Liquid Chromatography-multistage Mass Spectrometry Research Articles

Data-Independent Mass Spectrometry Approach for Screening and Identification of DNA Adducts.

Long-term exposures to environmental toxicants and endogenous electrophiles are causative factors for human diseases including cancer. DNA adducts reflect the internal exposure to genotoxicants and can serve as biomarkers for risk assessment. Liquid chromatography-multistage mass spectrometry (LC-MSn) is the most common method for biomonitoring DNA adducts, generally targeting single exposures and measuring up to several adducts. However, the data often provide limited evidence for a role of a chemical in the etiology of cancer. An "untargeted" method is required that captures global exposures to chemicals, by simultaneously detecting their DNA adducts in the genome; some of which may induce cancer-causing mutations. We established a wide selected ion monitoring tandem mass spectrometry (wide-SIM/MS2) screening method utilizing ultraperformance-LC nanoelectrospray ionization Orbitrap MSn with online trapping to enrich bulky, nonpolar adducts. Wide-SIM scan events are followed by MS2 scans to screen for modified nucleosides by coeluting peaks containing precursor and fragment ions differing by -116.0473 Da, attributed to the neutral loss of deoxyribose. Wide-SIM/MS2 was shown to be superior in sensitivity, specificity, and breadth of adduct coverage to other tested adductomic methods with detection possible at adduct levels as low as 4 per 109 nucleotides. Wide-SIM/MS2 data can be analyzed in a "targeted" fashion by generation of extracted ion chromatograms or in an "untargeted" fashion where a chromatographic peak-picking algorithm can be used to detect putative DNA adducts. Wide-SIM/MS2 successfully detected DNA adducts, derived from chemicals in the diet and traditional medicines and from lipid peroxidation products, in human prostate and renal specimens.

Stability behaviour of antiretroviral drugs and their combinations. 5: Characterization of novel degradation products of abacavir sulfate by mass and nuclear magnetic resonance spectrometry

In the present study, degradation behaviour of abacavir sulfate was evaluated in solution and solid stress conditions. Solution state studies resulted in formation of eleven degradation products; of which two were also formed on solid stress. The same were separated by high performance liquid chromatography. They were characterized using liquid chromatography-high resolution mass spectrometry, liquid chromatography-multistage mass spectrometry and hydrogen/deuterium exchange mass spectrometry data. Additionally, seven degradation products were isolated and subjected to 1D and 2D nuclear magnetic resonance studies for their structural confirmation.

Structural characterization of a vegetable oil‐based polyol through liquid chromatography multistage mass spectrometry

ABSTRACTA commercial vegetable oil‐based polyol for rigid polyurethane foams has been characterized by liquid chromatography‐electrospray ionization‐quadrupole ion trap mass spectrometry (LC‐ESI‐QIT‐MS). The absolute molecular weight (MW = 960) was measured by gel permeation chromatography (GPC) equipped with both refractive index (RI) detector and static laser light‐scattering detector (SLSD), which allowed further analysis by LC‐MS. The oligo‐polyol mixture was first separated in two elutes and then investigated by a deep multistage mass spectrometry (MSn) study and completed using NMR. The major constituents identified were regioisomers of propoxylated sucrose (nPO = 6–12), and the related esters of C16:0, C18:1, and C18:2 fatty acids had a mass ratio of 6:3:1. A comparison of fatty acids composition between the sample and palm oil demonstrated that the sample was initially prepared from the mixture of sucrose and palm oil by direct propoxylation. The MSn fragmentation studies validated the structure of propoxylated sucrose and the related fatty acids derivatives. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 255–262

Biotransformation and Rearrangement of Laromustine.

This review highlights the recent research into the biotransformations and rearrangement of the sulfonylhydrazine-alkylating agent laromustine. Incubation of [(14)C]laromustine with rat, dog, monkey, and human liver microsomes produced eight radioactive components (C-1 to C-8). There was little difference in the metabolite profile among the species examined, partly because NADPH was not required for the formation of most components, which instead involved decomposition and/or hydrolysis. The exception was C-7, a hydroxylated metabolite, largely formed by CYP2B6 and CYP3A4/5. Liquid chromatography-multistage mass spectrometry (LC-MS(n)) studies determined that collision-induced dissociation, and not biotransformation or enzyme catalysis, produced the unique mass spectral rearrangement. Accurate mass measurements performed with a Fourier-transform ion cyclotron resonance mass spectrometer (FTICR-MS) significantly aided determination of the elemental compositions of the fragments and in the case of laromustine revealed the possibility of rearrangement. Further, collision-induced dissociation produced the loss of nitrogen (N2) and methylsulfonyl and methyl isocyanate moieties. The rearrangement, metabolite/decomposition products, and conjugation reactions were analyzed utilizing hydrogen-deuterium exchange, exact mass, (13)C-labeled laromustine, nuclear magnetic resonance spectroscopy (NMR), and LC-MS(n) experiments to assist with the assignments of these fragments and possible mechanistic rearrangement. Such techniques produced valuable insights into these functions: 1) Cytochrome P450 is involved in C-7 formation but plays little or no role in the conversion of [(14)C]laromustine to C-1 through C-6 and C-8; 2) the relative abundance of individual degradation/metabolite products was not species-dependent; and 3) laromustine produces several reactive intermediates that may produce the toxicities seen in the clinical trials.

Stability behaviour of antiretroviral drugs and their combinations. 2: Characterization of interaction products of lamivudine and tenofovir disoproxil fumarate by mass and NMR spectrometry

This study focused on drug-drug interaction behaviour among lamivudine (3TC) and tenofovir disoproxil fumarate (TDF), the two anti-retroviral drugs. Apart from pre-known degradation products of individual drugs, a total of twelve interaction products were detected by high performance liquid chromatography (HPLC) using a C18 column as the stationary phase, and methanol and ammonium formate in gradient mode as the mobile phase. The same HPLC method was employed for liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-multi stage mass spectrometry (LC–MSn). For the characterization of interaction products, stability samples were subjected to LC-HRMS, LC–MSn and online hydrogen/deuterium exchange studies. Two isomeric interaction products were isolated and subjected to 1D and 2D nuclear magnetic resonance (NMR) studies. The collated information was utilized for the characterization of all twelve interaction products of the two drugs. Pathway of their formation was also outlined.

Preparative Separation and Structural Identification of Impurities of a New α2‑Adrenoceptor Agonist Using Stacking Injection, LC-MSn and LC-SPE-NMR

Identifying impurities in drug substances has become one of the most important issues in pharmaceutical analysis since it can have a significant impact on the efficacy of new pharmaceutical products. Due to the purity requirements, in this paper a new synthetic α2-adrenoceptor agonist, called LPSF-PT-31, was purified and its impurities were characterized by liquid chromatography-multistage mass spectrometry (LC-MSn) and liquid chromatography-solid phase extraction-nuclear magnetic resonance (LC-SPE-NMR). The purification step was conducted using a semi-preparative liquid chromatography and stacked injections as a new approach to drug purification. As a result, a total yield of 75% of the pure LPSF-PT-31 and 2.9 L day-1 in solvent reduction was obtained. The combination of semi-preparative stacking injection, LC-MSn, and LC-SPE-NMR, demonstrated to be efficient to purify active drugs and unambiguously identify its impurities. In addition, isolation and identification of drug impurities in the early stages of development can improve the synthetic pathway, preventing the formation of impurities or minimizing this formation to minimum levels.

Genome-wide mapping of 5-hydroxymethylcytosine in three rice cultivars reveals its preferential localization in transcriptionally silent transposable element genes.

5-Hydroxymethylcytosine (5hmC), a modified form of cytosine that is considered the sixth nucleobase in DNA, has been detected in mammals and is believed to play an important role in gene regulation. In this study, 5hmC modification was detected in rice by employing a dot-blot assay, and its levels was further quantified in DNA from different rice tissues using liquid chromatography-multistage mass spectrometry (LC-MS/MS/MS). The results showed large intertissue variation in 5hmC levels. The genome-wide profiles of 5hmC modification in three different rice cultivars were also obtained using a sensitive chemical labelling followed by a next-generation sequencing method. Thousands of 5hmC peaks were identified, and a comparison of the distributions of 5hmC among different rice cultivars revealed the specificity and conservation of 5hmC modification. The identified 5hmC peaks were significantly enriched in heterochromatin regions, and mainly located in transposable elements (TEs), especially around retrotransposons. The correlation analysis of 5hmC and gene expression data revealed a close association between 5hmC and silent TEs. These findings provide a resource for plant DNA 5hmC epigenetic studies and expand our knowledge of 5hmC modification.

Identification of the major degradation pathways of ticagrelor

Ticagrelor is a direct-acting and reversible P2Y12-adenosine diphosphate (ADP) receptor blocker used as antiplatelet drug. Forced degradation under various stress conditions was carried out. The degradation products have been detected and identified by high-pressure liquid chromatography multistage mass spectrometry (LC-MSn) along with high-resolution mass spectrometry. C18 XTerra MS column combined with a linear gradient mobile phase composed of a mixture of 10mM acetate ammonium/acetonitrile was shown suitable for drug and impurity determinations and validated as a stability indicating method. Structural elucidation of the degradation products relied on MSn studies and accurate mass measurements giving access to elemental compositions. Up to nine degradation products resulting from oxidation/auto-oxidation, S-dealkylation and N-dealkylation have been identified, covering a range of possible degradation pathways for derivatives with such functional groups. Kinetics was also studied in order to assess the molecule's shelf-life and to identify the most important degradation factors.

Identification and Characterisation of Phenolics fromIxora coccineaL. (Rubiaceae) by Liquid Chromatography Multi-stage Mass Spectrometry

Ixora coccinea L. leaves and stem are used in traditional Sudanese and Ayurvedic medicinal systems for the treatment of diarrhoea, fever, headache, skin diseases, eye trouble, wounds, sores and ulcers. Recent studies show that I. coccinea has anti-oxidant, anti-bacterial, anti-cancer, anti-inflammatory, analgaesic, anti-diarrhoeal, hepatoprotective, cardioprotective, anti-mutagenic, wound healing and anti-tumour activities. Ixora coccinea is a rich source of polyphenols such as proanthocyanidins, flavonoids, flavonoids glycosides and tannins. To develop a LC-MS(n) method for the identification and characterisation of phenolic compounds of I. coccinea L. leaves and stem. Aqueous methanolic (70% methanol) extracts of I. coccinea leaves and stem were used for LC-MS(n) to ensure efficient extraction of phenolics. A C18 amide reverse-phase HPLC column allowed separation of the phenolic compounds, including different isomers. For the LC-MS measurements, negative ion mode was used in order to obtain better tandem mass spectra and high-resolution mass spectra. The phenolics were identified by their typical UV absorptions at 254, 280 and 320 nm. All the flavonol glycosides showed a neutral loss of the glycan part; hydroxycinnamates showed loss of the cinnamoyl/cinnamic acid part; while proanthocyanidins showed a Diels-Alder fragment in negative ion mode mass spectra. It was possible to identify C-3 and C-7 flavonol glycosides by their order of elution and it was also possible to predict the glycosylation position in flavonol diglycosides from their tandem mass spectra.

Laser capture microdissection coupled with on-column extraction LC-MS(n) enables lipidomics of fluorescently labeled Drosophila neurons.

We have used laser capture microdissection (LCM) and fluorescence microscopy to isolate genetically labeled neurons from the Drosophila melanogaster brain. From native thin sections, regions of interest could be analyzed with a spatial resolution better than 50 μm. To exploit the specificity of LCM for lipidomics, catapulted tissue patches were directly collected on a reversed phase column and analyzed using an on-column extraction (OCE) that was directly coupled with liquid chromatography-multistage mass spectrometry (LC-MS(n)). With this approach, more than 50 membrane lipids belonging to 9 classes were quantified in tissue regions equivalent to a sample amount of 50 cells. Using this method, the limit of quantitation and the extraction efficiency could be estimated enabling a reliable evaluation of acquired lipid profiles. The lipid profiles of cell body- and synapse-enriched regions of the Drosophila brain were determined and found to be distinct. We argue that this workflow represents a tremendous improvement for tissue lipidomics by integrating genetics, fluorescence microscopy, LCM and LC-MS(n).

Rapid preconcentration for liquid chromatography-mass spectrometry assay of trace level neuropeptides.

Measurement of neuropeptides in the brain through in vivo microdialysis sampling provides direct correlation between neuropeptide concentration and brain function. Capillary liquid chromatography-multistage mass spectrometry (CLC-MS(n)) has proven to be effective at measuring endogenous neuropeptides in microdialysis samples. In the method, microliter samples are concentrated onto nanoliter volume packed beds before ionization and mass spectrometry analysis. The long times required for extensive preconcentration present a barrier to routine use because of the many samples that must be analyzed and instability of neuropeptides. In this study, we evaluated the capacity of 75μm inner diameter (i.d.) capillary column packed with 10μm reversed phase particles for increasing the throughput in CLC-MS(n) based neuropeptide measurement. Coupling a high injection flow rate for fast sample loading/desalting with a low elution flow rate to maintain detection sensitivity, this column has reduced analysis time from ∼30min to 3.8min for 5μL sample, with 3 pM limit of detection (LOD) for enkephalins and 10 pM LOD for dynorphin A1-8 in 5μL sample. The use of isotope-labeled internal standard lowered peptide signal variation to less than 5%. This method was validated for in vivo detection of Leu and Met enkephalin with microdialysate collected from rat globus pallidus. The improvement in speed and stability makes CLC-MS(n) measurement of neuropeptides in vivo more practical.

Aqueous Phototransformation of Diazepam and Related Human Metabolites under Simulated Sunlight

Phototransformation of the widely used benzodiazepine pharmaceuticals diazepam and human metabolites nordiazepam, temazepam and oxazepam under simulated sunlight in water was investigated. Photolysis experiments were conducted in the presence and absence of humic acids. Half-lives for each of the benzodiazepine pharmaceuticals were <200 h (under all conditions) suggesting that phototransformation is an important process for such chemicals in the photic zone of receiving waters. Due to the observed phototransformation of the benzodiazepines, significant emphasis was placed on identification of the photoproducts. A total of fourteen photoproducts, including benzophenones, acridinones and quinazolinones or quinazolines was identified and measured by liquid chromatography-multistage mass spectrometry (LC-MS(n)). Phototransformation studies were also undertaken on authentic samples of two of the identified photoproducts, 5-chloro-methylaminobenzophenone and 2-amino-5-chlorobenzophenone, in order to establish the phototransformation pathways. Interestingly, these two photoproducts showed relatively higher persistence than some of the benzodiazepines, suggesting that the fate and effects of photoproducts should also be incorporated into future risk assessments and environmental models of the fate of benzodiazepines.

Molecular level differentiation between end‐capped and intramolecular azofunctional oligo(ε‐caprolactone) positional isomers through liquid chromatography multistage mass spectrometry

AbstractEnd‐capped and intramolecular azofunctional oligocaprolactones were characterized at molecular level by liquid chromatography electrospray ionization mass spectrometry (ESI MS) and NMR spectroscopy. The Disperse Red 19 (DR19) azofunctional oligomers, DC, were synthesized by ring‐opening oligomerization of ε‐caprolactone (ε‐CL) initiated by the hydroxyl groups of DR19 azo dye. The reaction products consist of a minor fraction of end‐capped azo functional oligocaprolactone (α‐DC), that is, a single CL arm oligomer, and a major fraction of intramolecular azo functional oligocaprolactone (β‐DC), that is, a two CL arms oligomer. The chromatographic separation was used to discriminate between α‐DC and β‐DC, and the results were confirmed by MS/MS performed on an ESI ion trap instrument. The results supported by accurate mass data obtained for product ions using an ESI quadrupole time of flight instrument demonstrate the qualitative discrimination at the molecular level between intramolecular and end‐capped azofunctional oligoesters isomers through a relatively simple multistage mass spectrometry experiment. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012

Detailed molecular characterization of castor oil ethoxylates by liquid chromatography multistage mass spectrometry

The molecular characterization of castor oil ethoxylates (CASEOs) was studied by reverse-phase liquid chromatography (RPLC) mass spectrometry (MS) and multistage mass spectrometry (MS n ). The developed RPLC method allowed the separation of the various CASEO components, and especially, the baseline separation of multiple nominal isobars (same nominal mass) and isomers (same exact mass). MS and MS n were used for the determination and structure elucidation of various structures and for the discrimination of the isobars and isomers. Different ionization techniques and adduct ions were also tested for optimization of the MS detection and the MS n fragmentation. A unique fragmentation pathway of ricinoleic acid is proposed, which can be used as a marker of the polymerization process and the topology of ethoxylation in the CASEO. In addition, characteristic neutral losses of ricinoleic acid reveal its (terminal or internal) position in the molecule.

Comparison of extracted proteins of human stomach tumor and normal tissues with liquid chromatography-multistage mass spectrometry

Screening of tumor markers by proteomic technology is the research focus and key of early diagnosis of stomach cancer study. Aiming at the complexity of the extracted proteins from biological tissue, reversed-phase high performance liquid chromatography (RP-HPLC) was employed as one of the most efficient chromatographic methods. Based on the difference of hydrophobicity, RP-HPLC separation was performed to reduce the complexity of stomach cancer tissue and normal tissue samples, separately. By comparing the chromatograms, different components were collected. The fractions with the retention times from 45 min to 47 min were digested and identified by liquid chromatography-multistage mass spectrometry (LC-MS/MS). Nine common proteins were found in both tumor tissue and normal tissue. Six specific proteins were screened in normal tissue and seventeen specific proteins were found in tumor tissue under the same conditions. Two proteins with higher abundance in tumor tissue were selected for further investigation. These proteins provide more information for future drug target and drug pathway research by the analysis of biological information.

Liquid chromatography multi-stage mass spectrometry for the analysis of 5-hydroxymethylfurfural in foods

A new, simple and selective method for the analysis of 5-hydroxymethylfurfural (HMF) in foods by liquid chromatography coupled to ion trap multi-stage mass spectrometry (LC–MS n ) is proposed in the present study. Several chromatographic columns were tested and the best results were obtained using a phenyl fluorinated column. MS conditions were established using an atmospheric pressure chemical ionisation (APCI) source in the positive ionisation mode. MS/MS was used for quantitative analysis while MS 3 was required for confirmation purposes. Quality parameters such as day-to-day and run-to-run precision (RSD < 10%) and detection limit (133 ng g −1, 333 pg injected) were established. This method was successfully applied to the analysis of HMF content in several Spanish food samples from a local market.

Novel functionalised chlorins in sediments of the Messinian Vena del Gesso evaporitic sequence: Evidence for a facile route to reduction for biomarkers

Fossil pigment distributions from sediments of the Messinian Vena del Gesso, Italy, include two novel sedimentary chlorins as revealed by high performance liquid chromatography and liquid chromatography multistage mass spectrometry. The two novel chlorin components are meso-derivatives of chlorophyllone a and represent early stage diagenetic intermediates in the formation of bicycloalkano chlorin and porphyrin structures that occur in these and other carbonaceous sediments. Their presence indicates the operation of a facile route to reduction during early diagenesis.

In vitro synthesis of 1,N6‐etheno‐2′‐deoxyadenosine and 1,N2‐etheno‐2′‐deoxyguanosine by 2,4‐dinitrophenol and 1,3‐dinitropyrene in presence of a bacterial nitroreductase

The formation of covalent nitro-PAH DNA adducts and nitro-PAH mediated oxidative lesions are two possible mechanisms for the initiation of nitro-PAH carcinogenesis. Sixty-minute incubation of 1,3-dinitropyrene (100 microM) or 1,4-dinitrophenol (100 microM) with a mixture of 150 microM NADH, 0.5 units of E. coli nitroreductase, 100 microM linoleic acid, 0.5 mM ferrous iron, and 100 microM 2'-deoxyadenosine (2'-dA) or 100 microM 2'-deoxyguanosine (2'-dG) were analyzed by liquid chromatography multistage mass spectrometry. Mixtures of 1,N(6)-etheno-2'-deoxyadenosine (epsilondA) plus 4-oxo-2-nonenal (4-ONE) and 1,N(2)-etheno-2'-deoxyguanosine (epsilondG) plus 4-ONE could be detected from 2'-dA and 2'-dG, respectively. Addition of 2% propanol inhibited the formation of etheno adducts. Analyses of disappearance kinetics of dA and dG showed that dG was more rapidly eliminated than does dA (t[1/2] = 23.3 min and 98.3 min for dG and dA, respectively). Curves of formation kinetics revealed that the peak of epsilondG was at 55.6 min while that of epsilondA was at 186.9 min. These peaks represented 1.43% and 1.25% of the original dG and dA, respectively. In both cases, the peaks were followed by rapid degradations of etheno adducts. The results, obtained in this system, do not allow any extrapolation to realistic cellular responses; nevertheless, these data questioned the validity of the use of unsubstituted etheno adducts as reliable oxidative stress and nitro-PAH exposure biomarkers.

Distribution and significance of chlorophyll derivatives and oxidation products during the spring phytoplankton bloom in the Celtic Sea April 2002

The abundances and distributions of chlorophyll a and its common transformation products have been profiled by high performance liquid chromatography during the disruption, re-establishment, growth and senescence of a phytoplankton bloom in the Celtic Sea during April 2002. Transformation products of chlorophyll a indicative of herbivory of the phytoplankton bloom have been identified using atmospheric pressure chemical ionisation liquid chromatography-multistage mass spectrometry and chlorophyll oxidation products, formed by autoxidation, are present in increasing abundance in samples collected during the later stages of bloom development. The formation of oxidation products during the earliest stages in the chlorophyll transformation pathway identifies water column processes as sources of these early intermediates in the formation of geologic aetioporphyrins and/or cycloalkanoporphyrins.

Structural variations in derivatives of the bacteriochlorophylls of Chlorobiaceae: impact of stratigraphic resolution on depth profiles as revealed by methanolysis

Bacteriochlorophylls c and d, recovered from two sedimentary sequences, were converted to bacteriophaeophorbide methyl esters by methanolysis and analysed by atmospheric pressure chemical ionisation liquid chromatography-multistage mass spectrometry (APCI LC-MS n ). The distributions in both settings, a moderately consolidated sediment from Kirisjes Pond, Antarctica, and in a finely laminated microbial mat from Les Salines de la Trinitat, Spain, show significant variations within a narrow depth interval. The overall bacteriophaeophorbide c to d ratios in the two sediments are different, as are the ratios of particular C-3 1 diastereoisomers, indicating distinct differences between the bacterial communities that contributed to each sediment. Furthermore, a shift towards more extensive alkylation in homologues within each sediment is consistent either with changing environmental conditions in the depositional environments, or development-related changes in the structure of the bacterial community, leading to increased competition for light or nutrients.