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.     

Investigation of fragmentation behaviors of steroidal drugs with Li+, Na+, K+ adducts by tandem mass spectrometry aided with computational analysis

In this study, we have investigated the fragmentation of the widely used steroidal pharmaceutical drugs (n = 14), complexed by a singly charged proton or alkali metal ion (Li⁺, Na⁺, K⁺) using Ion trap and quadrupole time-of-flight mass spectrometers. Spectra were collected by LC-MS/MS analysis using system automated collision energy i.e., of 25–60 eV. Theoretical calculations were also calculated using DFT software. The metal complexes showed different fragmentation pathways not commonly observed for protonated compounds. There was a distinct difference observed in the relative intensities of some common fragments for free vs. metallated drugs. Some major fragments from protonated and lithium adducts showed close resemblance, while sodium and potassium adducts showed different fragments. Theoretical calculations showed a distinct difference in the position of attachment of proton and metals. This adducts ion fragmentation information will be helpful for the identification of these compounds in complex samples.     

Screening the life cycle of Schistosomamansoni using high-resolution mass spectrometry

Schistosomiasis is a common tropical disease caused by Schistosoma species Schistosomiasis' pathogenesis is known to vary according to the worms' strain. Moreover, high parasitical virulence is directly related to eggs release and granulomatous inflammation in the host's organs. This virulence might be influenced by different classes of molecules, such as lipids. Therefore, better understanding of the metabolic profile of these organisms is necessary, especially for an increased potential of unraveling strain virulence mechanisms and resistance to existing treatments. In this report, direct-infusion electrospray high-resolution mass spectrometry (ESI(+)-HRMS) along with the lipidomic platform were employed to rapidly characterize and differentiate two Brazilian S. mansoni strains (BH and SE) in three stages of their life cycle: eggs, miracidia and cercariae, with samples from experimental animals (Swiss/SPF mice). Furthermore, urine samples of the infected and uninfected mice were analyzed to assess the possibility of direct diagnosis. All samples were differentiated using multivariate data analysis, PCA, which helped electing markers from distinct lipid classes; phospholipids, diacylglycerols and triacylglycerols, for example, clearly presented different intensities in some stages and strains, as well as in urine samples. This indicates that biochemical characterization of S. mansoni may help narrowing-down the investigation of new therapeutic targets according to strain composition and aggressiveness of disease. Interestingly, lipid profile of infected mice urine varies when compared to control samples, indicating that direct diagnosis of schistosomiasis from urine may be feasible.     

Towards identification of traditional European and indigenous Australian paint binders using pyrolysis gas chromatography mass spectrometry

We report a pyrolysis GC–MS method capable of analysing Indigenous Australian and European binders typically used in the manufacture of culturally important painted works. Eleven different traditional European binders and ten different Indigenous Australian binders were examined. The method allows discrimination between highly complex and impure lipid, resin, polysaccharide, wax, and protein-based binders. Each was found to have characteristic pyrolysis products that were unique to the binder material, demonstrating the potential for differentiation of these binders on Australian Aboriginal artworks towards identification and conservation of cultural heritage.     

Dissimilarity analysis and automatic identification of monomethylalkanes from gas chromatography mass spectrometry data 1. Principle and protocols

Monomethylalkanes are common but important components in many naturally occurring and synthetic organic materials. Generally, this kind of compounds is routinely analyzed by gas chromatography mass spectrometry (GC–MS) and identified by the retention pattern or similarity matching to the reference mass spectral library. However, these identification approaches rely on the limited standard database or costly standard compounds. When unknown monomethylalkane is absent from the reference library, these approaches might be less useful. In this study, based on the fragmentation rules and empirical observation, many interesting mass spectral characteristics of monomethylalkanes were discovered and employed to infer the number of carbon atoms and methylated position. Combined with the retention pattern, a protocol was described for the identification of monomethylalkane analyzed by GC–MS. After tested by simulated data and GC–MS data of the gasoline sample, it was demonstrated that the developing approach could automatically and correctly identify monomethylalkanes in complicated GC–MS data.     

Ultra-high performance liquid chromatography tandem mass spectrometry for comprehensive analysis of urinary acylcarnitines

We report an enabling mass spectrometric method for the analysis of lipid metabolites in order to define better the lipid metabolome in terms of chemical diversity and generate fragment ion spectra of these metabolites as a potential resource for unknown metabolite identification. This work focuses on the analysis of one important class of lipid metabolites, the acylcarnitines. Current analytical methods have only detected and identified a limited number of these metabolites. The method described herein provides the most comprehensive acylcarnitine profile in urine of healthy individuals up to date. It involves an optimized solid phase extraction technique for selective analyte extraction using cartridges containing both lipophilic and cation-exchange properties. The captured analytes are then subjected to ultra-high performance liquid chromatography (UPLC) separation, followed by tandem mass spectrometry (MS/MS) analysis using information-dependent acquisitions and selected reaction monitoring (SRM). The urine of six healthy individuals was analyzed using this method. A total of 355 acylcarnitines were detected; only 43 of them have been previously reported in the urine of healthy individuals. Detection of this large number of acylcarnitines illustrates the great diversity of the lipid metabolome as well as the usefulness of the method for profiling acylcarnitines. Furthermore, the MS/MS spectra of the 355 acylcarnitines will be uploaded to a public human metabolome database as a mass spectrometric resource for unknown metabolite identification.     

Differential fragmentation patterns of pectin oligogalacturonides observed by nanoelectrospray quadrupole ion-trap mass spectrometry using automated spectra interpretation

Oligogalacturonides of different degrees of polymerization (DP) and methyl esterification (DE) were structurally analyzed by nanoESI quadrupole ion-trap mass spectrometry. The fragmentation patterns of the oligogalacturonides were compared using the program 'Virtual Expert Mass Spectrometrist' (VEMS) for structural annotation. In the analyzed oligogalacturonides of lower DP, the generation of C/Y ions, i.e. ions retaining the glycosidic oxygen, was higher than that of B/Z ions. In general, with oligogalacturonides of higher DP, the B/Z ions were generated more abundantly. Oligogalacturonides with free carboxylic acid groups underwent higher water loss compared to fully methyl-esterified oligogalacturonides under the same fragmentation conditions. Cross-ring cleavage, in which fragmentation occurs across the ring system of the galacturonate residue and signified by unique mass losses, was observed to be higher in fully methyl-esterified oligogalacturonides than in non-methyl-esterified ones. This study demonstrates the different fragmentation patterns of oligogalacturonides as influenced by the presence or absence of methyl ester groups. For a detailed analysis of unknown oligogalacturonides, cross-ring fragmentation gives more structural information than glycosidic bond cleavage. One implication of this is that more structural information is obtained when analyzing methyl-esterified oligogalacturonides than non-methyl-esterified ones in an ion-trap instrument. This is of particular importance in pectin chemistry, where mass spectrometry has become the technique of choice for structural determination. Although this study was not designed to explain the mechanisms of oligogalacturonide fragmentation, possible explanations for why non-methyl-esterified oligogalacturonides undergo more water loss than methyl-esterified ones will be postulated. In addition, the VEMS program was extended to automatically interpret and assign the fragment ions peaks generated in this study.     

Comprehensive analysis of chromatographic data by using PARAFAC2 and principal components analysis

The most straightforward method to analyze an obtained GC–MS dataset is to integrate those peaks that can be identified by their MS profile and to perform a Principal Component Analysis (PCA). This procedure has some important drawbacks, like baseline drifts being scarcely considered or the fact that integration boundaries are not always well defined (long tails, co-eluted peaks, etc.). To improve the methodology, and therefore, the chromatographic data analysis, this work proposes the modeling of the raw dataset by using PARAFAC2 algorithm in selected areas of the GC profile and using the obtained well-resolved chromatographic profiles to develop a further PCA model. With this working method, not only the problems arising from instrumental artifacts are overcome, but also the detection of new analytes is achieved as well as better understanding of the studied dataset is obtained. As a positive consequence of using the proposed working method human time and work are saved. To exemplify this methodology the aroma profile of 36 apples being ripened were studied. The benefits of the proposed methodology (PARAFAC2 + PCA) are shown in a practitioner perspective, being able to extrapolate the conclusions obtained here to other hyphenated chromatographic datasets.     

米格列奈及其3个异构体杂质的反相液相色谱分离及质谱碎裂分析

建立了超高效反相液相色谱-高分辨质谱方法以实现米格列奈及其3种异构体杂质的分离，以ACQUITY UPLC HSS T3（100 mm×2.1 mm，1.8 μ m）为色谱柱，以水-乙腈-正戊醇（75：25：1）（用甲酸调节pH至1.8）为流动相，流速为0.4 mL/min。根据Q Exactive四极杆/静电场轨道阱高分辨质谱的精确质量数及碎裂情况，发现了米格列奈及3种异构体存在碎片离子丰度的明显差异，确认其中两种为本次新发现的异构体杂质，并推断了米格列奈及3种异构体杂质可能的质谱裂解机理。经验证，该方法的灵敏度、重复性及线性均满足分析要求。在此基础上，对米格列奈异构体杂质的来源进行了探讨，发现异构体杂质1可在高温下降解产生，并对各企业的米格列奈钙原料样品进行了测定。     

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.     

Quantum chemical mass spectrometry: ab initio prediction of electron ionization mass spectra and identification of new fragmentation pathways

The electron ionization mass spectra of four organic compounds are predicted based on the results of quantum chemical calculations at the DFT/B3LYP/6‐311 + G* level of theory. This prediction is performed ‘ab initio’, i.e. without any prior knowledge of the thermodynamics or kinetics of the reactions under consideration. Using a set of rules determining which routes will be followed, the fragmentation of the molecules' bonds and the complete resulting fragmentation pathways are studied. The most likely fragmentation pathways are identified based on calculated reaction energies ΔE when bond cleavage is considered and on activation energies ΔE^? when rearrangements are taken into account; the final intensities of the peaks in the spectrum are estimated from these values. The main features observed in the experimental mass spectra are correctly predicted, as well as a number of minor peaks. In addition, the results of the calculations allow us to propose fragmentation pathways new to empirical mass spectrometry, which have been experimentally verified using tandem mass spectrometry measurements. Copyright © 2016 John Wiley & Sons, Ltd.     

Rapid automatic identification and quantification of compounds in complex matrices using comprehensive two-dimensional gas chromatography coupled to high resolution time-of-flight mass spectrometry with a peak sentinel tool

Comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC × GC–MS) is a powerful tool for comprehensive analysis of organic pollutants. In this study, we developed a powerful analytical method using GC × GC for rapid and accurate identification and quantification of compounds in environmental samples with complex matrices. Specifically, we have developed an automatic peak sentinel tool, T-SEN, with free programming software, R. The tool, which consists of a simple algorithm for on peak finding and peak shape identification, allows rapid screening of target compounds, even for large data sets from GC × GC coupled to high resolution time of flight mass spectrometry (HRTOFMS). The software tool automatically assigns and quantifies compounds that are listed in user databases. T-SEN works on a typical 64 bit workstation, and the reference calculation speed is 10–20 min for approximately 170 compounds for peak finding (five ion count setting) and integration from 1–2 GB of sample data acquired by GC × GC–HRTOFMS. We analyzed and quantified 17 PCDD/F congeners and 24 PCB congeners in a crude lake sediment extract by both GC × GC coupled to quadrupole mass spectrometry (qMS) and GC × GC–HRTOFMS with T-SEN. While GC × GC–qMS with T-SEN resulted in false identification and inaccurate quantification, GC × GC–HRTOFMS with T-SEN provided correct identification and accurate quantification of compounds without sample pre-treatment. The differences between the values measured by GC × GC–HRTOFMS with T-SEN and the certified values for the certified reference material ranged from 7.3 to 36.9% for compounds with concentrations above the limit of quantification. False positives/negatives were not observed, except for when co-elution occurred. The technique of GC × GC–HRTOFMS in combination with T-SEN provides rapid and accurate screening and represents a powerful new approach for comprehensive analysis.     

Profiling monoterpenol glycoconjugation in Vitis vinifera L. cv. Muscat of Alexandria using a novel putative compound database approach,high resolution mass spectrometry and collision induced dissociation fragmentation analysis

In this work we present a novel approach for the identification of plant metabolites using ultrahigh performance liquid chromatography coupled to accurate mass time-of-flight mass spectrometry. The workflow involves developing an in-house compound database consisting of exact masses of previously identified as well as putative compounds. The database is used to screen accurate mass spectrometry (MS) data to identify possible compound matches. Subsequent tandem MS data is acquired for possible matches and used for structural elucidation. The methodology is applied to profile monoterpene glycosides in Vitis vinifera cv. Muscat of Alexandria grape berries over three developmental stages. Monoterpenes are a subclass of terpenes, the largest class of plant secondary metabolites, and are found in two major forms in the plant, “bound” to one or more sugar moieties or “free” of said sugar moieties. In the free form, monoterpenes are noted for their fragrance and play important roles in plant defense and as attractants for pollinators. However, glycoconjugation renders these compounds odorless, and it is this form that the plant uses for monoterpene storage. In order to gain insight into monoterpene biochemistry and their fate in the plant an analysis of intact glycosides is essential. Eighteen monoterpene glycosides were identified including a monoterpene trisaccharide glycoside, which is tentatively identified here for this first time in any plant. Additionally, while previous studies have identified monoterpene malonylated glucosides in other grapevine tissue, we tentatively identify them for the first time in grape berries. This analytical approach can be readily applied to other plants and the workflow approach can also be used for other classes of compounds. This approach, in general, provides researchers with data to support the identification of putative compounds, which is especially useful when no standard is available.     

超高效液相色谱-串联四极杆质谱法分析婴儿乳粉中的唾液酸

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)测定婴儿配方乳粉中唾液酸含量的分析方法。利用酸水解方法释放出婴儿配方乳中的唾液酸,经HLB反相色谱固相萃取柱净化,采用BEH HILIC色谱柱分离,以0.1%甲酸水溶液和100%乙腈溶液作为流动相进行梯度洗脱,流速为0.25 mL/min,进样体积5 μL,柱温30℃,电喷雾质谱检测,正离子多反应监测模式进行定性和定量分析。结果表明:唾液酸在0.05~5.0 mg/L范围内与唾液酸峰面积的线性关系良好(R²=0.9989);以0.1、0.5、2.5和5.0 mg/L 4个添加水平进行添加回收试验,唾液酸的平均回收率为84.3%~98.9%,相对标准偏差为4.9%~8.2%;唾液酸的检出限为0.01 mg/L。该方法简单、快速、重复性好、灵敏度高,可广泛用于奶粉、牛奶及母乳中唾液酸含量的分析测定。     

Tandem mass spectrometric analysis of novel diquaternary ammonium gemini surfactants and their bromide adducts in electrospray-positive ion mode ionization

Gemini surfactants are cationic lipids which are utilized for both in vitro and in vivo gene delivery. Structurally, they are comprised of two hydrophobic tail regions with polar head termini that are attached to one another through a spacer region. Structural elucidation and characterization of 29 novel diquaternary ammonium gemini surfactant molecules were achieved using a quadrupole time-of-flight mass spectrometer (QqToF-MS) and a quadrupole-hexapole-quadrupole mass spectrometer (QhQ-MS). The tested compounds were categorized into four distinct structural families based upon the composition of the spacer region. Single stage (MS), tandem stage (MS/MS) and quasimulti-stage (quasi MS(3)) mass spectrometric analysis allowed for confirmation of each gemini surfactant's molecular composition and structure through the identification of common and unique product ions. Identification of similarities in the gemini surfactants' fragmentation behaviour resulted in the production of a universal fragmentation pathway that can assist in the future MS/MS analysis of novel quaternary ammonium gemini surfactants, with unique product ions being indicative of specific structural elements. Furthermore, evidence for the association of agemini surfactant with bromine counter ion was confirmed during MS analysis of tested gemini surfactants regardless of their chemical composition; previously, evidence for bromine and gemini surfactant association was only observed with compounds bearing short alkyl spacer regions. MS/MS analysis of the bromine adducts was also confirmatory to the molecular structure.Understanding the ionization and fragmentation behaviour of gemini surfactants, including bromine adducts, will allow for future qualitative and quantitative identification of these novel drug delivery agents within biological samples.     

Specific features of resonance electron capture mass spectra of ecdysteroid molecules

Specificity of the dissociative attachment of low-energy electrons to ecdysteroid molecules (viz., 20-hydroxyecdysone 2,3:20,22-diacetonide, 20-hydroxyecdysone 20,22-acetonide, and poststerone 2-acetate) was found, which is manifested as the formation of long-lived pseudo-molecular negative ions that appeared due to the elimination of the H₂ and H₂O molecules. These rearrangements are resulted from the formation of the system of conjugated double bonds in the ecdysteroid skeleton, stabilizing the lowest vacant molecular orbital.     

Use of ultra-performance liquid chromatography/time-of-flight mass spectrometry with nozzle-skimmer fragmentation for comprehensive quantitative analysis of secondary metabolites in Arabidopsis thaliana

This study sought to develop techniques for LC/MS-based metabolomics and to verify that an MS/MS spectral tag (MS2T) could be used in practical secondary metabolite profiling. The retention time (RT), precursor ions, and fragment ions generated by nozzle-skimmer fragmentation were determined using ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) and compared with the MS2T. A standard mix was analyzed with UPLC/TOF-MS under the same conditions as were used to construct the MS2T. The difference in RT for the standards was less than 0.15 min and the average RSD was about 2.8%, suggesting that the analysis was highly repeatable. Both precursor ions and fragment ions were observed when the cone voltage was 75 V. Experimental data and fragmentation pattern in the MS2T annotation list were highly similar. Wild-type and cas-1 mutant Arabidopsis thaliana samples treated with an elicitor were analyzed using UPLC/TOF-MS. Sixty-five peaks were successfully annotated. Fragment ions were observed with nozzle-skimmer fragmentation in 50 of 65 (77%) peaks. The reliability of annotation may have increased as a result of fragment ions. Results of multivariate analysis suggested that cas-1 was related to induction of the biosynthesis of these flavonoids. The devised method facilitated practical secondary metabolite profiling.     

液相色谱-电喷雾离子阱串联质谱同时分析烟草中的20种游离氨基酸

建立了液相色谱-电喷雾离子阱串联质谱(LC-ESI-IT-MS/MS)同时分析烟草中20种游离氨基酸的方法。烟草样品经萃取后过滤直接进样,无需进行衍生和固相萃取等其他前处理步骤。液相色谱采用HyPURITY C18反相色谱柱(200 mm×2.1 mm, 5 μm),采用1%(体积分数,下同)乙腈水溶液(含0.1%九氟戊酸)和90%乙腈水溶液(含0.1%九氟戊酸)为流动相进行梯度洗脱。结果表明,20种氨基酸的检出限(LOD)为0.01～0.05 μmol/L (S/N=3),线性相关系数均大于0.9977,峰面积测定的相对标准偏差(RSD)为0.78%～4.93%。该方法分析效率、灵敏度和选择性高,已成功应用于多种烟草样品中氨基酸的分析测定。