Characteristic Isotope Patterns Research Articles

Analysis of the Fragmentation Pathways for the Collision-Induced Dissociation of Protonated Cyclophosphamide: A Mass Spectrometry and Quantum Mechanical Study.

Cyclophosphamide is a well-known anticancer agent acting by means of DNA alkylation. Associated with its tumor selectivity, it also possesses a wide spectrum of toxicities. As the requirement of metabolic activation before cyclophosphamide exerts either its therapeutic or toxic effects is well recognized, research aiming at elucidating the pathways that lead to the activation of this drug is of key importance. This has created the necessity for developing an effective analytical method for detecting cyclophosphamide and its breakdown products. In this paper, an Acquity TQ tandem quadrupole mass spectrometer equipped with electrospray ionization in positive-ion mode was employed for detecting cyclophosphamide in its protonated form. The full-scan mass spectrum of cyclophosphamide shows two ion clusters displaying the characteristic isotopic pattern of two chlorine atoms and assigned as sodiated cyclophosphamide, [CP + Na]+, and protonated cyclophosphamide, [CP + H]+ or PCP. With the aid of quantum mechanical DFT calculation, free energy differences in the gas phase among PCP protomers were computed with respect to the most stable protomer being protonated on the 2-oxide oxygen of the 1,3,2-oxazaphosphorine-2-oxide ring. In addition, the interconversion mechanisms among the different protomers were also proposed by intercepting the corresponding transition states in the gas phase. Collision-induced dissociation (CID) of PCP generated six characteristic product ions. Fragmentation mechanisms were proposed and supported by computation. The calculated energy barriers for all of the located transition states were found to be accessible under the reported experimental conditions.

Characterization of the Time-Domain Isotopic Beat Patterns of Monoclonal Antibodies in Fourier Transform Mass Spectrometry.

The time-domain transients in the Fourier transform mass spectrometry (FTMS) analysis of monoclonal antibodies (mAbs) are known to exhibit characteristic isotopic beat patterns. These patterns are defined by the isotopic distributions of all gaseous mAb ions present in the FTMS mass analyzer, originating from single or multiple charge states, and from single or multiple proteoforms. For an isolated charge state of a single proteoform, the mAb isotopic beat pattern resembles narrow splashes of signal amplitude (beats), spaced periodically in the time-domain transient, with broad (often exceeding 1 s) "valleys" between them. Here, we reinforce the importance of isotopic beat patterns for the accurate interpretation and presentation of FTMS data in the analysis of mAbs and other large biopolymers. An updated, mAb-grade version of the transient-mediated FTMS data simulation and visualization tool, FTMS Simulator is introduced and benchmarked. We then apply this tool to evaluate the charge-state dependent characteristics of isotopic beats in mAbs analyses with modern models of Orbitrap and ion cyclotron resonance (ICR) FTMS instruments, including detection of higher-order harmonics. We demonstrate the impact of the isotopic beat patterns on the analytical characteristics of the resulting mass spectra of individual and overlapping mAb proteoforms. The results reported here detail highly nonlinear dependences of resolution and signal-to-noise ratio on the time-domain transient period, absorption or magnitude mode spectra representation, and apodization functions. The provided description and the demonstrated ability to routinely conduct accurate simulations of FTMS data for large biopolymers should aid the end-users of Orbitrap and ICR FTMS instruments in the analysis of mAbs and other biopolymers, including viruses.

Single Nucleotide Resolution RNA-Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow.

RNA–protein interactions mediate many intracellular processes. CLIR-MS (cross-linking of isotope-labeled RNA and tandem mass spectrometry) allows the identification of RNA–protein interaction sites at single nucleotide/amino acid resolution in a single experiment. Using isotopically labeled RNA segments for UV-light-induced cross-linking generates characteristic isotope patterns that constrain the sequence database searches, increasing spatial resolution. Whereas the use of segmentally isotopically labeled RNA is effective, it is technically involved and not applicable in some settings, e.g., in cell or tissue samples. Here we introduce an extension of the CLIR-MS workflow that uses unlabeled RNA during cross-linking and subsequently adds an isotopic label during sample preparation for MS analysis. After RNase and protease digests of a cross-linked complex, the nucleic acid part of a peptide–RNA conjugate is labeled using the enzyme T4 polynucleotide kinase and a 1:1 mixture of heavy 18O4-γ-ATP and light ATP. In this simple, one-step reaction, three heavy oxygen atoms are transferred from the γ-phosphate to the 5′-end of the RNA, introducing an isotopic shift of 6.01 Da that is detectable by mass spectrometry. We applied this approach to the RNA recognition motif (RRM) of the protein FOX1 in complex with its cognate binding substrate, FOX-binding element (FBE) RNA. We also labeled a single phosphate within an RNA and unambiguously determined the cross-linking site of the FOX1-RRM binding to FBE at single residue resolution on the RNA and protein level and used differential ATP labeling for relative quantification based on isotope dilution. Data are available via ProteomeXchange with the identifier PXD024010.

Systematic Identification of Proteins Binding with Cisplatin in Blood by Affinity Chromatography and a Four-Dimensional Proteomic Method.

Cisplatin is widely used for the treatment of various solid tumors. It is mainly administered by intravenous injection, and a substantial amount of the drug will bind to plasma proteins, a feature that is closely related to its pharmacokinetics, activity, toxicity, and side effects. However, due to the unique properties of platinum complexes and the complexity of the blood proteome, existing methods cannot systematically identify the binding proteome of cisplatin in blood. In this study, high-abundance protein separation and an ion mobility mass spectrometry-based 4D proteomic method were combined to systematically and comprehensively identify the binding proteins of cisplatin in blood. The characteristic isotope patterns of platinated peptides and a similarity algorithm were utilized to eliminate false-positive identification. Finally, 39 proteins were found to be platinated. Bioinformatics analysis showed that the identified proteins were mainly involved in the complement and coagulation cascade pathways. The binding ratio of some peptides with cisplatin was measured based on the area ratio of the free peptide using the parallel reaction monitoring method. This study provides a new method for systematically identifying binding proteins of metal drugs in blood, and the identified proteins might be helpful for understanding the toxicity of platinum anticancer drugs.

Boron Isotope Tag-Assisted Ultrahigh-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry for Discovery and Annotation of cis-Diol-Containing Metabolites.

cis-Diol-containing metabolites are widely distributed in living organisms, and they participate in the regulation of various important biological activities. The profiling of cis-diol-containing metabolites could help us in fully understanding their functions. In this work, based on the characteristic isotope pattern of boron, we employed a boronic acid reagent as the isotope tag to establish a sensitive and selective liquid chromatography-high-resolution mass spectrometry method for the screening and annotation of cis-diol-containing metabolites in biological samples. Boronic acid reagent 2-methyl-4-phenylaminomethylphenylboronic acid was used to label the cis-diol-containing metabolites in biological samples to improve the selectivity and MS sensitivity of cis-diol-containing metabolites. Based on the characteristic 0.996 Da mass difference of precursor ions and the peak intensity ratio of 1:4 originating from 10B and 11B natural isotopes, the potential cis-diol-containing metabolites were rapidly screened from biological samples. Potential cis-diol-containing metabolites were annotated by database searching and analysis of fragmentation patterns obtained by multistage MS (MSn) via collision-induced dissociation. Importantly, the cis-diol position could be readily resolved by the MS3 spectrum. With this method, a total of 45 cis-diol-containing metabolites were discovered in rice, including monoglycerides, polyhydroxy fatty acids, fatty alcohols, and so forth. Furthermore, the established method showed superiority in avoiding false-positive results in profiling cis-diol-containing metabolites.

Development of an accurate and direct method for the green food colorants detection

Color impression represents between 60 and 90% of the final acceptance/rejection choice made by consumers. Consequently, color additives are attribute standards for our daily life in any market and any culture. Currently, authorized natural green food colorants comprise several copper-chelated chlorophyll derivatives. Both the raw materials and the manufacturing processes for the acquisition of these green food colorants are numerous and diverse. Hence, each producer applies its own know-how to obtain ‘signature’ green colorant products. Indeed, the chlorophyll profile of these products is partially known and may substantially differ among batches, while their identification just by HPLC-UV–Vis is not complete. Native chlorophylls do not chelate copper. Therefore, we propose a fast and specific method for copper chlorophyll detection, as indicative (except in a few fermented foods) of probable green food colorant addition or “re-greening” with copper salts. The new method is based on the characteristic isotopic pattern of the copper chlorophyll derivatives and does not require the precise characterization of the corresponding chlorophyll structure. This accurate methodology, based on a specific HPLC-ESI/APCI-HRMS method assisted with powerful post-processing software, is versatile as it can be used for other metallo-chlorophyll complexes also applied to improve the green coloration of food products.

Stable Isotopic Labeling and Nontarget Identification of Nanogram/Liter Amino Contaminants in Water.

We report a new combinatorial approach of stable isotopic labeling (SIL)-solid phase extraction (SPE)-liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS/MS) for identification of amino-containing contaminants at trace levels in source water. The new SIL method requires small amounts of formaldehyde (CH2O) and deuterated formaldehyde (CD2O) to efficiently label ng/L amino compounds in 1 L of water and improves SPE recovery, enabling environmental analysis of trace amino-compounds. Isotopically methylated components were confirmed using LC-MS/MS based on their retention times, and characteristic isotope patterns of the molecular and product ions. Using the characteristic isotope patterns, we established a data prioritization process to identify the amino compounds in thousands of mass peaks in raw data. Analysis of a labeled authentic source water detected 8952 m/z peaks and tentatively identified 154 amino compounds. Our SIL-methylation prioritization approach effectively reduced the complexity of data. Manual spectrum interpretation identified 77 of the 154 components as amino acids and peptides. We confirmed 8 of the 77 compounds using commercially available standards to demonstrate the feasibility and reliability of our SIL-SPE-LC-HRMS/MS method for environmental analysis of trace amino-containing contaminants. The method can efficiently identify amino-precursors in source water, enabling other studies of nitrogenous disinfection byproduct formation.

Behavior of transition metal salts during the electrospray ionization process

A study of the behavior of transition metal salts during the electrospray ionization (ESI) process using a quadrupole ion trap mass spectrometer and novel ion source is reported. The unique results in both positive and negative modes for multiple salts of 28 different cations are discussed. In positive mode, the metal cations formed clusters with the solvents, following the pattern [M + (solvent)n]+, with maximum observed values for n of 5. In general, transition metals with more than one oxidation state, such as iron, cobalt, and chromium, produced more complicated mass spectra, because the ions formed also included multiples of the anion. In negative mode, the cations formed clusters with the anions of the salts, following the pattern [My + Xn]-. The relationship between y and n correlates to the oxidation state of the cation, with maximum observed values for y and n of 3 and 7, respectively. For some environmental applications, studies in negative mode demonstrate an alternative to specialized elemental analysis instrumentation such as ICP/MS or AMS, as well as complicated sample preparation, such as the use of chelating complexes. This is especially applicable for the analyses of mercury and lead, as these species form only a few, predictable ions. For instance, mercury present in seawater forms a single ion, [Hg + Cl3]-, which can be readily isolated from the sodium chloride clusters in the background with a non-specialized ion trap mass spectrometer using ESI, and can be verified by its characteristic isotopic pattern.

Desorption electrospray ionization-high resolution mass spectrometry for the analysis of unknown materials: The phytosanitary product case

In this work, we tested the potential of desorption electrospray ionization-high resolution mass spectrometry (DESI-HRMS) for the analysis of unknown materials. To this end, our study focused on analyzing an unknown complex sample suspected of being an adulterated phytosanitary product or a fraud. A simple and fast sample manipulation procedure (filter paper impregnated with the sample) was used for the chemical characterization of the phytosanitary product by DESI-HRMS(/MS). Kendrick Mass Defect (KMD) analysis was used to process the DESI high-resolution mass spectral data, allowing the identification of a low molecular weight polymer (polyethylene glycol) and the detection of other ions, which did not follow polymer distributions. The characteristic isotope patterns of these ions suggested the presence of organometallic compounds. Accurate mass measurements, isotope pattern fits and the structural information obtained by DESI-MS/HRMS (wide isolation window) allowed identifying the presence of triphenyltin, a biocide extensively used for agricultural purposes and restricted by the European Commission, in the phytosanitary product. The concentration of triphenyltin in the sample was 35%, which corresponds to ∼12% expressed as weight of tin, 120 times higher than the maximum legislated level.

Transformation products elucidation of forchlorfenuron in postharvest kiwifruit by time-of-flight mass spectrometry

Forchlorfenuron (1-(2-chloro-4-pyridyl)-3-phenylurea, FCF) is a plant growth regulator, being extensively used for increasing kiwifruit size. The toxicological properties of its may persist in their transformation products (TPs) or even higher toxicity than FCF. TPs elucidation of FCF in postharvest kiwifruit (Actinidia chinensis, Chinese gooseberry) by the liquid chromatography ionization hybrid ion trap and time-of-flight mass spectrometry (LC-ESI-IT-TOF/MS) in positive mode was the objective of the present study. Fifteen days after full bloom, kiwifruits were dipped for 5s with high dosage FCF solution (60 mg/L), so that sufficient peaks could be detected. The chemical structure of unknown TPs was analyzed in combination of functions of LCMS-IT-TOF, such as high-accurate MSn, formula predictor, metabolite structural analysis software MetID Solution, profiling solution metabolomics software, and neutral loss, characteristic isotopic patterns of chlorine, the fragmentation pattern and retention time of standard substances, nitrogen rule, chemical components of kiwifruit. Total 17 TPs were detected via comparisons of their accurate MSn data of commercial analytical standards and synthesized standards with high purity, such as 4-amino-2-chloropyridine, phenylurea, 2-hydroxy-FCF, 1-(2-chloro-6-((3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) pyridin-4-yl)-3-phenylurea, 1, 3-bis (2-chloropyridin-4-yl) urea, 1,3-diphenylurea, 1-(2-chloropyridin-4-yl)urea, FCF-2-O-β-D-glucoside, and so on. The major transformation pathways of FCF in kiwifruit were biochemical and photochemical cleavage pathway. The experimental results indicate that LCMS-IT-TOF is powerful and effective tool for identification of FCF TPs.

Detection of over 100 selenium metabolites in selenized yeast by liquid chromatography electrospray time-of-flight mass spectrometry.

The characterization of the selenometabolome of Selenized(Se)-yeast, that is the fraction of water soluble low-molecular weight Se-metabolites produced in Se-yeast is of paramount interest to expand the knowledge on the composition of this food supplement. In this work, we have applied liquid chromatography electrospray time-of-flight mass spectrometry (LC-TOFMS) to search for Se-species from the low molecular weight range fraction of the selenized yeast used for food supplements. Prior to LC-TOFMS, sample treatment consisted of ultrasound assisted water extraction followed by size exclusion fractionation assisted with off-line inductively coupled plasma mass spectrometry detection of isotope 82Se. The fraction corresponding to low-molecular weight species was subjected to LC-TOFMS using electrospray ionization in the positive ion mode. The detection of the suspected selenized species has been based on the information obtained from accurate mass measurements of both the protonated molecules and fragments from in-source CID fragmentation; along with the characteristic isotope pattern exhibited by the presence of Se. The approach enables the detection of 103 selenized species, most of them not previously reported, in the range from ca. 300-650Da. Besides the detection of selenium species, related sulphur derivate metabolites were detected based on the accurate mass shift due to the substitution of sulphur and selenium.

Investigation of biotransformation of selenium in plants using spectrometric methods

The aim of this research was to study the processes of biotransformation of selenium in plants such as garlic, radish sprouts and sunflower sprouts via identification of selenium-containing compounds as metabolites of inorganic selenium using mass spectrometry. Speciation analysis of selenium in extracts from plant samples was performed with the use of hyphenated high performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) method. Matching the retention times of sample compounds with standards allowed identification of Se-methyl-selenocysteine, selenomethionine, γ-glutamyl-Se-methylselenocysteine and inorganic SeO32−. However, registered chromatograms included additional 82Se signals which couldn't be identified due to the lack of standards.Qualitative analysis of unknown compounds was achieved using high-resolution mass spectrometer equipped with mass analyzer Orbitrap coupled to high performance liquid chromatography. Since selenium has six stable isotopes of different abundance in nature, mass spectra of have a very characteristic isotopic pattern. In order to elucidate the structure of unknown Se compounds, selected ions were subjected to the fragmentation. Following selenocompounds were identified an inorganic selenium metabolites in garlic, sunflower sprouts and/or radish sprouts: selenohomolanthionine, Se-methyl-selenocysteine, selenomethionine, selenomethionine oxide, deaminohydroxy-selenohomolanthionine, N-acetylcysteine-selenomethionine, γ-glutamyl-Se-methyl-selenocysteine, methylseleno-Se-pentose-hexose, Se-methyl-selenoglutathione, 2,3-dihydroxy-propionyl-selenocysteine-cysteine, methyltio-selenoglutathione, 2,3-dihydroxypropionyl-selenolanthionine and two Se-containing compounds with proposed molecular formula C10H18N2O6Se and C10H13N5O3Se. Moreover, the structure was proposed for one selenocompound found in sunflower sprouts which has not been reported so far.

Evolution of a mass spectrometry-grade protease with PTM-directed specificity

Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (kcat/KM = 6.9 × 105 M-1⋅s-1). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y1 ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.

Fabrication and Application of Isotopically Labelled Gold Arrays for Multiplexed Peptide Analysis.

A new array-based technology for the simultaneous capture, chemical labelling and mass spectrometry analysis of peptides is presented. Isotopically labelled self-assembled monolayer (SAM) gold arrays are constructed and used simultaneously to capture and label a range of peptides. The array-immobilised, labelled peptides were released by MALDI ablation, analysed by MALDI mass spectrometry and readily identified as labelled peptides from their characteristic isotope pattern. This new solid-phase array platform has the advantage of minimal sample manipulation and is suitable for multiple analyses of single protein digests on a single MALDI target plate.

Mass spectrometric studies on the interaction of cisplatin and insulin.

The interaction of antitumor drug, cisplatin (cis-[PtCl2(NH3)2], CDDP) with insulin from porcine pancreas has been investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high resolution hybrid ion trap/time-of-flight mass spectrometry (MALIDI-TOF/TOF-MS and ESI-IT/TOF MS). The MALDI-TOF/TOF-MS results demonstrated that the presence of cisplatin complex resulted in the reduction of the disulfide bond in porcine pancreas after the incubations of the two substances were performed in vitro. It indicated that the presence of cisplatin would destroy the native configuration of insulin, which may lead to the inactivation of insulin. High resolution mass values and the characteristic isotopic pattern of the platinated insulin ions allowed the analysis of platinated mono-, di- and triadducts of cisplatin and insulin in the incubations under different conditions. The laser-induced dissociation of the monoadduct obtained in MALDI source was carried out and one platinum was found to bind to insulin B chain was determined. The platinum binding sites were further identified to be the N terminus (B chain), cysteine 7 (B chain) and cysteine 19 (B chain) residues by electrospray ionization tandem mass spectrometry. The identification of the interaction between insulin and cisplatin broadens the horizon of the knowledge in the interaction of the proteins and metallodrugs.

An overview of methods using (13)C for improved compound identification in metabolomics and natural products.

Compound identification is a major bottleneck in metabolomics studies. In nuclear magnetic resonance (NMR) investigations, resonance overlap often hinders unambiguous database matching or de novo compound identification. In liquid chromatography-mass spectrometry (LC-MS), discriminating between biological signals and background artifacts and reliable determination of molecular formulae are not always straightforward. We have designed and implemented several NMR and LC-MS approaches that utilize 13C, either enriched or at natural abundance, in metabolomics applications. For LC-MS applications, we describe a technique called isotopic ratio outlier analysis (IROA), which utilizes samples that are isotopically labeled with 5% (test) and 95% (control) 13C. This labeling strategy leads to characteristic isotopic patterns that allow the differentiation of biological signals from artifacts and yield the exact number of carbons, significantly reducing possible molecular formulae. The relative abundance between the test and control samples for every IROA feature can be determined simply by integrating the peaks that arise from the 5 and 95% channels. For NMR applications, we describe two 13C-based approaches. For samples at natural abundance, we have developed a workflow to obtain 13C–13C and 13C–1H statistical correlations using 1D 13C and 1H NMR spectra. For samples that can be isotopically labeled, we describe another NMR approach to obtain direct 13C–13C spectroscopic correlations. These methods both provide extensive information about the carbon framework of compounds in the mixture for either database matching or de novo compound identification. We also discuss strategies in which 13C NMR can be used to identify unknown compounds from IROA experiments. By combining technologies with the same samples, we can identify important biomarkers and corresponding metabolites of interest.

Characterization of the designer drug bk-2C-B (2-amino-1-(bromo-dimethoxyphenyl)ethan-1-one) by gas chromatography/mass spectrometry without and with derivatization with 2,2,2-trichloroethyl chloroformate, liquid chromatography/high-resolution mass spectrometry, and nuclear magnetic resonance.

We describe the analytical characterization of the designer drug bk-2C-B, a cathinone derivative, contained in a seized tablet, in the absence of an analytical standard. The analytical techniques employed include gas chromatography/mass spectrometry (GC/MS), without and with derivatization with 2,2,2-trichloroethyl chloroformate, liquid chromatography/high-resolution-MS (LC/HRMS) with an Orbitrap® analyzer, and nuclear magnetic resonance (NMR). LC/HRMS measurements consisted of accurate mass measurements of MH(+) ionic species under full scan conditions; comparison of experimental and calculated MH(+) isotopic patterns; examination of the isotopic fine structure (IFS) of the M+1, M+2, M+3 isotopic peaks relative to the monoisotopic M+0 peak; study of MH(+) collision-induced dissociation (CID) product ions obtained in fragmentation experiments. GC/MS analysis gave highly informative EI mass spectra, particularly after the derivatization of bk-2C-B with 2,2,2-trichloroethyl chloroformate. The application of LC/HRMS, allowing for accurate mass measurements at 100,000 resolving power, greatly enhanced analytical capabilities in structural characterization of this new designer drug. HRMS allowed us to obtain the accurate mass measurements of bk-2C-B MH(+) ionic species, with a mass accuracy of 2.19 ppm; fully superimposable experimental and calculated MH(+) isotopic patterns, with RIA1 and RIA2 values <4%; the IFS of the M+1, M+2, M+3 isotopic peaks relative to the monoisotopic M+0 peak completely in accordance with theoretical values. These findings enabled us to obtain the elemental composition formula of the seized drug. Furthermore, characteristic MH(+) CID product ions enabled the characterization of the bk-2C-B molecular structure. The presence of (79)Br and (81)Br isotopes in the substance molecule produced a characteristic isotopic pattern in most MS spectra. Lastly, NMR spectra allowed us to obtain useful information about the position of substituents in the designer drug. The combination of all the analytical techniques employed allowed the characterization of the seized psychoactive substance, in spite of the lack of a reference standard.

Cytotoxic acyl amides from the soil fungus Gymnascella dankaliensis

The soil fungus Gymnascella dankaliensis was collected in the vicinity of the Giza pyramids, Egypt. When grown on solid rice medium the fungus yielded four new compounds including 11′-carboxygymnastatin N (1), gymnastatin S (2), dankamide (3), and aranorosin-2-methylether (4), the latter having been reported previously only as a semisynthetic compound. In addition, six known metabolites (5–10) were isolated. Addition of NaCl or KBr to the rice medium resulted in the accumulation of chlorinated or brominated compounds as indicated by LC–MS analysis due to the characteristic isotope patterns observed. From the rice medium spiked with 3.5% NaCl the known chlorinated compounds gymnastatin A (11) and gymnastatin B (12) were obtained. All isolated compounds were unambiguously structurally elucidated on the basis of comprehensive spectral analysis (1D and 2D NMR, and mass spectrometry), as well as by comparison with the literature. Compounds 4, 7 and 11 showed potent cytotoxicity against the murine lymphoma cell line L5178Y (IC50 values 0.44, 0.58 and 0.64μM, respectively), whereas 12 exhibited moderate activity with an IC50 value of 5.80μM.

Identification of xenobiotic metabolites from biological fluids using flow injection analysis high-resolution mass spectrometry and post-acquisition data filtering.

Concern for public health entails the need to evaluate the degree of exposure of population to toxicants. To do this, robust high-throughput approaches are required to be able to perform a large number of analyses in cohort studies. In this study, a data-filtering procedure was applied to mass spectral data acquired by direct analysis of biological fluids leading to rapid detection of metabolites in a model xenobiotic system. Flow injection analysis (FIA) coupled to negative electrospray ionization (ESI)-LTQ Orbitrap Fourier transform mass spectrometry was used to directly analyze urine of rats treated with vinclozolin. Tandem mass spectrometry (MS/MS) experiments were subsequently performed for confirmation of a new metabolite structure. The isotope filtering based on the difference between accurate masses of (35)Cl and (37)Cl was applied to the raw data for the specific detection of ions containing at least one chlorine atom. Seven metabolites of vinclozolin were manually identified thanks to the characteristic isotope pattern of dichlorinated compounds. A new metabolite of vinclozolin was detected for the first time and identified as a sulfate conjugate. The application of an isotope-filtering procedure allowed the selective extraction of pertinent signals from the data. The processed mass spectrum was greatly simplified, significantly facilitating the detection of the seven metabolites previously identified. The use of FIA-HRMS in combination with dedicated bio-informatics data processing is shown to be an efficient approach for the rapid detection of metabolites in biological fluids. This is a very promising high-throughput approach for rapid characterization of the exposure status to xenobiotics.

Silver dopants for targeted and untargeted direct analysis of unsaturated lipids via infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI).

Unsaturated lipids play a crucial role in cellular processes as signaling factors, membrane building blocks or energy storage molecules. However, adequate mass spectrometry imaging of this diverse group of molecules remains challenging. In this study we implemented silver cationization for direct analysis by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids and facilitate peak assignment. Trace amounts of silver nitrate were doped into the electrospray solvent of an IR-MALDESI imaging source coupled to an Orbitrap mass analyzer. Calcifediol was examined as a model compound to demonstrate the effect of silver dopants on sensitivity and assay robustness. Dried human serum spots were subsequently analyzed to compare Ag-doped solvents with previously described solvent compositions. Mass differences as well as ion abundance ratio filters were employed to interpret results based on the characteristic isotopic pattern of silver. Olefinic lipids were readily observed as silver adducts in IR-MALDESI analyses. Silver cationization decreased the limit of detection for calcifediol by at least one order of magnitude and was not affected in complex biological matrices. The ion abundance ratio and mass difference of [M + (107) Ag(+)](+) and [M + (109) Ag(+)](+) were successfully applied to facilitate the spectral assignment of silver adducts. Overall, silver cationization increased the analyte coverage in human serum by 43% compared with a standard IR-MALDESI approach. Silver cationization has been shown to enhance IR-MALDESI sensitivity and selectivity for unsaturated lipids, even when applied to complex samples. Increased compound coverage, enhanced robustness as well as the developed tools for peak assignment and mapping of isotopic patterns will clearly benefit future mass spectrometry imaging studies.