In-source Fragmentation Research Articles

Atmospheric pressure chemical ionization mass spectrometry of retro-carotenoids.

The single and double bonds of the polyene chain of the studied retro-carotenoids are located at the neighboring positions compared to those of regular carotenoids. Our mass spectrometry approach targeted at facilitating the characterization of retro-carotenoids as their structural diversity in nature is not yet fully elucidated. Moreover, extended π-electron systems endow several retro-carotenoids with exceptional colors from golden-orange to vibrant red that stimulate the food industry's interest. Atmospheric pressure chemical ionization-quadrupole time-of-flight-high-resolution mass spectrometry (APCI-QTOF-HRMS) experiments of the three structurally related retro-carotenoids rhodoxanthin, eschscholtzxanthone, and eschscholtzxanthin were performed to elucidate the formation of specific ion species compared to those of the common carotenoids lutein and zeaxanthin. Mass fragmentations of the aforementioned retro-carotenoids were unraveled using APCI-tandem mass spectrometry (MS/MS) in the negative and positive ion modes. Abundant in-source fragment ions [M + H - H2 O]+ of eschscholtzxanthin and eschscholtzxanthone were formed in the positive ion mode owing to the loss of water at the hydroxylated ε-rings. Eliminations of the ε-rings at the characteristic exocyclic double bonds at C-6,7 and C-6',7' were observed after the resonance-stabilized loss of water. Distinct product ions were yielded for all retro-carotenoids assessed because of the cleavage at their typical central single bond at C-15,15'. Detailed APCI-QTOF-HRMS analyses enabled a highly accurate detection of the most abundant ion species and respective signal intensity ratios of retro-carotenoids, facilitating their further screening and reliable identification in natural sources. Mass fragmentations of the studied retro-carotenoids were found to be substantially impacted by the extraordinary configuration of their polyene backbone.

Nontargeted High-Resolution Mass Spectrometric Workflow for the Detection of Butyrylcholinesterase-Derived Adducts with Organophosphorus Toxicants and Structural Characterization of Their Phosphyl Moiety after In-Source Fragmentation.

Organophosphorus (OP) nerve agents were used for chemical warfare, assassination, and attempted murder of individuals. Therefore, forensic methods are required to identify known and unknown incorporated OP poisons. Serum is tested for the presence of covalent reaction products (adducts) of the toxicant with, e.g., butyrylcholinesterase (BChE) typically by targeted analysis, thus only detecting known OP adducts. We herein present a nontargeted two-step mass spectrometry (MS)-based workflow taking advantage of a high-resolution (HR) Orbitrap mass spectrometer and its option for in-source collision-induced dissociation (IS-CID) highly valuable for the detection of unknown agents. BChE adducts are extracted by immunomagnetic separation and proteolyzed with pepsin yielding a phosphylated nonapeptide (NP) biomarker NP(OP). In step 1, the sample is separated by micro liquid chromatography (μLC) detecting the NP(OP) by nontargeted HR MS followed by data-dependent tandem-MS (ddMS2). Extracted ion chromatograms of diagnostic product ions at m/z 778.33661, 673.29402, and 602.25690 reveal the accurate mass of the NP(OP) precursor ion as well as the elemental composition of the adducted phosphyl moiety. Considering this information, a second μLC run is performed (step 2) for nonselective IS-CID of NP(OP) yielding the cleaved charged phosphyl moiety. This fragment ion is immediately subjected to targeted CID in parallel reaction monitoring (PRM). The accurate mass of its product ions allows the determination of their elemental composition and thus supports its structural elucidation. The described workflow was exemplarily applied to NP(OP) of three Tamelin esters and VX providing highly appropriate abilities for the detection of adducts even of unknown OP poisons like Novichok agents.

Structural characterization of phospholipids and sphingolipids by in-source fragmentation MALDI/TOF mass spectrometry.

Phospholipids (PLs) and sphingolipids (SLs) performcritical structural and biological functions in cells. The structure of these lipids, including the stereospecificity and double-bond position of fatty acyl (FA) chains, is critical in decoding lipid biology. In this study, we presented a simple in-source fragmentation (ISF) MALDI/TOF mass spectrometry method that affords complete structural characterization of PL and SL molecules. We analyzed several representative unsaturated lipid species including phosphatidylcholine (PC), plasmalogen PC (pPC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), cardiolipin (CL), sphingomyelin (SM), and ceramide (Cer). Fragment ions reflecting the FA chains at sn-1 and sn-2 position, and those characteristics of the head groups of different PL classes, are readily identified. Specific fragment ions from cleavages of the C-C bond immediately adjacent to the cis C=C double-bond position(s) of FA chains and the trans C=C double bond of the sphingosine constituents allow precise localization of double bonds. The identities of the exemplary product ions from vinylic, allylic, and double-bond cleavages were also verified by LIFT-TOF/TOF. Identification of individual PL species in the lipid mixture was also carried out with ISF-MALDI/TOF. Together, this approach provides a simple yet effective method for structural characterization of PLs and SLs without the additional modification on the instrument hardware, and serves as a simple tool forthe identification of lipids.

In-Source Fragmentation for the Identification of Compounds by CE-ESI-TOF in Human Plasma. L-Proline as Case Study.

One of the aims of untargeted metabolomics is searching for selective biomarkers of different pathophysiological conditions. Modified amino acids originated from the posttranslational modification of proteins play a key role as potential biomarkers; however, they are very often still classified as unknown after metabolite annotation. We have developed an analytical workflow for the targeted screening of these compounds using CE-ESI-MS. The workflow is based on the in-source fragmentation of molecules that produces diagnostic ions that we have collected in an open-source library. In this chapter, we describe in detail the strategy for the targeted screening of modified amino acids (MAAs), using as an example L-proline and its modified derivatives. We illustrate the strategy with two case studies in human plasma.

BH+/MH+-matching method for discovery of cis-diol-containing modified nucleosides in urine by ribose-targeted solid phase extraction followed by dual-mass spectrometry platform identification

Profiling of new modified nucleosides from urine plays an important role in exploring biomarkers for cancer. However, limitations from the nature of the compound, bio-sample, instrumentation, and analytical method pose great challenges to achieving a comprehensive analysis of urinary nucleosides. Herein, a method of BH+/MH+-matching (BH+, protonated nucleobase ion; MH+, protonated precursor ion) was developed to discover novel modified nucleosides from human urine samples based on solid phase extraction targeted toward specific modified nucleosides combined with ultra-performance liquid chromatography coupled with dual-mass spectrometry platforms. Firstly, nucleosides containing 2,3-diol structure on ribose were effectively enriched by PBA (Phenylboronic Acid) cartridges. Secondly, a novel method, “BH+/MH+-matching” was established to achieve rapid screening of modified nucleosides. Based on the in-source fragmentation pattern of nucleosides, a series of putative modified nucleosides were rationally designed and characterized by matching the daughter ion BH+ and its parent ion MH+ in UPLC-MSE spectra. Finally, as a complement to UPLC Q-TOF/MS, UPLC Q-Trap/MS was employed to validate the structure of putative compounds by MRM-IDA-EPI mode. Using the strategy, 12 new cis-diol-containing nucleoside analogs were successfully characterized, which were formed by modified base (m1A, m6A, m2,2,7G, ac4C) and modified ribose containing C5′-O-formylation or C5′-O-methylation. Taken together, the results demonstrated our strategy could efficiently support the rapid discovery of cis-diol-containing nucleosides with modifications on either ribose or base moiety (or both), which exhibited a promising perspective in the future application of biochemical analysis and clinical diagnosis.

Annotation of complex mass spectra by multi-layered analysis

Annotating electrospray small molecule mass spectra remains a challenging problem due to the multiple processes occurring during ionization. Although an [M+H]+ is often present, ions can be formed by reactions with other cations, background compounds and co-eluting species, and by in-source fragmentation. Even single analytes can produce multiple ion forms, many of which remain unidentified and may appear to be different species, affecting reproducibility, quantification and precursor selection in DDA experiments. Annotation usually compares differences between peaks to known adducts and losses but fails if key peaks are missing or if the peaks are from unexpected adducts. Further, isotopes are often assumed to be due to 13C and removed prior to analysis which can leave ‘orphan’ peaks if unusual elements are present. Here we describe an alternative multi-layered approach (MLA) which successively matches spectra to calculated target ion lists and reprocesses the residual ions. This allows the analyst to focus on the unknown ions and to progressively increase target list complexity since explained ions are removed. Target ion lists can be calculated from expected or observed masses and potential adducts or can be pre-defined lists, for example common contaminants. Using this approach on spectra of known standards we identified adducts with Ca, Al, Fe, Ba and possibly Mg and Sr. We also detected several compounds and adducts in a spectrum of co-eluting species from an LC-MS analysis.

Discovery of contaminants with antagonistic activity against retinoic acid receptor in house dust

Retinoic acid receptors (RARs) control reproduction and development in vertebrates, but little attention has been paid to anthropogenic chemicals exhibiting RAR agoniztic/antagonistic activity. Here we applied a His-RARα pull-down assay combined with high-resolution mass spectrometry to identify chemicals with RARα activity in house dust. After screening, a total of 540 peaks were retained as potential RARα ligands. The mass spectra of 14 chemicals matched with those in the database, of which triphenyl phosphate, galaxolidone, di(2-ethylhexyl) phthalate (DEHP), tris(2-ethylhexyl) phosphate (TEHP), and tris(2-butoxyethyl) phosphate were confirmed by their standards. While one chemical in the sample matched with monophenyl phosphate in the MS/MS database, its retention time was much higher than that of monophenyl phosphate standard, suggesting that it may be an in-source fragment. Its parent ion was finally identified to be m/z 399.2663 using a similarity analysis among chromatographic peaks of hundreds of ions at the same retention time in MS1 spectrum, and bis(2-ethylhexyl) phenyl phosphate (BEHPP) was identified. BEHPP, DEHP, and TEHP were for the first time identified to be RARα antagonists with IC50 values of 6556, 6600, and 2538 nM, respectively. This study improved structural annotation and filled the knowledge gap regarding widespread environmental contaminants with RAR antagonistic activity.

Evaluation and Optimization of APGC Parameters for the Analysis of Selected Hop Essential Oil Volatiles.

Hop essential oil is a mixture of several hundred volatile metabolites that quantitatively and qualitatively distinguish hop varieties. Given the commercial relevance of hops in the brewing industry and the complexity of hop oil, analytical tools enabling a comprehensive characterization of oil constituents are required. At this, atmospheric pressure chemical ionization interfaced to gas chromatography and high-resolution mass spectrometry (APGC–MS) is a promising option that combines soft ionization, high sensitivity, and high resolution. While high sensitivity is required to detect minor or trace-level volatile metabolites, soft ionization and high resolution enable the reliable identification of unknowns based on exact masses of the molecular ion or the protonated molecule. Twenty-two volatile metabolites typically found in hop oil were studied in respect to their APGC ionization behavior. For 15 compounds, APGC–MS did not yield high molecular ion or protonated molecule intensities and considerable in-source fragmentation was observed. APGC–MS parameter optimization (cone gas flow and cone voltage) was able to yield the maximum absolute intensity for the base peak. However, in-source fragmentation could not be prevented, leading to spectra with either the protonated molecule or a characteristic fragment ion as the base peak. APGC–MS operated under optimized parameters was applied to a hop essential oil sample to verify the effect of optimization. By estimating the limit of quantification for the 22 compounds, it is concluded that APGC–MS is well suited to analyze major, minor, and trace-level volatiles from hops.

Mapping Glyceride Species in Biodiesel by High-Temperature Gas Chromatography Combined with Chemical Ionization Mass Spectrometry.

Accurate and comprehensive identification of residual glycerides in biodiesel is an important part of fuel characterization due to the impact of glycerides on the fuel physicochemical properties. However, analysis of bound glycerol in biodiesel samples faces challenges due to lack of readily available standards of structurally complex glyceride species in nontraditional biodiesel feedstocks and a risk of misannotation in the presence of impurities in gas chromatographic separations. Here, we evaluate methane and isobutane chemical ionization-single quadrupole mass spectrometry combined with high-temperature gas chromatography separations for mapping monoacylglycerols, diacylglycerols, and triacylglycerols in biodiesel. Unlike electron impact ionization, which produces mostly in-source fragments, isobutane chemical ionization spectra of tetramethylsilyl-derivatized monoacylglycerols and diacylglycerols are dominated by molecular ions and M-SiO(CH3)3+ ions, which provide important diagnostic information. We demonstrate the utility of isobutane chemical ionization in identifying structurally complex glycerolipid standards as well as species in biodiesel samples from different plant and animal feedstocks.

Proteomics with Enhanced In-Source Fragmentation/Annotation: Applying XCMS-EISA Informatics and Q-MRM High-Sensitivity Quantification.

Enhanced in-source fragmentation/annotation (EISA) has recently been shown to produce fragment ions that match tandem mass spectrometry data across a wide range of small molecules. EISA has been developed to facilitate data-dependent acquisition (DDA), data-independent acquisiton (DIA), and multiple-reaction monitoring (MRM), enabling molecular identifications in untargeted metabolomics and targeted quantitative single-quadrupole MRM (Q-MRM) analyses. Here, EISA has been applied to peptide-based proteomic analysis using optimized in-source fragmentation to generate fragmentation patterns for a mixture of 38 peptides, which were comparable to the b- and y-type fragment ions typically observed in tandem MS experiments. The optimal in-source fragmentation conditions at which high-abundance peptide fragments and precursor ions coexist were compared with automated data-dependent acquisition (DDA) in the same quadrupole time-of-flight (QTOF-MS) mass spectrometer, generating a significantly higher fragment percentage of peptides from both singly and doubly charged b- and y-type fragment (b+, y+, b2+, and y2+) ions. Higher fragment percentages were also observed for these fragment ion series over linear ion trap instrumentation. An XCMS-EISA annotation/deconvolution program was developed, making use of the retention time and peak shape continuity between precursor fragment ions, to perform automated proteomic data analysis on the enhanced in-source fragments. Post-translational modification (PTM) characterization on peptides was demonstrated with EISA, producing fragment ions corresponding to a neutral loss of phosphoric acid with greater intensity than observed with DDA on a QTOF-MS. Moreover, Q-MRM demonstrated the ability to use EISA for peptide quantification. The availability of more sophisticated in-source fragmentation informatics, beyond XCMS-EISA, will further enable EISA for sensitive autonomous identification and Q-MRM quantitative analyses in proteomics.

The multicomponent characterization of Shuanghe decoction by dimension-enhanced data-independent HDMSE: Focusing on the performance comparison between MSE and HDMSE

The co-elution, pervasive isomers, and frequently occurring in-source fragmentation definitely hinder the comprehensive characterization of the multiple components from traditional Chinese medicine (TCM) by liquid chromatography/mass spectrometry. Herein, a dimension-enhanced strategy, by utilizing an ion mobility quadrupole time-of-flight mass spectrometer coupled to ultra-high performance liquid chromatography (UHPLC/IM-QTOF-MS) with the ability of offering four-dimensional information (retention time, Rt; drift time or collision cross section, CCS; accurate mass of precursors; and accurate MS2 product ions), is presented. Shuanghe Decoction (SHD) is a classic tonifying formula composed of nine herbs, however, little is known to its chemical complexity, hitherto. Good chromatographic separation was achieved on an HSS T3 column, and by the Vion™ IMS-QTOF high-resolution mass spectrometer, data-independent MSE and High-Definition MSE (HDMSE) in both the negative and positive electrospray ionization modes were employed for acquiring the collision-induced dissociation-MS2 data. A self-built database, including 642 known compounds, was established, which assisted to perform the automated peak annotation by UNIFI™. By reference to 55 compounds, totally 236 components were identified or tentatively characterized from SHD with 4D data recorded. The combination of negative and positive modes could yield more information complementary to structural elucidation. Compared with the conventional MSE, HDMSE exhibit of merits in the provision of CCS, better separation and detection those easily co-eluting components, and reduction of the false positives in the identification results. Conclusively, the combination of HDMSE acquisition, in-house library, and UNIFI-facilitated automated peak annotation, renders a potent approach dedicated to deconvoluting the chemical composition of complex systems like TCM formulae.

High-resolution imaging and identification of biomolecules using Nano-DESI coupled to ion mobility spectrometry

Simultaneous spatial localization and structural characterization of molecules in complex biological samples currently represents an analytical challenge for mass spectrometry imaging (MSI) techniques. In this study, we describe a novel experimental platform, which substantially expands the capabilities and enhances the depth of chemical information obtained in high spatial resolution MSI experiments performed using nanospray desorption electrospray ionization (nano-DESI). Specifically, we designed and constructed a portable nano-DESI MSI platform and coupled it with a drift tube ion mobility (IM) spectrometer-mass spectrometer. We demonstrate imaging of drift time-separated ions with a high spatial resolution of better than ∼25 μm using uterine tissues on day 4 of pregnancy in mice. Collision cross-section measurements provide unique molecular descriptors of molecules observed in nano-DESI-IM-MSI necessary for their unambiguous identification by comparison with databases. Meanwhile, isomer-specific imaging reveals variations in the isomeric composition across the tissue. Furthermore, IM separation efficiently eliminates isobaric and isomeric interferences originating from solvent peaks, overlapping isotopic peaks of endogenous molecules extracted from the tissue, and products of in-source fragmentation, which is critical to obtaining accurate concentration gradients in the sample using MSI. The structural information provided by the IM separation substantially expands the molecular specificity of high-resolution MSI necessary for unraveling the complexity of biological systems.

Advancing Structure Characterization of PS-80 by Charge-Reduced Mass Spectrometry and Software-Assisted Composition Analysis

The commercially available Polysorbate 80 (PS-80) is a highly heterogeneous product. It is a complex and structurally diverse mixture consisting of polymeric species containing polyoxyethylenes (POEs), fatty acid esters, with/or without a carbohydrate core. The core is primarily sorbitan, with some isosorbide and sorbitol. Depending on the sources of fatty acids and the degrees of esterification, multiple combinations of fatty acid esters are commonly observed. A number of POE intermediates, such as polyoxyethylene glycols, POE-sorbitans, POE-isosorbides, and an array of fatty acid esters from these intermediates remain in the raw material as well. The complex composition of PS-80 is difficult to control and poses a significant characterization challenge for its use in the pharmaceutical industry. Here, we present a novel solution for PS-80 characterization using ultra high-performance liquid chromatography coupled with charge-reduction high resolution mass spectrometry. Post column co-infusion of triethylamine focused the signal into mainly singly charged molecular ions and reduced the extent of in-source fragmentation, resulting in a simpler ion map and enhanced measurement of PS-80 species. The data processing workflow is designed to programmatically identify PS-80 component classes and reduce the burden of manually analyzing complex MS data. The 2-dimensional graphical representation of the data helps visualize these features. Together, these innovative methodologies enabled us to analyze components in PS-80 with unprecedented detail and shall be a useful tool to study formulation and stability of pharmaceutical preparations. The power of this approach was demonstrated by comparing the composition of PS-80 obtained from different vendors.

Alternating in-source fragmentation with single-stage high-resolution mass spectrometry with high annotation confidence in non-targeted metabolomics

Non-targeted metabolomics is increasingly applied in various applications for understanding biological processes and finding novel biomarkers in living organisms. However, high-confidence identity confirmation of metabolites in complex biological samples is still a significant bottleneck, especially when using single-stage mass analysers. In the current study, a complete workflow for alternating in-source fragmentation on a time-of-flight mass spectrometry (TOFMS) instrument for non-targeted metabolomics is presented. Hydrophilic interaction liquid chromatography (HILIC) was employed to assess polar metabolites in yeast following ESI parameter optimization using experimental design principles, which revealed the key influence of fragmentor voltage for this application. Datasets from alternating in-source fragmentation high resolution mass spectrometry (HRMS) were evaluated using open-source data processing tools combined with public reference mass spectral databases. The significant influence of the selected fragmentor voltages on the abundance of the primary analyte ion of interest and the extent of in-source fragmentation allowed an optimum selection of qualifier fragments for the different metabolites. The new acquisition and evaluation workflow was implemented for the non-targeted analysis of yeast extract samples whereby more than 130 metabolites were putatively annotated with more than 40% considered to be of high confidence. The presented workflow contains a fully elaborated acquisition and evaluation methodology using alternating in-source fragmentor voltages suitable for peak annotation and metabolite identity confirmation for non-targeted metabolomics applications performed on a single-stage HRMS platform.

Integration of Chemical Derivatization and in-Source Fragmentation Mass Spectrometry for High-Coverage Profiling of Submetabolomes.

In-source fragmentation-based high-resolution mass spectrometry (ISF-HRMS) is a potential analytical technique, which is usually used to profile some specific compounds that can generate diagnostic neutral loss (NL) or fragment ion (FI) in ion source inherently. However, the ISF-HRMS method does not work for those compounds that cannot inherently produce diagnostic NL or FI in ion source. In this study, a derivatization-based in-source fragmentation-information-dependent acquisition (DISF-IDA) strategy was proposed for profiling the metabolites with easily labeled functional groups (submetabolomes) by liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (LC-ESI-Q-TOF MS). As a proof-of-concept study, 36 carboxylated compounds labeled with N,N-dimethylethylenediamine (DMED) were selected as model compounds to examine performance of DISF-IDA strategy in screening the carboxylated metabolites and acquiring their MSn spectra. In ESI source, the DEMD-derived carboxylated compounds were fragmented to produce characteristic neutral losses of 45.0578, 63.0684, and/or 88.1000 Da that were further used as diagnostic features for screening the carboxylated metabolites by DISF-IDA-based LC-Q-TOF MS. Furthermore, high-resolution MSn spectra of the model compounds were also obtained within a single run of DISF-IDA-based LC-Q-TOF MS analysis, which contributed to the improvement of the annotation confidence. To further verify its applicability, DISF-IDA strategy was used for profiling carboxylated submetabolome in mice feces. Using this strategy, a total of 351 carboxylated metabolites were detected from mice feces, of which 178 metabolites (51% of the total) were positively or putatively identified. Moreover, DISF-IDA strategy was also demonstrated to be applicable for profiling other submetabolomes with easily labeled functional groups such as amino, carbonyl, and cis-diol groups. Overall, our proposed DISF-IDA strategy is a promising technique for high-coverage profiling of submetabolomes with easily labeled functional groups in biological samples.

Development of an LC-MS/MS Method for Measurement of Irinotecan and Its Major Metabolites in Plasma: Technical Considerations.

Irinotecan (CPT-11) is an important drug used in the treatment of several solid tumor types. To minimize its toxicity, therapeutic drug monitoring of CPT-11 and its major metabolites (SN-38, SN-38-glucuronide [SN-38G], and APC) has been proposed. We aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of CPT-11 and its major metabolites in plasma. Specimen preparation consisted of protein precipitation, evaporation, and reconstitution. Analyses were performed on a C18 column using reverse-phase gradient elution. Electrospray ionization and multiple reaction monitoring in positive mode were used for MS. The following heavy isotope-labeled internal standards were used: CPT-11 D10, SN-38 D3, SN-38G D3, and APC D3. We found that CPT-11, SN-38G, and APC eluted at ~4.6 to 4.7 minutes, and SN-38 eluted at ~5.1 to 5.2 minutes. A second peak for SN-38 was detected at ~4.6 to 4.7 minutes. Given that the structure of SN-38 is found in CPT-11, SN-38G, and APC, and in the CPT-11 D10 used here, in-source fragmentation was the likely cause. In addition, we found that a low-level SN-38 impurity was present in CPT-11 D10 and to a lesser extent in SN-38 D3. When developing methods for CPT-11 and its metabolites, it is important to consider the effects of in-source fragmentation and the choice of internal standards.

Single Quadrupole Multiple Fragment Ion Monitoring Quantitative Mass Spectrometry.

Single quadrupole mass spectrometry (MS) with enhanced in-source multiple fragment ion monitoring was designed to perform high sensitivity quantitative mass analyses. Enhanced in-source fragmentation amplifies fragmentation from traditional soft electrospray ionization producing fragment ions that have been found to be identical to those generated in tandem MS. We have combined enhanced in-source fragmentation data with criteria established by the European Union Commission Directive 2002/657/EC for electron ionization single quadrupole quantitative analysis to perform quantitative analyses. These experiments were performed on multiple types of complex samples that included a mixture of 50 standards, as well as cell and plasma extracts. The dynamic range for these quantitative analyses was comparable to triple quadrupole multiple reaction monitoring (MRM) analyses at up to 5 orders of magnitude with the cell and plasma extracts showing similar matrix effects across both platforms. Amino acid and fatty acid measurements performed from certified NIST 1950 plasma with isotopically labeled standards demonstrated accuracy in the range of 91-110% for the amino acids, 76-129% for the fatty acids, and good precision (coefficient of variation <10%). To enhance specificity, a newly developed correlated ion monitoring algorithm was designed to facilitate these analyses. This algorithm autonomously processes, aligns, filters, and compiles multiple ions within one chromatogram enabling both precursor and in-source fragment ions to be correlated within a single chromatogram, also enabling the detection of coeluting species based on precursor and fragment ion ratios. Single quadrupole instrumentation can provide MRM level quantitative performance by monitoring/correlating precursor and fragment ions facilitating high sensitivity analysis on existing single quadrupole instrumentation that are generally inexpensive, easy to operate, and technically less complex.

High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry

We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion Lumos. Following digestion to nucleosides, the DNA samples are analyzed at the rate of <1 min per injection with comparable detection limits of 0.63 ng/μL and 0.31 ng/μL, respectively. In contrast, the detection limits for 5 mC and 5hmC in state-of-art nano liquid chromatography (LC) coupled to online mass spectrometry (nLC-MS) are notably different (0.04 ng/μL and 2.5 ng/μL, respectively). The high sensitivity of DI-MS is achieved by optimizing sample buffer composition, the source fragmentation energy, and the radio frequency of the instrument ion funnel. DI-MS accurately reports the relative abundance of 5 mC and 5hmC over a range of 1%–7% (R2 > 0.98) and 0.13%–1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC and 5hmC is achieved by optimizing in-source fragmentation to obtain a population of up to 93% of just the nucleoside base. This protocol minimizes base dimer formation and partial base-deoxyribose dissociation in gas phase and greatly improves modified base quantitation. We also demonstrate that DI-MS overcomes biases in differential chromatographic retention and issues of sample degradation in the autosampler due to its high throughput. Finally, we present an application of our workflow to quantify DNA modifications on a batch of 81 samples in about 1.5 h.

ISFrag: De Novo Recognition of In-Source Fragments for Liquid Chromatography-Mass Spectrometry Data.

In-source fragmentation (ISF) is a naturally occurring phenomenon during electrospray ionization (ESI) in liquid chromatography-mass spectrometry (LC-MS) analysis. ISF leads to false metabolite annotation in untargeted metabolomics, prompting misinterpretation of the underlying biological mechanisms. Conventional metabolomic data cleaning mainly focuses on the annotation of adducts and isotopes, and the recognition of ISF features is mainly based on common neutral losses and the LC coelution pattern. In this work, we recognized three increasingly important patterns of ISF features, including (1) coeluting with their precursor ions, (2) being in the tandem MS (MS2) spectra of their precursor ions, and (3) sharing similar MS2 fragmentation patterns with their precursor ions. Based on these patterns, we developed an R package, ISFrag, to comprehensively recognize all possible ISF features from LC-MS data generated from full-scan, data-dependent acquisition, and data-independent acquisition modes without the assistance of common neutral loss information or MS2 spectral library. Tested using metabolite standards, we achieved a 100% correct recognition of level 1 ISF features and over 80% correct recognition for level 2 ISF features. Further application of ISFrag on untargeted metabolomics data allows us to identify ISF features that can potentially cause false metabolite annotation at an omics-scale. With the help of ISFrag, we performed a systematic investigation of how ISF features are influenced by different MS parameters, including capillary voltage, end plate offset, ion energy, and "collision energy". Our results show that while increasing energies can increase the number of real metabolic features and ISF features, the percentage of ISF features might not necessarily increase. Finally, using ISFrag, we created an ISF pathway to visualize the relationships between multiple ISF features that belong to the same precursor ion. ISFrag is freely available on GitHub (https://github.com/HuanLab/ISFrag).

Metabolic characterization of a potent natural neuroprotective agent dendrobine in vitro and in rats.

Dendrobine is the main sesquiterpene alkaloid of Dendrobium nobile Lindl, which exhibits potent neuroprotective activity. However, its metabolism and disposition are little known. In this study, we investigated the metabolic characteristics of dendrobine in vitro and in rats. The metabolic stability and temporal profile of metabolites formation of dendrobine were assayed in human/rat liver microsomal and S9 fractions. Dendrobine metabolites were separated and identified mainly by UPLC-Q/Orbitrap MS. After oral administration of dendrobine (50 mg/kg) to rats, the accumulative excretion rate of dendrobine in feces, urine, and bile was 0.27%, 0.52%, and 0.031%, respectively, and low systematic exposure of dendrobine (AUC0-∞ = 629.2 ± 56.4 ng·h/mL) was observed. We demonstrated that the elimination of dendrobine was very rapid in liver microsomal incubation (the in vitro elimination t1/2 in rat and human liver microsomes was 1.35 and 5.61 min, respectively). Dendrobine underwent rapid and extensive metabolism; cytochrome P450, especially CYP3A4, CYP2B6, and CYP2C19, were mainly responsible for its metabolism. Aldehyde dehydrogenase, alcohol dehydrogenase and aldehyde oxidase were involved in the formation of carboxylic acid metabolites. By the aid of in-source fragmentation screening, hydrogen/deuterium exchange experiment, post-acquisition processing software, and available reference standards, 50 metabolites were identified and characterized in liver microsomal incubation and in rats. The major metabolic pathways of dendrobine were N-demethylation, N-oxidation, and dehydrogenation, followed by hydroxylation and glucuronidation. Collectively, the metabolic fate of dendrobine elucidated in this study not only yields benefits for its subsequent metabolism study but also facilitates to better understanding the mode of action of dendrobine and evaluating the pharmacologic efficiency of the high exposure metabolites.