Analysis of long-chain fatty acyl coenzyme a thioesters by negative ion fast-atom bombardment mass spectrometry and tandem mass spectrometry

Long-chain acyl Coenzyme A (CoA) is essentially composed of three major chemical groups, fatty acyl-, phosphopantetheino-, and 3′, 5′,-adenosine diphospho-moieties. The negative ion fast-atom bombardment mass spectrometry spectra of long-chain acyl CoA thioesters were characterized by the formation of abundant [M ? H]^? and two distinct classes of fragment ions, one class which retained the acyl group and another class which is related to CoA that contains the phosphopantethene and adenine. The ions which retained the acyl group in the spectrum of palmitoyl CoA appeared at m/z 675, 657, 595, and 577 and were found to decompose by loss of alkylketene observed at m/z 357 and 339. Those ions which retained the adenine group were observed at m/z 426 and 408. In contrast to these ions observed following fast-atom bombardment ionization, tandem mass spectrometry of the [M ? H]^?, from palmitoyl CoA (m/z 1004), yielded the adenine-containing ions as major products and the acyl-containing ions were of low abundance or not detected. These results suggested that the formation of many characteristic ions observed in direct FAB analysis occurred during the desorption process. The unique relationship between ions which involved the transition from acyl-containing ions to only CoA-containing ions by the loss of alkylketene allowed the development of tandem mass spectrometry protocols for the analysis of acyl CoA mixtures. Precursor scans of either m/z 357 or 339 yielded the identification of each species in a complex mixture. Identification of specific species was obtained with a neutral loss scan of the mass for a specific alkylketene.     

Enhanced electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of long-chain polysaccharides

A novel strategy was developed to extend the application of electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to the analysis of long-chain polysaccharides. High molecular weight polydisperse maltodextrins (poly-alpha(1-4) glucose) and dextrans (poly-alpha(1-6) glucose) were chosen as model compounds in the present study. Increased ionization efficiency of these mixtures in the positive ion mode was achieved upon modification of their reducing end with nitrogen-containing groups. The derivatization method is based on the formation of a new C--N bond between 1,6-hexamethylenediamine (HMD) and the reducing end of the polysaccharide, which exists in solution as an equilibrium between the hemiacetal and the open-ring aldehyde form. To achieve the chemical modification of the reducing end, two synthetic pathways were developed: (i) coupling of HMD by reductive amination and (ii) oxidation of the hemiacetal to lactone, followed by ring opening by HMD to yield the maltodextrin lactonamide of 1,6-hexanediamine (HMMD). Amino-functionalized polysaccharides were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR-MS) in the positive ion mode by direct flow injection. The hexamethylenediamine (HMD) and maltodextrin lactonamide of 1,6-hexanediamine (HMMD) moieties provide increased proton affinities which dramatically improve the detection of the long-chain polysaccharides by FTICR-MS. The present approach allowed for identification of single components in mixtures with prominent heterogeneity in the degree of polymerization (DP), without the need for chromatographic separation prior to MS. The high mass accuracy was essential for the unambiguous characterization of the species observed in the analyzed mixtures. Furthermore, molecular components containing up to 42 glucose residues were detected, representing the largest polysaccharide chains analyzed so far by ESI FTICR-MS.     

Analysis of oligonucleotides by hydrophilic interaction liquid chromatography coupled to negative ion electrospray ionization mass spectrometry

Hydrophilic interaction liquid chromatography (HILIC) is here successfully coupled to negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of synthetic and chemically modified oligonucleotides. Separation was performed on a 2.1 mm × 100 mm PEEK ZIC^® HILIC column packed with hydrophilic stationary phase with a permanent zwitterionic functional group and a particle size of 3.5 μm with an average pore diameter of 200 Å. A method was developed to separate homogeneous and heterogeneous oligonucleotides as well as methylated oligonucleotides using a quaternary pumping system containing ammonium acetate and water with an acetonitrile gradient. Analyses of oligonucleotides were performed by LC/MS with a detection limit of 2.5 picomole (20 mer) with signal to noise ratio (S/N) of 4.12. The influence of the eluent composition, type of buffer and its concentration, and organic modifier were also evaluated. The HILIC LC/MS method presented in this paper used common, ‘MS friendly’, mobile phases achieving sensitive and selective oligonucleotide analysis.     

Characterization of oil sands naphthenic acids treated with ultraviolet and microwave radiation by negative ion electrospray Fourier transform ion cyclotron resonance mass spectrometry

Naphthenic acids (NAs) are concentrated in oil sand process water (OSPW) as a result of caustic oil sands extraction processes. There is considerable interest in methods for treatment of NAs in OSPW. Earlier work has shown that the combination of ultraviolet (UV) and microwave treatments in the laboratory was effective in reducing the concentration of classical NAs. Here we apply Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS) to further characterize NAs treated with (a) UV (254 nm) in the presence of TiO₂ catalyst; and/or (b) microwave irradiation (2.45 GHz). FT‐ICR MS was used to characterize the NA fraction before and after treatment. Acidic oxygen‐containing classes were most abundant in all samples whereas other heteroatomic classes were least abundant or not present in some samples. For example, the SO₂‐containing species were absent in UV‐ or combined UV‐ and microwave‐treated samples. The O₂ class was dominant in all samples, indicative of NAs. However, samples treated with UV and microwave radiation have a lower relative abundance of other heteroatomic classes. We observed O₂, S₁O₂, O₃, S₁O₃, O₄, O₅, and O₆ classes, whereas the species with relatively high O_n content, namely, the O₃, O₅, and O₆ classes, were present only in UV‐ and microwave‐treated samples. The relatively high O_n content is consistent with oxidation of the parent acids in treated samples. There may thus be potential implications for environmental forensics. For example, the monitoring of the ratio of SO₂:O₂ or tracking the relative abundances of O₂, O₃, O₄, O₅, and O₆ classes may provide insights for distinguishing naturally derived oil sands components from those that are process‐related in aquatic environments. Copyright © 2010 John Wiley & Sons, Ltd.     

A fragmentation study of isoflavones in negative electrospray ionization by MSn ion trap mass spectrometry and triple quadrupole mass spectrometry

This study has elucidated the fragmentation pathway for deprotonated isoflavones in electrospray ionization using MS(n) ion trap mass spectrometry and triple quadrupole mass spectrometry. Genistein-d(4) and daidzein-d(3) were used as references for the clarification of fragment structures. To confirm the relationship between precursor and product ions, some fragments were traced from MS(2) to MS(5). The previous literature for the structurally related flavones and flavanones located the loss of ketene (C(2)H(2)O) to ring C, whereas the present fragmentation study for isoflavones has shown that the loss of ketene occurs at ring A. In the further fragmentation of the [M-H-CH(3)](-*) radical anion of methoxylated isoflavones, loss of a hydrogen atom was commonly found. [M-H-CH(3)-CO-B-ring](-) is a characteristic fragment ion of glycitein and can be used to differentiate glycitein from its isomers. Neutral losses of CO and CO(2) were prominent in the fragmentation of deprotonated anions in ion trap mass spectrometry, whereas recyclization cleavage accounted for a very small proportion. In comparison with triple quadrupole mass spectrometry, ion trap MS(n) mass spectrometry has the advantage of better elucidation of the relationship between precursor and product ions.     

Analysis of curcuminoids by positive and negative electrospray ionization and tandem mass spectrometry

The curcuminoids are a group of diarylheptanoid molecules that possess important pharmacological activities, particularly acting as anti-inflammatory agents. The main purpose of this study was to investigate the fragmentation behavior of the three major curcuminoids in ion trap liquid chromatography/tandem mass spectrometry (LC/MS/MS). Both positive and negative mode electrospray ionization in tandem and multidimensional MS(n) experiments in quadrupole ion trap instruments and high-resolution and accurate mass MS and sustained off-resonance irradiation (SORI) MS/MS experiments in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer were used to elucidate the main fragmentation channels of these compounds. These experiments yielded essentially the same fragmentation results in both ion trap and ICR instruments for all three curcuminoids and for their phenolic monoacetates. Major and diagnostic fragment ions were identified and their origins are proposed.     

A systematic study of carboxylic acids in negative ion mode electrospray ionisation mass spectrometry providing a structural model for ion suppression

We report a systematic investigation of the effects and structural requirements for ion suppression in negative ion mode electrospray ionisation mass spectrometry of a series of carboxylic acids and present a structural model rationalising ion suppression effects.     

Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry

Eleven naturally occurring flavonoid aglycones, belonging to the representative flavone, flavonol, and flavanone types were separated by high performance liquid chromatography and analyzed on-line with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained, each compound was reinvestigated by direct loop injections using an ion trap mass spectrometer. The MSn spectra obtained allowed us to propose plausible schemes for their fragmentation supported by the analysis of five complementary synthetic flavonoid aglycones. The negative ion ESI-MS/MS behavior of the different aglycones investigated in this study revealed interesting differences when compared with the previously described patterns obtained using various ionization techniques in positive ion. Thus, concerning the retro Diels-Alder (RDA) fragmentation pathways, several structurally informative anions appeared highly specific of the negative ion mode. In addition, a new lactone-type structure, instead of a ketene, was proposed for a classic RDA diagnostic ion. We also observed unusual CO, CO2, and C3O2 losses which appear to be characteristic of the negative ion mode. All these results and these unusual neutral losses show that the negative ion mode was a powerful complementary tool of the positive ion mode for the structural characterization of flavonoid aglycones by ESI-MS/MS.     

Analysis of O-glucuronide conjugates in urine by electrospray ion trap mass spectrometry

The application of electrospray ionization (ESI) ion trap mass spectrometry in the characterization of O-glucuronide conjugates of some drugs in urine is described. The conjugated metabolites formed in rabbit and human were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by multi-stage mass spectrometry (MSⁿ) experiments in negative ion mode. The ESI mass spectra showed a deprotonated molecule [M–H]^–, which was chosen as precursor ion. Collision-induced dissociation (CID) of [M–H]^– in MSⁿ experiments resulted in the appearance of glucuronate ‘fingerprint’ ions at m/z 175 and 113 as well as prominent aglycone ions which were the same as those produced from authentic specimens. This information can be used to identify this type of compound directly without the need for derivatization or hydrolysis of enzymes, providing a rapid and specific method for guiding the isolation and characterization of similar compounds in complex matrices with LC/MS.     

Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids

Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix.     

Characterizing ion mobility and collision cross section of fatty acids using electrospray ion mobility mass spectrometry

This study investigated the ion mobility (IM) and the collision cross section (CCS) of fatty acids (FAs) using electrospray IM MS. The IM analysis of 18 FA ions showed intriguing differences among the saturated FAs, monounsaturated FAs, multi‐unsaturated FAs, and cis‐isomer/trans‐isomer with respect to the aliphatic tail chains. The length of aliphatic tail chain present in the ion structures had a strong influence on the differentiation of drift, while the number of double bond showed a weaker influence. The tiny drift differences between cis‐isomer and trans‐isomer were also observed. In the CCS measurements, two internal standards were involved in the mobility calibration and accuracy estimation. It insured our empirical CCS values were of high experimental precision (±0.35% or better) and accuracy (±0.25% or better). Moreover, the mass‐to‐charge ratio (m/z) – mobility plots obtained by ion mobility spectrometry with mass spectrometry analysis of FAs – was used to investigate the structural relationship between the molecules. Each series of FAs sharing a similar structure was aligned in the linear plot. Finally, the developed procedure was applied to the determination of FAs in rat adipose tissues, and it allowed the presence of 13 FAs to be confirmed with their exact masses and CCS values. These studies reveal the direct relationship between the behaviors in IM and the molecular structures and thus may provide further validations to the FA identification process. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of oligomannuronic acids and oligoguluronic acids by high-performance anion-exchange chromatography and electrospray ionization mass spectrometry

Oligomannuronic acids and oligoguluronic acids were prepared by enzymatic hydrolysis of alginate with alginate lyases. The oligosaccharides generated up to degree of polymerization 16 were characterized by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) and electrospray ionization mass spectrometry (ESI-MS). Acetate buffer linear gradients were used as mobile phases for separations of oligosaccharides. ESI-MS and HPAEC-PAD are very effective for the analysis and characterization of anionic oligosaccharides.     

Determination of fatty acid positions in native lipid A by positive and negative electrospray ionization mass spectrometry

Lipid A is the endotoxic principle of the lipopolysaccharide fraction from Gram-negative bacteria. It is involved in the elicitation of cytokine production that leads to massive inflammation and to septic shock as a lethal consequence. For this reason, the structural elucidation of lipopolysaccharides from toxic Gram-negative bacteria is an important and complicated task, mainly owing to its natural heterogeneity. Here, a new methodology to infer the distribution of the primary and secondary acyl residues is described, based on electrospray ionization mass spectrometry (ESI-MS) of intact lipid A under high cone voltage in order to achieve in-source fragmentation. Under these conditions, acyl fragmentation is induced and a different regioselective cleavage of secondary fatty acids is observed in positive and negative ESI-MS, allowing the rapid identification of the lipid A structure.     

Characterization and identification of isomeric flavonoid O-diglycosides from genus Citrus in negative electrospray ionization by ion trap mass spectrometry and time-of-flight mass spectrometry

Flavonoid O-diglycosides are important bioactive compounds from genus Citrus. They often occur as isomers, which makes the structural elucidation difficult. In the present study, the fragmentation behavior of six flavonoid O-diglycosides from genus Citrus was investigated using ion trap mass spectrometry in negative electrospray ionization (ESI) with loop injection. For the flavonoid O-rutinosides, [M − H − 308]⁻ ion was typically observed in the MS² spectrum, suggesting the loss of a rutinose. The fragmentation patterns of flavonoid O-neohesperidosides were more complicated in comparison with their rutinoside analogues. A major difference was found in the [M − H − 120]⁻ ion in the MS² spectrum, which was a common feature of all the flavonoid O-neohesperidosides. The previous literature for naringin located the loss of 120 Da to the glycan part, whereas the present study for naringin had shown that the [M − H − 120]⁻ ion was produced by a retro-Diels-Alder reaction in ring C, and this fragmentation pattern was confirmed by the accurate mass measurement using an orthogonal time-of-flight mass spectrometer. Combined with high performance liquid chromatography (HPLC) and diode array detection (DAD), the established approach to the structural identification of flavonoid O-diglycosides by ion trap mass spectrometry was applied to the analysis of extracts of two Chinese medicines derived from genus Citrus, namely Fructus aurantii and F. aurantii immaturus. According to the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by multistage mass spectrometry (MSⁿ) spectra, 13 flavonoid O-glycosides in F. aurantii and 12 flavonoid O-glycosides in F. a. immaturus were identified rapidly.     

Negative ion formation in electrospray mass spectrometry

Analytical and Chemical Sciences, Research Triangle Institute, Research Triangle Park, North Carolina, USA Negative ion electrospray (ES) operating on a single quadrupole mass spectrometer for the detection of low-molecular-weight molecules is discussed. The ES interface was operated at a positive cylindrical electrode potential to produce negative ions, and the results obtained were compared to the positive ion mode. As in the case of operation in the more common positive mode, negative ions with varying degrees of solvation and structurally relevant fragments can be obtained from a variety of solute species, including β-lactam antibiotics, aminoglycosides, aminocyclitols, tetracyclines, sulfonamides, nucleotides, peptides, and explosives. No fragmentation of parent species, except those from some labile explosives, was provided because low potential differences are applied between the capillary and the first skimmer, and electrical discharge is avoided in the gas phase. An increase in the capillary voltage resulted in collision-induced decomposition to produce structurally relevant fragment ions in both operation modes. An evaluation of representative chromatographic solvents indicated that 2-propanol added with oxygen in the ES bath gas is best suited to suppress electrical (corona) discharge phenomena in negative ion operation, whereas it aids in solution nebulization, desolvation, and transfer of ions in solution to the gas phase. For positive ion mode, no such precaution was necessary. Conditions that promote the formation of ions in solution usually improve ES response. Therefore, an increase in the solvent pH can increase the sensitivity in negative ion ES ionization. Negative ion ES offers the advantage of providing complementary structural information to help in the characterization of an unknown compound or to confirm a certain tentatively proposed structure. Nucleotides and explosives were best characterized in negative ion mode owing to the ease with which they form anions in solution, and they could be detected down to the l-pg /gML level.     

Screening and sequencing of complex sialylated and sulfated glycosphingolipid mixtures by negative ion electrospray Fourier transform ion cyclotron resonance mass spectrometry

A protocol for negative ion nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (-)nanoESI-FTICR MS, investigation of complex biological mixtures consisting of sialylated or sulfated glycosphingolipids (GSL) expressing high heterogeneity in the ceramide portion is described. Different instrumental and solvent conditions were explored and optimized to promote efficient ionization, reduce the in-source fragmentation and consequently enhance the detection of intact molecular species from complex mixtures. Using the novel optimized (-)nanoESI-FTICR MS protocol, a reliable and detailed compositional fingerprint of the polysialylated ganglioside mixture isolated from human brain was obtained. Sustained off-resonance irradiation collision-induced dissociation mass spectrometry (SORI-CID MS2) was introduced for the first time for structural elucidation of polysialylated gangliosides. Under well-defined conditions, an informative fragmentation pattern of the trisialylated ganglioside GT1 was obtained. The compositional mapping of a complex mixture of sulfated glucuronic acid containing neolacto-series GSLs extracted from bovine Cauda equina provided hard evidence upon previously described components and new structures not identified before by any other analytical method. Negative ion nanoESI-FTICR MS at 9.4 T is shown here to represent a valuable method in glycolipidomics, allowing a high resolution and mass accuracy detection of major and minor GSL glycoforms and identification of known and novel biologically relevant structures.     

Structural characterization by both positive and negative electrospray ion trap mass spectrometry of oligogalacturonates purified by high-performance anion-exchange chromatography

The off-line coupling of high-performance anion-exchange chromatography to electrospray ion trap mass spectrometry (ESI-IT-MS) is described. Two sets of isocratic conditions were optimised for the semi-preparative purification of oligogalacturonates of degree of polymerisation from 4 to 6 by monitoring eluates with either pulsed amperometric detection or evaporative light scattering detection in the presence of an online Dionex Carbohydrate Membrane Desalter (CMD). In these conditions, purified oligogalacturonate solutions were suitable, without further desalting steps, for infusion ESI-IT-MS experiments. This paper provides some interesting features of positive and negative ESI-IT-multiple MS (MSn) of these acidic oligosaccharides. The spectra acquired in both ion modes show characteristic fragments resulting from glycosidic bond and cross-ring cleavages. Under negative ionization conditions, the fragmentation of the singly-charged [M-H]- ions, as well as the Ci-, and Zi-, fragment ions through sequential MSn experiments, was always dominated by product ions from C- and Z-type glycosidic cleavages. All spectra also displayed 0.2 A-type cross-ring cleavage ions which carry linkage information. Collision-induced dissociation (CID) spectra of sodium-cationized species obtained under positive ionization conditions were more complex. Successive MSn experiments also led to the 0.2 A-type cross-ring cleavage ions observed together with B- and Y-type ions. The presence of the 0.2 A ion series was related to Mr 60 (C2H4O2) losses. Combined with the absence of the Mr 30 (CH2O) and the Mr 90 (C3H6O3) ions, these ions were indicative of 1-4 type glycosidic linkage.     

Positive ion electrospray ionization mass spectrometry of oligonucleotides

Positive ion electrospray ionization mass spectra have been obtained of deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), including transfer RNAs (77-mer, ～ 25 kDa). For several different solution conditions, the charge state distributions of DNA and RNA molecules were determined. It is postulated that the production of the multiply charged positive ions results from gas phase dissociation of complexes between nitrogen-containing bases and oligonucleotides.