Cyclable Variable Path Length Multilevel Structures for Lossless Ion Manipulations (SLIM) Platform for Enhanced Ion Mobility Separations.

Ion mobility-mass spectrometry (IMS-MS) is used to analyze complex samples and provide structural information on unknown compounds. As the complexity of samples increases, there is a need to improve the resolution of IMS-MS instruments to increase the rate of molecular identification. This work evaluated a cyclable and variable path length (and hence resolving power) multilevel Structures for Lossless Ion Manipulations (SLIM) platform to achieve a higher resolving power than what was previously possible. This new multilevel SLIM platform has eight separation levels connected by ion escalators, yielding a total path length of ∼88 m (∼11 m per level). Our new multilevel SLIM can also be operated in an "ion cycling" mode by utilizing a set of return ion escalators that transport ions from the eighth level back to the first, allowing even extendable path lengths (and higher IMS resolution). The platform has been improved to enhance ion transmission and IMS separation quality by reducing the spacing between SLIM boards. The board thickness was reduced to minimize the ions' escalator residence time. Compared to the previous generation, the new multilevel SLIM demonstrated better transmission for a set of phosphazene ions, especially for the low-mobility ions. For example, the transmission of m/z 2834 ions was improved by a factor of ∼3 in the new multilevel SLIM. The new multilevel SLIM achieved 49% better resolving powers for GRGDS1+ ions in 4 levels than our previous 4-level SLIM. The collision cross-section-based resolving power of the SLIM platform was tested using a pair of reverse sequence peptides (SDGRG1+, GRGDS1+). We achieved 1100 resolving power using 88 m of path length (i.e., 8 levels) and 1400 following an additional pass through the eight levels. Further evaluation of the multilevel SLIM demonstrated enhanced separation for positively and negatively charged brain total lipid extract samples. The new multilevel SLIM enables a tunable high resolving power for a wide range of ion mobilities and improved transmission for low-mobility ions.

Ion Mobility Separations Using Cocentric Architecture.

Ion mobility separations, especially using drift tube ion mobility spectrometers, are usually performed in linear channels, which can have a large footprint when extended to achieve higher resolving powers. In this work, we explored the performance of an ion mobility device with a curved architecture, which can have a more compact form. The cocentric ion mobility spectrometer (CoCIMS) manipulates ions between two cocentric surfaces containing a serpentine track. The mobility separation inside the CoCIMS is achieved using traveling waveforms (TWs). We initially evaluated the device using ion trajectory simulations using SIMION, which indicated that when ions traveled circularly inside the CoCIMS they resulted in similar resolving powers and transmitted m/z range as traveling in a straight path. We then performed experimental validation of the CoCIMS in conjunction with a TOF MS. The CoCIMS was made of two flexible printed circuit board materials folded into cocentric cylinders separated by a gap of 2.8 mm. The device was about 50 mm diameter ×152 mm long and provided 1.846 m of serpentine path length. Three sets of mixtures (Agilent tune mixture, tetraalkylammonium salts, and an eight-peptide mixture) and four traveling waveform profiles (square, sine, triangle, and sawtooth) were used. The sawtooth TW profile produced a slightly higher resolving power for the Agilent tuning mixture and tetraalkylammonium ions. The average resolving power for Agilent tune mixture ions ranged from 37 (using sawtooth TW) to 27 (using square TW). The average resolving powers ranged from 45 (sawtooth TW) to 31 (square TW) for tetraalkylammonium ions. The resolving power of the peptide mixture ions was similar among the four TW profiles and ranged from 51 to 56. The average percent error in TWCCS for the peptide mixture ions was about 0.4%. The new device showed promising results, but improvements are needed to further increase the resolving power.

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.

Evaluation of a Reference-Free Collision Cross Section Calibration Strategy for Proteomics Using SLIM-Based High-Resolution Ion Mobility Spectrometry-Mass Spectrometry.

Ion mobility spectrometry (IMS) is a gas-phase analytical technique that separates ions with different sizes and shapes and is compatible with mass spectrometry (MS) to provide an additional separation dimension. The rapid nature of the IMS separation combined with the high sensitivity of MS-based detection and the ability to derive structural information on analytes in the form of the property collision cross section (CCS) makes IMS particularly well-suited for characterizing complex samples in -omics applications. In such applications, the quality of CCS from IMS measurements is critical to confident annotation of the detected components in the complex -omics samples. However, most IMS instrumentation in mainstream use requires calibration to calculate CCS from measured arrival times, with the most notable exception being drift tube IMS measurements using multifield methods. The strategy for calibrating CCS values, particularly selection of appropriate calibrants, has important implications for CCS accuracy, reproducibility, and transferability between laboratories. The conventional approach to CCS calibration involves explicitly defining calibrants ahead of data acquisition and crucially relies upon availability of reference CCS values. In this work, we present a novel reference-free approach to CCS calibration which leverages trends among putatively identified features and computational CCS prediction to conduct calibrations post-data acquisition and without relying on explicitly defined calibrants. We demonstrated the utility of this reference-free CCS calibration strategy for proteomics application using high-resolution structures for lossless ion manipulations (SLIM)-based IMS-MS. We first validated the accuracy of CCS values using a set of synthetic peptides and then demonstrated using a complex peptide sample from cell lysate.

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.