Mass Spectrometric Fragmentation Pathways Research Articles

Degradation products of aflatoxin M1 (AFM1) formed by high voltage atmospheric cold plasma (HVACP) treatment

Cold plasma technology is a novel non-thermal technology that has shown promising results for food decontamination and improving food safety. This study is a continuation of a previous investigation of the treatment of AFM1-contaminated skim and whole milk samples by HVACP. Previous research has shown HVACP is effective in degrading aflatoxin M1 (AFM1) in milk. The goal of this study is to identify the degradation products of AFM1 after HVACP treatment in pure water. An HVACP direct treatment at 90 kV using modified air (MA65: 65% O2, 30% CO2, 5% N2) was performed for up to 5 min at room temperature on a 5.0 mL water sample in a Petri dish artificially contaminated with 2 μg/mL of AFM1. The degradants of AFM1 were analyzed and their molecular formulae were elucidated by using high-performance liquid-chromatography time-of-flight mass spectrometry (HPLC−TOF-MS). Three main degradation products were observed and based on mass spectrometric fragmentation pathways, chemical structures for the degradation products were tentatively assigned. According to the structure–bioactivity relationship of AFM1, the bioactivity of the AFM1 samples treated with HVACP was reduced due to the disappearance of the C8–C9 double bond in the furofuran ring in all of the degradation products.

2,4-dinitrophenylhydrazine capturing combined with mass defect filtering strategy to identify aliphatic aldehydes in biological samples

Aliphatic aldehydes are toxic substances that correlate with the onset of many diseases. However, up to date, the methods to identify aliphatic aldehydes in biological samples are less selectivity and/or robustness. In this study, a strategy based on 2,4-dinitrophenylhydrazine (DNPH) capturing combined with mass defect filtering (MDF) was established and validated to identify aliphatic aldehydes in two biological samples (serum of immunosuppressed rats and oxidative damaged cells). Firstly, the mass spectrometric characteristic ions (m/z 163.01, 163.02 and 191.04) and fragmentation pathways of aldehyde-DNPHs were acquired through analyzing the standard references. Then, biological samples were derivatized by DNPH, a routine reagent, and subsequently assessed on an ultra-performance liquid chromatography coupled time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). Thirdly, the raw chromatogram was processed by MDF method to obtain interference-free chromatogram. Fourthly, the aldehyde-DNPHs were characterized through investigating the mass spectrometric information of each peak referred to the identified characteristic ions and fragmentation pathways. Finally, 6 and 8 aliphatic aldehydes were exclusively identified in serum of immunosuppressed rats and supernatant of oxidative damaged cells. Among which, propanal and butanal were positively correlate with immunosuppression, while formalin was more relevant to oxidative stress. The results demonstrated that the established strategy could robustly characterize the aliphatic aldehydes in biological samples, which would be helpful to evaluate the physical conditions of subjects.

Chemical Constituent Analysis of Ranunculus sceleratus L. Using Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Orbitrap High-Resolution Mass Spectrometry.

Ranunculus sceleratus L.(RS) has shown various pharmacological effects in traditional Chinese medicine. In our previous study, the positive therapeutic effect on α-naphthylisothiocyanate induced intrahepatic cholestasis in rats was obtained using TianJiu treatment with fresh RS. However, the chemical profile of RS has not been clearly clarified, which impedes the research progress on the therapeutic effect of RS. Herein, an ultra-high performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method was developed to rapidly separate and identify multiple constituents in the 80% methanol extract of RS. A total of sixty-nine compounds (19 flavonoids, 22 organic acids, 6 coumarins, 4 lignans, 14 nitrogenous compounds, and 4 anthraquinones) were successfully characterized. A total of 12 of these compounds were unambiguously identified by standard samples. Their mass spectrometric fragmentation pathways were investigated. It is worth noting that flavonoids and lignans were identified for the first time in RS. In this study, we successfully provide the first comprehensive report on identifying major chemical constituents in RS by UHPLC-Q-Orbitrap HRMS. The obtained results enrich the RS chemical profile, paving the way for further phytochemical study, quality control, and pharmacological investigation of RS.

Systematic Characterization and Identification of Saikosaponins in Extracts From Bupleurum marginatum var. stenophyllum Using UPLC-PDA-Q/TOF-MS.

Saikosaponins comprise a large group of chemical components present in the Bupleurum species that have attracted attention in the field of medicine because of their significant biological activities. Due to the high polarity, structural similarity, and the presence of several isomers of this class of components, their structural identification is extremely challenging. In this study, the mass spectrometric fragmentation pathways, UV spectral features, and chromatographic behavior of different types of saikosaponins were investigated using 24 standard substances. Saikosaponins containing carbonyl groups (C=O) in the aglycone produced fragment ions by loss of 30 Da, and in addition, type IV saikosaponins could produce [aglycone−CH2OH−OH−H]− and [aglycone−H2O−H]− fragment ions through neutral losses at positions C16 and C17. The above characteristic ions can be used to identify saikosaponins. More notably, the identification process of saikosaponins was systematically summarized, and using this method, 109 saikosaponins were identified or tentatively characterized from the saikosaponins extract of Bupleurum marginatum var. stenophyllum (BMS) using UPLC-PDA-Q/TOF-MS with both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes, of which 25 were new compounds and 60 were first discovered from BMS. Further studies revealed that the saikosaponins profiles of BMS, Bupleurum chinense DC (BC), and Bupleurum marginatum Wall. ex DC (BMW) were very similar. This work is of great significance for the basic research of the Bupleurum species and provides strong technical support to solve the resource problems associated with Radix Bupleuri.

Chemical composition analysis of pomegranate seeds based on ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry

Pomegranate fruit has shown a variety of pharmacological effects, however, the bioactive constituents in pomegranate seeds have not been fully identified. To this end, the chemical compositions of pomegranate seeds were analyzed using ultra-high-performance liquid chromatography (UHPLC) coupled with quadrupole-Orbitrap high-resolution mass spectrometry (Q-Orbitrap HRMS). Prior to analysis, pomegranate seed samples were treated by ultrasonic/microwave-assisted extraction (UMAE). A total of 88 chemical compounds were tentatively identified based on accurate-mass measurements for precursor and fragment ions, including flavonoids, coumarins, phenolic acids, amino acids, and nucleosides. Their mass spectrometric fragmentation pathways were investigated. The study has revealed the nutritional value of pomegranate seeds, paving the way for future further applications of pomegranate seeds in nutrition and health care industry.

Characterization of stable and reactive metabolites of piperine formed on incubation with human liver microsomes.

Black pepper, though commonly employed as a spice, has many medicinal properties. It consists of volatile oils, alkaloids, pungent resins, etc., of which piperine is a major constituent. Though safe at low doses, piperine causes alteration in the activity of drug metabolising enzymes and transporters at high dose and is known to precipitate liver toxicity. It has a potential to form reactive metabolite(s) (RM) owing to the presence of structural alerts, such as methylenedioxyphenyl (MDP), α, β-unsaturated carbonyl group (Michael acceptor), and piperidine. The present study was designed to detect and characterize stable and RM(s) of piperine formed on in vitro incubation with human liver microsomes. The investigation of RMs was done with the aid of trapping agents, viz, glutathione (GSH) and N-acetylcysteine (NAC). The samples were analysed by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) using Thermo Scientific Q Exactive Plus Orbitrap. Full scan MS followed by data-dependent MS2 (Full MS-ddMS2 ) mode was used to establish mass spectrometric fragmentation pathways of protonated piperine and its metabolites. In total, four stable metabolites and their isomers (M1a-c, M2a-b, M3a-c, and M4a-b) were detected. Their formation involved removal of carbon (3, M1a-c), hydroxylation (2, M2a-b), hydroxylation with hydrogenation (3, M3a-c), and dehydrogenation (2, M4a-b). Out of these metabolites, M1, M2, and M3 are reported earlier in the literature, but their isomers and two M4 variants are novel. In addition, six novel conjugates of RMs, including three GSH conjugates of m/z 579 and three NAC conjugates of m/z 435, were also observed.

Separation and analysis of flavonoid chemical constituents in flowers of Juglans regia L. by ultra-high-performance liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry

The flowers of Juglans regia L. have demonstrated their medicinal value as a part of edible plants. In this study, an analytical methodology for identification of flavonoid chemical constituents in flowers of Juglans regia L. was established using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Thirty-six flavonoid compounds were identified based on highly accurate mass measurements, and the mass spectrometric fragmentation pathways of eleven representative compounds were proposed, which was helpful for the identification of different types of flavonoids. The study has laid the foundation for the research and development of effective drugs from flowers of Juglans regia L.

Tandem mass spectrometric analysis of S- and N-linked glutathione conjugates of pulegone and menthofuran and identification of P450 enzymes mediating their formation.

Menthofuran is a hepatotoxin and a major metabolite of pulegone, a monoterpene found in the essential oils of many mint species. It is bioactivated by cytochrome P450 (CYP) enzymes to reactive metabolites, which may further react with glutathione to form S-linked and N-linked conjugates. The tandem mass spectrometric (MS/MS) fragmentation pathways of rarely observed N-linked conjugates, and the differences to fragmentation of S-linked conjugates, have not been reported in the literature previously, although this information is essential to enable comprehensive MS/MS-based screening methods covering the both types of conjugates. (R)-(+)-Pulegone, (S)-(-)-pulegone, and menthofuran were incubated with a human liver S9 fraction with glutathione (GSH) as the trapping agent. Conjugates were searched with ultra-performance liquid chromatography (UPLC)/orbitrap MS and their MS/MS spectra were measured both in the negative and positive ionization polarities. Menthofuran was also incubated with recombinant human CYP enzymes and GSH to elucidate the CYPs responsible for the formation of the reactive metabolites. Four GSH conjugates of menthofuran were detected and identified as S- and N-linked conjugates based on MS/MS spectra. N-linked conjugates lacked the characteristic fragments of S-linked conjugates and commonly produced fragments that retained parts of glutamic acid. CYP1A2, 2B6 and 3A4 were observed to produce more GSH conjugates than other CYP isoforms. Furans can form reactive aldehydes that react in Schiff-base fashion with the free glutamyl-amine of GSH to form N-linked conjugates that have distinct MS/MS spectra from S-linked adducts. This should be taken into account when setting up LC/MS/MS-based detection of glutathione conjugates to screen for reactive metabolites, at least for compounds with a furan moiety. Neutral loss scanning of 178.0412 Da and 290.0573 Da in the positive ionization mode, or neutral loss scanning of 256.0695 Da and 290.0573 Da and precursor ion scanning of m/z 143.0462 in the negative ionization mode, is recommended. Copyright © 2016 John Wiley & Sons, Ltd.

Fragmentation pathways and structural characterization of 14 nerve agent compounds by electrospray ionization tandem mass spectrometry.

Organophosphate nerve agents (OPNAs) are some of the most widely used and proliferated chemical warfare agents. As evidenced by recent events in Syria, these compounds remain a serious military and terrorist threat to human health because of their toxicity and the ease with which they can be used, produced and stored. There are over 2,000 known, scheduled compounds derived from common parent structures with many more possible. To address medical, forensic, attribution, remediation and other requirements, laboratory systems have been established to provide the capability to analyze 'unknown' samples for the presence of these compounds. Liquid chromatography/mass spectrometric methods have been validated and are routinely used in the analysis of samples for a very limited number of these compounds, but limited data exist characterizing the electrospray ionization (ESI) and mass spectrometric fragmentation pathways of the compound families. This report describes results from direct infusion ESI/MS, ESI/MS(2) and ESI/MS(3) analysis of 14 G and V agents, the major OPNA families, using an AB Sciex 4000 QTrap. Using a range of conditions, spectra were acquired and characteristic fragments identified. The results demonstrated that the reproducible and predictable fragmentation of these compounds by ESI/MS, ESI/MS(2) and ESI/MS(3) can be used to describe systematic fragmentation pathways specific to compound structural class. These fragmentation pathways, in turn, may be useful as a predictive tool in the analysis of samples by screening and confirmatory laboratories to identify related compounds for which authentic standards are not readily available.

Elucidation of electron ionization mass spectrometric fragmentation pathways of trimethylsilyl ether derivatives of vicinal diols deriving from haplamine by collision‐induced dissociation gas chromatography/tandem mass spectrometry and 18O labelling

Formation of vicinal diols was observed after in vitro and in vivo studies of the natural product haplamine (9-methoxy-2,2-dimethyl-2,6-dihydropyrano[3,2-c]quinolin-5-one). These compounds, identified as trans- and cis-3,4-dihydroxy-9-methoxy-2,2-dimethyl-2,3,4,6-tetrahydropyrano[3,2-c]quinolin-5-ones and trans- and cis-3,4,9-trihydroxy-2,2-dimethyl-2,3,4,6-tetrahydropyrano[3,2-c]quinolin-5-ones, have a potential interest in oncology. It is therefore essential to elucidate their electron ionization mass spectrometric (EIMS) fragmentation pathways. EIMS fragmentation pathways of trimethylsilyl (TMS) derivatives of 3,4-dihydroxy- and 3,4,9-trihydroxyhaplamines were investigated. These pathways have been substantiated by: (i) comparison with EI mass spectra of structural homologues (silylated diols obtained from various chromenes and 1,2-dihydronaphthalene), (ii) low-energy collision-induced dissociation (CID) gas chromatography/tandem mass spectrometry (GC/MS/MS) and (iii) (18)O labelling. CID-MS/MS analyses and (18)O labelling demonstrated that EI mass spectral fragmentation of these TMS derivatives involves a transannular cleavage of the pyran ring with formation of a characteristic intense cyclic ion. The study of the mass spectra of TMS derivatives of different chromenes and 1,2-dihydroxynaphthalene allowed to confirm the proposed fragmentation pathways and to show that they act only when the pyran ring is present. Elimination of the neutral element [(CH3)2=C(H)OSi(CH3)3] and formation of cyclic ions play a key role during EI mass spectral fragmentation of the TMS derivatives of 3,4-dihydroxy- and 3,4,9-trihydroxyhaplamines. These fragmentation pathways could be generalized to TMS derivatives of cyclic compounds possessing vicinal diols close to a pyran ring.

Copper complexes formed by 3,5-bis(2,2′-bipyridin-4-ylethynyl)benzoic acid and its methyl and ethyl esters as studied by electrospray ionization mass spectrometry

Abstract Electrospray ionization mass spectrometry was used to study the complexes of ligands containing two bipyridine units, namely 3,5-bis(2,2-bipyridin-4-ylethynyl)benzoic acid (1) and its methyl and ethyl esters (2, 3), with copper cation, with CuCl2 as a source of copper. It was found that the type of complexes formed strongly depends on CuCl2 concentration. At lower CuCl2 concentration, the detected complexes were rather simple and some of them were formed upon electrospray ionization conditions e.g. ions [22+Cu2]2+ and [32+Cu2]2+ (complexes ligand-Cu(I) of stoichiometry 2:2) which are analogical to the well known, for quaterpyridine, helical complexes. At higher CuCl2 concentration, the detected complexes were more complicated, and most of them contained copper cations bridged by chlorides. The largest ions were [L2+Cu4Cl6]2+. The CID MS/MS spectra of these ions allowed determination of their mass spectrometric fragmentation pathways and as a consequence their structure elucidation.

Tandem mass spectrometric analysis of isosorbide‐1,4‐cyclohexane‐dicarboxylic acid polyester oligomer cations using ion‐trap mass spectrometry

Isosorbide is a promising biomass-derived molecule that can be used as a replacement for fossil resource-derived diol monomers used in polyester synthesis. Due to its increased use in sustainable development, it is useful to understand the tandem mass spectrometric (MS/MS) fragmentation pathways of the isosorbide-based copolymer as an aid to interpreting the MS/MS spectra of other isosorbide-containing copolymers. Collision-activated dissociation (CAD) experiments were performed on the sodiated/protonated molecules, [(AB)(n)A+Na(or H)](+), n = 2-5, of isosorbide (A)-1,4-cyclohexanedicarboxylic acid (B) oligomers formed by ion-trap electrospray ionization (ESI). Product ions arose from cleavage of the bonds between isosorbide and 1,4-cyclohexanedicarboxylic acid. In the MS/MS spectra, f(n)'' product ions were most abundant, followed by e(n) ions. McLafferty rearrangement appeared to provide the most facile pathway to yield the abundant f(n)'' and e(n) ions. In addition, a(n), b(n)'', f(n)''u(n)'', and en (+) ions were observed. Inductive cleavage and β-elimination were suggested to be the pathways involved in generating e(n)(+)- and e(n)/b(n)''-type ions, respectively. Based on the obtained CAD spectra, the alternating sequences of two copolymer building blocks, A and B, were unambiguously determined. The fragmentation pathways leading to the observed product ion types were also established.

Separation of isomeric bavachin and isobavachalcone in the Fructus Psoraleae by capillary electrophoresis–mass spectrometry

Bavachin and isobavachalcone are the isomeric compounds in the fructus Psoraleae. The ion trap mass spectrometric fragmentation pathways of the bavachin and isobavachalcone in negative ion mode were elucidated for the identification. A novel method for determination of isomeric bavachin and isobavachalcone has been developed by capillary electrophoresis coupled with mass spectrometric detection. The effects of several factors such as concentration, pH of ammonium acetate buffer, separation voltage, composition and flow rate of the sheath liquid were investigated. Under optimal conditions, the linear concentration range for bavachin and isobavachalcone were 0.8-100 μg/mL with the correlation coefficient of 0.996 and 0.995, respectively. Relative standard deviations of migration time and peak areas were lower than 5%. The limits of detection (signal/noise = 3) were 60 ng/mL. The proposed method can be successfully applied to the determination of bavachin and isobavachalcone in the fructus Psoraleae and six fructus Psoraleae-containing preparations.

Understanding of the mass spectrometric fragmentation pathways of a few potentially genotoxic haloaniline isomers in their protonated form by collision-induced dissociation

Collision-induced dissociation (CID) mass spectra of a few haloaniline isomers, (chloroanilines, dichloroanilines, difluoroanilines, chloro-fluoroanilines and bromo-fluoroanilines) were characterized. The mass spectral behaviour of difluoroanilines was different from those of the corresponding regioisomers of the other haloanilines. For all ortho regioisomers except difluoroanilines, CID mass spectra resulted in hydrogen halide as well as halogen radical loss. In the case of difluoroanilines, peaks corresponding to hydrogen fluoride loss were observed during the same process. Meta and para-haloanilines have the tendency to lose either ammonia or halogen radicals. Six regioisomers of dichloroanilines were subjected to hydrogen/deuterium exchange experiments in solution to determine the CID fragmentation pathways. From the experimental results we propose two fragmentation pathways for the dicholoroanilines: (a) formation of aza-biheterocyclic intermediate and (b) via heterolytic hydrogen transfer from the charged center. The demonstrated unique characteristics in CID mass fragmentation of haloanilines may be useful in identification and differentiation of isomers as impurities during chemical process development. A good use of the ortho effect is the significant differentiation between 2-chloro-4-fluoroaniline and 4-chloro-2-fluoroaniline by CID mass spectra.

Fragmentation investigation of brassinosteroid compounds by ion trap and quadrupole time‐of‐flight mass spectrometry

The fragmentation mechanisms of three types of brassinosteroids (BRs), 23,24-tris-epicastasterone, epicastasterone, tris-epicastasterone, 24-epibrassinolide and 6-deoxo-24-epicastasterone, have been extensively investigated by tandem mass spectrometry (MS(n), n = 1, 2, 3, 4, 5) with the assistance of high mass accuracy quadrupole time-of-flight mass spectrometry (QToF MS). The electrospray ionization (ESI) mass spectrometric fragmentation pathways of these five BRs were comprehensively elucidated for the first time. Cleavages of side chains, neutral losses of water or other molecules and opening of a ring induce the main fragmentation patterns. The results from the present study can potentially afford important guidance for the structural elucidation of different BRs and provide some fundamental data for metabolomic analysis of BRs.

Fragmentation study of two brassinolides by ion trap tandem mass spectrometry

The fragmentation pathways of two isomers of brassinosteroids, 28-homobrassinolide (28-h-BL) and 28-epihomobrassinolide (28-eh-BL), have been investigated by tandem mass spectrometry (MS n , n = 1, 2, 3, 4) with the electrospray ionization (ESI) source. The ESI mass spectrometric fragmentation pathways of protonated 28-h-BL and 28-eh-BL were comprehensively elucidated, and the principles revealed in this investigation could potentially be used to identify and distinguish brassinosteroids and their isomers.

Formation of Organometallic Species, [M – H]+ Ions and Radical Cations upon Mass Spectrometric Fragmentation of Mercury–Crown Ether Complexes

Mass spectrometric fragmentation pathways of [M + HgClO(4)](+) (M - crown ether molecule), determined by tandem mass spectrometry experiments, are discussed in detail. The decomposition of [M + HgClO(4)](+) proceeds along three fragmentation pathways: formation of [M - H](+) ions, formation of organometallic species, namely [M - H + Hg](+) ions, and formation of radical cations [M](+*). The factors influencing these processes, namely crown ether cavities and the presence of electron withdrawing/electron donor groups, have been discussed.

Can density functional theory (DFT) be used as an aid to a deeper understanding of tandem mass spectrometric fragmentation pathways?

Prediction of tandem mass spectrometric (MS/MS) fragmentation for non-peptidic molecules based on structure is of immense interest to the mass spectrometrist. If a reliable approach to MS/MS prediction could be achieved its impact within the pharmaceutical industry could be immense. Many publications have stressed that the fragmentation of a molecular ion or protonated molecule is a complex process that depends on many parameters, making prediction difficult. Commercial prediction software relies on a collection of general heuristic rules of fragmentation, which involve cleaving every bond in the structure to produce a list of 'expected' masses which can be compared with the experimental data. These approaches do not take into account the thermodynamic or molecular orbital effects that impact on the molecule at the point of protonation which could influence the potential sites of bond cleavage based on the structural motif. A series of compounds have been studied by examining the experimentally derived high-resolution MS/MS data and comparing it with the in silico modelling of the neutral and protonated structures. The effect that protonation at specific sites can have on the bond lengths has also been determined. We have calculated the thermodynamically most stable protonated species and have observed how that information can help predict the cleavage site for that ion. The data have shown that this use of in silico techniques could be a possible way to predict MS/MS spectra.

Automated molecular formula determination by tandem mass spectrometry (MS/MS)

Automated software was developed to analyze the molecular formula of organic molecules and peptides based on high-resolution MS/MS spectroscopic data. The software was validated with 96 compounds including a few small peptides in the mass range of 138-1569 Da containing the elements carbon, hydrogen, nitrogen and oxygen. A Micromass Waters Q-TOF Ultima Global mass spectrometer was used to measure the molecular masses of precursor and fragment ions. Our software assigned correct molecular formulas for 91 compounds, incorrect molecular formulas for 3 compounds, and no molecular formula for 2 compounds. The obtained 95% success rate indicates high reliability of the software. The mass accuracy of the precursor ion and the fragment ions, which is critical for the success of the analysis, was high, i.e. the accuracy and the precision of 850 data were 0.0012 Da and 0.0016 Da, respectively. For the precursor and fragment ions below 500 Da, 60% and 90% of the data showed accuracy within < or = 0.001 Da and < or = 0.002 Da, respectively. The precursor and fragment ions above 500 Da showed slightly lower accuracy, i.e. 40% and 70% of them showed accuracy within < or = 0.001 Da and < or = 0.002 Da, respectively. The molecular formulas of the precursor and the fragments were further used to analyze possible mass spectrometric fragmentation pathways, which would be a powerful tool in structural analysis and identification of small molecules. The method is valuable in the rapid screening and identification of small molecules such as the dereplication of natural products, characterization of drug metabolites, and identification of small peptide fragments in proteomics. The analysis was also extended to compounds that contain a chlorine or bromine atom.

Electrospray ionization tandem mass spectrometric analysis of ent‐6,7‐seco‐kaurane diterpenoids from the Isodon species

Electrospray ionization tandem mass spectrometry (ESI-MS(n)) using an ion trap instrument and accurate mass measurement on a quadrupole time-of-flight (Q-TOF) mass spectrometer has aided the structural characterization and differentiation of the enmein and spiro-lactone types of ent-6,7-seco-kaurane diterpenoids from Isodon species. The mass spectral fragmentation data from both techniques was compared to obtain the mass spectrometric fragmentation pathways of the ent-6,7-seco-kaurane diterpenoids with high confidence. The analysis revealed that losses of CH(2)O and CO(2) are the predominant process for the enmein type of ent-kauranes in negative ion mode, and the loss of CO(2) is typical for the spiro-lactone type in positive ion mode. In addition, compounds of the spiro-lactone type with a conserved core structure but different substituent groups, such as acetyl, hydroxyl, and aldehyde moiety, resulted in diagnostic product ions by means of successive losses of AcOH, H(2)O, and CO, respectively. The fragmentation knowledge will facilitate the analysis and identification of the ent-6,7-seco-kauranes in future plant research.