Collision Cross Section Values Research Articles

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

IntroductionIon 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. ObjectivesThe 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. MethodsLC-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. ResultsLC-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. ConclusionIn 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.

Improving confidence in lipidomic annotations by incorporating empirical ion mobility regression analysis and chemical class prediction.

Mass spectrometry-based untargeted lipidomics aims to globally characterize the lipids and lipid-like molecules in biological systems. Ion mobility increases coverage and confidence by offering an additional dimension of separation and a highly reproducible metric for feature annotation, the collision cross-section (CCS). We present a data processing workflow to increase confidence in molecular class annotations based on CCS values. This approach uses class-specific regression models built from a standardized CCS repository (the Unified CCS Compendium) in a parallel scheme that combines a new annotation filtering approach with a machine learning class prediction strategy. In a proof-of-concept study using murine brain lipid extracts, 883 lipids were assigned higher confidence identifications using the filtering approach, which reduced the tentative candidate lists by over 50% on average. An additional 192 unannotated compounds were assigned a predicted chemical class. All relevant source code is available at https://github.com/McLeanResearchGroup/CCS-filter. Supplementary data are available at Bioinformatics online.

Physicochemical property evaluation of modified oligonucleotides by traveling-wave ion mobility mass spectrometry.

Therapeutic oligonucleotides have molecular weights of more than 6000 Da. They typically contain chemically modified structures such as phosphorothioate (PS) and a locked nucleic acid (LNA). To determine the effect of the length and chemical modification on the physicochemical properties, various nucleic acids with different lengths and modified structures were analyzed using traveling-wave ion mobility mass spectrometry (TWIMS). The physicochemical characteristics of the modified oligonucleotides were determined using IM-MS. Each oligonucleotide was evaluated by confirming the multivalent charge state drift times, collision cross-section (CCS) values, and CCS widths. By plotting the m/z for oligonucleotides of different lengths and the CCS values at each charge state, a bottoming-out shape plot at one charge per 4.0-3.5 bases was confirmed. Moreover, significant differences were observed in the CCS values between the PS-modified and unmodified oligonucleotides. The PS-modified oligonucleotide showed a wider CCS range that was proportional to the PS modification ratio of the oligonucleotide sequence. The TWIMS results showed a correlation between the length and modification of oligonucleotides and the CCS values. In addition, it suggested that each charge state of the oligonucleotide ion has different physicochemical properties.

Tautomers and Rotamers of Curcumin: A Combined UV Spectroscopy, High-Performance Liquid Chromatography, Ion Mobility Mass Spectrometry, and Electronic Structure Theory Study.

The structures of tautomers and rotameric forms of curcumin, the bioactive compound present in spice plant turmeric, have been investigated using ion mobility mass spectrometry (IMMS) in conjunction with high-performance liquid chromatography (HPLC) and UV-visible spectroscopy. Two tautomeric forms of this β-diketone compound, keto-enol and diketo, have been chromatographically separated, and the electronic absorption spectra for these two tautomeric forms in methanol solution have been recorded separately for the first time. The molecular identity of the HPLC-separated solution fractions is established unambiguously by recording the mass and fragmentation spectra simultaneously. The ion mobility spectrum for the deprotonated curcumin anion, [Cur-H]-, corresponding to the diketo tautomer, displays only one peak (P), and the collision cross-section (CCS) value is measured to be 185.9 Å2. However, the ion mobility spectrum corresponding to the HPLC-separated keto-enol tautomer shows three distinctly separated peaks, P, Q, and R, with CCS values of 185.9, 194.8, and 203.4 Å2, respectively, whereby peak R appears to be the most intense one, followed by peaks P and Q. The theoretically calculated CCS values of different isomers of [Cur-H]-, optimized by electronic structure theory methods, display satisfactory correlation with the experimentally observed values, corroborating our assignments. The spectral attributes also indicate the occurrence of structural rearrangements in the electrospray ionization process. With the aid of the electronic structure calculation, low-energy pathways for the occurrence of the structural isomerization to surpass the energy barrier are suggested, which are consistent with the assignments of the peaks observed in the IM spectra.

Uncovering the behaviour of ions in the gas-phase to predict the ion mobility separation of isomeric steroid compounds

Bile acids are steroid compounds involved in biological mechanisms of neurodegenerative diseases making them potential biomarkers for diagnosis or treatment. These compounds exist as structural and conformational isomers, which hinder distinguishing them in physiological processes. We aimed to develop tandem mass spectrometry-ion mobility spectrometry (MS/MS-IMS) methodologies to explore and understand the behaviour of isomeric steroids in the gas-phase and rapidly separate them. Unlike previously published ion mobility data, various isomers were investigated in mixtures to better mimic complex (pre-) clinical samples. The experimental collision cross sections (CCS)s were compared to the theoretical CCS values for an in-depth analysis of isomeric ions' behaviour in the gas-phase. Based on density-functional theory, we identified the impact of adduct positioning on the 3D conformation of enantiomers, diastereomers and structural isomers. The curling of the large side chains hedged the small differences among the isomers and lowered the CCS values. On the other hand, fragmenting off the identical side branches as well as imposing the bending of the steroid ring resulted in ion mobility differentiation. Careful data evaluation revealed the tendency of isomers to form homo-cluster in the mixture solutions and assist the separation. Our fundamental and experimental findings enable the ion mobility separation of isomeric steroids to be predicted. The introduced rapid and optimal MS/MS-IMS analytical methodology can be applied to distinguish isomeric bile acids both in a solution and potentially in patients’ tissue samples, and consequently, reveal their molecular pathways.

Influence Exerted by the Solvent Effect on the Mobility Peak of 1,8-Naphthalic Anhydride in Ion Mobility Spectrometry.

The collision cross-section (CCS) values of ions determined by ion mobility-mass spectrometry (IM-MS) can be used to deduce the shape and size of the ions. For each compound, as well as its isomer or tautomer, a unique arrival time peak was obtained in extracted ion mobility (EIM) spectra, which corresponded to a specific CCS value. However, the generation of solvated ions by electrospray ionization (ESI) increases the number of mobility peaks, which makes the EIM spectra difficult to interpret. In this study, solvent clusters formed by acetonitrile and methanol around 1,8-naphthalic anhydride (1,8-NA) cations ([C12H6O3 + H]+1,8-NA) were investigated using trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOF MS). The effects of infusion flow rate, nebulizer gas pressure, drying gas rate, and drying gas temperature on the formation of solvent clusters from acetonitrile and methanolic solution were systematically studied. The formation of solvent clusters was observed with infusion flow rates increased, which was manifested by the larger experimental CCS values of [C12H6O3 + H]+1,8-NA. Acetonitrile tended to form solvent clusters around ions more readily than methanol. These solvent clusters were stable enough to be detected by TIMS, but they cannot survive under ion activation conditions of mass spectrometry (MS). Increasing the nebulizer gas pressure seems to be a better way to eliminate the formation of solvent clusters in TIMS-TOF MS and give a "cleaner" EIM spectra. The current research demonstrates that more attention should be paid to the solvent effect on CCS values and their interpretation.

Prediction of Collision Cross Section Values: Application to Non-Intentionally Added Substance Identification in Food Contact Materials.

The synthetic chemicals in food contact materials can migrate into food and endanger human health. In this study, the traveling wave collision cross section in nitrogen values of more than 400 chemicals in food contact materials were experimentally derived by traveling wave ion mobility spectrometry. A support vector machine-based collision cross section (CCS) prediction model was developed based on CCS values of food contact chemicals and a series of molecular descriptors. More than 92% of protonated and 81% of sodiated adducts showed a relative deviation below 5%. Median relative errors for protonated and sodiated molecules were 1.50 and 1.82%, respectively. The model was then applied to the structural annotation of oligomers migrating from polyamide adhesives. The identification confidence of 11 oligomers was improved by the direct comparison of the experimental data with the predicted CCS values. Finally, the challenges and opportunities of current machine-learning models on CCS prediction were also discussed.

Least absolute shrinkage and selection operator-based prediction of collision cross section values for ion mobility mass spectrometric analysis of lipids.

Collision cross section (CCS) values generated from ion mobility mass spectrometry (IM-MS) have commonly been employed to facilitate lipid identification. However, this is hindered by the limited available lipid standards. Recently, CCS values were predicted by means of computational calculations, though the prediction precision was generally not good and the predicted CCS values of the lipid isomers were almost identical. To address this challenge, a least absolute shrinkage and selection operator (LASSO)-based prediction method was developed for the prediction of lipids' CCS values in this study. In this method, an array of molecular descriptors were screened and optimized to reflect the subtle differences in structures among the different lipid isomers. The use of molecular descriptors together with a wealth of standard CCS values for the lipids (365 in total) significantly improved the accuracy and precision of the LASSO model. Its accuracy was externally validated with median relative errors (MREs) of <1.1% using an independent data set. This approach was demonstrated to allow differentiation of cis/trans and sn-positional isomers. The results also indicated that the LASSO-based prediction method could practically reduce false-positive identifications in IM-MS-based lipidomics.

Food authentication: truffle species classification by non-targeted lipidomics analyses using mass spectrometry assisted by ion mobility separation.

Truffles are appreciated as food all over the world because of their extraordinary aroma. However, quantities are limited and successful cultivation in plantations is very labor-intensive and expensive, or even impossible for some species. These factors make truffles a very valuable food, which is why it is particularly rewarding and tempting to declare inferior species of truffles as more expensive species and thereby counterfeit them. The various species differ in their aroma and thus in their culinary value, but the adulterations cannot be detected on the basis of pure morphology. For this reason, the objective of the present study was to develop a non-targeted lipidomics approach using ion mobility spectrometry-mass spectrometry to distinguish between the white truffle species Tuber magnatum and T. borchii as well as the black truffle species T. melanosporum, T. aestivum and T. indicum. Several hundred features were detected, which were present in significantly different concentrations in the studied truffle species. The most important of them were identified using MS/MS spectra and collision cross section (CCS) values. Some compounds were detected whose CCS values have not yet been published and may facilitate identification by other researchers in the future. Just a few marker substances are sufficient to be able to distinguish both black and white truffle species with 100% accuracy. These results can be used for the development of rapid tests, which in the best case will allow truffle analysis directly on-site.

Multi-CRAFTI: Relative Collision Cross Sections from Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Line Width Measurements.

Determination of collision cross sections (CCS) using the cross-sectional areas by the Fourier transform ion cyclotron resonance (CRAFTI) technique is limited by the requirement that accurate pressures in the trapping cell of the mass spectrometer must be known. Experiments must also be performed in the energetic hard-sphere regime such that ions decohere after single collisions with neutrals; this limits application to ions that are not much more massive than the neutrals. To mitigate these problems, we have resonantly excited two (or more) ions of different m/z to the same center-of-mass kinetic energy in a single experiment, subjecting them to identical neutral pressures. We term this approach "multi-CRAFTI". This facilitates measurement of relative CCS without requiring knowledge of the pressure and enables determination of absolute CCS using internal standards. Experiments with tetraalkylammonium ions yield CCS in reasonable agreement with the one-ion-at-a-time CRAFTI approach and with ion mobility spectrometry (IMS) when differences in collision energetics are taken into account (multi-CRAFTI generally yields smaller CCS than does IMS due to the higher collision energies employed in multi-CRAFTI). Comparison of multi-CRAFTI and IMS results with CCS calculated from structures computed at the M06-2X/6-31+G* level of theory using projection approximation or trajectory method values, respectively, indicates that the computed structures have CCS increasingly smaller than the experimental CCS as m/z increases, implying the computational model overestimates interactions between the alkyl arms. For ions that undergo similar collisional decoherence processes, relative CCS reach constant values at lower collision energies than do absolute CCS values, suggesting a means of increasing the accessible upper m/z limit by employing multi-CRAFTI.

A Deep Convolutional Neural Network for Prediction of Peptide Collision Cross Sections in Ion Mobility Spectrometry.

Most frequently, the identification of peptides in mass spectrometry-based proteomics is carried out using high-resolution tandem mass spectrometry. In order to increase the accuracy of analysis, additional information on the peptides such as chromatographic retention time and collision cross section in ion mobility spectrometry can be used. An accurate prediction of the collision cross section values allows erroneous candidates to be rejected using a comparison of the observed values and the predictions based on the amino acids sequence. Recently, a massive high-quality data set of peptide collision cross sections was released. This opens up an opportunity to apply the most sophisticated deep learning techniques for this task. Previously, it was shown that a recurrent neural network allows for predicting these values accurately. In this work, we present a deep convolutional neural network that enables us to predict these values more accurately compared with previous studies. We use a neural network with complex architecture that contains both convolutional and fully connected layers and comprehensive methods of converting a peptide to multi-channel 1D spatial data and vector. The source code and pre-trained model are available online.

Systematic Optimization of Ambient Ionization Ion Mobility Mass Spectrometry for Rapid Separation of Isomers.

Current methods typically used for metabolite screening and disease diagnosis often require extensive sample preparation, which increases analysis time and associated costs. While ambient ionization techniques enable the analysis of various samples in complex matrices with little or no sample preparation in a short time (typically within a minute), their reduced selectivity, even when coupled with high-resolution mass spectrometers, limits their application in certain fields. In this study, we have optimized the coupling of paper spray (PS) and leaf spray (LS) ambient ionization techniques with a commercially available ion mobility mass spectrometer (IM-MS) and demonstrated the separation of geometric and constitutional isomers. Ambient ionization techniques allow simultaneous introduction and ionization of samples, while background noise and matrix interference from paper and leaf substrates are filtered out by IM separation, resulting in high sensitivity and selectivity of the PS-IM-MS and LS-IM-MS workflows. In addition, we introduced a novel approach to perform single-field collision cross section (CCS) measurements, which resulted in CCS values that differ by 0.15% and 0.25% from traditional stepped-field and single-field methods, respectively. In addition, we used advanced computational tools to confidently identify analyte structures by comparing CCS values from experimental IM measurements and theoretical calculations. These results suggest that the coupling of ambient ionization methods with ion mobility techniques enables rapid, sensitive, and highly selective analysis that can be used in different fields, such as agrochemical screening and disease diagnostics.

Large-Scale Evaluation of Collision Cross Sections to Investigate Blood-Brain Barrier Permeation of Drugs.

Successful drug administration to the central nervous system requires accurate adjustment of the drugs’ molecular properties. Therefore, structure-derived descriptors of potential brain therapeutic agents are essential for an early evaluation of pharmacokinetics during drug development. The collision cross section (CCS) of molecules was recently introduced as a novel measurable parameter to describe blood-brain barrier (BBB) permeation. This descriptor combines molecular information about mass, structure, volume, branching and flexibility. As these chemical properties are known to influence cerebral pharmacokinetics, CCS determination of new drug candidates may provide important additional spatial information to support existing models of BBB penetration of drugs. Besides measuring CCS, calculation is also possible; but however, the reliability of computed CCS values for an evaluation of BBB permeation has not yet been fully investigated. In this work, prediction tools based on machine learning were used to compute CCS values of a large number of compounds listed in drug libraries as negative or positive with respect to brain penetration (BBB+ and BBB− compounds). Statistical evaluation of computed CCS and several other descriptors could prove the high value of CCS. Further, CCS-deduced maximum molecular size of BBB+ drugs matched the dimensions of BBB pores. A threshold for transcellular penetration and possible permeation through pore-like openings of cellular tight-junctions is suggested. In sum, CCS evaluation with modern in silico tools shows high potential for its use in the drug development process.

A Novel Approach to Characterize the Lipidome of Marine Archaeon Nitrosopumilus maritimus by Ion Mobility Mass Spectrometry.

Archaea are differentiated from the other two domains of life by their biomolecular characteristics. One such characteristic is the unique structure and composition of their lipids. Characterization of the whole set of lipids in a biological system (the lipidome) remains technologically challenging. This is because the lipidome is innately complex, and not all lipid species are extractable, separable, or ionizable by a single analytical method. Furthermore, lipids are structurally and chemically diverse. Many lipids are isobaric or isomeric and often indistinguishable by the measurement of mass or even their fragmentation spectra. Here we developed a novel analytical protocol based on liquid chromatography ion mobility mass spectrometry to enhance the coverage of the lipidome and characterize the conformations of archaeal lipids by their collision cross-sections (CCSs). The measurements of ion mobility revealed the gas-phase ion chemistry of representative archaeal lipids and provided further insights into their attributions to the adaptability of archaea to environmental stresses. A comprehensive characterization of the lipidome of mesophilic marine thaumarchaeon, Nitrosopumilus maritimus (strain SCM1) revealed potentially an unreported phosphate- and sulfate-containing lipid candidate by negative ionization analysis. It was the first time that experimentally derived CCS values of archaeal lipids were reported. Discrimination of crenarchaeol and its proposed stereoisomer was, however, not achieved with the resolving power of the SYNAPT G2 ion mobility system, and a high-resolution ion mobility system may be required for future work. Structural and spectral libraries of archaeal lipids were constructed in non-vendor-specific formats and are being made available to the community to promote research of Archaea by lipidomics.

Collision Cross Sections for Native Proteomics: Challenges and Opportunities.

Recent advancements place a comprehensive catalog of protein structure, oligomeric state, sequence, and modification status tentatively within reach, thus providing an unprecedented roadmap to therapies for many human diseases. To achieve this goal, revolutionary technologies capable of bridging key gaps in our ability to simultaneously measure protein composition and structure must be developed. Much of the current progress in this area has been catalyzed by mass spectrometry (MS) tools, which have become an indispensable resource for interrogating the structural proteome. For example, methods associated with native proteomics seek to comprehensively capture and quantify the endogenous assembly states for all proteins within an organism. Such technologies have often been partnered with ion mobility (IM) separation, from which collision cross section (CCS) information can be rapidly extracted to provide protein size information. IM technologies are also being developed that utilize CCS values to enhance the confidence of protein identification workflows derived from liquid chromatography-IM-MS analyses of enzymatically produced peptide mixtures. Such parallel advancements in technology beg the question: can CCS values prove similarly useful for the identification of intact proteins and their complexes in native proteomics? In this perspective, I examine current evidence and technology trends to explore the promise and limitations of such CCS information for the comprehensive analysis of multiprotein complexes from cellular mixtures.

Ion mobility spectrometry-tandem mass spectrometry strategies for the on-line monitoring of a continuous microflow reaction

Continuous flow chemistry is an efficient, sustainable and green approach for chemical synthesis that surpasses some of the limitations of the traditional batch chemistry. Along with the multiple advantages of a flow reactor, it could be directly connected to the analytical techniques for on-line monitoring of a chemical reaction and ensure the quality by design. Here, we aim to use ion mobility, mass and tandem mass spectrometry (IMS-MS and MS/MS) for the on-line analysis of a pharmaceutically relevant chemical reaction. We carried out a model hetero-Diels Alder reaction in a microflow reactor directly connected to the IMS-MS and MS/MS using either electrospray or atmospheric pressure photo ionization methods. We were able to monitor the reaction mechanism of the Diels Alder reaction and structurally characterize the reaction product and synthesis side-products. The chosen approach enabled identification of two isomers of the main reaction product. A new strategy to annotate the ion mobility spectrum in the absence of standard molecules was introduced and tested for its validity. This was achieved by determining the survival yield of each isomer upon ion mobility separation and density functional theory calculations. This approach was verified by comparing the theoretically driven collision cross section values to the experimental data. In this paper, we demonstrated the potential of combined IMS-MS and MS/MS on-line analysis platform to investigate, monitor and characterize structural isomers in the millisecond time scale.

Rapid Discrimination of Citrus reticulata 'Chachi' by Electrospray Ionization-Ion Mobility-High-Resolution Mass Spectrometry.

A common idea is that some dishonest businessmen often disguise Citrus reticulata Blanco varieties as Citrus reticulata ‘Chachi’, which places consumers at risk of economic losses. In this work, we combined high-resolution ion mobility (U-shaped mobility analyzer) with high-resolution mass spectrometry to rapidly distinguish Citrus reticulata ‘Chachi’ from other Citrus species. The samples were analyzed directly through simple extraction and the analytes were separated in one second. It only took about 1 min to perform a cycle of sample analysis and data acquisition. The results showed that polymethoxylated flavones and their isomers were separated easily by the ion mobility analyzer and preliminarily identified according to the accurate mass. Moreover, the collision cross-section values of all analytes, which could be used as auxiliary parameters to characterize and identify the compounds in the samples, were measured. Twenty-four samples were grouped as two clusters by multivariate analysis, which meant that Citrus reticulata ‘Chachi’ could be effectively differentiated. It was confirmed that the developed method had the potential to rapidly separate polymethoxylated flavones and distinguish between Citrus reticulata ‘Chachi’ and other Citrus reticulata Blanco varieties.

Separation of phosphorothioated oligonucleotide diastereomers using multiplexed drift tube ion mobility mass spectrometry

Hydrophilic interaction liquid chromatography (HILIC) coupled to drift tube ion mobility spectrometry (DTIMS) was used to separate diastereomers of five-unit oligonucleotides containing 0, 1, 2 or 3 phosphorothioate (PS) linkages. Multiplexed DTIMS (where ions are pulsed into the drift tube according to a pre-encoded sequence) and post-acquisition processing using an innovative demultiplexing tool were investigated. The electric field inside the drift tube was optimized to achieve the highest resolving power. The entrance voltage providing the best two-peak resolution was −1000V with 3-bit multiplexing.Under optimized conditions, the eight diastereomers of an oligonucleotide with three PS linkages (5′-TC∗G∗T∗G-3′) could be separated unambiguously. Indeed, those diastereomers differed in their collision cross section (CCS) values. The minimal CCS values difference between two adjacent diastereomers was 0.9% with maximal RSD on CCS values of 0.3%.The use of multiplexed ion mobility and the novel high-resolution demultiplexing tool represents a real breakthrough for resolution enhancement of diastereomers in linear DTIMS.

Optimization of a liquid chromatography-ion mobility-high resolution mass spectrometry platform for untargeted lipidomics and application to HepaRG cell extracts

Analytical methods to evaluate the lipidome of biological samples need to provide high data quality to ensure comprehensive profiling and reliable structural elucidation. In this perspective, liquid chromatography-high resolution mass spectrometry (LC-HRMS) is the state-of-the-art technique for lipidomic analysis of biological samples. There are thousands of lipids in most biological samples, and therefore separation methods before introduction to the mass spectrometer is key for relative quantitation and identification. Chromatographic methods differ across laboratories, without any consensus on the best methodologies. Therefore, we designed an experiment to determine the optimal LC methodology, and assessed the value of ion mobility for an additional dimension of separation. To apply an untargeted method for hypothesis generation focused on lipidomics, LC-HRMS parameters were optimized based on the measurement of 50 panel lipids covering key human metabolic pathways. Reversed-phase liquid chromatography columns were compared based on a quality scoring system considering the signal-to-noise ratio, peak shape, and retention factor. Furthermore, drift tube ion mobility spectrometry (DTIMS) was implemented to increase peak capacity and confidence during annotation by providing collision cross section (CCS) values for the analytes under investigation. However, hyphenating DTIMS to LC-HRMS may result in a reduced sensitivity due to impaired duty cycles. To increase the signal intensity, a Box-Behnken design (BBD) was used to optimize four key factors, i.e. drift entrance voltage, drift exit voltage, rear funnel entrance, and rear funnel exit voltages. Application of a maximized desirability function provided voltages for the above-mentioned parameters resulting in higher signal intensity compared to each combination of parameters used during the BBD. In addition, the influence of single pulse and Hadamard 4-bit multiplexed modes on signal intensity was explored and different trap filling and release times of ions were evaluated. The optimized LC-DTIM-HRMS platform was applied to extracts from HepaRG cells and resulted in 3912 high-quality features (<30% median relative standard deviation; n = 6, t = 24 h). From these features, 436 lipid species could be annotated (i.e., matching based on accurate mass <5 ppm, isotopic pattern, in-silico MS/MS fragmentation, and in-silico CCS database matching <3%). The application of LC-DTIM-HRMS for untargeted analysis workflows is growing and the platform optimization, as described here, can be used to guide the method development and CCS database comparison for high confidence lipid annotation.

Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC–Ion Mobility–HRMS

The detection and unambiguous identification of anabolic-androgenic steroid metabolites are essential in clinical, forensic, and antidoping analyses. Recently, sulfate phase II steroid metabolites have received increased attention in steroid metabolism and drug testing. In large part, this is because phase II steroid metabolites are excreted for an extended time, making them a potential long-term chemical marker of choice for tracking steroid misuse in sports. Comprehensive analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been used to detect and identify glucuronide and sulfate steroids in human urine with high sensitivity and reliability. However, LC-MS/MS identification strategies can be hindered by the fact that phase II steroid metabolites generate nonselective ion fragments across the different metabolite markers, limiting the confidence in metabolite identifications that rely on exact mass measurement and MS/MS information. Additionally, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is sometimes insufficient at fully resolving the analyte peaks from the sample matrix (commonly urine) chemical noise, further complicating accurate identification efforts. Therefore, we developed a liquid chromatography-ion mobility-high resolution mass spectrometry (LC-IM-HRMS) method to increase the peak capacity and utilize the IM-derived collision cross section (CCS) values as an additional molecular descriptor for increased selectivity and to improve identifications of intact steroid analyses at low concentrations.