Direct detection and quantitation of He@C60 by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry

In this paper, we report negative ion microelectrospray Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry of C60 samples containing approximately 1% 3He@C60 or 4He@C60. Resolving He@C60- and 4He@C60- from C60 containing 3 or 4 13C instead of 12C atoms is technically challenging, because the target species are present in low relative abundance and are very close in mass. Nevertheless, we achieve baseline resolution of 3He@C60- from 13C3(12C57-) and 4He@C60- from 13C4(12C56-) in single-scan mass spectra obtained in broadband mode without preisolation of the ions of interest. The results constitute the first direct mass spectrometric observation of endohedral helium in a fullerene sample at this (low) level of incorporation. The results also demonstrate the feasibility of determining the extent of He incorporation from the FT-ICR mass spectral peak heights. The present measurements are in agreement with those obtained by the pyrolysis method [1-3]. Although limited in sensitivity, the mass spectral method is faster and easier than pyrolysis.     

Assigning product ions from complex MS/MS spectra: The importance of mass uncertainty and resolving power

This study offers a unique insight into the mass accuracy and resolving power requirements in MS/MS analyses of complex product ion spectra. In the examples presented here, accurate mass assignments were often difficult because of multiple isobaric interferences and centroid mass shifts. The question then arose whether the resolving power of a medium-resolution quadrupole time-of flight (QqTOF) is sufficient or high-resolution Fourier-transform ion cyclotron resonance (FT-ICR) is required for unambiguous assignments of elemental compositions. For the comparison, two paralytic shellfish poisons (PSP), saxitoxin (STX) and neosaxitoxin (NEO), with molecular weights of 299 and 315 g x mol(-1), respectively, were chosen because of the high peak density in their MS/MS spectra. The assessment of QqTOF collision-induced dissociation spectra and FT-ICR infrared multiphoton dissociation spectra revealed that several intrinsic dissociation pathways leading to isobaric fragment ions could not be resolved with the QqTOF instrument and required FT-ICR to distinguish very close mass differences. The second major source of interferences was M + 1 species originating from coactivated 13C12Cc-1 ion contributions of the protonated molecules of the PSPs. The problem in QqTOF MS results from internal mass calibration when the MH+ ions of analyte and mass calibrant are activated at the same time in the collision or trapping cell. Although FT-ICR MS readily resolved these interfering species, the QqTOF did not provide resolving power >20,000 (full width at half maximum) required to separate most isobaric species. We were able to develop a semi-internal QqTOF calibration technique that activated only the isolated 12C isotope species of the protonated molecules, thus reducing the M + 1 interferences significantly. In terms of overall automated elemental formulas assignment, FT-ICR MS achieved the first formula hit for 100% of the product ions, whereas the QqTOF MS hit rate was only 56 and 65% for STX and NEO product ions, respectively. External mass calibration from commercial FT-ICR and QqTOF instruments gave similar results.     

A quality index for reference mass spectra

A numerical rating for evaluating the quality of reference mass spectra used for retrieval and interpretive applications is proposed. The overall quality index is the product of seven quality factors: (1) source of the spectrum; (2) ionization conditions; (3) higher molecular weight impurities; (4) illogical neutral losses; (5) isotopic abundance accuracy; (6) number of peaks; and (7) lower mass limit of peaks. For quality factor 6 the number of peaks of abundances ≥1% was found to correspond to 15 more than the number of atoms in the molecule with a correlation coefficient of 0.482. The program has been found useful in preparing a data base of 41429 mass spectra; for the majority of spectra judged to be unsatisfactory by the program this arose from a low quality factor 6 value (too few peaks).     

低温煤焦油常渣C5沥青质的分离与表征

从低温煤焦油中蒸馏提取出大于350℃的常渣,以正戊烷、正戊烷+5%乙醇、正戊烷+5%异丙醇三种溶剂沉淀沥青质。通过1H-NMR、13C-NMR、傅里叶变换离子回旋共振质谱仪(FT-ICR MS)、元素分析和相对分子量测定等手段对重组分沥青质的结构参数、杂原子化合物的分布等方面进行了分析,构建含杂原子的平均分子结构模型。结果表明,乙醇和异丙醇的加入可以显著降低C5-沥青质的收率,所得沥青质的芳香性增强,平均分子量增大,为更高缩合度的具有较短侧链的结构;极性溶剂的加入没有改变沥青质中杂原子化合物的类型,但是O1和O2中一部分低缩合度的化合物转移到可溶质中,O3-O6化合物的相对丰度增大,表明混合溶剂有利于萃取低缩合度的化合物。     

Identification of the 'wrong' active pharmaceutical ingredient in a counterfeit Halfan drug product using accurate mass electrospray ionisation mass spectrometry,accurate mass tandem mass spectrometry and liquid chromatography/mass spectrometry

Methodology is presented for identifying an unknown active (pharmaceutical) ingredient (AI) in a counterfeit drug product. A range of mass spectrometric techniques, i.e., accurate mass mass spectrometry, tandem mass spectrometry (MS/MS) and liquid chromatography/mass spectrometry (LC/MS), has been employed to determine the AI in a counterfeit Halfan suspension, an antimalarial drug. In particular, use of LockSpray accurate mass MS/MS allowed identification of parts of the molecule from fragments, hence limiting the number of possible elemental compositions for the nominal mass of 278 found for the AI in the counterfeit product. The analysis of the isotope pattern observed for the protonated molecule further reduced the number of possible elemental compositions. A literature search for readily commercially available compounds of molecular formula C(12)H(14)N(4)O(2)S suggested that the AI was either sulfamethazine or sulfisomidine. An LC/MS separation of those two compounds and reference MS/MS spectra obtained for sulfamethazine and sulfisomidine led to the conclusion that the AI in the counterfeit Halfan suspension is sulfamethazine, which is an antibacterial agent.     

The computer processing and interpretation of mass spectral information. II—computation of the degree of isotope labelling in organic compounds

The program ISOMETA which processes the low resulution mass spectra and determines the degree of labelling in the molecular and fragment ions is described. The degree of labelling for any stable isotope (D, 13C, 15N, 18O, etc.) in compounds of known molecular formulae can be estimated. The compound may contain any number of elements and atoms of each element. The mass spectra are processed in terms of peak groups of the molecular and fragment ions which have been identified as having one composition. The program is written in ALGOL, runs on a BESM-6 computer and requires 6.5K core memory.     

Petroleomics: advanced molecular probe for petroleum heavy ends

To look into complex mixtures of petroleum heavy ends at the molecular level, ultra high-resolution mass spectrometry, i.e. resolving power > 50,000, is needed to resolve overlapping components for accurate determination of molecular composition of individual components. Recent progress in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) incorporated with soft ionization techniques adaptable to liquid chromatography enables analysis of petroleum high ends, i.e., heavy oils, residua and asphaltenes. FT-ICR MS at the Future Fuels Institute of Florida State University and the National High Magnetic Field Laboratory (NHMFL) routinely provides 1,000,000 resolving power at 400 Da, with root mean square (rms) mass measurement accuracy between 30 and 500 ppb for 5000-30,000 identified species in a single mass spectrum. Phase correction of the detected ion signal increases resolving power 40-100%, improving mass accuracy up to twofold. Overlapping ionic species that differ in mass by as little as one electron mass (548 μDa) can be resolved. A database of more than 100,000 components of different elemental composition has been generated at NHMFL.     

Molecular structure of fulvic acids by electrospray with quadrupole time-of-flight mass spectrometry

Characterisation of the molecular structure of aquatic fulvic acids (FA) has been performed using a quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an electrospray ionisation interface. Molecular masses centred around 450 Da and sinusoidal spectral distributions have been obtained for all fulvic acids. Tandem mass spectrometry (MS/MS) experiments showed losses of 18 Da (H(2)O) and 44 Da (CO(2)), and possible molecular structures were determined for the first time to our knowledge. A methodology is reported for evaluating the average elemental composition of FA from high-resolution mass spectra by processing post-acquisition data calculations using molecular size distributions and atomic compositions of ions. The results are found to be consistent with elemental analysis data.     

Reaction of hydrogen gas with C60 at elevated pressure and temperature: hydrogenation and cage fragmentation

Products of the reaction of C(60) with H(2) gas have been monitored by high-resolution atmospheric pressure photoionization Fourier transform ion cyclotron resonance mass spectrometry (APPI FT-ICR MS), X-ray diffraction, and IR spectroscopy as a function of hydrogenation period. Samples were synthesized at 673 K and 120 bar hydrogen pressure for hydrogenation periods between 300 and 5000 min, resulting in the formation of hydrofullerene mixtures with hydrogen content ranging from 1.6 to 5.3 wt %. Highly reduced C(60)H(x) (x > 36-40) and products of their fragmentation were identified in these samples by APPI FT-ICR MS. A sharp change in structure was observed for samples with at least 5.0 wt % of hydrogen. Low-mass (300-500 Da) hydrogenation products not observed by prior field desorption (FD) FT-ICR MS were detected by APPI FT-ICR MS and their elemental compositions obtained for the first time. Synthetic and analytical fragmentation pathways are discussed.     

Isotope ratio monitoring of small molecules and macromolecules by liquid chromatography coupled to isotope ratio mass spectrometry

In the field of isotope ratio mass spectrometry, the introduction of an interface allowing the connection of liquid chromatography (LC) and isotope ratio mass spectrometry (IRMS) has opened a range of new perspectives. The LC interface is based on a chemical oxidation, producing CO2 from organic molecules. While first results were obtained from the analysis of low molecular weight compounds, the application of compound-specific isotope analysis by irm-LC/MS to other molecules, in particular biomolecules, is presented here. The influence of the LC flow rate on the CO2 signal and on the observed delta13C values is demonstrated. The limits of quantification for angiotensin III and for leucine were 100 and 38 pmol, respectively, with a standard deviation of the delta13C values better than 0.4 per thousand. Also, accuracy and precision of delta13C values for elemental analyser-IRMS and flow injection analysis-IRMS (FIA-LC/MS) were compared. For compounds with molecular weights ranging from 131 to 66,390 Da, precision was better than 0.3 per thousand, and accuracy varied from 0.1 to 0.7 per thousand. In a second part of the work, a two-dimensional (2D)-LC method for the separation of 15 underivatised amino acids is demonstrated; the precision of delta13C values for several amino acids by irm-LC/MS was better than 0.3 per thousand at natural abundance. For labelled mixtures, the coefficient of variation was between 1% at 0.07 atom % excess (APE) for threonine and alanine, and around 10% at 0.03 APE for valine and phenylalanine. The application of irm-LC/MS to the determination of the isotopic enrichment of 13C-threonine in an extract of rat colon mucosa demonstrated a precision of 0.5 per thousand, or 0.001 atom %.     

In vitro metabolism of ethoxidine by human CYP1A1 and rat microsomes: identification of metabolites by high-performance liquid chromatography combined with electrospray tandem mass spectrometry and accurate mass measurements by time-of-flight mass spectrometry

Ethoxidine (N-methyl-12-ethoxy-2,3,8,9-tetramethoxybenzo[c]phenanthridinium methylsulfonate salt) is a synthetic 2-methoxy-12-ethoxy derivative of the natural alkaloid fagaronine. This new inhibitor of DNA-topoisomerase I is considered as a potential antitumor agent with higher in vitro activity than fagaronine. In order to further improve the efficiency of ethoxidine, its in vitro biotransformation by hepatic monooxygenases and the structures of its metabolites were investigated by high-performance liquid chromatography (HPLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) and accurate mass measurement by time-of-flight mass spectrometry (TOFMS). When ethoxidine was incubated with BNF-treated rat liver microsomes or with cells expressing different recombinant human cytochrome P450, the same four ethoxidine metabolites (m(1)-m(4)) were detected and were formed exclusively by CYP1A1. The structures of these metabolites were assigned from ESI-MS/MS mass spectra and compared with those of ethoxidine derivatives. Accurate mass measurements of in-source ESI-TOFMS fragment ions exhibited successive neutral losses of C(2)H(4) and CO for ethoxidine and its metabolites. Whereas a 15 Da loss (methyl radical) was observed for the metabolites m(1)-m(4) containing a quaternary ammonium group, a 16 Da loss (methane) was observed for ethoxidine and could have resulted from the presence of two methoxy groups at adjacent positions (C-2 and C-3). The proposed oxidative modifications of ethoxidine were further confirmed by determination of the number of exchangeable hydrogen atoms and by the proposed elemental compositions of the metabolites based on accurate mass measurements by TOFMS. Two major metabolites resulted from O-demethylation of ethoxidine; one was tentatively identified as 12-ethoxyfagaronine (m(3)) and the second as an O-demethylated ethoxidine isomer (m(4)). Two polar metabolites were shown to be O-demethylated (m(1)) and hydroxylated (m(2)) derivatives of 12-ethoxyfagaronine. When 12-ethoxyfagaronine was incubated under the same conditions as ethoxidine, m(2) was formed, thus supporting the proposal that 12-ethoxyfagaronine is the primary oxidative product of ethoxidine.     

Towards analytically useful two-dimensional Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) achieves high resolution and mass accuracy, allowing the identification of the raw chemical formulae of ions in complex samples. Using ion isolation and fragmentation (MS/MS), we can obtain more structural information, but MS/MS is time- and sample-consuming because each ion must be isolated before fragmentation. In 1987, Pfändler et al. proposed an experiment for 2D FT-ICR MS in order to fragment ions without isolating them and to visualize the fragmentations of complex samples in a single 2D mass spectrum, like 2D NMR spectroscopy. Because of limitations of electronics and computers, few studies have been conducted with this technique. The improvement of modern computers and the use of digital electronics for FT-ICR hardware now make it possible to acquire 2D mass spectra over a broad mass range. The original experiments used in-cell collision-induced dissociation, which caused a loss of resolution. Gas-free fragmentation modes such as infrared multiphoton dissociation and electron capture dissociation allow one to measure high-resolution 2D mass spectra. Consequently, there is renewed interest to develop 2D FT-ICR MS into an efficient analytical method. Improvements introduced in 2D NMR spectroscopy can also be transposed to 2D FT-ICR MS. We describe the history of 2D FT-ICR MS, introduce recent improvements, and present analytical applications to map the fragmentation of peptides. Finally, we provide a glossary which defines a few keywords for the 2D FT-ICR MS field.     

Evaluation of the spectral accuracy of mass spectrometers using compounds containing Cl or Br atoms

Current procedures for the evaluation of spectral accuracy of mass spectrometers are limited by the lack of certified isotopic reference materials and the high uncertainty in the isotopic composition of natural abundance molecules. The calculated uncertainties in the ratio M + 1/M for natural abundance molecules containing any number of C, H, N and/or O atoms are close to 5% relative because of the natural variability of the isotopic composition of carbon. So, we have developed two alternative measurement procedures with much lower theoretical uncertainties for a better evaluation of spectral accuracy in both single and triple quadrupole analysers. The first method is based on the measurement of the M + 2/M, M + 4/M + 2, etc. ratios for halogenated organic compounds containing either Cl or Br. The theoretical uncertainties for these ratios because of natural variability are in the order of 0.3 to 1.0% making them suitable for the evaluation of spectral accuracy with the additional advantage that there is no need to take into account other limitations such as cluster purity or poor mass resolution. This procedure was applied to the evaluation of a single quadrupole GC‐MS instruments using natural abundance PCB and PBDE standards with satisfactory results. The second method can be applied to tandem instruments and takes advantage of the loss of two halogen atoms when PCB and PBDE standards are fragmented by Collision Induced Dissociation. Theoretical SRM transition ratios can be calculated as a pure combinatorial probability with theoretical uncertainties lower than 0.1%. By combining PCBs and PBDEs with different number of halogen atoms, a mass range from 100 to 700 u and abundance ratios from 0.1 to 10 can be evaluated. The use of penta‐chlorinated PCBs and/or penta‐brominated PBDEs is finally recommended for the evaluation of spectral accuracy of mass spectrometers with the EI source. Copyright © 2016 John Wiley & Sons, Ltd.     

A combined nuclear magnetic resonance and computational study of monohydroxyflavones applied to product ion mass spectra

A method is presented for the estimation of 13C-chemical shifts for carbon atoms in protonated and deprotonated molecules; in principle, this method can be applied to ions in general. Experimental 13C-chemical shifts were found to vary linearly with computed atomic charges using the PM3 method. Pseudo-13C-chemical shifts for atoms in protonated and deprotonated molecules can be estimated from computed atomic charges for such atoms using the above linear relationship. The pseudo-13C-chemical shifts obtained were applied to the rationalization of product ion mass spectra of protonated and deprotonated molecules of flavone and 3-, 5-, 6-, 7-, 2'-, 3'-, and 4'-hydroxyflavones, where product ion formation is due to either cross-ring cleavage of the C-ring (retro-Diels-Alder reaction) or to cleavage of a C-ring bond followed by loss of either a small neutral molecule or a radical. The total product ion abundance ratio of C-ring cross cleavage to C-ring bond cleavage, gamma, varied by a factor of 660 for deprotonated monohydroxyflavones, i.e., from 0.014:1 to 9.27:1. The magnitude of gamma, which is dependent on the relative bond orders within the C-ring of the protonated and deprotonated molecules of monohydroxyflavones, can be rationalized on the basis of the magnitudes of the 13C- and 1H-chemical shifts as determined by nuclear magnetic resonance spectroscopy.     

Tandem mass spectrometric accurate mass performance of time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry: a case study with pyridine derivatives

The interpretation of mass spectra is a key process during compound identification, and the combination of tandem mass spectrometry (MS/MS) with high-accuracy mass measurements may deliver crucial information on the identity of a compound. Obtaining accurate mass data of fragment ions in MS/MS reveals the particular problem of mass calibration when a lockmass, which is frequently used to obtain accurate masses in MS, is absent. An alternative technique is to recalibrate the MS/MS spectrum using a reference MS/MS spectrum acquired under the same conditions. We have tested and validated this approach using a hybrid quadrupole/orthogonal acceleration reflectron-type time-of-flight (TOF) mass spectrometer. The results were compared with those obtained under similar conditions on a Fourier transform ion cyclotron resonance (FT-ICR) instrument. We found that the mass accuracy observed with such an "external" recalibration on the TOF instrument in MS/MS is identical to what can be obtained on a similar instrument operating in one-dimensional MS mode using the lockmass technique. However, mass accuracy in both cases is one order of magnitude inferior to that obtained using FTMS, and also inferior to that observed using sector field MS when operated at comparable resolution. Nevertheless, for small (<200 Da) molecules, this mass accuracy was still sufficient to have the "true" elemental composition identified as the first hit in about 70% of all cases. It was possible to elucidate the fragmentation mechanism of eight azaheterocycles containing a pyridine moiety, where the accurate mass data from the TOF instrument allowed distinction between two alternative fragmentation pathways.     

Improving mass accuracy of high performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of intact antibodies

The glycosylation profile of intact antibody due to the galactose and fucose heterogeneity in the N-linked sugars was determined with instrument resolution of 5000 and 10,000. After deconvolution of electrospray ionization mass spectra to complete convergence, several extra peaks appeared in addition to the peaks observed in the original mass spectra. The artificial peaks were avoided if deconvolution was stopped after a smaller number of iterations. A standard antibody was used as an external calibrant to minimize mass measurement errors during long-period experiments. Precision of four consecutive LC/MS measurements of the same antibody was 10 ppm (+/-1.5 Da). By using this approach, the masses of 11 intact antibodies were measured. All antibodies containing N-terminal glutamines had a negative mass shift due to the formation of pyroglutamate (-17 Da). Although the pyroglutamate variant of intact antibody was not resolved from the unmodified variant, this modification led to a mass shift proportional to the percentage of N-terminal pyroglutamate. By accurately measuring the mass shift we were able to quantify the abundance of pyroglutamic acid on intact antibodies. Mass accuracy in measuring different antibodies was below 30 ppm (+/-4 Da). The accurate mass measurement can be an effective tool for monitoring chemical degradations in therapeutic antibodies.     

Recherches dans la série des cyclitols XXXVII Etude par spectrométrie de masse

Low resolution mass spectra of cyclohexane-triols, -tetrols, -pentols and -hexols and of some of their deuterium labelled derivatives have been measured. The results indicate that for some geometrical isomers quantitative differences between ion intensities are significant, allowing to deduce the stereochemistry of the molecule from its mass spectrum. In all these compounds the electron impact induced elimination of water occurs mainly by interaction between OH groups. There is a correlation between the relative abundance of (M – H₂O)⁺ and (M ?2 H₂O)⁺ ions and the number of axial hydroxyls in the more stable conformation of the molecule. Difficulties encountered in deducing fragmentation patterns, and determination of number and position of deuterium atoms in the molecule of ms-inositol are discussed.     

Characterization of a single crystal cubic Prussian blue Co8(tacn)8(CN)12 cluster by ion trap and Fourier transform ion cyclotron resonance mass spectrometry with microelectrospray ionization

A single crystal of Co₈(tacn)₈(CN)₁₂ has been characterized by microelectrospray ionization mass spectrometry. The spectra obtained by use of Fourier transform ion cyclotron resonance (FT-ICR), ion trap and quadrupole mass spectrometers show the +4, +3, and +2 charge states of the cluster. With the aid of a 9.4 tesla FT-ICR mass spectrometer it was possible to resolve the isotope pattern for each individual charge state. The data collected suggest that microelectrospray renders spectra which are more specific to the intact molecule, whereas more fragmentation is induced under normal electrospray conditions. The present data suggest that microelectrospray is a powerful tool for characterization of Prussian blue complexes.     

Accurate mass measurement of low molecular weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We report the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the accurate measurement of mass of low molecular weight compounds (smaller than 1500 Da), a linear peptide, two types of cyclic depsipeptides, a polyhydroxy-macrocyclic lactone, and two prenylated flavonoids, with delayed extraction in the reflector mode. The performance of the MALDI-TOF instrument was less than those of fast atom bombardment and Fourier-transform ion cyclotron resonance mass spectrometry instruments and insufficient to give acceptable accuracy for literature reporting. Nevertheless, when combined with NMR spectrometry and/or amino acid analysis to give information on the numbers of carbon atoms and index of hydrogen deficiency, MALDI was useful for determination of the elemental composition of the low molecular weight compounds available in small quantities.     

Fourier transform ion cyclotron resonance mass spectrometry in the speciation of high molecular weight sulfur heterocycles in vacuum gas oils of different boiling ranges

The analysis of sulfur aromatics in vacuum gas oils (VGO) distilled from an Iranian light crude oil is discussed. The VGOs were fractionated into three boiling ranges, 390–460, 460–520, and 520–550 °C, and were analyzed using liquid chromatographic separation on a Pd(II)-bonded stationary phase followed by identification with electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). It was possible to detect a large number of thiophenes, including a substantial number of isomers, in the three VGO fractions. Separation on the palladium phase and inclusion of sulfur-selective derivatization makes electrospray ionization of these nonpolar compounds possible. An elemental composition can be assigned to a large number of S₁ compounds without ambiguity in the presence of abundant hydrocarbons. With an increase in boiling temperature, an increase in the size of the aromatic system and the number of side chain carbon atoms was observed. In addition, the masses of higher magnitude shifted toward larger aromatic systems with an increase in boiling range. A comparison of FT-ICR MS and comprehensive gas chromatography is also given. This work is dedicated to Prof. B. Krebs on the occasion of his 70th birthday.