The role of solvation on the conformational landscape of α-synuclein.

Native ion mobility mass spectrometry has been used extensively to characterize ensembles of intrinsically disordered protein (IDP) conformers, but the extent to which the gaseous measurements provide realistic pictures of the solution conformations for such flexible proteins remains unclear. Therefore, we systematically studied the relationship between the solution and gaseous structural ensembles by measuring electrospray charge state and collision cross section (CCS) distributions for cationic and anionic forms of α-synuclein (αSN), an anionic protein in solution, as well as directly probed gas phase residue to residue distances via ion/ion reactions between gaseous α-synuclein cations and disulfonic acid linkers that form strong electrostatic bonds. We also combined results from in-solution protein crosslinking identified from native tandem mass spectrometry (MS/MS) with an initial αSN ensemble generated computationally by IDPConformerGenerator to generate an experimentally restrained solution ensemble of αSN. CCS distributions were directly calculated for the solution ensembles determined by NMR and compared to predicted gaseous conformers. While charge state and collision cross section distributions are useful for qualitatively describing the relative structural dynamics of proteins and major conformational changes induced by changes to solution states, the predicted and measured gas phase conformers include subpopulations that are significantly different than those expected from completely "freezing" solution conformations and preserving them in the gas phase. However, insights were gained on the various roles of solvent in stabilizing various conformers for extremely dynamic proteins like α-synuclein.

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 (≤30 min), 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 the resolving 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.

Zinc Ammonio-dodecaborates: Synthesis, Lewis Acid Strength, and Reactivity.

Two series of zinc salts, [EtZn][A] and Zn[A]2, with weakly coordinating anions [A]- as counterions have been prepared, and their activities as catalysts for hydrosilylation reactions of 1-hexene, benzophenone, and acetophenone have been investigated. The counterions and per- and partially chlorinated 1-ammonio-closo-dodecaborate anions [Me3NB12Cl11]- [1]-, [Pr3NB12H5Cl6]- [2]-, [Bu3NB12H4Cl7]- [3]-, and [Hex3NB12H5Cl6]- [4]- were chosen as potential and more readily available alternatives to carborate anions such as [CHB11Cl11]- and [HexCB11Cl11]-. The basicity of anion [4]- was determined as being close to that of the triflimide anion [N(SO2CF3)2]-, and the fluoride ion affinities (FIAs) of compounds [EtZn][2] and Zn[2]2 are lower than those of the Lewis acids B(C6F5)3 and Zn[HexCB11Cl11]2. The higher anion basicity and the resulting lower Lewis acidity of the zinc centers result in low activity in 1-hexene hydrosilylation catalysis and only moderate activity in the hydrosilylation catalysis of benzophenone and acetophenone.

ß-Cyclodextrin Encapsulation of Synthetic AHLs: Drug Delivery Implications and Quorum-Quenching Exploits.

Many bacteria, such as Pseudomonas aeruginosa, regulate phenotypic switching in a population density-dependent manner through a phenomenon known as quorum sensing (QS). For Gram-negative bacteria, QS relies on the synthesis, transmission, and perception of low-molecular-weight signal molecules that are predominantly N-acyl-l-homoserine lactones (AHLs). Efforts to disrupt AHL-mediated QS have largely focused on the development of synthetic AHL analogues (SAHLAs) that are structurally similar to native AHLs. However, like AHLs, these molecules tend to be hydrophobic and are poorly soluble under aqueous conditions. Water-soluble macrocycles, such as cyclodextrins (CDs), that encapsulate hydrophobic guests have long been used by both the agricultural and pharmaceutical industries to overcome the solubility issues associated with hydrophobic compounds of interest. Conveniently, CDs have also demonstrated anti-AHL-mediated QS effects. Here, using fluorescence spectroscopy, NMR spectrometry, and mass spectrometry, we evaluate the affinity of SAHLAs, as well as their hydrolysis products, for ß-CD inclusion. We also evaluated the ability of these complexes to inhibit wild-type P. aeruginosa virulence in a Caenorhabditis elegans host infection study, for the first time. Our efforts confirm the potential of ß-CDs for the improved delivery of SAHLAs at the host/microbial interface, expanding the utility of this approach as a strategy for probing and controlling QS.

Ion Mobility Spectrometry with High Ion Utilization Efficiency Using Traveling Wave-Based Structures for Lossless Ion Manipulations.

Ion packets introduced from gates, ion funnel traps, and other conventional ion injection mechanisms produce ion pulse widths typically around a few microseconds or less for ion mobility spectrometry (IMS)-based separations on the order of 100 milliseconds. When such ion injection techniques are coupled with ultralong path length traveling wave (TW)-based IMS separations (i.e., on the order of seconds) using structures for lossless ion manipulations (SLIMs), typically very low ion utilization efficiency is achieved for continuous ion sources [e.g., electrospray ionization (ESI)]. Even with the ability to trap and accumulate much larger populations of ions than being conventionally feasible over longer time periods in SLIM devices, the subsequent long separations lead to overall low ion utilization. Here, we report the use of a highly flexible SLIM arrangement, enabling concurrent ion accumulation and separation and achieving near-complete ion utilization with ESI. We characterize the ion accumulation process in SLIM, demonstrate >98% ion utilization, and show both increased signal intensities and measurement throughput. This approach is envisioned to have broad utility to applications, for example, involving the fast detection of trace chemical species.

Rapid Quantitation of 25-Hydroxyvitamin D2 and D3 in Human Serum Using Liquid Chromatography/Drift Tube Ion Mobility-Mass Spectrometry.

Ion mobility was integrated with liquid chromatography/high resolution mass spectrometry (LC/IM-HRMS) to quantify 25-hydroxyvitamin D (25OHD) in human serum. It has previously been shown that 25OHD adopts two gas-phase conformations which are resolved using ion mobility; in contrast, the inactive epimer, 3-epi-25-hydroxyvitamin D (epi25OHD), only adopts one. Interference from epi25OHD was eliminated by filtering the chromatogram to retain the drift time that corresponds to the unique gas-phase conformation of 25OHD. Although ion mobility separates the epimers, some chromatography is required to separate compounds which interfere with ionization or fall at the same nominal m/z. Standards were prepared in 4% albumin solutions and compared against commercial serum quality controls. Standards and quality controls were analyzed and validated using a 2 min LC/IM-MS method. 25-Hydroxyvitamin D3 and D2 were quantified over the range between 2 and 500 ng/mL with bias and precision within 15%. When epi25OHD was spiked into quality control samples, no significant bias was introduced, and analysis of 30 patient samples shows good agreement between this LC/IM-MS and traditional LC/MS/MS methods. This work shows that ion mobility can be incorporated with liquid chromatography and mass spectrometry for rapid quantitation of 25OHD in human serum.

Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses.

Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100 nL/min nanoLC separation were fractionated into ~ 60 nanowells on a microfluidic glass chip using an in-house-developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses. Graphical abstract A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.

Isolation of Tryptanthrin and Reassessment of Evidence for Its Isobaric Isostere Wrightiadione in Plants of the Wrightia Genus.

A series of Wrightia hanleyi extracts was screened for activity against Mycobacterium tuberculosis H37Rv. One active fraction contained a compound that initially appeared to be either the isoflavonoid wrightiadione or the alkaloid tryptanthrin, both of which have been previously reported in other Wrightia species. Characterization by NMR and MS, as well as evaluation of the literature describing these compounds, led to the conclusion that wrightiadione (1) was misidentified in the first report of its isolation from W. tomentosa in 1992 and again in 2015 when reported in W. pubescens and W. religiosa. Instead, the molecule described in these reports and in the present work is almost certainly the isobaric (same nominal mass) and isosteric (same number of atoms, valency, and shape) tryptanthrin (2), a well-known quinazolinone alkaloid found in a variety of plants including Wrightia species. Tryptanthrin (2) is also accessible synthetically via several routes and has been thoroughly characterized. Wrightiadione (1) has been synthesized and characterized and may have useful biological activity; however, this compound can no longer be said to be known to exist in Nature. To our knowledge, this misidentification of wrightiadione (1) has heretofore been unrecognized.

Rapid Ion Mobility Separations of Bile Acid Isomers Using Cyclodextrin Adducts and Structures for Lossless Ion Manipulations.

Bile acids (BAs) constitute an important class of steroid metabolites often displaying changes associated with disease states and other health conditions. Current analyses for these structurally similar compounds are limited by a lack of sensitivity and long separation times with often poor isomeric resolution. To overcome these challenges and provide rapid analyses for the BA isomers, we utilized cyclodextrin adducts in conjunction with novel ion mobility (IM) separation capabilities provided by structures for lossless ion manipulations (SLIM). Cyclodextrin was found to interact with both the tauro- and glyco-conjugated BA isomers studied, forming rigid noncovalent host-guest inclusion complexes. Without the use of cyclodextrin adducts, the BA isomers were found to be nearly identical in their respective mobilities and thus unable to be baseline resolved. Each separation of the cyclodextrin-bile acid host-guest inclusion complex was performed in less than 1 s, providing a much more rapid alternative to current liquid chromatography-based separations. SLIM provided capabilities for the accumulation of larger ion populations and IM peak compression that resulted in much higher resolution separations and increased signal intensities for the BA isomers studied.

Improved Sensitivity and Separations for Phosphopeptides using Online Liquid Chromotography Coupled with Structures for Lossless Ion Manipulations Ion Mobility-Mass Spectrometry.

Phosphoproteomics greatly augments proteomics and holds tremendous potential for insights into the modulation of biological systems for various disease states. However, numerous challenges hinder conventional methods in terms of measurement sensitivity, throughput, quantification, and capabilities for confident phosphopeptide and phosphosite identification. In this work, we report the first example of integrating structures for lossless ion manipulations ion mobility-mass spectrometry (SLIM IM-MS) with online reversed-phase liquid chromatography (LC) to evaluate its potential for addressing the aforementioned challenges. A mixture of 51 heavy-labeled phosphopeptides was analyzed with a SLIM IM module having integrated ion accumulation and long-path separation regions. The SLIM IM-MS provided limits of detection as low as 50-100 pM (50-100 amol/µL) for several phosphopeptides, with the potential for significant further improvements. In addition, conventionally problematic phosphopeptide isomers could be resolved following an 18 m SLIM IM separation. The 2-D LC-IM peak capacity was estimated as ∼9000 for a 90 min LC separation coupled to an 18 m SLIM IM separation, considerably higher than LC alone and providing a basis for both improved identification and quantification, with additional gains projected with the future use of longer path SLIM IM separations. Thus, LC-SLIM IM-MS offers great potential for improving the sensitivity, separation, and throughput of phosphoproteomics analyses.

Distinguishing enantiomeric amino acids with chiral cyclodextrin adducts and structures for lossless ion manipulations.

Enantiomeric molecular evaluations remain an enormous challenge for current analytical techniques. To date, derivatization strategies and long separation times are generally required in these studies, and the development and implementation of new approaches are needed to increase speed and distinguish currently unresolvable compounds. Herein, we describe a method using chiral cyclodextrin adducts and structures for lossless ion manipulations (SLIM) and serpentine ultralong path with extended routing (SUPER) ion mobility (IM) to achieve rapid, high resolution separations of d and l enantiomeric amino acids. In the analyses, a chiral cyclodextrin is added to each sample. Two cyclodextrins were found to complex each amino acid molecule (i.e. potentially sandwiching the amino acid in their cavities) and forming host-guest noncovalent complexes that were distinct for each d and l amino acid pair studied and thus separable with IM in SLIM devices. The SLIM was also used to accumulate much larger ion populations than previously feasible for evaluation and therefore allow enantiomeric measurements of higher sensitivity, with gains in resolution from our ultralong path separation capabilities, than previously reported by any other IM-based approach.

Cation-Dependent Conformations in 25-Hydroxyvitamin D3-Cation Adducts Measured by Ion Mobility-Mass Spectrometry and Theoretical Modeling.

Ion mobility-mass spectrometry is a useful tool in separation of biological isomers, including clinically relevant analytes such as 25-hydroxyvitamin D3 (25OHD3) and its epimer, 3-epi-25-hydroxyvitamin D3 (epi25OHD3). Previous research indicates that these epimers adopt different gas-phase sodiated monomer structures, either the "open" or "closed" conformer, which allow 25OHD3 to be readily resolved in mixtures. In the current work, alternative metal cation adducts are investigated for their relative effects on the ratio of "open" and "closed conformers. Alkali and alkaline earth metal adducts caused changes in the 25OHD3 conformer ratio, where the proportion of the "open" conformer generally increases with the size of the metal cation in a given group. As such, the ratio of the "open" conformer, which is unique to 25OHD3 and absent for its epimer, can be increased from approximately 1:1 for the sodiated monomer to greater than 8:1 for the barium adduct. Molecular modeling and energy calculations agree with the experimental results, indicating that the Gibbs free energy of conversion from the "closed" to the "open" conformation decreased with increasing cation size, correlating with the variation in ratio between the conformers. This work demonstrates the effect of cation adducts on gas-phase conformations of small, flexible molecules and offers an additional strategy for resolution of clinically relevant epimers.

Ion Mobility in Clinical Analysis: Current Progress and Future Perspectives.

BACKGROUND: Ion mobility spectrometry (IMS) is a rapid separation tool that can be coupled with several sampling/ionization methods, other separation techniques (e.g., chromatography), and various detectors (e.g., mass spectrometry). This technique has become increasingly used in the last 2 decades for applications ranging from illicit drug and chemical warfare agent detection to structural characterization of biological macromolecules such as proteins. Because of its rapid speed of analysis, IMS has recently been investigated for its potential use in clinical laboratories. CONTENT: This review article first provides a brief introduction to ion mobility operating principles and instrumentation. Several current applications will then be detailed, including investigation of rapid ambient sampling from exhaled breath and other volatile compounds and mass spectrometric imaging for localization of target compounds. Additionally, current ion mobility research in relevant fields (i.e., metabolomics) will be discussed as it pertains to potential future application in clinical settings. SUMMARY: This review article provides the authors' perspective on the future of ion mobility implementation in the clinical setting, with a focus on ambient sampling methods that allow IMS to be used as a "bedside" standalone technique for rapid disease screening and methods for improving the analysis of complex biological samples such as blood plasma and urine.

Improved separation of fentanyl isomers using metal cation adducts and high-resolution ion mobility-mass spectrometry.

Fentanyl is a potent synthetic opioid that has attracted significant attention due to its illegal production and distribution, resulting in misuse, overdose, and fatalities. Because numerous fentanyl analogs, including structural isomers, with different potency have been discovered in the field, there is a critical need to continue developing analytical methodologies capable of accurate identification in forensic and clinical laboratories. This study aimed to develop a rapid method for detecting and separating fentanyl isomers based on ion mobility-mass spectrometry (IM-MS), where IM separates gas-phase ions based on differences in their size, shape, and charge. Several strategies for improved differentiation were implemented, including using unconventional cation adducts (e.g., alkali and transition metals) and data post-processing by high-resolution demultiplexing. A collection of collision cross section (CCS) values for the various metal ion adducts was gathered, which can be used to improve confidence of identification in future samples. Notable examples, such as [M + Cu]+ and [M + Ag]+ adducts, contributed to significant improvement of resolution between isomers. Furthermore, the addition of high-resolution post-processing provided resolving power of >150, which constitutes a significant increase in comparison with the normal 50-60 obtained with low-resolution drift tube instruments. Collectively, these improved separation strategies allowed for confident detection and subsequent quantitative analysis. The optimized IM-MS method resulted in quantification of fentanyl in human urine with limits of detection and quantification of 13 pg/mL and 40 pg/mL, respectively.

Separation and Characterization of Synthetic Cannabinoid Metabolite Isomers Using SLIM High-Resolution Ion Mobility-Tandem Mass Spectrometry (HRIM-MS/MS).

Synthetic cannabinoids, a subclass of new psychoactive substances (NPS), are laboratory-made substances that are chemically similar to those found naturally in the cannabis plant. Many of these substances are illicitly manufactured and have been associated with severe health problems, prompting a need to develop analytical methods capable of characterizing both known and previously undetected compounds. This work focuses on a novel Structures for Lossless Ion Manipulations (SLIM) IM-MS approach to the differentiation and structural characterization of synthetic cannabinoid metabolites, specifically MDA-19/BUTINACA, JWH-018, and JWH-250 isomer groups. These different compound classes are structurally very similar, differing only in the position of one or a few functional groups; this yielded similarity in measured collision cross section (CCS) values. However, the high resolution of SLIM IM provided adequate separation of many of these isomers, such as sodiated JWH-250 metabolites N-4-OH, N-5-OH, and 5-OH, which displayed CCS of 187.5, 182.5, and 202.3 Å2, respectively. In challenging cases where baseline separation was precluded due to nearly identical CCS, such as for JWH-018 isomers, simple derivatization by dansyl chloride selectively reacted with the 6-OH compound to provide differentiation of all isomers using a combination of CCS and m/z. Finally, the opportunity to use this method for structural elucidation of unknowns was demonstrated by using SLIM IM mobility-aligned MS/MS fragmentation. Different MDA-19/BUTINACA isomers were first mobility separated and could then be individually activated, yielding unique fragments for both targeted identification and structural determination. Overall, the described SLIM IM-MS/MS workflow provides significant potential as a rapid screening tool for the characterization of emerging NPS such as synthetic cannabinoids and their metabolites.

Rapid differentiation of xylazine metabolites using SLIM IM-MS.

Xylazine represents an increased threat to the recreational drug market. In this study, we present a rapid strategy for identifying xylazine and differentiating its common isomeric metabolites using Structures for Lossless Ion Manipulations (SLIM) ion mobility coupled to high-resolution/tandem mass spectrometry (IM-HRMS/MS). Chemical derivatization using dansyl chloride also assisted with separations and led to identification of resolvable reaction product atropisomers.

SLIM-Based High-Resolution Ion Mobility Reveals New Structural Insights into Isomeric Vitamin D Metabolites and their Isotopologues.

Testing for vitamin D deficiency remains a high-volume clinical assay, much of which is done using mass spectrometry-based methods to alleviate challenges in selectivity associated with immunoassays. Ion mobility-mass spectrometry (IM-MS) has been proposed as a rapid alternative to traditional LC-MS/MS methods, but understanding the structural ensemble that contributes to the ion mobility behavior of this molecular class is critical. Herein we demonstrate the first application of high-resolution Structures for Lossless Ion Manipulations (SLIM) IM separations of several groups of isomeric vitamin D metabolites. Despite previous IM studies of these molecules, the high resolving power of SLIM (Rp ∼ 200) has revealed additional conformations for several of the compounds. The highly similar collision cross sections (CCS), some differing by as little as 0.7%, precluded adequate characterization with low-resolution IM techniques where, in some cases, wider than expected peak widths and/or subtle shoulders may have hinted at their presence. Importantly, these newly resolved peaks often provided a unique mobility that could be used to separate isomers and provides potential for their use in quantification. Lastly, the contribution of isotopic labeling to arrival time distribution for commonly used 13C- and deuterium-labeled internal standards was explored. Minor shifts of ∼0.2-0.3% were observed, and in some instances these shifts were specific to the conformer being measured (i.e., "closed" vs "open"). Accounting for these shifts is important during raw data extraction to ensure reproducible peak area integration, which will be a critical consideration in future quantitative applications.

Characterization of Bile Acid Isomers and the Implementation of High-Resolution Demultiplexing with Ion Mobility-Mass Spectrometry.

Bile acids (BAs) are a complex suite of clinically relevant metabolites that include many isomers. Liquid chromatography coupled to mass spectrometry (LC-MS) is an increasingly popular technique due to its high specificity and sensitivity; nonetheless, acquisition times are generally 10-20 min, and isomers are not always resolved. In this study, the application of ion mobility (IM) spectrometry coupled to MS was investigated to separate, characterize, and measure BAs. A subset of 16 BAs was studied, including three groups of isomers belonging to unconjugated, glycine-conjugated, and taurine-conjugated BA classes. A variety of strategies were explored to increase BA isomer separation such as changing the drift gas, measuring different ionic species (i.e., multimers and cationized species), and enhancing the instrumental resolving power. In general, Ar, N2, and CO2 provided the best peak shape, resolving power (Rp), and separation, especially CO2; He and SF6 were less preferable. Furthermore, measuring dimers versus monomers improved isomer separation due to enhanced gas-phase structural differences. A variety of cation adducts other than sodium were characterized. Mobility arrival times and isomer separation were affected by the choice of adduct, which was shown to be used to target certain BAs. Finally, a novel workflow that involves high-resolution demultiplexing in combination with dipivaloylmethane ion-neutral clusters was implemented to improve Rp dramatically. A maximum Rp increase was observed with lower IM field strengths to obtain longer drift times, increasing Rp from 52 to 187. A combination of these separation enhancement strategies demonstrates great potential for rapid BA analysis.

Development of a Rapid, Targeted LC-IM-MS Method for Anabolic Steroids.

Anabolic steroids are of high biological interest due to their involvement in human development and disease progression. Additionally, they are banned in sport due to their performance-enhancing characteristics. Analytical challenges associated with their measurement stem from structural heterogeneity, poor ionization efficiency, and low natural abundance. Their importance in a variety of clinically relevant assays has prompted the consideration of integrating ion mobility spectrometry (IMS) into existing LC-MS assays, due primarily to its speed and structure-based separation capability. Herein we have optimized a rapid (2 min) targeted LC-IM-MS method for the detection and quantification of 40 anabolic steroids and their metabolites. First, a steroid-specific calibrant mixture was developed to cover the full range of retention time, mobility, and accurate mass. Importantly, this use of this calibrant mixture provided robust and reproducible measurements based on collision cross section (CCS) with interday reproducibility of <0.5%. Furthermore, the combined separation power of LC coupled to IM provided comprehensive differentiation of isomers/isobars within 6 different isobaric groups. Multiplexed IM acquisition also provided improved limits of detection, which were well below 1 ng/mL in almost all compounds measured. This method was also capable of steroid profiling, providing quantitative ratios (e.g., testosterone/epitestosterone, androsterone/etiocholanolone, etc.). Lastly, phase II steroid metabolites were probed in lieu of hydrolysis to demonstrate the ability to separate those analytes and provide information beyond total steroid concentration. This method has tremendous potential for rapid analysis of steroid profiles in human urine spanning a variety of applications from developmental disorders to doping in sport.

Targeted glucocorticoid analysis using ion mobility-mass spectrometry (IM-MS).

Introduction: Ion mobility-mass spectrometry (IM-MS) is an emerging technique in the -omics fields that has broad potential applicability to the clinical lab. As a rapid, gas-phase structure-based separation technique, IM-MS offers promise in isomer separations and can be easily combined with existing LC-MS methods (i.e., LC-IM-MS). Several experimental conditions, including analyte cation adducts and drift composition further provide a means to tune separations for global and/or targeted applications. Objectives: The primary objective of this study was to demonstrate the utility of IM-MS under a range of experimental conditions for detection of glucocorticoids, and specifically for the separation of several isomeric pairs. Methods: LC-IM-MS was used to characterize 16 glucocorticoids including three isomer pairs: cortisone/prednisolone, betamethasone/dexamethasone, and flunisolide/triamcinolone acetonide. Collision cross section (CCS) values were measured for all common adducts (e.g., protonated and sodiated) using both step-field and single-field methods. Alternative alkali, alkaline earth, and transition metals were introduced, such that their adducts could also be measured. Finally, four different drift gases (helium, nitrogen, argon, and carbon dioxide) were compared for their relative separation capability. Results: LC-IM-MS offered a robust, multidimensional separation technique that allowed for the 16 glucocorticoids to be analyzed and separated in three-dimensions (retention time, CCS, and m/z). Despite the relatively modest resolution of isomer pairs under standard conditions (i.e., nitrogen drift gas, sodiated ions, etc.), improvements were observed for alkaline earth and transition metals (notable barium adducts) and in carbon dioxide drift gas. Conclusion: In summary, LC-IM-MS offers potential as a clinical method due to its ease of coupling with traditional LC-MS methods and its promise for tuning separations to better resolve targeted and/or global isomers in complex biological samples.