Multiple-stage Mass Spectrometry Research Articles

Identification of hydroxyquinazoline alkaloids from Justicia adhatoda L. leaves, a traditional natural remedy with NF-κB and AP-1-mediated anti-inflammatory properties and antioxidant activity

Ethnopharmacological relevanceJusticia adhatoda L. is used as traditional medicine in Nepal to treat cough, asthma, and inflammatory disorders, and is indicated as “Asuro”. Leaves are used worldwide as herbal medicine due to cardiotonic, expectorant, anti-asthmatic, and bronchodilatory properties. The aim of this work was to study the phytochemical composition of leaves of Nepalese J. adhatoda and assess their anti-inflammatory and antioxidant properties in vitro. Materials and methodsSecondary metabolites were extracted from dried leaves using methanol (JAME: J. adhatoda methanol extract). They were analysed by means of liquid chromatography coupled with multiple-stage mass spectrometry (LC-MSn). Anti-inflammatory potential was determined by the NF-κB and AP-1 inhibition assay, and DPPH, ABTS, and β-carotene bleaching assays were performed to assess its antioxidant properties. ResultsJAME is a rich source of secondary metabolites, especially quinazoline alkaloids such as vasicine, vasicinone, vasicoline, and adhatodine. 7-Hydroxy derivatives of peganidine, vasicolinone, and adhatodine were also identified by means of MSn data and are here reported in J. adhatoda for the first time. JAME inhibited NF-κB and AP-1 expression in THP-1 cells to a greater extent than the positive control prednisolone. A moderate radical-quenching property was observed in DPPH and ABTS assays, but the anti-carotene bleaching activity was significantly higher than the reference BHT. ConclusionsTo the best of our knowledge, this is the first insight into the phytochemical composition of Asuro leaves from Nepal and their bioactivity. Our results will contribute to the valorisation of this medicinal species still widely used in the traditional and complementary medicine.

Exploring the effects of post-distillation and post-supercritical CO2 extraction on chemical profile and biological activities of two Salvia species (S. chrysophylla and S. microstegia)

In this paper the extraction of phytoconstituents from Salvia chrysophylla and S. microstegia aerial parts was performed using maceration, supercritical carbon dioxide extraction (SCO2) and hydrodistillation. Additionally, the residual material from hydrodistillation and SCO2 was evaluated for the content in secondary metabolites. To achieve these goals Nuclear Magnetic Resonance (NMR) spectroscopy, liquid chromatography coupled with diode array and multiple stage mass spectrometry (LC-DAD-MSn) and liquid chromatography coupled with high resolution quadrupole time of flight mass spectrometry (LC-HR-QTOF) were used for the analysis of non-volatile compounds. Gas chromatography coupled with mass spectrometry (GC–MS) was adopted for the essential oil analysis. Main constituents were rosmarinic acid and luteolin-7-O-glucopyranoside, in S. microstegia while sclareol and salvigenin in S. crisophylla. Regarding essential oil composition terpine-4-ol-acetate and caryophyllene oxide were the most abundant constituents in S. microstegia and S. chrysophylla respectively. Subsequently, we determined the antioxidant capacity using different chemical assays. To establish potential other bioactivities inhibition on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-amylase and α-glucosidase, was examined. The findings revealed the water extract (WE) and methanol total extract (TE) of S. microstegia extracts, exhibited higher total phenolic contents and antioxidant activity compared to S. chrysophylla extracts. Moreover, both S. microstegia and S. chrysophylla essential oils EOs displayed significant phosphomolybdenum (PBD) activity, and their acetylcholinesterase (AChE) inhibitory activity was notably elevated and nearly equivalent, measuring 2.79 mg GALAE/g and 2.78 mg GALAE/g, respectively. These findings underscore the potential for diverse applications of these plant extracts in various industries, including pharmaceuticals and cosmeceuticals.

Lipidomic analysis of Porphyromonas gingivalis reveals novel glycerol bisphosphoceramide, phosphatidyl-, and phosphoglycerol dipeptide lipid families

Porphyromonas gingivalis, like other members of the phylum Bacteroidetes (synonym Bacteroidota), synthesizes several classes of dihydroceramides and peptidolipids. Using a similar strategy as that recently used to delimit the lipidome of its close relative Bacteroides fragilis, we applied linear ion trap multiple-stage mass spectrometry (linear ion trap MSn) with high-resolution mass spectrometry, to structurally characterize the complete lipidome of P. gingivalis and compare it to B. fragilis. This analysis discovered that the P. gingivalis lipidome consists of several previously unidentified lipid families, including dihydroceramide-1-phosphophate, acylated dihydroceramide-1-phosphophate, phosphoglycerol glycylserine lipid, and bis(phosphodihydroceramide) glycerol. Interestingly, we also found a novel sphingolipid family containing a polyunsaturated long–chain base, and a new lipoglycylserine phosphatic acid containing unsaturated acyl chains not reported for the lipid family. The comprehensive coverage of the lipidome of P. gingivalis conducted in this study has revealed more than 140 lipid species including several novel lipids in over 20 lipid families/subfamilies.

Lipidome of the Bacteroides Genus Containing New Peptidolipid and Sphingolipid Families Revealed by Multiple-Stage Mass Spectrometry.

The anaerobic bacteria of the Bacteroides fragilis group including Bacteroides thetaiotaomicron, B. fragilis, Bacteroides vulgatus, and Bacteroides ovatus in genus Bacteroides are among the most commonly found human gut microbiota. They are generally commensal but are also opportunistic pathogens. Both the inner and outer membranes of the Bacteroides cell envelope contain abundant lipids with diversified structures, and dissection of the lipid composition of the inner and outer membrane fractions is important for understanding the biogenesis of this multilaminate wall structure. Here, we describe mass spectrometry-based approaches to delineate in detail the lipidome of the membrane and the outer membrane vesicle of the bacteria cells. We identified 15 lipid class/subclasses (>100 molecular species), including sphingolipid families [dihydroceramide (DHC), glycylseryl (GS) DHC, DHC-phosphoinositolphosphoryl-DHC (DHC-PIP-DHC), ethanolamine phosphorylceramide, inositol phosphorylceramide (IPC), serine phosphorylceramide, ceramide-1-phosphate, and glycosyl ceramide], phospholipids [phosphatidylethanolamine, phosphatidylinositol (PI), and phosphatidylserine], peptide lipids (GS-, S-, and G-lipids) and cholesterol sulfate, of which several have not been reported previously, or have similar structures to those found in Porphyromonas gingivalis, the periodontopathic bacterium in oral microbiota. The new DHC-PIPs-DHC lipid family is found only in B. vulgatus, which, however, lacks the PI lipid family. The galactosyl ceramide family is exclusively present in B. fragilis, which nevertheless lacks IPC and PI lipids. The lipidomes as revealed in this study demonstrate the lipid diversity among the various strains and the utility of multiple-stage mass spectrometry (MSn) with high-resolution mass spectrometry in the structural elucidation of complex lipids.

Hydroxyl transfer versus cyclization reaction in the gas phase: Sequential loss of NH3 and CH2CO from protonated phenylalanine derivatives.

Collisional activation of protonated phenylalanine derivatives deamination products leads to hydroxyl skeletal rearrangement versus cyclization reaction, and to form hydroxylbenzyl cation via elimination of CH2CO. To better clarify this unusual fragmentation reaction, accurate mass measurements experiments, native isotope experiments, multiple-stage mass spectrometry experiments, different substituents experiments, and density functional theory (DFT) calculations were carried out to investigate the dissociation mechanistic pathways of protonated phenylalanine derivatives deamination products. In route 1, a three-membered ring-opening reaction and a 1,3-hydroxyl transfer (from the carbonyl carbon atom to the interposition carbon atom of carbonyl) occurs to form 3-hydroxy-1-oxo-3-phenylpropan-1-ylium, followed by dissociation to lose CH2CO to give hydroxy (phenyl)methylium. In route 2, a successive cyclization rearrangement reaction and proton transfer occur to form a 2-hydroxylphenylpropionyl cation or protonated 2-hydroxy-4H-benzopyran, followed by dissociation to lose CH2CO or CH≡COH to give 2-hydroxylbenzyl cation. In route 3, a successive hydroxyl transfer (from the carbonyl carbon atom to the ortho carbon atom on benzene) and two stepwise proton transfer (1,2-proton transfer to the ipso-carbon atom of the phenyl ring followed by 1,3-proton transfer to the ortho carbon atom of carbonyl) occurs to form a 2-hydroxylphenylpropionyl cation, which subsequently dissociates to form 2-hydroxylbenzyl cation by elimination of CH2CO. DFT calculations suggested that route 1 was more favorable than route 2 and route 3 from a thermodynamic point of view.

Characterization of the (Poly)Phenolic Fraction of Fig Peel: Comparison among Twelve Cultivars Harvested in Tuscany.

(1) Background: The fig tree (Ficus carica L.) is widely cultivated in the Mediterranean area and it produces fruits largely consumed in the Mediterranean diet. Previous studies have shown that this fruit represents a rich source of (poly)phenols, which are mainly located in the peel rather than the pulp. In our study, fig peel derived from twelve different cultivars located in Tuscany was assessed for its (poly)phenol profile. (2) Methods: The (poly)phenol characterization was performed through ultra-high performance liquid chromatography coupled to multiple-stage mass spectrometry. (3) Results: Twenty-eight (poly)phenolic compounds were quantified in the investigated fig peel. It was possible to observe an interesting variability in the (poly)phenol content among the twelve cultivars of fig peel. Rutin and 5-caffeoylquinic acid were the main compounds in the greenish fig peel, while cyanidin-3-O-rutinoside was the main component in the dark-violet fig peel. (4) Conclusions: fig peel could be used as a (poly)phenol-rich ingredient in several food products to increase the bioactive compound content of foods. Moreover, dark-violet peel could be considered potentially suitable as a natural food colorant.

Characterization of Mycobacterium tuberculosis Mycolic Acids by Multiple-Stage Linear Ion-Trap Mass Spectrometry.

Mycobacterium tuberculosis (Mtb) cells are known to synthesize very long chain (C60-90) structurally complex mycolic acids with various functional groups. In this study, we applied linear ion-trap (LIT) multiple-stage mass spectrometry (MSn), combined with high-resolution mass spectrometry to study the mechanisms underlying the fragmentation processes of mycolic acid standards desorbed as lithiated adduct ions by ESI. This is followed by structural characterization of a Mtb mycolic acid family (Bovine strain). Using the insight fragmentation processes gained from the study, we are able to achieve a near complete characterization of the whole mycolic acid family, revealing the identity of the α-alkyl chain, the location of the functional groups including methyl, methoxy, and keto groups along the meroaldehyde chain in each lipid species. This study showcased the power of LIT MSn toward structural determination of complex lipids in a mixture, which would be otherwise very difficult to define using other analytical techniques.

HILIC-ESI-MS analysis of phosphatidic acid methyl esters artificially generated during lipid extraction from microgreen crops.

The uncontrolled activation of endogenous enzymes may introduce both qualitative and quantitative artefacts when lipids are extracted from vegetal matrices. In the present study, a method based on hydrophilic interaction liquid chromatography coupled either to high‐resolution/accuracy Fourier‐transform mass spectrometry (HILIC‐ESI‐FTMS) or to linear ion trap multiple stage mass spectrometry (HILIC‐ESI‐MS n , with n = 2 and 3) with electrospray ionization was developed to unveil one of those artefacts. Specifically, the artificial generation of methyl esters of phosphatidic acids (MPA), catalysed by endogenous phospholipase D (PLD) during lipid extraction from five oleaginous microgreen crops (chia, soy, flax, sunflower and rapeseed), was studied. Phosphatidylcholines (PC) and phosphatidylglycerols (PG) were found to be the most relevant precursors of MPA among glycerophospholipids (GPLs), being involved in a transphosphatidylation process catalysed by PLD and having methanol as a coreactant. The combination of MS2 and MS3 measurements enabled the unambiguous recognition of MPA from their fragmentation pathways, leading to distinguish them from isobaric PA including a further CH2 group on their side chains. PLD was also found to catalyse the hydrolysis of PC and PG to phosphatidic acids (PAs). The described transformations were confirmed by the remarkable decrease of MPA abundance observed when isopropanol, known to inhibit PLD, was tentatively adopted instead of water during the homogenization of microgreens. The unequivocal identification of MPA might be exploited to assess if GPL alterations are actually triggered by endogenous PLD during lipid extractions from specific vegetal tissues.

In-depth triacylglycerol profiling using MS3 Q-Trap mass spectrometry

Total triacylglycerol (TAG) level is a key clinical marker of metabolic and cardiovascular diseases. However, the roles of individual TAGs have not been thoroughly explored in part due to their extreme structural complexity. We present a targeted mass spectrometry-based method combining multiple reaction monitoring (MRM) and multiple stage mass spectrometry (MS3) for the comprehensive qualitative and semiquantitative profiling of TAGs. This method referred as TriP-MS3 – triacylglycerol profiling using MS3 – screens for more than 6,700 TAG species in a fully automated fashion. TriP-MS3 demonstrated excellent reproducibility (median interday CV ∼ 0.15) and linearity (median R2 = 0.978) and detected 285 individual TAG species in human plasma. The semiquantitative accuracy of the method was validated by comparison with a state-of-the-art reverse phase liquid chromatography (RPLC)-MS (R2 = 0.83), which is the most commonly used approach for TAGs profiling. Finally, we demonstrate the utility and the versatility of the method by characterizing the effects of a fatty acid desaturase inhibitor on TAG profiles in vitro and by profiling TAGs in Caenorhabditis elegans.

Characterization of Triterpene Saponins from Gypsophila arrostii by Electrospray Ionization Ion Trap Multiple-Stage Mass Spectrometry

Saponins are natural surfactant compounds whose aqueous solutions can form stable foam and micellar solutions. Gypsophila arrostii Guss., Caryophyllaceae, represents the main source of saponins for industrial applications, such as vaccine adjuvants. Due to the complex and amphiphilic nature of triterpene saponins, these compounds require sophisticated techniques for their isolation and characterization. The hydromethanolic saponin fractions from the roots of the analyzed plant material were investigated by electrospray ionization ion trap multiple-stage mass spectrometry (ESI-IT-MS) in negative mode. According to the m/z values for the deprotonated molecule [M-H]− and the characteristic product ions, four known Gypsophila-type saponins were successfully characterized. In addition, four undescribed saponin compounds were detected by the registered ions [M-H]−.

Screening of Alkaloid-Producing Endophytic Penicillium Strains from Amazon Medicinal Plants by Electrospray Ionization Mass Spectrometry (ESI-MS) and Principal Component Analysis (PCA)

The genus Penicillium is among the most promising alkaloid-producing fungal and therefore plays an important role in terms of producing molecules with biotechnological potential. Thus, in order to identify alkaloid-producing fungi, 25 endophytic Penicillium strains previously isolated from Amazon medicinal plants were subject to an integrative approach based on direct infusion positive electrospray ionization mass spectrometry (ESI-MS) and principal component analysis (PCA). The multivariate analysis pointed paxiline (1), glandicoline B (2), roquefortine C (3), and oxaline (5) as responsible for the segregation of three promising alkaloid-producing groups, been these groups constituted for P. chrysogenum, P. oxalicum, P. paxilli, and P. rubens strains. These alkaloids and the glandicoline A (4) were tentatively identified by multiple-stage mass spectrometry. In addition, compounds 1 and 2 were isolated and confirmed by using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Overall, the chemical profile analysis by ESI-MS along with PCA provided a simple and effective approach to screening alkaloid-producing Penicillium strains for biotechnological applications.

Characterization of the Uncommon Lipid Families in Corynebacterium glutamicum by Mass Spectrometry.

This book chapter provides readers the step-by-step instruction for cell growth, lipid isolation, and lipid analysis to obtain the lipidome of Corynebacterium glutamicum (C. glutamicum) in the genus Corynebacterium, a biotechnologically important bacterium. We separate thelipid families by preparative HPLC with an analytical C-8 column, followed by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high-resolution mass measurement to define the structures of cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), α-D-mannopyranosyl-(1→4)-α-D-glucuronyl diacylglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), and acyl trehalose monomycolate (acyl-TMM) whose structures have been previously mis-assigned or not defined by mass spectrometric means. We also define the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in the cellwall. The similarity of the lipidome to that in the Mycobacterium genera is consistent with the notion that Corynebacterium and Mycobacterium are gram-positive bacteria belonging to the suborder Corynebacterineae.

Coffee (Coffea arabica L.) by-Products as a Source of Carotenoids and Phenolic Compounds—Evaluation of Varieties With Different Peel Color

Analysis of pulp and peels of Arabica coffee varieties with different external fruit color allowed the identification of 16 phenolic compounds using high-performance liquid chromatography with diode array detection and electrospray ionization multiple-stage mass spectrometry (HPLC-DAD-ESI-MSn). Nine chlorogenic acids, three flavan-3-ols, the xanthone mangiferin, the flavonol rutin and two anthocyanins were tentatively identified and quantified. 5-O-Caffeoylquinic acid together with a putative (epi)catechin hexoside were the predominant phenolics detected in the coffee varieties analyzed in this work. Whereas 3- and 4-caffeoylquinic acids, as well as 5-O-feruloylquinic acid were consistently found in higher quantities in the pulp than in the peels when individual varieties were compared, the opposite was found for the other phenolic compounds detected. Complementary, GC-MS after alkaline hydrolysis and trimethylsilylation permitted the identification of more than 30 constituents, including phenolic compounds and other benzenoids, caffeine, and diverse carboxylic acids. Detected anthocyanins were cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside. Anthocyanin contents in orange-colored berries were lower than those in red fruits, while no anthocyanins were found in yellow-colored fruit. Among non-anthocyanin pigments, we found β-carotene and lutein in all varieties, along with other chloroplast-specific carotenoids in some accessions. In addition, saponification evidenced the presence of several xanthophyll esters. Both, chlorophyll a and b, were detected in the peels of all varieties, while only chlorophyll b was observed in the pulp. Thus, the color of yellow-peeled varieties is due to carotenoids, while that of orange and red-peeled varieties is due to both carotenoids as well as low and high levels of anthocyanins, respectively. Present results point out to the potential use of by-products of particular coffee varieties with differences in the external fruit color as a source of distinctive bioactive compounds, including anthocyanins and carotenoids, with health benefits.

Unveiling the biodiversity of lipid species in Corynebacteria- characterization of the uncommon lipid families in C. glutamicum and pathogen C. striatum by mass spectrometry

Uncommon lipids in biotechnologically important Corynebacterium glutamicum and pathogen Corynebacterium striatum in genus Corynebacterium are isolated and identified by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high resolution mass measurement. We redefined several lipid structures that were previously mis-assigned or not defined, including cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), (α-d-mannopyranosyl)-(1 → 4)-(α-D-glucuronyl diacyglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), acyl trehalose monomycolate (acyl-TMM). We also report the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in which many isomeric structures are present. The LIT MSn approaches afford identification of the functional group, the fatty acid substituents and their regiospecificity in the molecules, revealing the biodiversities of the lipid species in two Corynebacterium strains that have played very different and important roles in human nutrition and health.

Targeted analysis of ceramides and cerebrosides in yellow lupin seeds by reversed-phase liquid chromatography coupled to electrospray ionization and multistage mass spectrometry

Ceramides (Cer) and cerebrosides are important sphingolipids (SL) involved in many biological processes. Herein, the SL content of yellow lupin seeds (Lupinus luteus) was determined by liquid–liquid extraction, mild alkaline hydrolysis (1 h at 37 °C) and reversed-phase liquid chromatography with negative electrospray ionization coupled to either an orbital-trap (Fourier-transformed, FT) or linear ion-trap (LIT) mass spectrometry (RPLC-ESI/MS). The chemical identity of SL including the sugar residues and sphingoid backbone (SB) was inferred by collision-induced dissociation multiple-stage mass spectrometry (MSn, n = 2,3). Up to 52 Cer and 47 cerebrosides were successfully recognized and quantified in sample extracts of L. luteus seeds also counting isobaric species. As reported for other vegetables, a hydroxylated SB was observed whereby the N-acyl chains showed a high occurrence of very-long-chain moieties; phytosphingosine and 4-hydroxy-8-sphingenine were the predominant SB paired mainly with oleic acid (C18:1), hydroxylated behenic acid (C22:0;1) and hydroxylated lignoceric acid (C24:0;1).

Electrospray ionization tandem mass spectrometry of monoketone curcuminoids.

Although monoketone curcuminoids (MKCs) have been largely investigated due to their biological activities, data on the gas-phase fragmentation reactions of protonated MKCs under collision-induced dissociation (CID) conditions are still scarce. Here, we combined electrospray ionization tandem mass spectrometry (ESI-MS/MS) data, multiple-stage mass spectrometry (MSn ), deuterium exchange experiments, accurate-mass data, and thermochemical data estimated by computational chemistry to elucidate and to rationalize the fragmentation pathways of eleven synthetic MKCs. The MKCs were synthesized by Claisen-Schmidt condensation under basic (1-9) or acidic (10-11) conditions. ESI-CID-MS/MS analyses and deuterium-exchange experiments were carried out on a triple quadrupole mass spectrometer. MSn analyses on an ion trap mass spectrometer helped to elucidate the fragmentation pathways. Accurate-mass data and thermochemical data, obtained at the B3LYP/6-31+G(d,p) level of theory, were used to support the ion structures. The most intense product ions were the benzyl ions ([C7 H2 R1 R2 R3 R4 R5 ]+ ) and the acylium ions ([M + H - C8 H3 R1 R2 R3 R4 R5 ]+ ), which originated directly from the precursor ion as a result of two competitive hydrogen rearrangements. Product ions [M + H - H2 O]+ and [M + H - C6 HR1 R2 R3 R4 R5 ]+ , which are formed after Nazarov cyclization, were also common to all the analyzed compounds. In addition, •Br and •Cl eliminations were diagnostic for the presence of these halogen atoms at the aromatic ring, whereas •CH3 eliminations were useful to identify the methyl and methoxy groups attached to this same ring. Nazarov cyclization in the gas phase occurred for all the investigated MKCs and did not depend on the presence of the hydroxyl group at the aromatic ring. However, the presence and the position of a hydroxyl group at the aromatic rings played a key role in the Nazarov cyclization mechanism. Our results reinforce some aspects of the fragmentation pathways previously published for 1,5-bis-(2-methoxyphenyl)-1,4-pentadien-3-one and 1,5-bis-(2-hydroxyphenyl)-1,4-pentadien-3-one. The alternative fragmentation mechanism proposed herein can explain the fragmentation of a wider diversity of monoketone curcuminoids.

Characterisation of phenylethanoid glycosides by multiple-stage mass spectrometry.

Although phenylethanoid glycosides (PhGs) occur widely in plants, their characterisation by liquid chromatography/mass spectrometry (LC/MS) is less well studied than other phenolic glycosides such as flavonoid glycosides. The multiple-stage mass spectrometry (MSn ) experiments required to improve the annotation of common verbascoside-type PhGs are described here. Deprotonated, ammoniated and sodiated molecules of nine PhGs were subjected to low-energy collision-induced dissociation (CID) in a hybrid ion trap/orbitrap mass spectrometer. Most experiments were recorded at nominal mass using the linear ion trap analyser for wider applicability in the plant metabolomics community. Data interpretation was supported by high-resolution orbitrap scanning of product ions. Comparative data was acquired on the same instrument by performing higher-energy collisional dissociation (HCD) in the C-trap. Low-energy CID-MS2 of the deprotonated and ammoniated molecules generated diagnostic product ions from which the molecular masses of the phenolic acid and phenylethanoid moieties, respectively, could be determined. The sugar at C-3' of the core glucose was preferentially lost from the sodiated molecule following CID-MS2 , while CID-MSn produced a sodiated product ion from ring cleavage of the core glucose bearing the sugar at C-6'. Evidence of a disaccharide substitution came from a sodiated disaccharide residue in CID-MSn spectra. The consistency of PhG dissociation following low-energy CID-MSn of various ions is sufficient to enable annotation of verbascoside-type PhGs in LC/MS analyses of crude plant extracts. This can be achieved on a low-resolution instrument capable of MSn .

Structural Determination of a New Peptidolipid Family from Rhodococcus opacus and the Pathogen Rhodococcus equi by Multiple Stage Mass Spectrometry.

The cell walls of the genus Rhodococcus including the pathogenic bacterium Rhodococcus equi (R. equi) and biotechnologically important bacterium Rhodococcus opacus (R. opacus) contain an abundant peptidolipid (or termed lipopeptide) family whose structures have not been reported previously. Here, we describe a linear ion-trap multiple-stage mass spectrometric (LIT MSn) approach with high resolution mass spectrometry (HRMS), in conjunction with NMR spectroscopy, chemical reactions, and GC/MS analysis to define the structures of these compounds. We employed LIT MSn (n = 2-8) on the [M + Na]+ ion species to establish the peptide sequence, the identity of the fatty acyl substituent, and its location within the molecule, while NMR spectroscopy and GC/MS were used to recognize the Leu and Ile moieties. The major new lipopeptide found in R. opacus is defined as C17H35CH(OH)CH2CO-NHLeu-Ser-Leu-Ile-Thr-Ile-PheCOOH, where a β-OH fatty acyl (C18-C22) substituent is attached to the N-terminal of the LSLITIF peptide chain via a NH-CO bond. By contrast, the main peptidolipids found in R. equi belong to the cyclopeptidolipid family, which possesses the same peptide sequence and lipid chain, but the β-OH group of the fatty acyl moiety and the C-terminus of the peptide (i.e., the -COOH) are cyclized by an ester bond formation to a lactone, with a structure similar to iturin-A (Peypoux, F. et al. Biochemistry 1978, 17, 3992-3996). The antibiotic activity test of these new lipids did not reveal an activity against any of seven microorganisms tested.

Ozonation of rivaroxaban production waste water and comparison of generated transformation products with known in vivo and in vitro metabolites

During production of rivaroxaban, an orally administrated anticoagulant, process waste water is generated at several stages of manufacturing. Due to low biodegradability in conventional waste water treatment plants, it is currently incinerated. Thus, new pre-treatment methods for rivaroxaban-containing waste water could facilitate its subsequent biological processing. In this study, ozonation was investigated as the pre-treatment method, with special emphasis on the elimination of the target compound and the generation of its main transformation products. These were determined by high performance liquid chromatography – high resolution mass spectrometry (HPLC-HRMS). Our results show that the concentration of rivaroxaban in the ozonated waste water can be effectively reduced (below detection limit) under acidic conditions, whereas neutral or basic reaction environment proved less suitable. Four main transformation products were identified and their concentrations determined. Rivaroxaban and its most prominent transformation product acetoxamide were elucidated in multiple-stage mass spectrometry (MSn) experiments. Transformation products derived from the degradation of rivaroxaban were then compared to its natural (animal and human) metabolites as well as other known derivatives. In contrast to previously published rivaroxaban-derived metabolites, the presented ozonation experiments resulted in new transformation products.

Advances in structure elucidation of low molecular weight carbohydrates by liquid chromatography-multiple-stage mass spectrometry analysis

Oligosaccharides are gaining importance because of their beneficial properties in human health. They normally appear in natural and synthetic products as complex mixtures of different monomeric units, glycosidic linkages and degrees of polymerization, being disaccharides and trisaccharides usually the most abundant ones. Although liquid chromatography-mass spectrometry is a useful technique for oligosaccharides analysis, the similarity of their structures makes difficult their characterization. Moreover, there is still scarce information about the relationship between carbohydrate chemical structure, mass spectra and chromatographic data. Then, in this work, chromatographic parameters for 23 disaccharides with different linkages and monomeric units (glucose, galactose, mannose and fructose) were determined using porous graphitized and hydrophilic interaction liquid chromatography columns. Moreover, diagnostic ions of these disaccharides obtained by tandem mass spectra (MS2) were established by stepwise linear discriminant analysis. The relationship between carbohydrate chemical structure and their chromatographic retention data and characteristic ions obtained by multiple-stage mass spectrometry (MSn) was successful in establishing some specific criteria that allowed the characterization of trisaccharides with different structural features.