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.

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.

A Dual-Gated Structures for Lossless Ion Manipulations-Ion Mobility Orbitrap Mass Spectrometry Platform for Combined Ultra-High-Resolution Molecular Analysis.

High-resolution ion mobility spectrometry-mass spectrometry (HR-IMS-MS) instruments have enormously advanced the ability to characterize complex biological mixtures. Unfortunately, HR-IMS and HR-MS measurements are typically performed independently due to mismatches in analysis time scales. Here, we overcome this limitation by using a dual-gated ion injection approach to couple an 11 m path length structures for lossless ion manipulations (SLIM) module to a Q-Exactive Plus Orbitrap MS platform. The dual-gate setup was implemented by placing one ion gate before the SLIM module and a second ion gate after the module. The dual-gated ion injection approach allowed the new SLIM-Orbitrap platform to simultaneously perform an 11 m SLIM separation, Orbitrap mass analysis using the highest selectable mass resolution setting (up to 140 k), and high-energy collision-induced dissociation (HCD) in ∼25 min over an m/z range of ∼1500 amu. The SLIM-Orbitrap platform was initially characterized using a mixture of standard phosphazene cations and demonstrated an average SLIM CCS resolving power (RpCCS) of ∼218 and an SLIM peak capacity of ∼156, while simultaneously obtaining high mass resolutions. SLIM-Orbitrap analysis with fragmentation was then performed on mixtures of standard peptides and two reverse peptides (SDGRG1+, GRGDS1+, and RpCCS = 305) to demonstrate the utility of combined HR-IMS-MS/MS measurements for peptide identification. Our new HR-IMS-MS/MS capability was further demonstrated by analyzing a complex lipid mixture and showcasing SLIM separations on isobaric lipids. This new SLIM-Orbitrap platform demonstrates a critical new capability for proteomics and lipidomics applications, and the high-resolution multimodal data obtained using this system establish the foundation for reference-free identification of unknown ion structures.

Development of a Structure for Lossless Ion Manipulations (SLIM) High Charge Capacity Array of Traps.

Enhancing the sensitivity of low-abundance ions in a complex mixture without sacrificing measurement throughput is highly desirable. This work demonstrates a way to greatly improve the sensitivity of ion mobility (IM)-selected ions by accumulating them in an array of high-capacity ion traps located inside a novel structures for lossless ion manipulations ion mobility spectrometer (SLIM-IMS) module. The array of ion traps used in this work consisted of seven independently controllable traps. Each trap was 386 mm long and possessed a charge capacity of ∼4.5 × 108 charges, with a linear range extending to ∼2.5 × 108 charges. Each ion trap could be used to extract a peak (or ions over a mobility range) from an ion mobility separation based on arrival time. Ions could be stored without losses for long times (>100 s) and then released all at once or one trap at a time. It was possible to accumulate large ion populations by extracting and storing ions over repeated IM separations. Enrichment of up to seven individual ion distributions could be performed using the seven independently controllable ion traps. Additionally, the ion trapping process effectively compressed ion populations into narrow peaks, which provides a greatly improved basis for subsequent ion manipulations. The array of high charge capacity ion traps provides a flexible addition to SLIM and a powerful tool for IMS-MS applications requiring high sensitivity.

SimELIT: A Novel GUI-Based Comprehensive Ion Trajectory Simulation Software for Mass Spectrometry.

Ion trajectory simulation in mass spectrometry systems from injection to detection is technically challenging but very important for better understanding the ion dynamics in instrument development. Here, we present SimELIT (Simulator of Eulerian and Lagrangian Ion Trajectories), a novel ion trajectory simulation platform. SimELIT is built upon a suite of multiphysics solvers compiled into OpenFOAM (an open-source numerical solver library particularly used for computational mechanics), with a simple web-based graphical user interface (GUI) allowing users to define the details of OpenFOAM cases and run simulations. SimELIT is a modular program and can provide extensions of physics (e.g., gas flows, electrodynamic fields) and thus enable ion trajectory simulations from the ion source to detector. The current version (SimELIT) provides two numerical solvers for ion trajectory simulations─(1) a Lagrangian particle tracker in vacuum and (2) a Eulerian ion density solver in background gas in the presence of electric fields. Here, we describe the architecture of SimELIT, including its use of Docker and the React Framework, and demonstrate the computation of ion trajectories of multiple m/z values in a static/linear voltage drop in vacuum (across a 1 m long flight tube). Further, the drift motion of ions under 1 Torr pressure conditions in a static background (N2) gas through a 20 V/cm static electric field is shown. The results produced from SimELIT were compared with SIMION and theoretical estimates. In addition, we report the computation of ion trajectories in electrodynamic fields within a planar FAIMS device operating at atmospheric pressure.

On the relationships between resolution, dimensionless stability, pseudopotential well depth, acceptance, and transmission in mass filters.

The relationships between resolution, stability, pseudopotential well depth, acceptance aperture, and transmission for sinusoidal quadrupole mass filters are examined graphically and mathematically. Simple linear or power relationships are revealed. Comparison of these quantities plotted against resolving power show that the pseudopotential well depth correlates well with the mass filter transmission. Pseudopotential well depth is directly proportional to the product of dimensionless stability well depth and the AC voltage. This relationship extends to all quadrupoles regardless of operational zone because it is rooted in stability. Ion transmission and sensitivity scale directly with the pseudopotential well depth. Resolving power and pseudopotential well depth increase when operating in higher stability zones for all types of mass filters. Unfortunately, for fixed frequency sine wave mass filters, the increased resolving power and pseudopotential well depth are accompanied by a significant reduction of the mass range and increased in fringe field effects. For these reasons, sine mass filter operation in higher stability zones has been reported but not commercially produced. In contrast, for rectangular wave mass filter operation, there is no mass range limitation in any stability zone. The fringe field does not increase because the AC voltage is constant and does not change within a single stability zone or between them. A DC voltage is also unnecessary to access any zone. The high resolution and sensitivity of rectangular wave mass filters that can be gained by operation in higher stability zones without mass limit and limited fringe field restrictions suggest a bright and expansive future for this technology.

Implementing Digital-Waveform Technology for Extended m/z Range Operation on a Native Dual-Quadrupole FT-IM-Orbitrap Mass Spectrometer.

Here, we describe a digital-waveform dual-quadrupole mass spectrometer that enhances the performance of our drift tube FT-IMS high-resolution Orbitrap mass spectrometer (MS). The dual-quadrupole analyzer enhances the instrument capabilities for studies of large protein and protein complexes. The first quadrupole (q) provides a means for performing low-energy collisional activation of ions to reduce or eliminate noncovalent adducts, viz., salts, buffers, detergents, and/or endogenous ligands. The second quadrupole (Q) is used to mass-select ions of interest for further interrogation by ion mobility spectrometry and/or collision-induced dissociation (CID). Q is operated using digital-waveform technology (DWT) to improve the mass selection compared to that achieved using traditional sinusoidal waveforms at floated DC potentials (>500 V DC). DWT allows for increased precision of the waveform for a fraction of the cost of conventional RF drivers and with readily programmable operation and precision (Hoffman, N. M. . A comparison-based digital-waveform generator for high-resolution duty cycle. Review of Scientific Instruments 2018, 89, 084101).

Improving Signal to Noise Ratios in Ion Mobility Spectrometry and Structures for Lossless Ion Manipulations (SLIM) using a High Dynamic Range Analog-to-Digital Converter.

Signal digitization is a commonly overlooked part of ion mobility-mass spectrometry (IMS-MS) workflows, yet it greatly affects signal-to-noise ratio and MS resolution measurements. Here, we report on the integration of a 2 GS/s, 14-bit ADC with structures for lossless ion manipulations (SLIM-IMS-MS) and compare the performance to a commonly used 8-bit ADC. The 14-bit ADC provided a reduction in the digitized noise by a factor of ∼6, owing largely to the use of smaller bit sizes. The low baseline allowed threshold voltage levels to be set very close to the MCP baseline voltage, allowing for as much signal to be acquired as possible without overloading or excessive digitization of MCP baseline noise. Analyses of Agilent tuning mixture ions and a mixture of heavy labeled phosphopeptides showed that the 14-bit ADC provided a ∼1.5-2× signal-to-noise (S/N) increase for high intensity ions, such as the Agilent tuning mixture ions and the 2+ and 3+ charge states of many phosphopeptide constituents. However, signal enhancements were as much as 10-fold for low intensity ions, and the 14-bit ADC enabled discernible signal intensities otherwise lost using an 8-bit digitizer. Additionally, the 14-bit ADC required ∼14-fold fewer mass spectra to be averaged to produce a mass spectrum with a similar S/N as the 8-bit ADC, demonstrating ∼10× higher measurement throughput. The high resolution, low baseline, and fast speed of the new 14-bit ADC enables high performance digitization of MS, IMS-MS, and SLIM-IMS-MS spectra and provides a much better picture of analyte profiles in complex mixtures.

Will the Digital Mass Filter Be the Next High-Resolution High-Mass Analyzer?

Mass filter operation in higher stability zones is known to provide better resolution. Unfortunately, for sine driven instruments, higher stability zone operation reduces the accessible mass range and increases the degenerative effects of fringe fields. Conversely, digitally driven mass filters do not suffer from loss of mass range, and the fringe field effects do not increase significantly by switching stability zones because the AC voltage is always constant and the DC voltage is always zero. This work catalogues 12 stability zones that are accessible with the new digital waveform generation technology. These zones have theoretical baseline resolving powers that range from 22 to 1 300 000 with pseudopotential well depths that range from 3.5 to 43 V. Operation in higher stability zones also has the advantage of aligned axial stability wells. That alignment maximizes the pseudopotential well depth for each higher stability zone, making them more than an order of magnitude greater than the standard ∼0.2 V well of a sine filter operating in the first stability zone at unit resolution. Increased pseudopotential well depth correlates with better ion transmission and sensitivity. Our theoretical examination suggests that the digital mass filter can obtain both high resolution and high sensitivity with essentially unlimited mass range.

Computational evaluation of a new digital tandem quadrupole mass filter.

A tandem mass filter consists of two low-resolution mass filters arranged in series that operate with a small offset between their mass windows. In principle, the overlap of the two individual mass windows defines the tandem window. Tandem operation provides improved resolution and transmission compared to a single mass filter operated with the same mass window. The improvement in transmission is owed to the larger acceptance of the low-resolution quadrupoles. The tandem filter resolution and transmission are adjusted by changing the amount of offset separating the mass windows of the individual filters. Sine wave systems create this offset through voltage changes. Digital tandem mass filters depart from convention because they do not change voltage. The tandem mass window is created when the individual filters are operated with two slightly different duty cycles. Both quadrupoles operate at the same frequency, phase, and voltage. When the frequency, phase, and voltage of each quadrupole are identical, there theoretically are no changes to the Mathieu parameters to cause ion excitation and loss during transition between the quadrupole pair. The work presented here shows that a fixed AC voltage digital tandem mass filter can only operate in higher stability zones. However, unlike sine mass filters, the mass range of a digital system is not limited. This makes the digital tandem mass filter feasible as a commercial product. For the tandem digital mode to be successful, the duty cycles of each quadrupole must be precisely controlled because the duty cycle differences required to shift the mass windows are small. The creation of these mass window offsets requires waveform generation that can obtain high duty cycle resolution. Our method of generating waveforms can meet this demand; however, modifications to our current printed circuit board must be made. These modifications are minor and will be discussed.

Quantifying the operation of sinusoidal mass filters.

Even though sinusoidal quadrupole mass filters have been around for more than 50 years, the relationships defining resolution, resolving power, and transmission from the applied voltages have not been rigorously quantified or discussed. Traditional quadrupole mass filter theory implies that voltages are scanned at constant direct current (DC) to alternating current (AC) voltage ratios with the scanline passing through the origin of the voltage stability diagram. A prominent feature of constant voltage ratio scans is constant baseline theoretical resolving power (m/Δm) that is the same for all masses. Commercial quadrupole instruments rarely scan at constant resolving power because ion transmission increases with mass. Instead, they scan at constant resolution, meaning that the mass window width is fixed. Constant resolution mass scans are preferred because ion transmission does not change with mass. Commercial mass filter systems create constant resolution scans by linearly changing the DC and AC voltages at a fixed ratio in the presence of an additional negative DC voltage offset. This manuscript systematically quantifies the effects of the DC and AC voltages on resolution, resolving power, pseudopotential well depth, and transmission. To quantify these properties, recently developed spreadsheet tools that calculate the laboratory frame stability of ions from the matrix solutions of Hill's equation were used. Voltage scanning methods and their effects on theoretically determined transmission and sensitivity will be discussed.

New tools for theoretical comparison of rectangular and sine wave operation of ion traps, guides and mass filters.

The first completely digital quadrupole mass filter was recently introduced. There is now a need to understand and demonstrate the benefits of digital operation and compare them to the commercial standards. Our work to date has demonstrated that sine and square wave operation are very similar because of their similar stability diagrams and because they use a direct current (DC) potential between the electrode pairs to narrow and limit the mass range. In contrast, rectangular wave-operated digital mass filters and ion traps narrow and limit the stable mass range with the waveform duty cycle without the need for a DC potential. To understand and compare the differences between rectangular and sinusoidal modes of operation, our group has developed new spreadsheet tools that permit calculation of the m/z versus frequency space stability diagrams with the application of a DC potential between the electrode sets for rectangular and sine waveforms, and plots of the pseudopotential well depth against the entire range of stable m/zs for rectangular and sine waveforms. Our spreadsheets were used to make comparisons between fixed-frequency variable-voltage and fixed-voltage variable-frequency modes of operation. They provide a comprehensive companion tool for operating in the laboratory frame and are tunable to the instrument. This manuscript introduces these tools as it compares sine and rectangular wave modes of operation and provides a basis for understanding the advantages and disadvantages of digital operation relative to conventional technology.

Digital Mass Analysis in a Linear Ion Trap without Auxiliary Waveforms.

Mass analysis in a linear ion trap is traditionally performed using resonant ejection induced by auxiliary waveforms. For sinusoidally driven ion traps without resonant ejection, resolution and sensitivity are poor because mass-selected instability yields excitation along both the x and y axes simultaneously. Digital ion traps, on the other hand, have the advantage of duty cycle manipulation that can be used to change the ion excitation along the x and y axes. Consequently, the duty cycle can be used to enhance the resolution and sensitivity for mass-selected instability in a linear ion trap without the application of an auxiliary waveform. This work introduces and explores mass-selected instability in a linear trap without the use of auxiliary waveforms.

Computational evaluation of mass filter acceptance and transmittance influenced by developing fields: An application of the plane method to investigate prefilter efficacy for rectangular wave operated mass filters.

The acceptance of quadrupole mass filters is improved when the alternating current (AC) and direct current (DC) fields are developed separately. Physically, this is achieved when a short RF only quadrupole (prefilter) is situated directly ahead of the mass filter. The acceptance gained by a system operating with a prefilter can be observed as an increase in sensitivity over conventional operation. Frequency dynamic duty cycle based rectangular waveform driven (rectangular wave) mass filters, a recent development, currently do not operate with prefilters. Little is known about the influence of duty cycle changes on the acceptance of rectangular wave mass filters. The sensitivity gain seen by conventional systems operating with prefilters indicates that the sensitivity of duty cycle based rectangular wave systems should increase comparably. The objective of this work was to determine prefilter efficacy for nonspecific rectangular wave mass filter systems. In this work, the plane method of acceptance was used to model the change to the acceptance and transmittance of sine and rectangular waveform driven mass filters under different modes of field development. Both systems indicated a fourfold increase in sensitivity when the mass filtering DC or duty cycle was delayed.

Tutorial and comprehensive computational study of acceptance and transmission of sinusoidal and digital ion guides.

A quadrupoles acceptance is a measure of its ability to catch ions with certain trajectories. One way to calculate acceptance is the method of ellipses. The method arose partly from a simplification that trajectories could be calculated for an electrode axis independently of others. It has been used to calculate the acceptance and transmission of sine-driven quadrupole mass filters for over 50 years. Although the method is straightforward, it is generally described with little detail or presented as a confusing string of equations. As such, it may not be decipherable by all practitioners. For this reason, the first half of this paper presents a practical explanation of the method of ellipses and the concepts that make it work. Only equations necessary to describe the method are introduced. The tutorial also prepares the reader for the second half, which presents an alternative approach for calculating acceptance based on an array of initial trajectories. The alternative approach is used to compare the acceptance of simplified sinusoidal and digital ion guides. The method of ellipses was applied to validate results of the new approach for calculation of acceptance.

Digital mass filter analysis in stability zones A and B.

Digitally driven mass filter analysis is an advancing field. This work presents a tutorial of digital waveforms, stability diagrams, and pseudopotential well plots. Experimental results on digitally driven mass filter analysis in stability zones A and B are also shown. This work explains duty cycle manipulation of the waveforms to axially trap and eject ions from linear quadrupoles and how to change and distort the stability diagrams to create mass filters and their effects on the pseudopotential well depth. It discusses the sensitivity and resolution that can be obtained and what limits these benchmarks. It reveals the advantages of mass filter operation without any added direct current potential between the quadrupole electrodes (a = 0).

Digital Waveform Technology and the Next Generation of Mass Spectrometers.

Ion traps and guides are integral parts of current commercial mass spectrometers. They are currently operated with sinusoidal waveform technology that has been developed over many years. Recently, digital waveform technology has begun to emerge and promises to supplant its older cousin because it presents new capabilities that result from the ability to instantaneously switch the frequency and duty cycle of the waveforms. This manuscript examines these capabilities and reveals their uses and effects on instrumentation. Graphical Abstract ᅟ.