Recommendations for reporting ion mobility Mass Spectrometry measurements.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

Combined hydrophilic interaction liquid chromatography-scanning field asymmetric waveform ion mobility spectrometry-time-of-flight mass spectrometry for untargeted metabolomics.

Untargeted metabolite profiling of biological samples is a challenge for analytical science due to the high degree of complexity of biofluids. Isobaric species may also not be resolved using mass spectrometry alone. As a result of these factors, many potential biomarkers may not be detected or are masked by co-eluting interferences in conventional LC-MS metabolomic analyses. In this study, a comprehensive liquid chromatography-mass spectrometry workflow incorporating a fast-scanning miniaturised high-field asymmetric waveform ion mobility spectrometry separation (LC-FAIMS-MS) is applied to the untargeted metabolomic analysis of human urine. The time-of-flight mass spectrometer used in the study was scanned at a rate of 20 scans s-1 enabling a FAIMS CF spectrum to be acquired within a 1-s scan time, maintaining an adequate number of data points across each LC peak. The developed method is demonstrated to be able to resolve co-eluting isomeric species and shows good reproducibility (%RSD < 4.9%). The nested datasets obtained for fresh, aged, and QC urine samples were submitted for multivariate statistical analysis. Seventy unique biomarker ions showing a statistically significant difference between fresh and aged urine were identified with optimal transmission CF values obtained across the full CF spectrum. The potential of using FAIMS to select ions for in-source collision-induced dissociation is demonstrated for FAIMS-selected methylxanthine ions yielding characteristic fragment ion species indicative of the precursor. Graphical abstract.

Increasing Peak Capacity in Nontargeted Omics Applications by Combining Full Scan Field Asymmetric Waveform Ion Mobility Spectrometry with Liquid Chromatography-Mass Spectrometry.

Full scan field asymmetric waveform ion mobility spectrometry (FAIMS) combined with liquid chromatography and mass spectrometry (LC-FAIMS-MS) is shown to enhance peak capacity for omics applications. A miniaturized FAIMS device capable of rapid compensation field scanning has been incorporated into an ultrahigh performance liquid chromatography (UHPLC) and time-of-flight mass spectrometry analysis, allowing the acquisition of full scan FAIMS and MS nested data sets within the time scale of a UHPLC peak. Proof of principle for the potential of scanning LC-FAIMS-MS in omics applications is demonstrated for the nontargeted profiling of human urine using a HILIC column. The high level of orthogonality between FAIMS and MS provides additional unique compound identifiers with detection of features based on retention time, FAIMS dispersion field and compensation field (DF and CF), and mass-to-charge (m/z). Extracted FAIMS full scan data can be matched to standards to aid the identification of unknown analytes. The peak capacity for features detected in human urine using LC-FAIMS-MS was increased approximately threefold compared to LC-MS alone due to a combination of the reduction of chemical noise and separation of coeluting isobaric species across the entire analytical space. The use of FAIMS-selected in source collision induced dissociation (FISCID) yields fragmentation of ions, which reduces sample complexity associated with overlapping fragmentation patterns and provides structural information on the selected precursor ions.

Rapid Analysis of Anabolic Steroid Metabolites in Urine by Combining Field Asymmetric Waveform Ion Mobility Spectrometry with Liquid Chromatography and Mass Spectrometry.

The combination of field asymmetric waveform ion mobility spectrometry with liquid chromatography-mass spectrometry (LC-FAIMS-MS) has been developed for the analysis of glucuronide and sulfate metabolites of seven anabolic-androgenic steroids in urine. Separation by FAIMS-MS was investigated in positive ion mode for selected cationic adducts (H+, NH4+, Na+, K+, and Cs+). LC-FAIMS-MS analysis of the doubly sodiated adducts ([M + 2Na - H]+) of isobaric and coeluting steroid metabolites allowed their rapid (8 min) qualitative and quantitative determination in spiked urine using hydrophilic interaction liquid chromatography prior to FAIMS-MS separation, with discrimination >95% achieved between the steroids investigated. A quantitative evaluation of the LC-FAIMS-MS method was performed giving limits of detection in the range 1-6 ng mL-1, limits of quantification in the range 3-20 ng mL-1, with reproducibility (%RSD < 10%; n = 6) and linearity (R2 > 0.99). The LC-FAIMS-MS method demonstrates increases in signal-to-noise ratios for the doubly sodiated steroid metabolites in unspiked urine (>250%) by the reduction of isobaric interferences from the matrix. An alternative or additional tool for identification of the steroid metabolites is based on the observations of different patterns of sodium acetate clusters that are characteristic for each metabolite.

Direct Analysis of Oil Additives by High-Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry Combined with Electrospray Ionization and Desorption Electrospray Ionization.

The analysis of corrosion inhibitors in the presence and absence of an oil matrix is reported using electrospray ionization (ESI) and desorption electrospray ionization (DESI), hyphenated with miniaturized high-field asymmetric waveform ion mobility spectrometry (FAIMS) and mass spectrometry (MS). The target analytes were successfully ionized in solution by ESI and directly from steel surfaces using DESI ambient ionization at levels ≥0.0004% w/w (4 ppm) in oil. Differences in the mass spectral profiles observed for the additive/oil mixture are attributed to differences between the ESI and DESI ionization processes. The use of FAIMS improved selectivity for ESI generated analyte ions through reduction in the chemical noise resulting from the oil matrix. DESI enabled the direct, rapid, native state interrogation of oil samples on steel surfaces without sample pretreatment, and the hyphenation of DESI with the miniaturized FAIMS enhanced the relative analyte responses of the surface-active corrosion inhibitors.

Identification of plasma protease derived metabolites of glucagon and their formation under typical laboratory sample handling conditions.

RATIONALE: Glucagon modulates glucose production, and it is also a biomarker for several pathologies. It is known to be unstable in human plasma, and consequently stabilisers are often added to samples, although these are not particularly effective. Despite this, there have not been any studies to identify in vitro plasma protease derived metabolites; such a study is described here. Knowledge of metabolism should allow the development of more effective sample stabilisation strategies. METHODS: Several novel metabolites resulting from the incubation of glucagon in human plasma were identified using high-resolution mass spectrometry with positive electrospray ionisation. Tandem mass spectrometric (MS/MS) scans were acquired for additional confirmation using a QTRAP. Separation was performed using reversed-phase ultra-high-performance liquid chromatography. The formation of these metabolites was investigated during a time-course experiment and under specific stress conditions representative of typical laboratory handling conditions. Clinical samples were also screened for metabolites. RESULTS: Glucagon(3-29) and [pGlu](3) glucagon(3-29) were the major metabolites detected, both of which were also present in clinical samples. We also identified two oxidised forms of [pGlu](3) glucagon(3-29) as well as glucagon(19-29), or 'miniglucagon', along with the novel metabolites glucagon(20-29) and glucagon(21-29). The relative levels of these metabolites varied throughout the time-course experiment, and under the application of the different sample handling conditions. Aprotinin stabilisation of samples had negligible effect on metabolite formation. CONCLUSIONS: Novel plasma protease metabolites of glucagon have been confirmed, and their formation characterised over a time-course experiment and under typical laboratory handling conditions. These metabolites could be monitored to assess the effectiveness of new sample stabilisation strategies, and further investigations into their formation could suggest specific enzyme inhibitors to use to increase sample stability. In addition the potential of the metabolites to affect immunochemistry-based assays as a result of cross-reactivity could be investigated.

Direct determination of urinary creatinine by reactive-thermal desorption-extractive electrospray-ion mobility-tandem mass spectrometry.

A direct, ambient ionization method has been developed for the determination of creatinine in urine that combines derivatization and thermal desorption with extractive electrospray ionization and ion mobility-mass spectrometry. The volatility of creatinine was enhanced by a rapid on-probe aqueous acylation reaction, using a custom-made thermal desorption probe, allowing thermal desorption and ionization of the monoacylated derivative. The monoacyl creatinine [M + H](+) ion (m/z 156) was subjected to mass-to-charge selection and collision induced dissociation to remove the acyl group, generating the protonated creatinine [M + H](+) product ion at m/z 114 before an ion mobility separation was applied to reduce chemical noise. Stable isotope dilution using creatinine-d3 as internal standard was used for quantitative measurements. The direct on-probe derivatization allows high sample throughput with a typical cycle time of 1 min per sample. The method shows good linearity (R(2) = 0.986) and repeatability (%RSD 8-10%) in the range of 0.25-2.0 mg/mL. The creatinine concentrations in diluted urine samples from a healthy individual were determined to contain a mean concentration of 1.44 mg/mL creatinine with a precision (%RSD) of 9.9%. The reactive ambient ionization approach demonstrated here has potential for the determination of involatile analytes in urine and other biofluids.

Direct detection of a sulfonate ester genotoxic impurity by atmospheric-pressure thermal desorption-extractive electrospray-mass spectrometry.

A direct, ambient ionization method has been developed using atmospheric pressure thermal desorption-extractive electrospray-mass spectrometry (AP/TD-EESI-MS) for the detection of the genotoxic impurity (GTI) methyl p-toluenesulfonate (MTS) in a surrogate pharmaceutical matrix. A custom-made thermal desorption probe was used to the desorb and vaporize MTS from the solid state, by rapid heating to 200 °C then cooling to ambient temperature, with a cycle time of 6 min. The detection of MTS using EESI with a sodium acetate doped solvent to generate the [MTS+Na](+) adduct ion provided a significant sensitivity enhancement relative to the [M+H](+) ion generated using a 0.1% formic acid solvent modifier. The MTS detection limit is over an order of magnitude below the long-term daily threshold of toxicological concern (TTC) of 1.5 µg/g and the potential for quantitative analysis has been determined using starch as a surrogate active pharmaceutical ingredient (API).

The quantitative surface analysis of an antioxidant additive in a lubricant oil matrix by desorption electrospray ionization mass spectrometry.

RATIONALE: Chemical additives are incorporated into commercial lubricant oils to modify the physical and chemical properties of the lubricant. The quantitative analysis of additives in oil-based lubricants deposited on a surface without extraction of the sample from the surface presents a challenge. The potential of desorption electrospray ionization mass spectrometry (DESI-MS) for the quantitative surface analysis of an oil additive in a complex oil lubricant matrix without sample extraction has been evaluated. METHODS: The quantitative surface analysis of the antioxidant additive octyl (4-hydroxy-3,5-di-tert-butylphenyl)propionate in an oil lubricant matrix was carried out by DESI-MS in the presence of 2-(pentyloxy)ethyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate as an internal standard. A quadrupole/time-of-flight mass spectrometer fitted with an in-house modified ion source enabling non-proximal DESI-MS was used for the analyses. RESULTS: An eight-point calibration curve ranging from 1 to 80 µg/spot of octyl (4-hydroxy-3,5-di-tert-butylphenyl)propionate in an oil lubricant matrix and in the presence of the internal standard was used to determine the quantitative response of the DESI-MS method. The sensitivity and repeatability of the technique were assessed by conducting replicate analyses at each concentration. The limit of detection was determined to be 11 ng/mm(2) additive on spot with relative standard deviations in the range 3-14%. CONCLUSIONS: The application of DESI-MS to the direct, quantitative surface analysis of a commercial lubricant additive in a native oil lubricant matrix is demonstrated.

Gas-phase and solution studies of three resorcin[4]arene derivatives using electrospray time-of-flight mass spectrometry.

Electrospray ionisation mass spectrometry (ESI-MS) has been used to study the relative gas-phase proton and alkali metal (Li, Na, K and Cs) binding affinities of three different resorcin[4]arenes using the kinetic method. Collision-induced dissociation (CID) was used to study the fragmentation of resorcin[4]arene heterodimer sandwich complexes, allowing the relative binding affinity order to be established. All the alkali metal cations have the same gas-phase binding affinity order with the resorcin[4]arene host molecules. At collision energies of > or = 13eV, one of the [resorcin[4]arene+Metal]+, (Metal = Li, Na, K) ions fragmented through break-up of the resorcin[4]arene, whilst the other host resorcin[4]arene remained intact, causing an apparent change in binding affinity at high collision energy. This effect was not observed with caesium, since all complex ions dissociated readily under CID by displacement of the caesium cation. The binding affinity for the protonated resorcin[4]arenes was found to be different from the alkali metal cation binding affinity because of the higher proton affinity of the nitrogen-containing resorcin[4]arenes. It is shown that resorcin[4]arenes containing an oxazine ring can be converted into a ring-opened derivative via an Eschweiler-CLarke reaction in the presence of formic acid. A second ring-opening process also occurs, including a hydrolysis reaction that results in apparent Losses of 12 mass units from the intact resorcin[4]arene. Both these reactions occur in solution before mass spectrometric investigation and cannot be achieved by CID. This observation was confirmed by inducing the Eschweiter-CLarke reaction in a model benzoxazine compound.

Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry.

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 µg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).

Direct extraction of urinary analytes from undeveloped reversed-phase thin layer chromatography plates using a solvent gradient combined with on-line electrospray ionisation ion mobility-mass spectrometry.

The direct extraction of urinary analytes deposited on reversed-phase thin-layer chromatography (RP-TLC) plates is demonstrated using a solvent gradient extraction procedure without prior chromatographic development. The surface sample probe TLC-MS interface used for the gradient extraction is compared to direct loop injection into the electrospray ion source for biofluid profiling. The gradient elution is shown to enhance ion intensities, as urinary salts are eluted in aqueous formic acid in the early part of the gradient reducing ion suppression. The retention of urinary components on the C18 RP-TLC plate was confirmed by monitoring analyte responses with, and without, an aqueous wash phase prior to the solvent gradient extraction. The use of gradient elution allows fractionation of the complex biological matrix as a result of differential retention of urine components on the undeveloped RP-TLC plate. The direct gradient analysis of TLC plates has also been combined with ion mobility-mass spectrometry to further resolve the complex urinary profile and identify co-eluting compounds.

Real-time reaction monitoring using ion mobility-mass spectrometry.

The potential of ion mobility (IM) spectrometry in combination with mass spectrometry (MS) for real-time reaction monitoring is reported. The combined IM-MS approach using electrospray ionization affords gas-phase analyte characterization based on both mass-to-charge (m/z) ratio and gas-phase ion mobility (drift time). The use of IM-MS analysis is demonstrated for the monitoring of the reaction products formed when 7-fluoro-6-hydroxy-2-methylindole is deprotonated by aqueous sodium hydroxide. Real-time reaction monitoring was carried out over a period of several hours, with the reaction mixture sampled and analysed at intervals of several minutes. Product ion relative intensity is enhanced selectively in the ion mobility-selected mass spectrum, compared to mass spectrometry alone. The combined IM-MS approach has potential as a rapid and selective technique to aid pharmaceutical process control and for the elucidation of reaction mechanism.

Determination of testosterone and epitestosterone glucuronides in urine by ultra performance liquid chromatography-ion mobility-mass spectrometry.

UPLC-ion mobility spectrometry separations combined with mass spectrometry (UPLC-IM-MS) and tandem mass spectrometry (UPLC-IM-MS/MS) have been investigated for the simultaneous determination of testosterone and epitestosterone glucuronides in urine. The glucuronide epimers of testosterone and epitestosterone were separated by ion mobility spectrometry prior to mass analysis on the basis of differences in their collision cross sections, which have been measured in nitrogen. Combining ion mobility separation with UPLC/MS enhances the analysis of these low-abundance steroids in urine by selective interrogation of specific retention time, mass-to-charge and mobility regions. Detection limits for the UPLC-IM-MS/MS analysis of TG and ETG were 9.9 ng mL(-1) and 98 ng mL(-1) respectively, equivalent to 0.7 ng mL(-1) and 7.4 ng mL(-1) in urine, with linear dynamic ranges corresponding to 0.7-108 ng mL(-1) and 7.4-147 ng mL(-1) in urine. Repeatability (%RSD) for urine extracts was 0.64% and 2.31% for TG and ETG respectively.

Application of mass spectrometry-based proteomics techniques for the detection of protein doping in sports.

Mass spectrometry-based proteomic approaches have been used to develop methodologies capable of detecting the abuse of protein therapeutics such as recombinant human erythropoietin and recombinant human growth hormone. Existing detection methods use antibody-based approaches that, although effective, suffer from long assay development times and specificity issues. The application of liquid chromatography with tandem mass spectrometry and selected reaction-monitoring-based analysis has demonstrated the ability to detect and quantify existing protein therapeutics in plasma. Furthermore, the multiplexing capability of selected reaction-monitoring analysis has also aided in the detection of multiple downstream biomarkers in a single analysis, requiring less sample than existing immunological techniques. The flexibility of mass spectrometric instrumentation has shown that the technique is capable of detecting the abuse of novel and existing protein therapeutics, and has a vital role in the fight to keep sports drug-free.

The use of shift reagents in ion mobility-mass spectrometry: studies on the complexation of an active pharmaceutical ingredient with polyethylene glycol excipients.

Gas-phase ion mobility studies of mixtures containing polyethylene glycols (PEG) and an active pharmaceutical ingredient (API), lamivudine, have been carried out using electrospray ionization-ion mobility spectrometry-quadrupole-time-of-flight mass spectrometry (ESI-IMS-Q-TOF). In addition to protonated and cationized PEG oligomers, a series of high molecular weight ions were observed and identified as noncovalent complexes formed between lamivudine and PEG oligomers. The noncovalent complex ions were dissociated using collision induced dissociation (CID) after separation in the ion mobility drift tube to recover the protonated lamivudine free from interfering matrix ions and with a drift time associated with the precursor complex. The potential of PEG excipients to act as "shift reagents," which enhance selectivity by moving the mass/mobility locus to an area of the spectrum away from interferences, is demonstrated for the analysis of lamivudine in a Combivir formulation containing PEG and lamivudine.

Estimation of peptide concentration by a modified bicinchoninic acid assay.

Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 degrees C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 degrees C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.

Direct analysis of pharmaceutical formulations from non-bonded reversed-phase thin-layer chromatography plates by desorption electrospray ionisation ion mobility mass spectrometry.

The direct analysis of pharmaceutical formulations and active ingredients from non-bonded reversed-phase thin layer chromatography (RP-TLC) plates by desorption electrospray ionisation (DESI) combined with ion mobility mass spectrometry (IM-MS) is reported. The analysis of formulations containing analgesic (paracetamol), decongestant (ephedrine), opiate (codeine) and stimulant (caffeine) active pharmaceutical ingredients is described, with and without chromatographic development to separate the active ingredients from the excipient formulation. Selectivity was enhanced by combining ion mobility and mass spectrometry to characterise the desorbed gas-phase analyte ions on the basis of mass-to-charge ratio (m/z) and gas-phase ion mobility (drift time). The solvent composition of the DESI spray using a step gradient was varied to optimise the desorption of active pharmaceutical ingredients from the RP-TLC plates. The combined RP-TLC/DESI-IM-MS approach has potential as a rapid and selective technique for pharmaceutical analysis by orthogonal gas-phase electrophoretic and mass-to-charge separation.

An approach to enhancing coverage of the urinary metabonome using liquid chromatography-ion mobility-mass spectrometry.

The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC-IM-MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge (m/z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 micros) during each IMS separation (approximately 13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC-MS. Preliminary results indicate that spectral quality is improved when using LC-IM-MS, compared to direct injection IM-MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC-IM-MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).

On-line capillary column immobilised metal affinity chromatography/electrospray ionisation mass spectrometry for the selective analysis of histidine-containing peptides.

Capillary column immobilised metal affinity chromatography (IMAC) has been combined on-line with electrospray ionisation/quadrupole time-of-flight mass spectrometry for the fractionation of histidine-containing peptides. IMAC beads (Poros 20MC, 20 microm) containing imidodiacetate chelating groups on a cross-linked poly(styrene-divinylbenzene) support were packed into a fused silica column (250 microm i.d.), which was interfaced to the electrospray ion source of the spectrometer. A Cu(II) activated column was used to isolate histidine-containing peptides from tryptic and other peptide mixtures with an average breakthrough of 9.1%, to reduce the complexity of the mass spectral analysis. The analysis cycle time was reduced to less than 15 min, at an optimum flow rate of 7.5 microL/min, without sacrificing peptide selectivity. Direct coupling of capillary IMAC with MS allows on-line separation, using MS compatible loading and elution buffers, and detection in a high-throughput fashion when compared to off-line strategies.