Sodiation as a tool for enhancing the diagnostic value of MALDI‐TOF/TOF‐MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono‐ and diesters. For rapid fingerprinting of these esters, matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF/TOF‐MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono‐ and diester palmitate standards in MALDI‐TOF/TOF‐MS showed that sodium adduct parent masses [M + Na]⁺ gave much simpler MS² spectra than radical / protonated [M]^+● / [M + H]⁺ parents. [M + Na]⁺ fragments yielded diagnostic polyene‐specific eliminations and fatty acid neutral losses, whereas [M]^+● / [M + H]⁺ fragmentation resulted in a multitude of non‐diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na]⁺ ionization by addition of sodium acetate, and best signal‐to‐noise ratios were obtained in the 0.1 to 1.0 mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI‐TOF/TOF‐MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono‐ and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all‐trans esterified esters found in LC were identified with MALDI‐TOF/TOF‐MS, with the exception of two minor monoesters. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous Determination Baicalin and Forsythin in Shuanghuanglian Oral Liquid by HPLC/DAD and LC‐ESI‐MS

Rapid, simple and reliable HPLC/DAD and LC‐ESI‐MS methods for the simultaneous determination of baicalin and forsythin in the traditional Chinese medicinal preparation Shuanghuanglian oral liquid were described and validated. The separation condition for HPLC/DAD was optimized using a BDS hypersil C18 column (Thermo, 2.1 × 150 mm, particle size 5 μm) by gradient elution using methanol‐0.2 % ammonium acetate as the mobile phase. The suitable detection wavelength was set at 277 nm for the quantitative analysis of baicalin and forsythin in this method. Some operational parameters of the ESI interface were optimized, negative m/z 445[M?H]^? for baicalin and negative m/z 593[M+CH₃COO]^? for forsythin, positive m/z 447[M+H]⁺ for baicalin and positive m/z 552[M+NH 4]⁺ for forsythin, respectively. These HPLC/DAD and LC‐ESI‐MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). These methods can be used as a complementary method for the commercial quality control of Shuanghuanglian oral liquid and its pharmaceutical preparations.     

HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin

Spin‐labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2‐H configurations of these spin‐labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high‐performance liquid chromatography‐diode array detection (HPLC‐DAD) and a high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 position. In the HPLC‐ESI/MS spectra, each pair of diastereoisomers of the spin‐labeled derivatives in the mixture was directly confirmed and identified by [M+H]⁺ ions and ion ratios of relative abundance of [M‐ROH+H]⁺ (ion 397) to [M+H]⁺. When the [M‐ROH+H]⁺ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M‐ROH+H]⁺ (ion 397) to [M+H]⁺, [A+H]⁺ (ion 313) to [M‐ROH+H]⁺, [A+H‐OCH₃]⁺ (ion 282) to [M‐ROH+H]⁺ and [M‐ROH‐ArH+H]⁺ (ion 229) to [M‐ROH+H]⁺ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 in the mixture.     

Gas‐phase fragmentation of γ‐lactone derivatives by electrospray ionization tandem mass spectrometry

Fragmentation reactions of β‐hydroxymethyl‐, β‐acetoxymethyl‐ and β‐benzyloxymethyl‐butenolides and the corresponding γ‐butyrolactones were investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) using collision‐induced dissociation (CID). This study revealed that loss of H₂O [M + H ?18]⁺ is the main fragmentation process for β‐hydroxymethylbutenolide (1) and β‐hydroxymethyl‐γ‐butyrolactone (2). Loss of ketene ([M + H ?42]⁺) is the major fragmentation process for protonated β‐acetoxymethyl‐γ‐butyrolactone (4), but not for β‐acetoxymethylbutenolide (3). The benzyl cation (m/z 91) is the major ion in the ESI‐MS/MS spectra of β‐benzyloxymethylbutenolide (5) and β‐benzyloxymethyl‐γ‐butyrolactone (6). The different side chain at the β‐position and the double bond presence afforded some product ions that can be important for the structural identification of each compound. The energetic aspects involved in the protonation and gas‐phase fragmentation processes were interpreted on the basis of thermochemical data obtained by computational quantum chemistry. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of α‐acyloxyhydroperoxy aldehydes with electrospray ionization–tandem mass spectrometry (ESI‐MSn)

A series of α‐acyloxyhydroperoxy aldehydes was analyzed with direct infusion electrospray ionization tandem mass spectrometry (ESI/MSⁿ) as well as liquid chromatography coupled with the mass spectrometry (LC/MS). Standards of α‐acyloxyhydroperoxy aldehydes were prepared by liquid‐phase ozonolysis of cyclohexene in the presence of carboxylic acids. Stabilized Criegee intermediate (SCI), a by‐product of the ozone attack on the cyclohexene double bond, reacted with the selected carboxylic acids (SCI scavengers) leading to the formation of α‐acyloxyhydroperoxy aldehydes. Ionization conditions were optimized. [M + H]⁺ ions were not formed in ESI; consequently, α‐acyloxyhydroperoxy aldehydes were identified as their ammonia adducts for the first time. On the other hand, atmospheric‐pressure chemical ionization has led to decomposition of the compounds of interest. Analysis of the mass spectra (MS² and MS³) of the [M + NH₄]⁺ ions allowed recognizing the fragmentation pathways, common for all of the compounds under study. In order to get detailed insights into the fragmentation mechanism, a number of isotopically labeled analogs were also studied. To confirm that the fragmentation mechanism allows predicting the mass spectrum of different α‐acyloxyhydroperoxy aldehydes, ozonolysis of α‐pinene, a very important secondary organic aerosol precursor, was carried out. Spectra of the two ammonium cationized α‐acyloxyhydroperoxy aldehydes prepared with α‐pinene, cis‐pinonic acid as well as pinic acid were predicted very accurately. Possible applications of the method developed for the analysis of α‐acyloxyhydroperoxy aldehydes in SOA samples, as well as other compounds containing hydroperoxide moiety are discussed. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives using electrospray ionization multi‐stage mass spectrometry

N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro‐inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion‐trap multi‐stage mass spectrometry (MSⁿ). The MS² collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N‐Fmoc/Z‐amino acids, dipeptide and tripeptide acids show the [M + Na‐NH₂CHO]⁺ ion, arising from the loss of formamide, as the base peak. Differently, the MS² CID spectra of [M + Na]⁺ ion of all the N‐Boc derivatives yield the abundant [M + Na‐C₄H₈]⁺ and [M + Na‐Boc + H]⁺ ions because of the loss of isobutylene and CO₂ from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N‐protected dipeptidyl and tripeptidyl‐N′‐formamides is provided by MS² and subsequent MSⁿ experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M‐H]^?, [M + HCOO]^? and [M + Cl]^? ions, the presence of in‐source CID fragment ions deriving from the involvement of the N‐protecting group. Furthermore, MSⁿ spectra of [M + Cl]^? ion of N‐protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C‐terminal gem‐diamino residue. The present paper represents an initial attempt to study the ESI‐MS behavior of these important intermediates and lays the groundwork for structural‐based studies on more complex partially modified retro‐inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

One‐Electron Oxidation and Sensitivity of Uric Acid in On‐Line Electrochemistry and in Electrospray Ionization Mass Spectrometry

The sensitivity of detection of uric acid (H₂U) in positive ion mode electrospray ionization mass spectrometry (ESI MS) was enhanced by uric acid oxidation during electrospray ionization. With a carrier solution of pH 6.3>pK_a1=5.4 of H₂U, protonated unoxidized uric acid [H₂U+H]⁺ (m/z 169) was detected together with the protonated uric acid dimer [2H₂U+H]⁺ (m/z 337). The dimer likely forms by 1e^? oxidation of urate (HU^?) followed by rapid radical dimerization. A covalent structure of the dimer was verified by H/D exchange experiments. Efficiency of 2e^?, 2H⁺ oxidation of uric acid is low during ESI in pH 6.3 carrier solution and improves when a low on‐line electrochemical cell voltage is floated on the high voltage of the ES in on‐line electrochemistry ESI MS (EC/ESI MS). The intensity of the uric acid dimer decreases with an increase in the low applied voltage. In a carrier solution with 0.1 M KOH, pH 12.7>pK_a2=9.8 of H₂U, allantoin (Allnt) (MW 158.04), the final 2e^?, 2H⁺ oxidation product of uric acid, was detected as a potassium complex [K(Allnt)+K]⁺ (m/z 235) and the [2H₂U+H]⁺ dimer was not detected. In direct ESI MS analysis of 1000‐fold diluted urine [NaHU+H]⁺ (pK_sp NaHU=4.6) was detected in 40/60 (vol%) water/methanol, 1 mM NH₄Ac, pH ca. 6.3 carrier solution. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity in ESI and EC/ESI MS measurements of uric acid.     

Gas‐phase fragmentation reactions of protonated benzofuran‐ and dihydrobenzofuran‐type neolignans investigated by accurate‐mass electrospray ionization tandem mass spectrometry

We have investigated gas‐phase fragmentation reactions of protonated benzofuran neolignans (BNs) and dihydrobenzofuran neolignans (DBNs) by accurate‐mass electrospray ionization tandem and multiple‐stage (MSⁿ) mass spectrometry combined with thermochemical data estimated by Computational Chemistry. Most of the protonated compounds fragment into product ions B ([M + H–MeOH]⁺), C ([ B –MeOH]⁺), D ([ C –CO]⁺), and E ([ D –CO]⁺) upon collision‐induced dissociation (CID). However, we identified a series of diagnostic ions and associated them with specific structural features. In the case of compounds displaying an acetoxy group at C‐4, product ion C produces diagnostic ions K ([ C –C₂H₂O]⁺), L ([ K –CO]⁺), and P ([ L –CO]⁺). Formation of product ions H ([ D –H₂O]⁺) and M ([ H –CO]⁺) is associated with the hydroxyl group at C‐3 and C‐3′, whereas product ions N ([ D –MeOH]⁺) and O ([ N –MeOH]⁺) indicate a methoxyl group at the same positions. Finally, product ions F ([ A –C₂H₂O]⁺), Q ([ A –C₃H₆O₂]⁺), I ([ A –C₆H₆O]⁺), and J ([ I –MeOH]⁺) for DBNs and product ion G ([ B –C₂H₂O]⁺) for BNs diagnose a saturated bond between C‐7′ and C‐8′. We used these structure‐fragmentation relationships in combination with deuterium exchange experiments, MSⁿ data, and Computational Chemistry to elucidate the gas‐phase fragmentation pathways of these compounds. These results could help to elucidate DBN and BN metabolites in in vivo and in vitro studies on the basis of electrospray ionization ESI‐CID‐MS/MS data only.     

Dereplication of known pregnane glycosides and structural characterization of novel pregnanes in Marsdenia tenacissima by high‐performance liquid chromatography and electrospray ionization‐tandem mass spectrometry

In the search for novel natural products in plants, particularly those with potential bioactivity, it is important to efficiently distinguish novel compounds from previously isolated, known compounds, a process known as dereplication. In this study, electrospray ionization‐multiple stage tandem mass spectrometry (ESI‐MSⁿ) was used to study the behaviour of 12 pregnane glycosides and genins previously isolated from Marsdenia tenacissima, a traditional Chinese medicinal plant, as a basis for dereplication of compounds in a plant extract. In addition to [M + Na]⁺ and [M + NH₄]⁺ ions, a characteristic [M‐glycosyl + H]⁺ ion was observed in full‐scan mode with in‐source fragmentation. Sequential in‐trap collision‐induced dissociation of [M + Na]⁺ ions from 11,12‐diesters revealed consistent preferred losses of substituents first from C‐12, then from C‐11, followed by losses of monosaccharide fragments from the C‐3 tri‐ and tetrasaccharide substituents. A crude methanol extract of M. tenacissima stems was analysed using high‐performance liquid chromatography coupled to ESI‐MS. Several previously isolated pregnane glycosides were dereplicated, and the presence of an additional nine novel pregnane glycosides is predicted on the basis of the primary and fragment ions observed, including two with a previously unreported C₄H₇O C‐11/C‐12 substituent of pregnane glycosides. This study is the first report of prediction of the structures of novel pregnane glycosides in a crude plant extract by a combination of in‐source fragmentation and in‐trap collision‐induced dissociation and supports the usefulness of LC‐ESI‐MSⁿ not only for dereplication of active compounds in extracts of medicinal plants but also for detecting the presence of novel related compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

The transformation from 2°‐amine to 3°‐amine of cyclam ring alters the fragmentation patterns of 1‐tosylcytosine‐cyclam conjugates

The novel N‐1‐sulfonylcytosine‐cyclam conjugates 1 and 2 conjugates are ionized by electrospray ionization mass spectrometry (ESI MS) in positive and negative modes (ES⁺ and ES⁻) as singly protonated/deprotonated species or as singly or doubly charged metal complexes. Their structure and fragmentation behavior is examined by collision induced experiments. It was observed that the structure of the conjugate dictated the mode of the ionization: 1 was analyzed in ES⁻ mode while 2 in positive mode. Complexation with metal ions did not have the influence on the ionization mode. Zn²⁺ and Cu²⁺ complexes with ligand 1 followed the similar fragmentation pattern in negative ionization mode. The transformation from 2°‐amine in 1 to 3°‐amine of cyclam ring in 2 leads to the different fragmentation patterns due to the modification of the protonation priority which changed the fragmentation channels within the conjugate itself. Cu²⁺ ions formed complexes practically immediately, and the priority had the cyclam portion of the ligand 2 . The structure of the formed Zn²⁺ complexes with ligand 2 depended on the number of 3° amines within the cyclam portion of the conjugate and the ratio of the metal:ligand used. The cleavage of the cyclam ring of metal complexes is driven by the formation of the fragment that suited the coordinating demand of the metal ions and the collision energy applied. Finally, it was shown that the structure of the cyclam conjugate dictates the fragmentation reactions and not the metal ions.     

Detection of oxygen addition peaks for terpendole E and related indole–diterpene alkaloids in a positive‐mode ESI‐MS

This report describes that a regular positive electrospray ionization mass spectrometry (MS) analysis of terpendoles often causes unexpected oxygen additions to form [M + H + O]⁺ and [M + H + 2O]⁺, which might be a troublesome in the characterization of new natural analogues. The intensities of [M + H + O]⁺ and [M + H + 2O]⁺ among terpendoles were unpredictable and fluctuated largely. Simple electrochemical oxidation in electrospray ionization was insufficient to explain the phenomenon. So we studied factors to form [M + H + O]⁺ and [M + H + 2O]⁺ using terpendole E and natural terpendoles together with some model indole alkaloids. Similar oxygen addition was observed for 1,2,3,4‐tetrahydrocyclopent[b]indole, which is corresponding to the substructure of terpendole E. In tandem MS experiments, a major fragment ion at m/z 130 from protonated terpendole E was assigned to the substructure containing indole. When the [M + H + O]⁺ was selected as a precursor ion, the ion shifted to m/z 146. The same 16 Da shift of fragments was also observed for 1,2,3,4‐tetrahydrocyclopent[b]indole, indicating that the oxygen addition of terpendole E took place at the indole portion. However, the oxygen addition was absent for some terpendoles, even whose structure resembles terpendole E. The breakdown curves characterized the tandem MS features of terpendoles. Preferential dissociation into m/z 130 suggested the protonation tendency at the indole site. Terpendoles that are preferentially protonated at indole tend to form oxygen addition peaks, suggesting that the protonation feature contributes to the oxygen additions in some degrees. © 2014 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons, Ltd.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

Electrospray ionization and atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry of antioxidants applied in lubricants

The aim of this study was to investigate the utility of ion trap mass spectrometry (ITMS) in combination with the two desorption/ionization methods, electrospray (ESI) and atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI), for the detection of antioxidants which are applied in lubricants. These experiments should form the base for future investigations of antioxidants in tribologically formed thin layers on the surface of frictional systems. Seventeen different antioxidants were selected out of the group of hindered phenolic and aromatic aminic compounds. Practically all antioxidants could be characterized by positive ion ESI‐ and AP‐MALDI‐ITMS, forming various types/species of molecular ions (e.g. [M]^{+ .} , [M+H]⁺, [M+Na]⁺ or [M–2H+H]⁺). A few compounds could be analyzed by negative ion ESI‐MS, too, but none by negative ion AP‐MALDI‐MS. The influence of target materials in AP‐MALDI‐MS (gold‐ and titanium nitride (TiN)‐covered stainless steel, micro‐diamond‐covered hard metal, hand‐polished and sand‐blasted stainless steel targets) with respect to the molecular ion intensity and type of molecular ion of two selected antioxidants was evaluated. The surface properties are of particular interest because in friction tests different materials with different surface characteristics are used. However, the MS results indicate that optimal target surfaces have to be found for individual antioxidants in AP‐MALDI‐MS but in general smooth surfaces were superior to rough surfaces. Finally the gold‐covered stainless steel MALDI target provided the best mass spectra and was selected for all the antioxidants investigated. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation of 2‐aroylbenzofuran derivatives by electrospray ionization tandem mass spectrometry

We investigated the gas‐phase fragmentation reactions of a series of 2‐aroylbenzofuran derivatives by electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The most intense fragment ions were the acylium ions m/z 105 and [M+H–C₆H₆]⁺, which originated directly from the precursor ion as a result of 2 competitive hydrogen rearrangements. Eliminations of CO and CO₂ from [M+H–C₆H₆]⁺ were also common fragmentation processes to all the analyzed compounds. In addition, eliminations of the radicals •Br and •Cl were diagnostic for halogen atoms at aromatic ring A, whereas eliminations of •CH₃ and CH₂O were useful to identify the methoxyl group attached to this same ring. We used thermochemical data, obtained at the B3LYP/6‐31+G(d) level of theory, to rationalize the fragmentation pathways and to elucidate the formation of E , which involved simultaneous elimination of 2 CO molecules from B .     

Antioxidant Pathways and One‐Electron Oxidation of Dopamine and Cysteine in Electrospray and On‐Line Electrochemistry Electrospray Ionization Mass Spectrometry

Dopamine [DA]⁺ (m/z 154), DA dimer [2DA‐H]⁺ (m/z 307) and DA quinone [DAQ]⁺ (m/z 152) are detected in positive ion mode electrospray ionization mass spectrometry (ESI MS) of dopamine in 50/1/49 (vol%) water/acetic acid/methanol. H/D exchange experiments support a covalent structure of DA dimer. Thus, ESI of DA may involve 1e^?, 1H⁺ oxidation processes followed by rapid radical dimerization. The DA quinone signal is low in ESI MS, which indicates a low efficiency of the 2e^?, 2H⁺ oxidation reaction. On‐line electrochemistry ESI MS (EC/ESI MS) with low electrochemical cell voltage floated on high ES voltage increases electrospray current and improves sensitivity for DA. The DA quinone signal increases and DA dimer signal decreases. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity of ESI and EC/ESI MS measurements. A DA quinone‐cysteine adduct [DAQ+Cys]⁺ was detected in solutions of DA with cysteine (Cys). ESI MS and EC/ESI MS indicate formation of the DA quinone‐cysteine adduct by 1e^? pathway. Oxidation pathways in ESI MS are relevant to biological reactivity of DA and Cys.     

Formation of cyclic acylphosphoramidates in mass spectra of N‐monoalkyloxyphosphoryl amino acids using electrospray ionization tandem mass spectrometry

The fragmentation reactions of N‐monoalkyloxyphosphoryl amino acids (N‐MAP‐AAs) were studied by electrospray ionization tandem mass spectrometry (ESI‐MS). The sodiated cyclic acylphosphoramidates (CAPAs) were formed through a characteristic pentacoordinate phosphate participated rearrangement reaction in the positive‐ion ESI‐MS/MS and HR‐MS/MS of N‐MAP‐AAs, in which the fragmentation patterns were clearly different from those observed in the corresponding ESI‐MS/MS of N‐dialkyloxyphosphoryl amino acids/peptides and N‐phosphono amino acids. The formation of CAPAs depended on the chemical structures of N‐terminal phosphoryl groups, such as alkyloxy group, negative charge and alkali metal ion. A possible integrated rearrangement mechanism for both P‐N to P‐O phosphoryl group migration and formation of CAPAs was proposed. The fragmentation patterns of CAPAs as novel intermediates in gas phase were also investigated. In addition, it was found that the formation of α‐amino acid CAPAs was more favorable than β‐ or γ‐CAPAs in gas phase, which was consistent with previous solution‐phase experiments. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis and development of structure-fragmentation relationships in withanolides using an electrospray ionization quadropole time-of-flight tandem mass spectrometry hybrid instrument

Structural elucidation and gas‐phase fragmentation of ten withanolides (steroidal lactones) were studied using a positive ion electrospray ionization quadropole time‐of‐flight mass spectrometry (ESI‐QqTOF‐MS/MS) hybrid instrument. Withanolides form an important class of plant secondary metabolites, known to possess a variety of biological activities. Withanolides which possess hydroxyl groups at C‐4, C‐5, C‐17, C‐20, and C‐27, and an epoxy group at C‐5/C‐6, were evaluated to determine the characteristic fragments and their possible pathways. ESI‐QqTOF‐MS (positive ion mode) showed the presence of the protonated molecules [M + H]⁺. Low‐energy collision‐induced dissociation tandem mass spectrometric (CID‐MS/MS) analysis of the protonated molecule [M + H]⁺ indicated multiple losses of water and the removal of the C‐17‐substituted lactone moiety affording the [M + H–Lac]⁺ product ion as the predominant pathways. However, withanolides containing a hydroxyl group at C‐24 of the lactone moiety showed a different fragmentation pathway, which include the loss of steroidal part as a neutral molecule, with highly diagnostic ions at m/z 95 and 67 being generated from the cleavage of lactone moiety. Our results also determined the influence of the presence and positions of hydroxyl and epoxy groups on product ion formation and stability. Moreover, the knowledge of the fragmentation pattern was utilized in rapid identification of withanolides by the LC/MS/MS analysis of a Withania somnifera extract. Copyright © 2010 John Wiley & Sons, Ltd.     

LC/ESI‐MS/MS characterisation of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain

Lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain showed an interesting anti‐adhesion activity against biofilm formation of human pathogenic bacterial strains. The chemical characterisation of the crude extract of V9T14 strain was first developed through electrospray ionisation mass spectrometry (ESI‐MS) and ESI‐MS/MS direct infusions: two sets of molecular ion species belonging to the fengycin and surfactin families were revealed and their structures defined, interpreting their product ion spectra. The LC/ESI‐MS analysis of the crude extract allowed to separate in different chromatogram ranges the homologues and the isoforms of the two lipopeptide families. The extract was then fractionated by silica gel chromatography in two main fractions, I and II. The purified biosurfactants were analysed through a new, rapid and suitable LC/ESI‐MS/MS method, which allowed characterising the composition and the structures of the produced lipopeptides. LC/ESI‐MS/MS analysis of fraction I showed the presence of C₁₃, C₁₄ and C₁₅ surfactin homologues, whose structures were confirmed by the product ion spectra of the sodiated molecules [M + Na]⁺ at m/z 1030, 1044 and 1058. LC/ESI‐MS/MS analysis of fraction II confirmed the presence of two main fengycin isoforms, with the protonated molecules [M + H]⁺ at m/z 1478 and 1506 corresponding to C₁₇ fengycin A and C₁₇ fengycin B, respectively. Other homologues (C₁₄ to C₁₆) were revealed and confirmed as belonging to fengycin A or B according to the retention times and the product ions generated, although with the same nominal mass. Finally, a relative percentage content of each homologue for both lipopeptides families in the whole extract was proposed. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid screening and identification of lycodine‐type alkaloids in Lycopodiaceae and Huperziaceae plants by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Lycodine‐type alkaloids have gained significant interest owing to their unique skeletal characteristics and acetylcholinesterase activity. This study established a rapid and reliable method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐ESI‐Q/TOF‐MS/MS) for comprehensive characterization of lycodine‐type alkaloids for the first time. The lycodine‐type alkaloids were detected successfully from Lycopodiastrum casuarinoides, Huperzia serrata and Phlegmarirus carinatus in seven plants of the Lycopodiaceae and Huperziaceae families, based on the established characteristic MS fragmentation of five known alkaloids. Furthermore, a total of 13 lycodine‐type alkaloids were identified, of which three pairs of isomers were structurally characterized and differentiated. This study further improves mass analysis of lycodine‐type alkaloids and demonstrates the superiority of UPLC with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of other trace active compounds. Copyright © 2016 John Wiley & Sons, Ltd.     

两类三组份反应产物：1,3-恶嗪及嘧啶-2-酮类化合物的质谱学行为研究

电喷雾质谱被应用于分辨2-氨基-1,3-恶嗪及六氢化-4-苯基-吡喃[2,3-d]嘧啶-2-酮的杂环结构。两类化合物均为三组份反应的产物,且其杂环的结构很难用NMR判断。实验首次系统研究了两类化合物的质谱学行为（包括氘代实验和高分辨质谱研究）,发现前者在CID实验中丢失CH2N2和HCNO,而后者为直接丢失尿素。这些特征丢失为该类衍生物的结构判断,尤其是高通量的合成产物分析提供了重要的依据。