much greater intensities in the lower mobility distributions. This observation demonstrates that many fragments of fentanyls predominantly originate from one of two different gas-phase structures (suggestive of protomers). Furthermore, increasing the water concentration in the ESI solution increased the intensity of the lower mobility distribution relative to the higher mobility distribution, which further supports that fentanyls exist as two gas-phase protomers. Our observations on the IM and MS/MS properties of fentanyls can be exploited to positively differentiate fentanyls from other compounds without requiring reference libraries and will hopefully assist first responders and law enforcement in combating new and emerging fentanyls.

Spectral Quality Assessment Strategy Based on the 13C-Isotopic Pattern for the Fourier Transform Ion Cyclotron Resonance Mass Spectra of Dissolved Organic Matter.

The interpretation of data and optimization spectral acquisition of dissolved organic matter (DOM) by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) have been challenging due to the varied instrument performances among laboratories and the complex chemical characteristics of DOM. However, a universal spectral optimization strategy for FT-ICR MS spectra is still unavailable. The results of this study showed that the number, intensity, and resolving power of all assigned peaks increased with the ion accumulation time (IAT) and DOM concentrations within a reasonable range. The space-charge effect induced by the excess ions in the ICR cell can deteriorate the data quality of the FT-ICR MS spectra, which could be inspected by examining the mass errors and intensity deviation of the monoisotopic and 13C-isotopic peaks based on the 13C-isotopic pattern. The maximum absolute mass error and 13C-isotopic pattern-based intensity deviation are two critical criteria for inspecting the space-charge effect, which was suggested to be 2.0 ppm and 20%, respectively. Therefore, a novel strategy based on the 13C-isotopic pattern has been proposed in this study to optimize the FT-ICR MS spectra of DOM based on their wide occurrence of monoisotopic and 13C-isotopic signals. This optimization strategy has laid the fundamentals for the method development of FT-ICR MS and could be extended to different FT-ICR MS instruments and various organic complex mixtures.

Variability of Microcystin-LR Standards Available from Seven Commercial Vendors.

Microcystins (MCs) are a large group of heptapeptide cyanobacterial toxins commonly produced in harmful algal blooms (HABs) and associated with adverse health effects in wildlife, livestock, pets, and humans. MC chemical standards are extracted from cyanobacteria biomass rather than produced synthetically and are used in water assessment methods and toxicological studies. MC standards are generally supplied in less than 1 mg quantities, and verification of the mass can only be accomplished by analytical chemistry methods using a certified reference of the specific MC for comparison. Analytical quantification of MCs in environmental samples and toxicology studies using accurate doses of test chemicals administered to experimental animals rely on the availability and accuracy of chemical standards. To check the accuracy and purity of available standards, seven individual microcystin-LR (MCLR) standards were purchased from separate commercial vendors and analyzed to determine the actual mass supplied and identify the presence of potential contaminants. To determine the effect of varying toxin mass in toxicological studies, each MCLR standard was administered to CD-1 mice in doses based on mass purchased, by a single 40 µg/kg intraperitoneal injection. The measured mass purchased varied from the vendor label mass by more than 35% for two of the seven MCLR standards. Contaminants, including trifluoroacetic acid (TFA), were identified in four of the seven samples. Comparative in vivo hepatotoxicity between vendor samples closely reflected the actual amount of MCLR present in each standard and demonstrated the toxicological impact of varying cyanotoxin mass.

Characterization of large intact protein ions by mass spectrometry: What directions should we follow?

Theoretically, the gas-phase interrogation of whole proteoforms via mass spectrometry, known as top-down proteomics, bypasses the protein inference problem that afflicts peptide-centric proteomic approaches. Despite this obvious advantage, the application of top-down proteomics remains rare, mainly due to limited throughput and difficulty of analyzing proteins >30 kDa. Here we will discuss some of the problems encountered during the characterization of large proteoforms, and guided by a combination of theoretical background and experimental evidence we will describe some innovative data acquisition strategies and novel mass spectrometry technologies that can at least partially overcome such limitations.

Mass shift in mass spectrometry imaging: comprehensive analysis and practical corrective workflow.

MALDI mass spectrometry imaging (MSI) allows the mapping and the tentative identification of compounds based on their m/z value. In typical MSI, a spectrum is taken at incremental 2D coordinates (pixels) across a sample surface. Single pixel mass spectra show the resolving power of the mass analyzer. Mass shift, i.e., variations of the m/z of the same ion(s), may occur from one pixel to another. The superposition of shifted masses from individual pixels peaks apparently degrades the resolution and the mass accuracy in the average spectrum. This leads to low confidence annotations and biased localization in the image. Besides the intrinsic performances of the analyzer, the sample properties (local composition, thickness, matrix deposition) and the calibration method are sources of mass shift. Here, we report a critical analysis and recommendations to mitigate these sources of mass shift. Mass shift 2D distributions were mapped to illustrate its effect and explore systematically its origin. Adapting the sample preparation, carefully selecting the data acquisition settings, and wisely applying post-processing methods (i.e., m/z realignment or individual m/z recalibration pixel by pixel) are key factors to lower the mass shift and to improve image quality and annotations. A recommended workflow, resulting from a comprehensive analysis, was successfully applied to several complex samples acquired on both MALDI ToF and MALDI FT-ICR instruments.

A feasible strategy to improve confident elemental composition determination of compounds in complex organic mixture such as natural organic matter by FTICR-MS without internal calibration.

Confident elemental composition determination of compounds in complex samples such as natural organic matter (NOM) by ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is challenging due to the interference between multiple components in these samples during detection. Here the performance of Solarix 15T-FTICR-MS in terms of accurate relative natural isotope abundance (RIA) and mass measurements for elemental composition determination of compounds in complex samples such as NOM was systematically evaluated. The optimal sweep excitation power values ranging from 20% to 22% was found to significantly diminish the underestimation of RIA measurement for 13C1 peaks of NOM components by FTICR-MS. Random error was found to be one of the main sources for the RIA errors of 13C1 peaks with S/N ratios <25. The mean averaged RIA errors of less than 10% could be obtained by averaging the measured RIAs of each 13C1 peaks in five replicated runs. By adjusting the total ion abundance of NOM complex sample between 3.8-E7 and 1.4-E8 which was simultaneously similar to that of external calibrant during detection, mass errors of lower than 1 ppm for NOM components with m/z lower than 700 Da could be obtained without internal calibration. Meanwhile, a linear correlation between mass errors of ions in NOM complex sample and their m/z values could be obtained. The mass error deviation derived from the linearity was firstly used as new criterion to reduce the number of false formula candidates. A novel strategy of combination of high mass accuracy, high spectral accuracy, and mass error deviation for elemental composition determination of unknown compounds in complex sample such as NOM by FTICR-MS was proposed and applied for different complex samples. Compared to the traditional method, about one fold increasement in the number of the unique formula assignments for measured ions was obtained by using our strategy.

Exploration of Rapid Evaporative-Ionization Mass Spectrometry as a Shotgun Approach for the Comprehensive Characterization of Kigelia Africana (Lam) Benth. Fruit.

Rapid evaporative-ionization mass spectrometry (REIMS) coupled with an electroknife as a sampling device was recently employed in many application fields to obtain a rapid characterization of different samples without any need for extraction or cleanup procedures. In the present research, REIMS was used to obtain a metabolic profiling of the Kigelia africana fruit, thus extending the applicability of such a technique to the investigation of phytochemical constituents. In particular, the advantages of REIMS linked to a typical electrosurgical handpiece were applied for a comprehensive screening of this botanical species, by exploiting the mass accuracy and tandem MS capabilities of a quadrupole-time of flight analyzer. Then, 78 biomolecules were positively identified, including phenols, fatty acids and phospholipids. In the last decade, Kigelia africana (Lam.) Benth. fruit has attracted special interest for its drug-like properties, e.g., its use for infertility treatments and as anti-tumor agent, as well as against fungal and bacterial infections, diabetes, and inflammatory processes. Many of these properties are currently correlated to the presence of phenolic compounds, also detected in the present study, while the native lipid composition is here reported for the first time and could open new directions in the evaluation of therapeutic activity.

Mass Accuracy Check Using Common Background Peaks for Improving Metabolome Data Quality in Chemical Isotope Labeling LC-MS.

Chemical isotope labeling (CIL) LC-MS is a highly sensitive and quantitative method for metabolome analysis. Because of a large number of peaks detectable in a sample and the need of running many samples in a metabolomics project, any significant change in mass measurement accuracy during the whole period of running samples can adversely affect the downstream peak alignment and quantitative analysis. Herein, we report a rapid method to check the mass accuracy of individual spectra in each CIL LC-MS run in order to flag up any run containing spectra with accuracy drift that falls outside the expected error. The flagged run may be re-run or discarded before merging with other runs for peak alignment and analysis. This method is based on the observation that some background signals are commonly detected in almost all spectra collected in CIL LC-MS runs. A mass accuracy check (MAC) software program has been developed to first find the common background mass peaks and then use them as mass references to calculate any mass shifts over the course of multiple sample runs. Using a metabolome dataset of 324 human cerebrospinal fluid (CSF) samples and 35 quality control (QC) samples produced by CIL LC-MS, we show that this accuracy check method can streamline the initial raw data processing for downstream analysis in metabolomics.

A systematic study of molecular ion intensity and mass accuracy in low energy electron ionisation using gas chromatography-quadrupole time-of-flight mass spectrometry.

Molecular ions, which contain accurate mass information, are valuable for providing elemental composition elucidation. Under the most common electron ionisation (EI) condition (electron energy, 70 eV and temperature, 230 °C), molecular ions are often in relatively low intensities or completely unapparent. In this research, low energy EI source parameters (electron energy and temperature) in a gas chromatography-quadrupole time of flight (GC-QTOF) were systematically studied to evaluate their correlative impact on the intensity and mass accuracy of molecular ions. Lower temperatures were generally associated with higher molecular ion intensities under various EI energies. Apart from compounds with more chemically stable molecular ion structures, the lowest electron energy (12 eV) corresponded to higher intensities. On the other hand, mass accuracy appeared to be mostly constant (≤5 ppm) at different temperatures, while improvement was observed with the use of lower electron energies (12 eV). Moreover, the effect of compound concentration on molecular ion intensity and mass accuracy was studied from 50 to 5000 ppm, and the compound-specific concentration profiles were constructed. Finally, it was found that higher column flow rates corresponded to higher intensities, while the response under 12 eV was higher than that of 70 eV. Mass accuracy remained approximately constant across different flow rates. Therefore, these findings suggest that the use of low energy EI may be a viable approach for the preservation of molecular ions.

Ion traps in modern mass spectrometry.

This review is devoted to trapping mass spectrometry wherein ions are confined by electromagnetic fields for prolonged periods of time within limited volume, with mass measurement taking place within the same volume. Three major types of trapping mass spectrometers are discussed, specifically radiofrequency ion trap, Fourier transform ion cyclotron resonance and Orbitrap. While these three branches are intricately interwoven with each other over their recent history, they also differ greatly in their fundamentals, roots and historical origin. This diversity is reflected also in the difference of viewpoints from which each of these directions is addressed in this review. Following the theme of the issue, we focus on developments mainly associated with the country of Germany but, at the same time, we use this review as an illustration of the rapidly increasing globalization of science and expanding multi-national collaborations.

A Method for Defining the Position of Ion Formation in a MALDI TOFMS by Analysis of the Laser Image on the Sample Surface.

A method is developed to determine the position of ion formation along the flight axis of a MALDI TOFMS instrument using the image of the laser on the sample surface. Previous work (JASMS 2018, 29, 422-434) showed that misalignment of the sample stage in a Bruker Autoflex III MALDI TOFMS as well as multiple insertions/mountings of the target plate and differences in target plate shape itself produced reproducible changes in the measured ion time-of-flight which could be attributed to changes in the position of ion formation along the instrument flight axis. Here, a small but reproducible change in the position of the laser in the sample-viewing camera image was observed, with the movement depending on both the sample position and target plate used. Using the change in coordinates of the laser position in the camera image and the known angle of incidence of the laser on the sample surface, the initial z-axis position of the ion at different locations on the plate can be calculated, exactly defining changes in the ion flight path length and the distance between the sample plate and first extraction plate/grid with sample position on the target plate. A correction method is developed to correct the time-of-flight values collected from different locations on the sample plate using the laser images, with the relative standard deviation (RSD) being reduced from 23 ppm to below 6 ppm. The laser images, along with the measured target plate heights, are also used to calculate the misalignment of the sample stage. Graphical Abstract.

Novel automated workflow for spectral alignment and mass calibration in MS imaging using a sputtered Ag nanolayer.

Mass spectrometry imaging (MSI) is a technique that can map analyte spatial distribution directly onto a tissue section. This enables the spatial correlation of molecular entities with a tissue morphology to be investigated. Analyte annotation in MSI is intrinsically linked to the mass accuracy of the data. Mass accuracy and analyte identification are determined by such factors as the experimental set up and the data processing workflow. We present an MSI data processing workflow that uses a label-free approach to compensate for mass shifts. The algorithms developed were designed to perform efficiently even for datasets much larger than computer's memory. Herein, we present the application of the developed processing workflow to a dataset with more than 13.000 pixels and ∼50.000 mass channels. We assessed the overall mass accuracy in the range m/z 400 to 1200 using silver and gold sputtered nanolayers. With our novel processing workflow we were able to obtain mass errors as low as 5 ppm using a TOF instrument.

Diagnosing and Correcting Mass Accuracy and Signal Intensity Error Due to Initial Ion Position Variations in a MALDI TOFMS.

Frustrated by worse than expected error for both peak area and time-of-flight (TOF) in matrix assisted laser desorption ionization (MALDI) experiments using samples prepared by electrospray deposition, it was finally determined that there was a correlation between sample location on the target plate and the measured TOF/peak area. Variations in both TOF and peak area were found to be due to small differences in the initial position of ions formed in the source region of the TOF mass spectrometer. These differences arise largely from misalignment of the instrument sample stage, with a smaller contribution arising from the non-ideal shape of the target plates used. By physically measuring the target plates used and comparing TOF data collected from three different instruments, an estimate of the magnitude and direction of the sample stage misalignment was determined for each of the instruments. A correction method was developed to correct the TOFs and peak areas obtained for a given combination of target plate and instrument. Two correction factors are determined, one by initially collecting spectra from each sample position used and another by using spectra from a single position for each set of samples on a target plate. For TOF and mass values, use of the correction factor reduced the error by a factor of 4, with the relative standard deviation (RSD) of the corrected masses being reduced to 12-24 ppm. For the peak areas, the RSD was reduced from 28% to 16% for samples deposited twice onto two target plates over two days. Graphical Abstract.

Liquid chromatography - quadrupole Orbitrap mass spectrometry method for selected pharmaceuticals in water samples.

This study developed an analytical approach for sub-ppb level detection and confirmation of 13 pharmaceuticals and personal care products (PPCPs) in water samples using ultra high pressure liquid chromatography hyphenated with a quadrupole Orbitrap mass spectrometer (UHPLC- Q-Orbitrap-MS). Sample preparation was performed by using solid phase extraction (SPE) employing hydrophilic-lipophilic balance cartridges, with elution of sorbed analytes using methanol. Acceptable automatic gain control (AGC) target and maximum injection time (IT) were 1×106 and 200ms, respectively, resulting in a mass accuracy <2ppm. High response signals with sufficient data points per peaks (20-30) were obtained whilst maintaining high resolution of approximately 70,000 full width at half maximum. Extracted ion chromatograms provided quantitative analysis with linearity (R2) ranging from 0.9875 to 0.9993 and method detection limits ranging from 0.01-0.61ngmL-1. Compounds were further analysed by MS/MS analysis, with the MS operated in parallel reaction monitoring (PRM) mode under precursor ion analysis intervals and collision energies chosen for the different PPCPs. The developed method was applied to analyse water samples obtained from sources in Victoria, Australia.

Param-Medic: A Tool for Improving MS/MS Database Search Yield by Optimizing Parameter Settings.

In shotgun proteomics analysis, user-specified parameters are critical to database search performance and therefore to the yield of confident peptide-spectrum matches (PSMs). Two of the most important parameters are related to the accuracy of the mass spectrometer. Precursor mass tolerance defines the peptide candidates considered for each spectrum. Fragment mass tolerance or bin size determines how close observed and theoretical fragments must be to be considered a match. For either of these two parameters, too wide a setting yields randomly high-scoring false PSMs, whereas too narrow a setting erroneously excludes true PSMs, in both cases, lowering the yield of peptides detected at a given false discovery rate. We describe a strategy for inferring optimal search parameters by assembling and analyzing pairs of spectra that are likely to have been generated by the same peptide ion to infer precursor and fragment mass error. This strategy does not rely on a database search, making it usable in a wide variety of settings. In our experiments on data from a variety of instruments including Orbitrap and Q-TOF acquisitions, this strategy yields more high-confidence PSMs than using settings based on instrument defaults or determined by experts. Param-Medic is open-source and cross-platform. It is available as a standalone tool ( http://noble.gs.washington.edu/proj/param-medic/ ) and has been integrated into the Crux proteomics toolkit ( http://crux.ms ), providing automatic parameter selection for the Comet and Tide search engines.

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry at the Cyclotron Frequency.

The phenomenon of ion cyclotron resonance allows for determining mass-to-charge ratio, m/z, of an ensemble of ions by means of measurements of their cyclotron frequency, ω c . In Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), the ω c quantity is usually unavailable for direct measurements: the resonant state is located close to the reduced cyclotron frequency (ω+), whereas the ω c and the corresponding m/z values may be calculated via theoretical derivation from an experimental estimate of the ω+ quantity. Here, we describe an experimental observation of a new resonant state, which is located close to the ω c frequency and is established because of azimuthally-dependent trapping electric fields of the recently developed ICR cells with narrow aperture detection electrodes. We show that in mass spectra, peaks close to ω+ frequencies can be reduced to negligible levels relative to peaks close to ω c frequencies. Due to reduced errors with which the ω c quantity is obtained, the new resonance provides a means of cyclotron frequency measurements with precision greater than that achieved when ω+ frequency peaks are employed. The described phenomenon may be considered for a development into an FT-ICR MS technology with increased mass accuracy for applications in basic research, life, and environmental sciences. Graphical Abstract ᅟ.

Improved procedure for dendrimer-based mass calibration in matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry.

A procedure is described that results in a substantial increase in signal intensity and in improved accuracy of positive-ion mass calibration when using commercially available kits of monodisperse dendrimers (SpheriCal(®)) in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The peak intensities are amplified by an admixture of 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) matrix to the kits comprising of 9-nitroanthracene matrix, sodium trifluoroacetate, and four dendrimers. Boosted ion formation then permits lower laser fluence to be used and thus yields enhanced mass resolution. Further, the number of reference peaks is doubled by doping the sample preparation with cesium ions. This results in four [M+Cs](+) ion signals in addition to four [M+Na](+) ion signals provided by the standard kit. Overall, the modified procedure notably reduces the consumption of the expensive calibration standard kits, while it increases mass resolution and enables the use of an advanced calibration algorithm requiring at least six reference peaks. Graphical abstract A dendrimer-based mass calibration for MALDI-TOF-MS can be improved by adding a DCTB matrix and doping the sample preparation with Cs(+) ions. Having eight rather than just four reference peaks reduces the average mass error of the calibration curve about fivefold.