Collision-induced Dissociation Product Ion Spectra Research Articles

Predicting collision-induced dissociation spectra: Semi-empirical calculations as a rapid and effective tool in software-aided mass spectral interpretation

Fifteen molecules were modelled using quantum chemistry, prior to interpreting their collision-induced dissociation (CID) product ion spectra, in a 'blind trial' to establish if calculated protonation-induced bond elongation could be used to predict which bonds cleaved during CID. Bond elongation has the potential to be used as a descriptor predicting bond cleavage. The 15 molecules were modelled with respect to protonation-induced bond length changes using Density Functional Theory (DFT). Significant bond elongations were highlighted to flag potential bond cleavages. CID product ion spectra, obtained using positive ion electrospray ionisation (Waters Synapt G1), were interpreted to establish if observed bond cleavages correlated with calculated bond elongations. Calculations were also undertaken using AM1 (Austin Model 1) to see if this rapid approach gave similar results to the computationally demanding DFT. The AM1-calculated bond elongations were found to be similar to those generated by DFT. All the polarised bonds observed to cleave (n = 82) had been calculated to elongate significantly. Protonation, possibly via proton migration, on the most electronegative atom in the bond appeared to initiate cleavage, leading to a 100% success rate in predicting the bonds that broke as a result of protonation on a heteroatom. Cleavage of carbon-carbon bonds was not predicted. Cleavage of the polarised bonds appears to result from protonation on the more electronegative atom of the bond, inducing conformational changes leading to bond weakening. AM1-calculated bond length changes act as a descriptor for predicting bond cleavage. However, the impetus for cleavage of the unpolarised bonds may be product ion stability rather than bond weakening.

The low‐energy collision‐induced dissociation product ion spectra of protonated beta‐blockers reveal an analogy to fragmentation behaviour under electron ionisation conditions

The low‐energy collision‐induced dissociation product ion spectra of protonated beta‐blockers reveal an analogy to fragmentation behaviour under electron ionisation conditions

Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin D3 in serum of infants and adults

Since the introduction of liquid chromatography-mass spectrometry (LC/MS) for assessing vitamin D status, it has been shown that the C-3 epimer can account for a significant proportion of circulating 25-hydroxyvitamin D₃ (25OHD₃) concentrations in infants. However, some question whether monitoring a single MS transition at a chromatographic retention time typical for 3-epi-25OHD₃ sufficiently warrants conclusions about the identity of the substance generating the signal. Therefore, we aimed to substantiate the evidence for 3-epi-25OHD₃ in infants by collision induced dissociation (CID)-MS/MS product ion scans. A second objective was mass spectrometric investigation of the presence and prevalence of the 3-epi metabolite in serum from adults. Serum samples from six infants and 32 adults were studied using an ultra performance LC/tandem MS (UPLC/MS/MS) method designed to separate the 3-epi-25OHD₃ from 25OHD₃. Samples were submitted to liquid/liquid extraction and Sephadex LH-20 fractionation, prior to column-switching UPLC with MS/MS recording of CID product ion spectra of the [M+H](+) precursor ion. The respective standards were analyzed under identical UPLC/MS/MS conditions for comparison. In the chromatograms of all samples, two peaks eluted with retention characteristics and spectra closely matching those observed for the 25OHD₃ and the 3-epi standards. The percentage of the 3-epi metabolite relative to 25OHD₃ in infants ranged from 15% to 41%, and in adults from 2.5% to 17%. This preliminary finding suggests that the prevalence of 3-epi-25OHD₃ in serum of infants is considerable, and that even in adults the concentrations of this form should not be neglected.

Development of a targeted adductomic method for the determination of polycyclic aromatic hydrocarbon DNA adducts using online column-switching liquid chromatography/tandem mass spectrometry

Human exposure to polycyclic aromatic hydrocarbons (PAHs) from sources such as industrial or urban air pollution, tobacco smoke and cooked food is not confined to a single compound, but instead to mixtures of different PAHs. The interaction of different PAHs may lead to additive, synergistic or antagonistic effects in terms of DNA adduct formation and carcinogenic activity resulting from changes in metabolic activation to reactive intermediates and DNA repair. The development of a targeted DNA adductomic approach using liquid chromatography/tandem mass spectrometry (LC/MS/MS) incorporating software-based peak picking and integration for the assessment of exposure to mixtures of PAHs is described. For method development PAH-modified DNA samples were obtained by reaction of the anti-dihydrodiol epoxide metabolites of benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,l]pyrene (DB[a,l]P) and dibenz[a,h]anthracene with calf thymus DNA in vitro and enzymatically hydrolysed to 2'-deoxynucleosides. Positive LC/electrospray ionisation (ESI)-MS/MS collision-induced dissociation product ion spectra data showed that the majority of adducts displayed a common fragmentation for the neutral loss of 116 u (2'-deoxyribose) resulting in a major product ion derived from the adducted base. The exception was the DB[a,l]P dihydrodiol epoxide adduct of 2'-deoxyadenosine which resulted in major product ions derived from the PAH moiety being detected. Specific detection of mixtures of PAH-adducted 2'-deoxynucleosides was achieved using online column-switching LC/MS/MS in conjunction with selected reaction monitoring (SRM) of the [M+H](+) to [M+H-116](+) transition plus product ions derived from the PAH moiety for improved sensitivity of detection and a comparison was made to detection by constant neutral loss scanning. In conclusion, different PAH DNA adducts were detected by employing SRM [M+H-116](+) transitions or constant neutral loss scanning. However, for improved sensitivity of detection optimised SRM transitions relating to the PAH moiety product ions are required for certain PAH DNA adducts for the development of targeted DNA adductomic methods.

A Rapid Methodology for the Characterization of Dialkyl Tertiary Amine-N-Oxide Metabolites Using Structurally Dependent Dissociation Pathways and Reconstructed Ion Current Chromatograms

A high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) approach to the characterization of dialkyl tertiary amine-N-oxides is presented. The methodology is based upon forming reconstructed ion current chromatograms (RICCs) of m/z values of product ions known to form through diagnostic losses from dialkyl tertiary amine-N-oxides. The diagnostic losses of N,N-dimethylhydroxylamine and N,N-diethylhydroxylamine were identified through the analysis of a structurally diverse library of compounds by ESI-low-energy collision-induced dissociation (CID)-MS/MS using quadrupole ion trap-mass spectrometry (QIT-MS) and quadrupole time-of-flight-mass spectrometry (QqTOF-MS). The library consisted of dialkyl tertiary amine-containing commercially available pharmaceuticals, along with a number of model, synthetic N-oxides. The loss of the nitrogen-containing group was observed in 89% of the low-energy CID product ion spectra acquired using various collision energies. Further, the resultant product ions, formed through the loss of the nitrogen-containing group, were shown to be unstable because of the observation of second-generation dissociation. These observations regarding gas-phase ion chemistry could be useful to developers of in silico programs for fragmentation prediction by allowing the creation of improved algorithms and models for predicting dissociation. Using the information derived from the library analysis, the characterization methodology was developed and demonstrated using tetracaine. The approach is rapid, MS/MS platform independent, utilizes existing technology, and could be automated. Further, it is definitive and overcomes the limitations of other tools for N-oxide identification by localizing the site of oxidation. Thus, it provides a useful addition to the existing approaches for metabolite identification.

Detection of phosphodiester adducts formed by the reaction of benzo[a]pyrene diol epoxide with 2'-deoxynucleotides using collision-induced dissociation electrospray ionization tandem mass spectrometry

In this study, we investigated the products formed following the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE) with 2′-deoxynucleoside 3′-monophosphates. The B[a]PDE plus 2′-deoxynucleotide reaction mixtures were purified using solid phase extraction (SPE) and subjected to HPLC with fluorescence detection. Fractions corresponding to reaction product peaks were collected and desalted using SPE prior to analysis for the presence of molecular ions corresponding to m/z 648, 632, 608 and 623 [M−H]− consistent with B[a]PDE adducted (either on the base or phosphate group) 2′-deoxynucleotides of guanine, adenine, cytosine and thymine, respectively, using LC-ESI-MS/MS collision-induced dissociation (CID). Reaction products were identified having CID product ion spectra containing product ions at m/z 452, 436 and 412 [(B[a]Ptriol+base)−H]−, resulting from cleavage of the glycosidic bond between the 2′-deoxyribose and base, corresponding to B[a]PDE adducts of guanine, adenine and cytosine, respectively. Further reaction products were identified having unique CID product ion spectra characteristic of B[a]PDE adduct formation with the phosphate group of the 2′-deoxynucleotide. The presence of product ions at m/z 399 and 497 were observed for all four 2′-deoxynucleotides, corresponding to [(B[a]Ptriol+phosphate)−H]− and [(2′-deoxyribose+phosphate+B[a]Ptriol)−H]−, respectively. In conclusion, this investigation provides the first direct evidence for the formation of phosphodiester adducts by B[a]PDE following reaction with 2′-deoxynucleotides.

Metabolism, Distribution and Excretion of a Selective N-Methyl-d-Aspartate Receptor Antagonist, Traxoprodil, in Rats and Dogs

Disposition of traxoprodil ({1-[2-hydroxy-2-(4-hydroxy-phenyl)-1-methyl-ethyl]-4-phenyl-piperidin-4-ol}mesylate; TRX), a selective antagonist of the N-methyl-d-aspartate class of glutamate receptors, was investigated in rats and dogs after administration of a single i.v. bolus dose of [(14)C]TRX. Total mean recoveries of the radiocarbon were 92.5 and 88.2% from rats and dogs, respectively. Excretion of radioactivity was rapid and nearly complete within 48 h after dosing in both species. Whole-body autoradioluminography study suggested that TRX radioactivity was retained more by uveal tissues, kidney, and liver than by other tissues. TRX is extensively metabolized in rats and dogs since only 8 to 15% of the administered radioactivity was excreted as unchanged drug in the urine of these species. The metabolic pathways included aromatic hydroxylation at the phenylpiperidinol moiety, hydroxylation at the hydroxyphenyl ring, and O-glucuronidation. There were notable species-related qualitative and quantitative differences in the metabolism of TRX in rats and dogs. The hydroxylation at the 3-position of the phenol ring followed by methylation of the resulting catechol intermediate and subsequent conjugation were identified as the main metabolic pathways in dogs. In contrast, formation of the major metabolites in rats was due to oxidation at the 4'-position of the phenylpiperidinol moiety followed by further oxidation and phase II conjugation. TRX glucuronide conjugate was identified as the major circulating component in rats, whereas the glucuronide and sulfate conjugates of O-methyl catechol metabolite were the major metabolites in dog plasma. The site of conjugation of regioisomeric glucuronides was established from the differences in the collision-induced dissociation product ion spectra of their methylated products.

Detection of altrenogest and its metabolites in post administration horse urine using liquid chromatography tandem mass spectrometry—increased sensitivity by chemical derivatisation of the glucuronic acid conjugate

Altrenogest (17alpha-allyl-17beta-hydroxyestra-4,9,11-trien-3-one) is a steroid used for the control of estrus in horses. This drug can potentially be abused in racehorses as the occurrence of estrus can alter their performance. This work describes an analytical method based on liquid chromatography-tandem mass spectrometry for the detection of altrenogest in horse urine down to a concentration of 13 pg/mL (0.042 nM). Furthermore, the qualitative aspect of metabolism of altrenogest in the horse has been studied. The main transformations that were found for this species were conjugation with glucuronic acid and sulfate. These phase II metabolites were identified by molecular mass and by comparison of their collision-induced dissociation product ion spectra with that of the synthetic aglycone at positive and negative potential, respectively. No phase I metabolites were discovered. In order to increase the ionisation in positive electrospray, a derivatisation procedure forming a basic oxime was tested. This process significantly increased the detection sensitivity for altrenogest glucuronide in horse urine.

A Comparative Study of the Fragmentation of Neutral Lactooligosaccharides in Negative-Ion Mode by UV-MALDI-TOF and UV-MALDI Ion-Trap/TOF Mass Spectrometry

Structure analyses of underivatized neutral lacto oligosaccharides are systematically performed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) and UV-MALDI ion-trap time-of-flight mass spectrometry (ion-trap/TOF MS) acquired in negative-ion mode. Interestingly, their fragmentation significantly differ each other. In postsource decay (PSD) in UV-MALDI TOF MS, cross-ring cleavage at the reducing terminal predominates. On the other hand, glycosyl bond cleavage (C-type fragmentation) takes place preferentially in collision induced dissociation (CID) in UV-MALDI ion-trap/TOF MS. The cross-ring cleavage in PSD similar to that in in-source decay occurs via a prompt reaction path characteristic of the UV-MALDI process itself. The product ion spectra of UV-MALDI ion-trap/TOF MS are similar to the electrospray ionization (ESI) ion-trap or quadrupole/TOF CID product ion spectra. During ion-trap/TOF MS experiments, the deprotonated molecular ions survive for several tens of milliseconds after CID event because the high internal energy chlorinated precursor ions are cooled by collisional cooling in the ion trap. The results obtained suggest that the PSD from the chlorinated precursor ion in UV-MALDI TOF MS might proceed as a two-step reaction; in the first, a high internal energy deprotonated molecular ion is generated as a reaction intermediate during the flight in the drift tube, and in the second, the rapid decomposition from the deprotonated molecular ion takes place.

Information-Dependent Acquisition-Mediated LC−MS/MS Screening Procedure with Semiquantitative Potential

The development of a LC-MS/MS general unknown screening procedure for toxicologically relevant substances in blood samples by means of information-dependent acquisition on a Q-TOF is reported. IDA is an artificial intelligence-based product ion scan mode providing automatic "on-the-fly" MS to MS/MS switching. By performing information-dependent scanning at two different fragmentation energies, two collision-induced dissociation product ion spectra for each of the detected compounds are generated. As such, information-rich MS/MS spectra are obtained from precursor ions not known beforehand. In addition, limitation of the MS/MS acquisition time to an acceptable minimum resulted in an almost instantaneous switch back to the MS mode. As such, this approach provided MS chromatograms that still could be of use for semiquantitative purposes. Since the switching intensity threshold, unequivocally related to the background noise, proved a critical parameter, the solid-phase extraction procedure, the liquid chromatographic conditions, and the mass spectrometric parameters all were optimized to the advantage of information-dependent acquisition. Finally, the screening procedure we developed was benchmarked, on one hand, qualitatively against the results obtained from traditional GUS approaches in a number of routine toxicological laboratories (20 samples) and, on the other hand, quantitatively with respect to its potential against established LC-MS/MS methods (7 samples). The procedure performed very well from a qualitative point of view; almost all of the drugs detected by the conventional techniques were identified, as well as additional drugs that were not previously reported. The procedure proved well-suited for an initial semiquantitative assessment, as is customary in, for example, forensic toxicology before accurate intoxication levels are determined using targeted analytical analyses.

Identification of glucuronide conjugates of ketobemidone and its phase I metabolites in human urine utilizing accurate mass and tandem time-of-flight mass spectrometry.

The glucuronide conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone were identified in human urine post-intravenous administration of Ketogan Novum. The human urine was extracted on a mixed-mode solid-phase micro-column before analysis with liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and tandem MS (MS/MS). Accurate mass and collision-induced dissociation product ion spectra were used for identification of the glucuronide conjugates. Two different TOF mass spectrometers were used and the accurate mass measurements were performed on three separate days with each instrument. The accuracy of the mass measurements was better than 2.1 ppm for two out of three conjugates and the inter-day relative standard deviation was within +/-0.00049%. The MS/MS fragmentation patterns of the conjugates were in accordance with those of the synthetic aglycones and included peaks originating from the [M + H](+) ion of the respective aglycone.

High sensitivity analysis of phenylthiohydantoin amino acid derivatives by electrospray mass spectrometry

A new methodology has been developed for high sensitivity electrospray ionization mass spectrometric analyses of phenylthiohydantoin (PTH) amino acid derivatives. Key components of the methodology are the use of a solvent system consisting of methanol/dichloromethane (1:1 v/v) containing 5-mM lithium triflate, a stainless steel electrode having a relatively large surface area, and a microscale electrospray nozzle that provides for stable electrospray at flow rates in the range of 100–500 nL/min. A linear response for the absolute signal intensity of the protonated molecule was observed for a number of derivatives over the concentration range of 50–1000 fmol/µL. For all except the arginine derivative, there was a decrease in the signal intensity with increasing flow rate with 100–300 nL/min being optimum. Collision induced dissociation (CID) product ion spectra were obtained for 21 derivatives including carboxymethyl cysteine and dehydrothreonine. Leucine and isoleucine can be distinquished on the basis of their CID product ion spectra. A subfemtomole detection limit was demonstrated for the phenylalanine PTH derivative in a selected reaction monitoring (SRM) experiment. Samples from an automated Edman microsequencer run have been analyzed using the new technique and compared to results obtained by conventional high-performance liquid chromatography analysis with UV detection. This work demonstrates the feasibility of using mass spectrometry to identify and quantitate the products generated by automated protein microsequencing using standard Edman degradation chemistry.

Collision induced decomposition of peptides. Choice of collision parameters

Collision-induced dissociation product ion spectra of a series of doubly charged tryptic peptide ions produced by electrospray ionization were obtained by triple-quadrupole tandem mass spectrometry. The sequence information content of the product ion spectra was explored as a function of collision energy and collision-cell gas pressure for parent ions with molecular masses ranging from 300 to 2000 u. The energy range (at a given pressure) in which the degree of fragmentation is acceptable was found to be narrow for parent ions of a given mass, and the optimal collision energy was observed to exhibit a strong linear correlation with parent ion mass. This observed correlation opens the way for on-line software-controled selection of optimal mass spectrometric conditions in the enzymatic digestion-liquid chromatography-tandem mass spectrometric strategy of amino acid sequencing of proteins.

Collision‐induced dissociation in a tandem time‐of‐flight mass spectrometer with two single‐stage reflectrons

AbstractA tandem time‐of‐flight mass spectrometer was developed to be used for the study of collision‐induced dissociation (CID) products of peptides and small proteins. The instrument incorporates two single‐stage reflectrons, the first to energy focus the precursor ions to be fragmented and the second to both energy focus and disperse the product ions formed in the collision cell. Single‐stage rather than two‐stage reflectrons were used because the overall transmission is higher, interferences with grid wire fields are minimized and the mass calibration of product ion spectra is exactly linear with time. The CID product ion spectra of rhodamine and several peptides were acquired and found to exhibit very similar fragmentation patterns to those acquired in the B/E linked scan mode of a magnetic sector mass spectrometer.