Exploring the Conformational Landscape of Poly(l-lysine) Dendrimers Using Ion Mobility Mass Spectrometry.

Ion mobility mass spectrometry (IM-MS) measures the mass, size, and shape of ions in the same experiment, and structural information is provided via collision cross-section (CCS) values. The majority of commercially available IM-MS instrumentation relies on the use of CCS calibrants, and here, we present data from a family of poly(l-lysine) dendrimers and explore their suitability for this purpose. In order to test these compounds, we employed three different IM-MS platforms (Agilent 6560 IM-QToF, Waters Synapt G2, and a home-built variable temperature drift tube IM-MS) and used them to investigate six different generations of dendrimers in two buffer gases (helium and nitrogen). Each molecule gives a highly discrete CCS distribution suggestive of single conformers for each m/z value. The DTCCSN2 values of this series of molecules (molecular weight: 330-16,214 Da) range from 182 to 2941 Å2, which spans the CCS range that would be found by many synthetic molecules including supramolecular compounds and many biopolymers. The CCS values for each charge state were highly reproducible in day-to-day analysis on each instrument, although we found small variations in the absolute CCS values between instruments. The rigidity of each dendrimer was probed using collisionally activated and high-temperature IM-MS experiments, where no evidence for a significant CCS change ensued. Taken together, this data indicates that these polymers are candidates for CCS calibration and could also help to reconcile differences found in CCS measurements on different instrument geometries.

Stochasticity of poly(2-oxazoline) oligomer hydrolysis determined by tandem mass spectrometry.

Understanding modification of synthetic polymer structures is necessary for their accurate synthesis and potential applications. In this contribution, a series of partially hydrolyzed poly(2-oxazoline) species were produced forming poly[(2-polyoxazoline)-co-(ethylenimine)] (P(EtOx-co-EI)) copolymers; EI being the hydrolyzed product of Ox. Bulk mass spectrometry (MS) measurements accurately measured the EI content. Tandem mass spectrometry analysis of the EI content in the copolymer samples determined the distribution of each monomer within the copolymer and corresponded to a theoretically modelled random distribution. The EI distribution across the polymers was shown to be effected by the choice of terminus, with a permanent hydrolysis event observed at an OH terminus. A neighbouring group effect wasn't observed at the polymer length analysed (approximately 25-mer species), suggesting that previously observed neighbouring group effects require a larger polymer chain. Although clearly useful for random polymer distribution this approach may be applied to many systems containing non-specific modifications to determine if they are directed or random locations across peptides, proteins, polymers, and nucleic acids.

Characterization Across a Dispersity: Polymer Mass Spectrometry in the Second Dimension.

Due to the natural dispersity that is present in synthetic polymers, an added complexity is always present in the analysis of polymeric species. Tandem mass spectrometry analysis requires the isolation of individual precursors before a fragmentation event to allow the unambiguous characterization of these species and is not viable at certain levels of complexity due to achievable isolation widths. Two-dimensional mass spectrometry (2DMS) fragments ions and correlates fragments with their corresponding precursors without the need for isolation. In this study, 2DMS electron capture dissociation (ECD) fragmentation of a polyoxazoline and polyacrylamide species was carried out, resulting in the analysis of byproducts and individual polymer species without the use of chromatographic techniques. This study shows that 2DMS ECD is a powerful tool for the analysis of polyacrylamide and polyoxazoline species and offers a new dimension in the characterization of polymers.

Electron Capture Dissociation of Trithiocarbonate-Terminated Acrylamide Homo- and Copolymers: A Terminus-Directed Mechanism?

The structure and sequence elucidation of complex homo- and copolymers is key for further understanding polymers, polymer synthesis, and polymer interactions in biological processes. In this contribution, poly(dimethylacrylamide) homo- and dimethylacrylamide/4-acryloylmorpholine block copolymers were synthesized and analyzed by electron capture dissociation (ECD) and Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry. Double-resonance experiments were carried out, providing a better understanding of the fragmentation process. A novel radical dissociation process is presented, and electron capture caused a specific cleavage at the terminal butyl-trithiocarbonate group, which initiated a free radical dissociation process.

Coupling Electron Capture Dissociation and the Modified Kendrick Mass Defect for Sequencing of a Poly(2-ethyl-2-oxazoline) Polymer.

With increasing focus on the structural elucidation of polymers, advanced tandem mass spectrometry techniques will play a crucial role in the characterization of these compounds. In this contribution, synthesis and analysis of methyl-initiated and xanthate-terminated poly(2-ethyl-2-oxazoline) using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) was achieved. Electron capture dissociation (ECD) produced full end group characterization as well as backbone fragmentation including complete sequence coverage of the polymer. A method of fragment ion characterization is also presented with the use of the high-resolution-modified Kendrick mass defect plots as a means of grouping fragments from the same fragmentation pathways together. This type of data processing is applicable to all tandem mass spectrometry techniques for polymer analysis but is made more effective with high mass accuracy methods. ECD FT-ICR MS demonstrates its promising role as a structural characterization technique for polyoxazoline species.

Characterisation of phosphorylated nucleotides by collisional and electron-based tandem mass spectrometry.

RATIONALE: Tandem mass spectrometry of phosphorylated ions can often yield a limited number of product ions owing to the labile nature of phosphate groups. Developing techniques to improve dissociation for this type of ion has implications for the structural characterisation of many different phosphorylated ions, such as those from nucleotides, pharmaceutical compounds, peptides and polymers. METHODS: Solutions of adenosine monophosphate, diphosphate and triphosphate (AMP, ADP and ATP) were studied in a hybrid linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Precursor ions with an overall single positive charge, including protonated nucleotides or nucleotide cations containing one, two or three sodium atoms, were isolated for tandem mass spectrometry. Collision-induced dissociation (CID) was performed in the linear ion trap, with electron-induced dissociation (EID) being conducted in the FTICR cell. RESULTS: EID resulted in many product ions not seen in CID. EID product ion spectra were seen to vary for AMP, ADP and ATP when the nucleotide cation contained zero, one, two or three sodiums. Precursor cations that contain two or three sodiums mainly formed product ions derived from the phosphate group. Conversely, when a precursor ion containing no sodium underwent EID, product ions mainly relating to the non-phosphate end of the ion were observed. The number of phosphate groups was not seen to greatly affect either CID or EID product ion spectra. CONCLUSIONS: The presence of sodium in a precursor ion directs electron-induced bond dissociation, thus enabling targeted, and therefore tuneable, fragmentation of groups within that precursor ion. For all precursor ions, the most useful product ion spectra were obtained by EID for a precursor ion containing one sodium, with bond dissociation occurring across the entire nucleotide cation. The findings of this study can be used to improve the structural elucidation of many phosphorylated molecules by broadening the range of product ions achievable. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Electrochemical flow injection analysis of hydrazine in an excess of an active pharmaceutical ingredient: achieving pharmaceutical detection limits electrochemically.

The quantification of genotoxic impurities (GIs) such as hydrazine (HZ) is of critical importance in the pharmaceutical industry in order to uphold drug safety. HZ is a particularly intractable GI and its detection represents a significant technical challenge. Here, we present, for the first time, the use of electrochemical analysis to achieve the required detection limits by the pharmaceutical industry for the detection of HZ in the presence of a large excess of a common active pharmaceutical ingredient (API), acetaminophen (ACM) which itself is redox active, typical of many APIs. A flow injection analysis approach with electrochemical detection (FIA-EC) is utilized, in conjunction with a coplanar boron doped diamond (BDD) microband electrode, insulated in an insulating diamond platform for durability and integrated into a two piece flow cell. In order to separate the electrochemical signature for HZ such that it is not obscured by that of the ACM (present in excess), the BDD electrode is functionalized with Pt nanoparticles (NPs) to significantly shift the half wave potential for HZ oxidation to less positive potentials. Microstereolithography was used to fabricate flow cells with defined hydrodynamics which minimize dispersion of the analyte and optimize detection sensitivity. Importantly, the Pt NPs were shown to be stable under flow, and a limit of detection of 64.5 nM or 0.274 ppm for HZ with respect to the ACM, present in excess, was achieved. This represents the first electrochemical approach which surpasses the required detection limits set by the pharmaceutical industry for HZ detection in the presence of an API and paves the wave for online analysis and application to other GI and API systems.

The Competitive influence of Li+, Na+, K+, Ag+, and H+ on the fragmentation of a PEGylated polymeric excipient.

The collisionally activated dissociation (CAD) and electron capture dissociation (ECD) of doubly charged tocopheryl polyethylene glycol succinate (TPGS) have been examined. Li(+), Na(+), K(+), Ag(+), and H(+) were selected in the study, and the competitive influence of each ion was investigated by fragmenting TPGS attached with two different cations, [M + X1 + X2](2+) (X1 and X2 refer to Li(+), Na(+), K(+), Ag(+), H(+)). For metallic adducts, CAD results show that the dissociation of ionic adducts from the precursor is most likely depending on the binding strength, where the affinity of each ion to the TPGS is in the order of Ag(+) ≈ Li(+) ˃ Na(+) ˃ K(+). Introducing more strongly bound adducts increases fragmentation. During ECD, however, the silver cation is lost most easily compared with the other alkali metal ions, but silver also shows a dominant role in producing fragmentations. Moreover, the charge carriers are lost in an order (Ag(+) ˃ Na(+) ˃ K(+) ≥ Li(+) where the loss of Ag is most easily) that appears to correlate with the standard reduction potential of the metallic ions (Ag(+) ˃ Na(+) ˃ K(+) ˃ Li(+)). The ECD results suggest that the reduction potential of the charge carrier could be an important factor influencing the fragmentation, where the ion with a high reduction potential is more effective in capturing electrons, but may also be lost easily before leading to any fragmentation. Finally, a proton has the weakest binding with the TPGS according to the CAD results, and its dissociation in ECD follows the order of the reduction potential (Ag(+) ˃ H(+) ˃ Na(+) ˃ K(+) > Li(+)).

Enhanced performance in the determination of ibuprofen 1-ß-O-acyl glucuronide in urine by combining high field asymmetric waveform ion mobility spectrometry with liquid chromatography-time-of-flight mass spectrometry.

The incorporation of a chip-based high field asymmetric waveform ion mobility spectrometry (FAIMS) separation in the ultra (high)-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) determination of the (R/S) ibuprofen 1-ß-O-acyl glucuronide metabolite in urine is reported. UHPLC-FAIMS-HRMS reduced matrix chemical noise, improved the limit of quantitation approximately two-fold and increased the linear dynamic range compared to the determination of the metabolite without FAIMS separation. A quantitative evaluation of the prototype UHPLC-FAIMS-HRMS system showed better reproducibility for the drug metabolite (%RSD 2.7%) at biologically relevant concentrations in urine. In-source collision induced dissociation of the FAIMS-selected deprotonated metabolite was used to fragment the ion prior to mass analysis, enhancing selectivity by removing co-eluting species and aiding the qualitative identification of the metabolite by increasing the signal-to-noise ratio of the fragment ions.

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).

Using electron induced dissociation (EID) on an LC time-scale to characterize a mixture of analogous small organic molecules.

LC ESI FTICR MS of a sample of cediranib identified this pharmaceutical target molecule plus an additional 10 compounds of interest, all of which were less than 10% total ion current (TIC) peak intensity relative to cediranib. LC FTICR tandem mass spectrometry using electron induced dissociation (EID) has been achieved and has proven to be the best way to generate useful product ion information for all of these singly protonated molecules. Cediranib [M + H](+) fragmented by EID to give 29 product ions whereas QTOF-CID generated only one very intense product ion, and linear ion trap-CID, which generated 10 product ions, but all with poor S/N. Twenty-six of the EID product ions were unique to this fragmentation technique alone. By considering the complementary LC-EID and LC-CID data together, all 10 unknown compounds were structurally characterized and proven to be analogous to cediranib. Of particular importance, EID produced unique product ion information for one of the low level cediranib analogues that enabled full characterization of the molecule such that the presence of an extra propylpyrrolidine group was discovered and proven to be located on the pyrrolidine ring of cediranib, solving an analytical problem that could not be solved by collision induced dissociation (CID). Thus, it has been demonstrated that EID is in harmony with the chromatography duty-cycle and the dynamic concentration range of synthetic compounds containing trace impurities, providing crucial analytical information that cannot be obtained by more traditional methodologies.

Electron-induced dissociation of singly charged organic cations as a tool for structural characterization of pharmaceutical type molecules.

Collision-induced dissociation (CID) and electron-induced dissociation (EID) have been investigated for a selection of small, singly charged organic molecules of pharmaceutical interest. Comparison of these techniques has shown that EID carried out on an FTICR MS and CID performed on a linear ion trap MS produce complementary data. In a study of 33 molecule-cations, EID generated over 300 product ions compared to 190 product ions by CID with an average of only 3 product ions per precursor ion common to both tandem MS techniques. Even multiple stages of CID failed to generate many of the product ions observed following EID. The charge carrying species is also shown to have a very significant effect on the degree of fragmentation and types of product ion resulting from EID. Protonated species behave much like the ammonium adduct with suggestion of a hydrogen atom from the charge carrying species strongly affecting the fragmentation mechanism. Sodium and potassium are retained by nearly every product ion formed from [M + Na](+) or [M + K](+) and provide information to complement the EID of [M + H](+) or [M + NH(4)](+). In summary, EID is proven to be a fitting partner to CID in the structural elucidation of small singly charged ions and by studying EID of a molecule-ion holding different charge carrying species, an even greater depth of detail can be obtained for functional groups commonly used in synthetic chemistry.

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.

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).

Accurate mass measurement for the determination of elemental formula--a tutorial.

The application of accurate mass measurement for the determination of elemental formula has its origin in the 1950s and for many years was only carried out using magnetic sector mass spectrometers. The availability of such measurements was limited due to the cost and complexity of the instrumentation and the need for considerable expertise to acquire and interpret the spectra. In recent years the incredible pace of instrumental development has changed this, particularly with the renaissance of time of flight mass spectrometry. This has resulted in instrumentation capable of making accurate mass measurements in a robust fashion becoming available to most practitioners of (mass spectrometry) MS, without some of the earlier technical challenges and at lower cost. In this review the variety of accurate mass measurement instrumentation and techniques and their relative capabilities are discussed, along with a range of applications requiring the determination of elemental formula.

Reproducible product-ion tandem mass spectra on various liquid chromatography/mass spectrometry instruments for the development of spectral libraries.

The results of the comparison of product-ion tandem mass (MS/MS) spectra recorded on three ion trap mass spectrometers, a triple quadrupole mass spectrometer and a Fourier transform ion cyclotron resonance mass spectrometer are reported. The spectra were recorded in accordance with a simple experimental protocol, which involved the collision-induced dissociation (CID) attenuation of the abundance of the [M+H]+ ion to between 10 and 50% of its original abundance. The degree of similarity between the spectra from four of the mass spectrometers was calculated off-line by comparing the five most abundant ions from the spectrum on each instrument. A percentage fit value (20% for each ion that matches) was calculated by comparing each spectrum against the spectra recorded for the same compound on each instrument. The percentage of the inter-library pairwise comparisons (total = 434) that matched to > or = 60% ranged from 64-89%, depending on the instrument pair. A blind trial was also undertaken using five unknown compounds resulting in 1670 pairwise comparisons with the library entries. The blind trial produced no false positives and correct identifications in all cases. The results of the study have established the basis for the construction of a transferable product-ion MS/MS library.

Intercomparison study on accurate mass measurement of small molecules in mass spectrometry.

The results from an intercomparison of accurate mass measurement of a small molecule (molecular weight 475 Da) across a broad range of mass spectrometers are reported. The intercomparison was designed to evaluate the relative capabilities and the optimum methodology of the diverse range of mass spectrometers currently used to record accurate mass measurements. The data will be used as a basis for developing guidance on accurate mass measurement. The need for guidance has resulted from the continued growth in the use of accurate mass measurements for assignment of elemental formula in the chemical and biochemical industries. This has been fuelled by a number of factors and includes the rapid pace of instrument development, which has enabled accurate mass measurements to be made in a less costly, yet robust fashion. The data from the intercomparison will allow us to compare those protocols that produced excellent accuracy and precision with those that produced poorer accuracy and/or precision for each type of mass spectrometer. The key points for best practice will then be established from this comparison for each type of mass spectrometer and accurate mass measurement technique. A compound was sent to the participating laboratories (in the UK, Europe, and USA), the identity of which was not revealed. Each laboratory was asked to record a minimum of five repeat mass measurements of the molecular species using their local protocols and their preferred ionization technique or techniques. To the best of our knowledge there were no interfering (unresolved) ions that originated from the sample. A questionnaire was also completed with the experimental work. The information from the questionnaires was used to evaluate the protocols used to record the measurements. Forty-five laboratories have reported their results. To summarize the performance of mass spectrometers in the intercomparison, magnetic sector field mass spectrometers used in peak matching mode and FTMS reported the highest mean mass measurement accuracy (88 and 83%, respectively, achieved < or =1 ppm). Magnetic sector field mass spectrometers used in voltage scanning produced 60% of the mean mass measurements with accuracy < or =1 ppm. Magnetic sector field mass spectrometers used in magnet scanning modes, quadrupole-TOF and TOF instruments generally achieved mean mass measurement accuracy between 5 and 10 ppm. The two low resolution triple quadrupoles used in the inter-comparison produced mean mass measurement accuracy of <2 ppm. The precision of the data from each instrument and experiment type is an important consideration when evaluating their relative capabilities. Using both the precision and accuracy, it will be possible to define the uncertainty associated with the elemental formulae derived from accurate mass measurements. Therefore, a thorough statistical evaluation of the data is underway and will be presented in a subsequent publication.

Evaluation of protocols for reproducible electrospray in-source collisionally induced dissociation on various liquid chromatography/mass spectrometry instruments and the development of spectral libraries.

Mass spectral libraries provide a tool for identifying unknown compounds using both molecular weight and fragmentation information. Mass spectrometers with electrospray ionisation (ESI) and atmospheric chemical ionisation (ApCI) sources have the capability to produce data of this type using in-source collisionally induced dissociation (CID), and in-source CID libraries can be created. Due to the variation in electrospray source design from different instrument manufacturers, the production of reproducible in-source CID spectra that can be used in libraries for all instrument types is not a trivial task. To date, the evaluation of the production of in-source CID libraries has tended to focus on similar instruments from one manufacturer. The studies have also tended to focus on specific compound classes, with a limited molecular weight range.This report describes the findings from the investigation of protocols for the creation of mass spectral libraries using ESI in-source CID on six instruments from four different manufacturers. The overall goal was to create a spectral library for the identification of unknowns. The library could then be applied across all manufacturers' electrospray instruments. Two different experimental protocols were attempted. The first used a tuning compound to establish standard ESI source conditions, with fixed fragmentation potentials. The second involved the attenuation of the [M + H](+) ion to a known degree. A diverse range of compounds (pharmaceutical, photographic, pesticides) was tested to establish the reproducibility of the spectra on the six instruments. Both protocols produced spectra on the various instruments that in many cases were very similar. In other examples, the spectra differed not only in their relative ion abundances, but also in terms of the spectral content. Important observations regarding the effect of ion source design are also reported. The degree of spectral reproducibility was calculated off-line by comparing the five most abundant ions (20% for each ion that matches) from each spectrum on each instrument. This approach was adopted, as we do not possess a software package that met our requirements for spectral comparison. Match factors (% fit) were calculated by comparing each spectrum against the spectra recorded for the same compound and then for all other compounds, on each instrument. The % fit values derived by the off-line approach gave a clear view of the spectral reproducibility from instrument to instrument and also discriminated the spectra of the various compounds from each other. The applicability of this approach was tested using a blind trial in which several compounds were presented as unknowns, their in-source CID spectra recorded and the five-ion approach used for identification.