Automatic recalibration and processing of tandem mass spectra using formula annotation

High accuracy, high resolution tandem mass spectrometry (MS/MS) is becoming more common in analytical applications, yet databases of these spectra remain limited. Databases require good quality spectra with sufficient compound information, but processing, calibration, noise reduction and retrieval of compound information are time‐consuming tasks that prevent many contributions. We present a comprehensive workflow for the automatic processing of MS/MS using formula annotation for recalibration and cleanup to generate high quality spectra of standard compounds for upload to MassBank ( www.massbank.jp ). Compound information is retrieved via Internet services. Reference standards of 70 pesticides were measured at various collision energies on an LTQ‐Orbitrap XL to develop and evaluate the workflow. A total of 944 resulting spectra are now available on MassBank. Evidence of nitrogen adduct formation during MS/MS fragmentation processes was found, highlighting the benefits high accuracy MS/MS offers for spectral interpretation. A database of recalibrated, cleaned‐up spectra resulted in the most correct spectra ranked in first place, regardless of whether the search spectra were recalibrated or not, whereas the average rank of the correct molecular formula was improved from 2.55 (uncalibrated) to 1.53 when using recalibrated MS/MS data. The workflow is available as an R package RMassBank capable of generating MassBank records from raw MS and MS/MS data and can be adjusted to process data acquired with different settings and instruments. This workflow is a vital step towards addressing the need for more high quality, high accuracy MS/MS spectra in spectral databases and provides important information for spectral interpretation. Copyright © 2012 John Wiley & Sons, Ltd.     

MassBank: a public repository for sharing mass spectral data for life sciences

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron‐ionization mass spectrometry(EI‐MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)‐MSⁿ data of 2337 authentic compounds of metabolites, 11 545 EI‐MS and 834 other‐MS data of 10 286 volatile natural and synthetic compounds, and 3045 ESI‐MS² data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI‐MS² data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass‐to‐charge ratio are optimized to the ESI‐MS² data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI‐MS² data on an identical compound under different collision‐induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21–23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data. Copyright © 2010 John Wiley & Sons, Ltd.     

Towards a full reference library of MS(n) spectra. Testing of a library containing 3126 MS2 spectra of 1743 compounds

A library consisting of 3766 MS(n) spectra of 1743 compounds, including 3126 MS2 spectra acquired mainly using ion trap (IT) and triple-quadrupole (QqQ) instruments, was composed of numerous collections/sources. Ionization techniques were mainly electrospray ionization and also atmospheric pressure chemical ionization and chemical ionization. The library was tested for the performance in identification of unknowns, and in this context this work is believed to be the largest of all known tests of product-ion mass spectral libraries. The MS2 spectra of the same compounds from different collections were in turn divided into spectra of 'unknown' and reference compounds. For each particular compound, library searches were performed resulting in selection by taking into account the best matches for each spectral collection/source. Within each collection/source, replicate MS2 spectra differed in the collision energy used. Overall, there were up to 950 search results giving the best match factors and their ranks in corresponding hit lists. In general, the correct answers were obtained as the 1st rank in up to 60% of the search results when retrieved with (on average) 2.2 'unknown' and 6.2 reference replicates per compound. With two or more replicates of both 'unknown' and reference spectra (the average numbers of replicates were 4.0 and 7.8, respectively), the fraction of correct answers in the 1st rank increased to 77%. This value is close to the performance of established electron ionization mass spectra libraries (up to 79%) found by other workers. The hypothesis that MS2 spectra better match reference spectra acquired using the same type of tandem mass spectrometer (IT or QqQ) was neither strongly proved nor rejected here. The present work shows that MS2 spectral libraries containing sufficiently numerous different entries for each compound are sufficiently efficient for identification of unknowns and suitable for use with different tandem mass spectrometers.     

On the inter‐instrument and inter‐laboratory transferability of a tandem mass spectral reference library: 1. Results of an Austrian multicenter study

The inter‐instrument and inter‐laboratory transferability of a tandem mass spectral reference library originally built on a quadrupole‐quadrupole‐time‐of‐flight instrument was examined. The library consisted of 3759 MS/MS spectra collected from 402 reference compounds applying several different collision‐energy values for fragmentation. In the course of the multicenter study, 22 test compounds were sent to three different laboratories, where 418 tandem mass spectra were acquired using four different instruments from two manufacturers. The study covered the following types of tandem mass spectrometers: quadrupole‐quadrupole‐time‐of‐flight, quadrupole‐quadrupole‐linear ion trap, quadrupole‐quadrupole‐quadrupole, and linear ion trap‐Fourier transform ion cyclotron resonance mass spectrometer. In each participating laboratory, optimized instrumental parameters were gathered solely from routinely applied workflows. No standardization procedure was applied to increase the inter‐instrument comparability of MS/MS spectra. The acquired tandem mass spectra were matched against the established reference library using a sophisticated matching algorithm, which is presented in detail in a companion paper. Correct answers, meaning that the correct compound was retrieved as top hit, were obtained in 98.1% of cases. For the remaining 1.9% of spectra, the correct compound was matched at second rank. The observed high percentage of correct assignments clearly suggests that the developed mass spectral library search approach is to a large extent platform independent. Copyright © 2009 John Wiley & Sons, Ltd.     

Towards a full reference library of MSn spectra. II: A perspective from the library of pesticide spectra extracted from the literature/Internet

To gain perspective on building full transferable libraries of MSⁿ spectra from their diverse/numerous collections, a new library was built from 1723 MS^>1 spectra (mainly MS² spectra) of 490 pesticides and related compounds. Spectra acquired on different types of tandem instruments in various experimental conditions were extracted from 168 literature articles and Internet sites. Testing of the library was based on searches where 'unknown' and reference spectra originated from different sources (mainly from different laboratories) were cross‐compared. The NIST 05 MS² library was added to the reference spectra. The library searches were performed with all the test spectra or were divided into different subsamples containing (a) various numbers of replicate spectra of test compounds or (b) spectra acquired from different instrument types. Thus, the dependence of true/false search (identification) result rates on different factors was explored. The percentage of 1^st rank correct identifications (true positives) for the only 'unknown' mass spectrum and two and more reference spectra and matching precursor ion m/z values was 89%. For qualified matches, above the cut‐off match factor, that rate decreased to 80%. The corresponding rates based on the best match for two and more 'unknown' and reference spectral replicates were 89–94%. For quadrupole instruments, the rates were even higher: 91–95% (one 'unknown' spectrum) and 90–100% (two and more such spectra). This study shows that MS² spectral libraries generated from the numerous literature/Internet sources are not less efficient for the goal of identification of unknown compounds including pesticides than very common EI‐MS¹ libraries and are almost as efficient as the most productive from current MS² spectral databases. Such libraries may be used as individual reference databases or supplements to large experimental spectral collections covering many groups of abundant compounds and different types of tandem mass spectrometers. Copyright © 2011 John Wiley & Sons, Ltd.     

Is nontarget screening of emerging contaminants by LC-HRMS successful? A plea for compound libraries and computer tools

This review focuses on the possibilities and limits of nontarget screening of emerging contaminants, with emphasis on recent applications and developments in data evaluation and compound identification by liquid chromatography-high-resolution mass spectrometry (HRMS). The general workflow includes determination of the elemental composition from accurate mass, a further search for the molecular formula in compound libraries or general chemical databases, and a ranking of the proposed structures using further information, e.g., from mass spectrometry (MS) fragmentation and retention times. The success of nontarget screening is in some way limited to the preselection of relevant compounds from a large data set. Recently developed approaches show that statistical analysis in combination with suspect and nontarget screening are useful methods to preselect relevant compounds. Currently, the unequivocal identification of unknowns still requires information from an authentic standard which has to be measured or is already available in user-defined MS/MS reference databases or libraries containing HRMS spectral information and retention times. In this context, we discuss the advantages and future needs of publicly available MS and MS/MS reference databases and libraries which have mostly been created for the metabolomic field. A big step forward has been achieved with computer-based tools when no MS library or MS database entry is found for a compound. The numerous search results from a large chemical database can be condensed to only a few by in silico fragmentation. This has been demonstrated for selected compounds and metabolites in recent publications. Still, only very few compounds have been identified or tentatively identified in environmental samples by nontarget screening. The availability of comprehensive MS libraries with a focus on environmental contaminants would tremendously improve the situation.     

Testing an alternative search algorithm for compound identification with the ‘Wiley Registry of Tandem Mass Spectral Data,MSforID’

A tandem mass spectral database system consists of a library of reference spectra and a search program. State‐of‐the‐art search programs show a high tolerance for variability in compound‐specific fragmentation patterns produced by collision‐induced decomposition and enable sensitive and specific ‘identity search’. In this communication, performance characteristics of two search algorithms combined with the ‘Wiley Registry of Tandem Mass Spectral Data, MSforID’ (Wiley Registry MSMS, John Wiley and Sons, Hoboken, NJ, USA) were evaluated. The search algorithms tested were the MSMS search algorithm implemented in the NIST MS Search program 2.0g (NIST, Gaithersburg, MD, USA) and the MSforID algorithm (John Wiley and Sons, Hoboken, NJ, USA). Sample spectra were acquired on different instruments and, thus, covered a broad range of possible experimental conditions or were generated in silico. For each algorithm, more than 30 000 matches were performed. Statistical evaluation of the library search results revealed that principally both search algorithms can be combined with the Wiley Registry MSMS to create a reliable identification tool. It appears, however, that a higher degree of spectral similarity is necessary to obtain a correct match with the NIST MS Search program. This characteristic of the NIST MS Search program has a positive effect on specificity as it helps to avoid false positive matches (type I errors), but reduces sensitivity. Thus, particularly with sample spectra acquired on instruments differing in their setup from tandem‐in‐space type fragmentation, a comparably higher number of false negative matches (type II errors) were observed by searching the Wiley Registry MSMS. Copyright © 2013 John Wiley & Sons, Ltd.     

ESI-MS/MS library of 1,253 compounds for application in forensic and clinical toxicology

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) library which contains over 5,600 spectra of 1,253 compounds relevant in clinical and forensic toxicology has been developed using a hybrid tandem mass spectrometer with a linear ion trap. Pure compound solutions—in some cases solutions made of tablets—were prepared and 1 to 2,000 ng of each compound were injected into the system using standard reversed-phase analytical columns with gradient elution. To obtain maximum mass spectral information enhanced product ion spectra were acquired with positive and/or negative ionization at low, medium, and high collision energies and additionally applying collision energy spread. In this mode, all product ions generated by the different collision energies are trapped in the linear ion trap prior to their detection. The applicability of the library for other types of hybrid tandem mass spectrometers with a linear ion trap of the same manufacturer as well as a standard triple-quadrupole tandem mass spectrometer has been investigated with a selection of compounds. The spectra of the developed library can be used to create methods for target analysis, either screening methods or quantitative procedures by generating transitions for multiple reaction monitoring. For those procedures, suitable transitions and convenient collision energies are selected from the library. It also has been utilized to identify compounds with a multi target screening approach for clinical and forensic toxicology with a standardized and automated system. The novel aspects compared to our former library produced with a standard triple-quadrupole mass spectrometer are the enlargement of the ESI-MS/MS library and the additional acquisition of spectra with collision energy spread.     

On the inter‐instrument and the inter‐laboratory transferability of a tandem mass spectral reference library. 3. Focus on ion trap and upfront CID

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the ‘Wiley Registry of Tandem Mass Spectral Data, MSforID’. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem‐in‐space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem‐in‐time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time‐of‐flight (TOF), quadrupole–hexapole–TOF, linear ion trap (LIT), 3‐D ion trap and LIT–Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with ‘relative average match probability’ >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool. Copyright © 2012 John Wiley & Sons, Ltd.     

Computational mass spectrometry for metabolomics: Identification of metabolites and small molecules

The identification of compounds from mass spectrometry (MS) data is still seen as a major bottleneck in the interpretation of MS data. This is particularly the case for the identification of small compounds such as metabolites, where until recently little progress has been made. Here we review the available approaches to annotation and identification of chemical compounds based on electrospray ionization (ESI-MS) data. The methods are not limited to metabolomics applications, but are applicable to any small compounds amenable to MS analysis. Starting with the definition of identification, we focus on the analysis of tandem mass and MS(n) spectra, which can provide a wealth of structural information. Searching in libraries of reference spectra provides the most reliable source of identification, especially if measured on comparable instruments. We review several choices for the distance functions. The identification without reference spectra is even more challenging, because it requires approaches to interpret tandem mass spectra with regard to the molecular structure. Both commercial and free tools are capable of mining general-purpose compound libraries, and identifying candidate compounds. The holy grail of computational mass spectrometry is the de novo deduction of structure hypotheses for compounds, where method development has only started thus far. In a case study, we apply several of the available methods to the three compounds, kaempferol, reserpine, and verapamil, and investigate whether this results in reliable identifications.     

Use of quadrupole time-of-flight mass spectrometry in the elucidation of unknown compounds present in environmental water

Target compound monitoring is often not sufficient to assess the quality of water, as many of the unknown micro-contaminants present might be a threat to the environment and human health. In this work, the potential of hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS) coupled to liquid chromatography (LC) in the elucidation of unknown compounds in environmental water samples has been explored. Based on accurate mass measurement, possible elemental compositions for the precursor ions were calculated. Using model compounds, a useful strategy was developed, enabling determination and evaluation of potential molecular formulae for the detected unknowns. The possibility of performing tandem mass spectrometric (MS/MS) acquisitions to obtain product ion spectra in accurate mass mode also helped to elucidate the structures of these unknowns or to detect some functional groups, to further evaluate potential formulae. The remaining formulae were searched against available databases such as the Merck Index and the NIST library. Where standards were commercially available, retention time and MS/MS data were both also used as confirmatory tools. The approach developed was applied for the identification of unknown compounds in different types of water. To improve sensitivity, environmental water samples were preconcentrated on-line in a polymeric cartridge by direct injection of 2 mL water into the SPE-LC/MS/MS system. For three unknowns, structures were proposed and confirmed with standards. Although other compounds could not be unequivocally identified based on the data available within this study, details about the possible structures of some are given.     

基于解卷积报告软件数据库的GC/MS方法快速确证玩具中160多种有毒有害有机物质

自主建立了基于解卷积报告软件的含有160多种有毒有害有机物质的数据库,作为标准的GC/MS数据库来确证化合物,不用1 min解卷积报告软件即可帮助确证谱图中隐藏的上述有毒有害有机物质.该方法已经在众多玩具有机化学安全检测项目中得到了应用,不仅在某检测项目目标化合物如禁用偶氮物质等确证方面全面优于目前基于GC/MS化学工...     

Development of a dedicated peptide tandem mass spectral library for conservation science

In recent years, the use of liquid chromatography tandem mass spectrometry (LC–MS/MS) on tryptic digests of cultural heritage objects has attracted much attention. It allows for unambiguous identification of peptides and proteins, and even in complex mixtures species-specific identification becomes feasible with minimal sample consumption. Determination of the peptides is commonly based on theoretical cleavage of known protein sequences and on comparison of the expected peptide fragments with those found in the MS/MS spectra. In this approach, complex computer programs, such as Mascot, perform well identifying known proteins, but fail when protein sequences are unknown or incomplete. Often, when trying to distinguish evolutionarily well preserved collagens of different species, Mascot lacks the required specificity. Complementary and often more accurate information on the proteins can be obtained using a reference library of MS/MS spectra of species-specific peptides. Therefore, a library dedicated to various sources of proteins in works of art was set up, with an initial focus on collagen rich materials. This paper discusses the construction and the advantages of this spectral library for conservation science, and its application on a number of samples from historical works of art.     

Approaches towards the automated interpretation and prediction of electrospray tandem mass spectra of non-peptidic combinatorial compounds

Combinatorial chemistry is widely used within the pharmaceutical industry as a means of rapid identification of potential drugs. With the growth of combinatorial libraries, mass spectrometry (MS) became the key analytical technique because of its speed of analysis, sensitivity, accuracy and ability to be coupled with other analytical techniques. In the majority of cases, electrospray mass spectrometry (ES-MS) has become the default ionisation technique. However, due to the absence of fragment ions in the resulting spectra, tandem mass spectrometry (MS/MS) is required to provide structural information for the identification of an unknown analyte. This work discusses the first steps of an investigation into the fragmentation pathways taking place in electrospray tandem mass spectrometry. The ultimate goal for this project is to set general fragmentation rules for non-peptidic, pharmaceutical, combinatorial compounds. As an aid, an artificial intelligence (AI) software package is used to facilitate interpretation of the spectra. This initial study has focused on determining the fragmentation rules for some classes of compound types that fit the remit as outlined above. Based on studies carried out on several combinatorial libraries of these compounds, it was established that different classes of drug molecules follow unique fragmentation pathways. In addition to these general observations, the specific ionisation processes and the fragmentation pathways involved in the electrospray mass spectra of these systems were explored. The ultimate goal will be to incorporate our findings into the computer program and allow identification of an unknown, non-peptidic compound following insertion of its ES-MS/MS spectrum into the AI package. The work herein demonstrates the potential benefit of such an approach in addressing the issue of high-throughput, automated MS/MS data interpretation.     

Metabolite profiling and beyond: approaches for the rapid processing and annotation of human blood serum mass spectrometry data

In this paper, we describe data processing and metabolite identification approaches which lead to a rapid and semi-automated interpretation of metabolomics experiments. Data from metabolite fingerprinting using LC-ESI-Q-TOF/MS were processed with several open-source software packages, including XCMS and CAMERA to detect features and group features into compound spectra. Next, we describe the automatic scheduling of tandem mass spectrometry (MS) acquisitions to acquire a large number of MS/MS spectra, and the subsequent processing and computer-assisted annotation towards identification using the R packages MetShot, Rdisop, and the MetFusion application. We also implement a simple retention time prediction model using predicted lipophilicity logD, which predicts retention times within 42 s (6 min gradient) for most compounds in our setup. We putatively identified 44 common metabolites including several amino acids and phospholipids at metabolomics standards initiative (MSI) levels two and three and confirmed the majority of them by comparison with authentic standards at MSI level one. To aid both data integration within and data sharing between laboratories, we integrated data from two labs and mapped retention times between the chromatographic systems. Despite the different MS instrumentation and different chromatographic gradient programs, the mapped retention times agree within 26 s (20 min gradient) for 90 % of the mapped features.

Combinatorial synthesis, reversed-phase and normal-phase high-performance liquid chromatography elution data and liquid chromatography/positive atmospheric pressure chemical ionization tandem mass spectra of methoxylated and glycosylated resveratrol analogues

Analyses of methoxylated and glycosylated stilbenes remain scarce in the literature because of the commercial unavailability of these compounds. Here a library of 22 compounds was synthesized by combinatorial chemistry. Their elution profiles were compared on three different columns (C18, C8, and silica) with those of seven commercial resveratrol analogues and two viniferins. The spectra recorded by liquid chromatography/positive atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI(+)-MS/MS) are discussed and recommendations made for easier identification of new stilbenes.     

Screening and identification of unknown contaminants in water with liquid chromatography and quadrupole-orthogonal acceleration-time-of-flight tandem mass spectrometry

In order to assess and maintain the quality of surface waters, target compound monitoring is often not sufficient. Many unknown micro-contaminants are present in water, originating in municipal, industrial or agricultural effluents. Some of these might pose a risk to drinking water production and consequently to human health. The possibilities of screening surface water and identification of these non-target water pollutants with modern data acquisition possibilities of hybrid quadrupole-orthogonal acceleration time of flight mass spectrometers (Q-TOF), such as data-dependent MS to MS/MS switching were investigated. Using model compounds, a procedure for the liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening of water extracts was developed, enabling the detection and identification of compounds at levels < or = 0.25 microg/l in surface water. Based on the accurate mass the elemental compositions for the precursor and product ions are calculated. The calculated chemical formulae are searched against the Merck index, the NIST library, an own database containing about 2,500 water pollutants (pesticides and other contaminants) as well as a CI-CID library containing tandem MS spectra of about 100 water contaminants. The developed approach was applied for the identification of unknown compounds, present in native surface water extract. For three of these compounds, structures were proposed. Confirmation of the proposed structures with standards was beyond the scope of this study.     

How to distinguish between feruloyl quinic acids and isoferuloyl quinic acids by liquid chromatography/tandem mass spectrometry

All four regioisomers of feruloyl quinic acid and isoferuloyl quinic acid were synthesized and a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method developed that resolves all eight regioisomers. All eight regioisomers can be readily distinguished by their MS/MS spectra in the negative ion mode, illustrating the power of tandem mass spectrometry to elucidate the structures of regioisomeric compounds. Compound identification is possible, either by direct comparison of spectral fingerprints or by rational probing of diagnostic fragment ions, thus allowing the identification of these important classes of natural products and potential human metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP® LC-MS/MS system and library searching

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a “scheduled” survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP^® hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization–MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography–mass spectrometry, and liquid chromatography–diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).     

Structural investigation and elucidation of new communesins from a marine‐derived Penicillium expansum Link by liquid chromatography/electrospray ionization mass spectrometry

Penicillium expansum is a ubiquitous species for which there are only few reports for chemical investigation in marine environments. Among the numerous secondary metabolites produced by this species, communesins represent a new class of cytotoxic and insecticidal indole alkaloids. In this study, we investigated a marine P. expansum strain exhibiting neuroactivity on a Diptera larvae bioassay. Bio‐guided purification led to the isolation and the identification of communesin B as the main active compound by HRMS and ¹H and ¹³C NMR. Liquid chromatography analyses with detection by electrospray ionization coupled with tandem mass spectrometry (LC/ESI‐MS/MS) and high‐resolution tandem mass spectrometry (LC/HRMS/MS) allowed the identification and characterization of four other known communesins (A, D, E and F) in the crude extract. A fragmentation model for dimethyl epoxide communesins was proposed after detailed interpretation of their MS/MS spectra. Further analyses of the extract using the modelled fragmentations led to the detection of seven new communesins found as minor compounds. Chemical structural elucidation of these new derivatives is discussed based on their fragmentation characteristics. Copyright © 2009 John Wiley & Sons, Ltd.