Enhanced pseudotargeted analysis using a segment data dependent acquisition strategy by liquid chromatography–tandem mass spectrometry for a metabolomics study of liquiritin in the treatment of depression

An enhanced pseudotargeted method using a segment data‐dependent acquisition mode based on ultra‐high performance liquid chromatography–tandem mass spectrometry was developed. This segment data dependent acquisition‐based pseudotargeted method could improve the detection of co‐eluted ions and extend the coverage of analytes. A set of 502 multiple reaction monitoring channels were obtained by this segment strategy, which was twice the number created by the traditional data‐dependent acquisition mode. Compared with the untargeted method, the pseudotargeted profiling demonstrated higher sensitivity and higher precision. More than 90% of the metabolites detected by the enhanced pseudotargeted method had relative standard deviations less than 15%. The segment data dependent acquisition‐based pseudotargeted method was successfully applied to the metabolomics study of the depressed rats with the treatment of liquiritin. Forty‐seven differential metabolites were screened and five metabolic pathways were found to be related to depression including retinol metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, terpenoid backbone biosynthesis, and lysine degradation. The segment data dependent acquisition‐based pseudotargeted method widened the coverage of metabolites with good sensitivity and precision, which exhibited great potential in the discovery of differential metabolites in metabolomics studies.     

Ultra-high performance liquid chromatography/ion mobility time-of-flight mass spectrometry-based untargeted metabolomics combined with quantitative assay unveiled the metabolic difference among the root,leaf, and flower bud of Panax notoginseng

Despite Panax notoginseng (Sanchi: the root and rhizome) is globally popular serving as the source of food additives, health-care products, and traditional Chinese medicines (TCMs), the saponin difference between the root (PNR) and two aerial parts (leaf, PNL; flower bud, PNF) that can be vicariously used, remains unclear. Authentication of Sanchi, particular from the Chinese patent medicines (CPMs), poses great challenges. Ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS)-based untargeted metabolomics and quantitative assay by UHPLC-UV were utilized to establish the “Identification Markers” for Sanchi. Targeted monitoring of multiple identification markers was performed for authenticating Sanchi simultaneously from 15 different CPMs. Dimension-enhanced profiling by UHPLC/IM-QTOF-MS in the negative high-definition MS^E (HDMS^E) mode and in-house library-driven peak annotation could characterize totally 328 ginsenosides (133 from PNR, 125 from PNL, and 161 from PNF). Multivariate statistical analysis of the PNR/PNL/PNF samples (45 batches) identified 27 potential markers. Five major markers (notoginsenoside R1, ginsenosides Rg1, -Rb1, -Rb2, and -Rb3) thereof were quantitatively assayed by a fully validated UHPLC-UV (detected at 203 nm) approach. The application of selective ion monitoring (SIM) of 12 differential saponins coupled with UHPLC separation could precisely identify Sanchi from 15 different CPMs (45 batches). Holistic difference in ginsenosides among three parts of P. notoginseng was unveiled, and the markers deduced may assist to identify the illicit substitution of leaf or flower as the root in the TCM compound formulae. Conclusively, the integration of untargeted metabolomics and quantitative analysis can provide reliable information enabling the precise authentication of TCM.     

Analytical techniques for single-cell metabolomics: state of the art and trends

Single-cell metabolomics is an emerging field that addresses fundamental biological questions and allows one to observe metabolic phenomena in heterogeneous populations of single cells. In this review, we assess the suitability of different detection techniques and present considerations on sample preparation for single-cell metabolomics. Although targeted analysis of single cells can readily be conducted using fluorescent probes and optical instruments (microscopes, fluorescence detectors), a comprehensive metabolomic approach requires a powerful label-free method, such as mass spectrometry (MS). Mass-spectrometric techniques applied to study small molecules in single cells include electrospray MS, matrix-assisted laser desorption/ionization MS, and secondary ion MS. Sample preparation is an important aspect to be taken into account during further development of methods for single-cell metabolomics.     

Evidence for linkage position determination in known feruloylated mono- and disaccharides using electrospray ion trap mass spectrometry

Various feruloylated arabinose- and galactose-containing mono- and disaccharides with known linkage configurations (2-O-(trans-feruloyl)-L-arabinopyranose, 5-O-(trans-feruloyl)-L-arabinofuranose, O-[2-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->5)-L-arabinofuranose, and O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose) were analyzed by electrospray ionization mass spectrometry using an ion trap or a quadrupole time-of-flight (Q-TOF) mass analyzer. Collision-induced dissociation (CID) experiments using the two mass analyzers generated similar tandem mass spectrometric (MS/MS) fragmentation patterns. However, the ester-bond cleavage ions were more abundant using the Q-TOF mass analyzer. Compared with the positive ion mode, the negative ion mode produces simpler and more useful CID product-ion patterns. For arabinose-containing feruloylated compounds, results obtained with both analyzers show that it is possible to assign the location of the feruloyl group to the O-2 or O-5 of arabinosyl residues. In the characterization of the 2-O-feruloyl and 5-O-feruloyl linkages, the relative abundance of the cross-ring fragment ions at m/z 265 (-60 u or -62 u after 18O-labelling) and at m/z 217 (-108 u or -110 u after 18O-labelling) play a relevant role. For galactose-containing feruloylated compounds, losses of 60, 90 and 120 Da observed in MS3 experiment correspond to the production of 0,2A1, 0,3A1 and (0,2A1-60 Da) cross-ring cleavage ions, respectively, fixing the location of feruloyl group at the O-6 of the galactose residue.     

A pseudotargeted method based on sequential window acquisition of all theoretical spectra mass spectrometry acquisition and its application in quality assessment of traditional Chinese medicine preparation-Yuanhu Zhitong tablet

Quality control plays a key role in the application of Chinese materia medica, especially in the preparation of traditional Chinese medicine. A pseudotargeted analysis method using an ultra-high-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry that was operated in the sequential window acquisition of all theoretical spectra mode was proposed to explore the chemical markers of traditional Chinese medicine preparation. Full-scan-based untargeted analysis was applied to extract the target ions. After data preprocessing, 302 target ions were extracted and used for the subsequent sequential window acquisition of all theoretical spectra analyses. The established sequential window acquisition of all theoretical spectra-based pseudotargeted approaches exhibited good repeatability and a wide linear range. The established method was successfully applied to discover analytical markers for the Yuanhu Zhitong tablet. After multivariate statistical analysis, 94 potential markers were identified. Ten markers were annotated by matching accurate m/z and product ion information obtained from previous reports. It is clearly indicated that the pseudotargeted analysis could make a great contribution to the quality assessment of traditional Chinese medicine preparation as a newly emerging technique.     

N-linked oligosaccharide analysis of rat brain Thy-1 by liquid chromatography with graphitized carbon column/ion trap-Fourier transform ion cyclotron resonance mass spectrometry in positive and negative ion modes

We have previously described the site-specific glycosylation analysis of rat brain Thy-1 by LC/multistage tandem mass spectrometry (MS(n)) using proteinase-digested Thy-1. In the present study, detailed structures of oligosaccharides released from Thy-1 were elucidated by mass spectrometric oligosaccharide profiling using LC/MS with a graphitized carbon column (GCC-LC/MS). First, using model oligosaccharides, we improved the oligosaccharide profiling by ion trap mass spectrometry (IT-MS) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Sequential scanning of a full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in positive ion mode, and a subsequent full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in negative ion mode enabled the monosaccharide composition analysis as well as profiling and sequencing of both neutral and acidic oligosaccharides in a single analysis. The improved oligosaccharide profiling was applied to elucidation of N-linked oligosaccharides from Thy-1 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was demonstrated that Thy-1 possesses a significant variety of N-linked oligosaccharides, including Lewis a/x, Lewis b/y, and disialylated structure as a partial structure. Our method could be applicable to analysis of a small abundance of glycoproteins, and could become a powerful tool for glycoproteomics.     

Evaluation of automated single mass spectrometry to tandem mass spectrometry function switching for comprehensive drug profiling analysis using a quadrupole time-of-flight mass spectrometer

A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.     

Metabolomics approach for determining growth-specific metabolites based on Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) is the best MS technology for obtaining exact mass measurements owing to its great resolution and accuracy, and several outstanding FT-ICR/MS-based metabolomics approaches have been reported. A reliable annotation scheme is needed to deal with direct-infusion FT-ICR/MS metabolic profiling. Correlation analyses can help us not only uncover relations between the ions but also annotate the ions originated from identical metabolites (metabolite derivative ions). In the present study, we propose a procedure for metabolite annotation on direct-infusion FT-ICR/MS by taking into consideration the classification of metabolite-derived ions using correlation analyses. Integrated analysis based on information of isotope relations, fragmentation patterns by MS/MS analysis, co-occurring metabolites, and database searches (KNApSAcK and KEGG) can make it possible to annotate ions as metabolites and estimate cellular conditions based on metabolite composition. A total of 220 detected ions were classified into 174 metabolite derivative groups and 72 ions were assigned to candidate metabolites in the present work. Finally, metabolic profiling has been able to distinguish between the growth stages with the aid of PCA. The constructed model using PLS regression for OD₆₀₀ values as a function of metabolic profiles is very useful for identifying to what degree the ions contribute to the growth stages. Ten phospholipids which largely influence the constructed model are highly abundant in the cells. Our analyses reveal that global modification of those phospholipids occurs as E. coli enters the stationary phase. Thus, the integrated approach involving correlation analyses, metabolic profiling, and database searching is efficient for high-throughput metabolomics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Profiling of grape monoterpene glycosides (aroma precursors) by ultra‐high performance‐liquid chromatography‐high resolution mass spectrometry (UHPLC/QTOF)

A ‘suspect screening analysis’ method for grape metabolomics by ultra‐high performance‐liquid chromatography (UHPLC) and high‐resolution quadrupole‐time of flight (QTOF) mass spectrometry was recently developed. This method was applied to study grape monoterpene glycosides, the main grape aroma precursors. Since standard compounds were not available, they were tentatively identified by overlapping various analytical approaches, in agreement with the indications recommended in mass spectrometry (MS)‐based metabolomics. Accurate mass and isotopic pattern, MS/MS fragmentation, correlation between fragments observed and putative structures and between liquid chromatography coupled with mass spectrometry (LC/MS) and gas chromatography/mass spectrometry signals were studied. Seventeen monoterpene glycosides were identified without performing the hydrolytic artifacts commonly used to study these compounds which may affect sample profile. This is the first time that a detailed study of these aroma precursors has been carried out by direct LC/MS analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Automated neutral loss and data dependent energy resolved "pseudo MS3" for the targeted identification, characterization and quantitative analysis of methionine- containing peptides

A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary high- performance liquid chromatography and automated data dependent neutral loss scan mode tandem mass spectrometry (MS/MS) and "pseudo multiple mass spectrometry (MS(3))" product ion scans in a triple quadrupole mass spectrometer has been developed for the "targeted" gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. Selective gas-phase "enrichment" and identification is performed via neutral loss scan mode MS/MS, by low energy collision-induced dissociation of the derivatized methionine side chain, resulting in the formation of a single characteristic product ion. Structural characterization of identified peptides is then achieved by automatically subjecting the characteristic neutral loss product ion to further dissociation by data dependent product ion scan mode pseudo MS(3) under higher collision energy conditions. Quantitative analysis is achieved by measurement of the abundances of characteristic product ions formed by sequential neutral loss scan mode MS/MS experiments from "light" ((12)C) and "heavy" ((13)C) stable isotope encoded fixed-charge derivatized peptides. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method employed here was able to achieve accurate quantification for individual peptides at levels as low as 100 fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest.     

电喷雾离子源正离子模式下环烯醚萜苷的质谱裂解行为

利用高分辨率四极杆-飞行时间串联质谱(Q-TOF MS/MS)对环烯醚萜苷同系组分7,8-环戊烯型和环戊烷型环烯醚萜苷在电喷雾正离子(ESI⁺)模式下的质谱裂解行为进行了研究. 在ESI⁺模式下, 环烯醚萜苷主要的质谱裂解途径是脱去母环上的功能基团, 如丢失H₂O, CO₂, CH₃OH, CH₃COOH和糖单元部分等, 由于它们均为葡萄糖苷, 所以共有碎片离子[Glc+Na]⁺(m/z 185.0). 环烯醚萜苷母核环上半缩醛结构的异构化造成二氢吡喃环的断裂, 但未发现与苷元部分在负离子(ESI^-)模式下相同的其它断裂. 环烯醚萜苷在ESI⁺模式下的断裂途径特征性不如其在ESI^-模式下的明显, 且灵敏度比后者低.     

Diagnostic filtering to screen polycyclic polyprenylated acylphloroglucinols from Garcinia oblongifolia by ultrahigh performance liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry

A novel multistage MS approach, insource collision-induced dissociation (CID) combined with Time Aligned Parallel (TAP) fragmentation, was established to study the fragmentation behavior of polycyclic polyprenylated acylphloroglucinols (PPAPs), which could provide a more reliable fragmentation relationship between precursor and daughter ions. The diagnostic ions for different subtypes of PPAPs and their fragmentation behaviors have been summarized. Moreover, a new and reliable multidimensional analytical workflow that combines ultrahigh performance liquid chromatography (UHPLC), data-independent mass spectrometry (MS^E), and tandem MS with ion mobility (IM) has been optimized and established for the analysis of PPAPs in the plant Garcinia oblongifolia by diagnostic filtering. Diagnostic fragment ions were used to selectively screen PPAPs from extracts, whereas IM coupled to MS was used to maximize the peak capacity. Under the optimized UHPLC-IM-MS^E and UHPLC-IM-MS/MS method, 140 PPAPs were detected from the crude extract of G. oblongifolia, and 10 of them were unambiguously identified by comparing them to the reference compounds. Among those PPAPs, 7 pairs of coeluting isobaric PPAPs that were indistinguishable by conventional UHPLC-HRMS alone, were further resolved using UHPLC-IM-MS. It is anticipated that the proposed method will be extended to the rapid screening and characterization of the other targeted or untargeted compounds, especially these coeluting isomers in complex samples.     

Metabolomic characterization of semen from asthenozoospermic patients using ultra-high-performance liquid chromatography–tandem quadrupole time-of-flight mass spectrometry

Asthenozoospermia (AS) is a common factor of male infertility, and its pathogenesis remains unclear. The purpose of this study was to investigate the differential seminal plasma metabolic pattern in asthenozoospermic men and to identify potential biomarkers in relation to spermatogenic dysfunction using sensitive ultra-high-performance liquid chromatography–tandem quadruple time-of-flight MS (UHPLC–Q-TOF/MS). The samples of seminal plasma from patients with AS (n = 20) and healthy controls (n = 20) were checked and differentiated by UHPLC–Q-TOF/MS. Compared with the control group, the AS group showed a total of nine significantly different metabolites, including increases in creatinine, uric acid, N⁶-methyladenosine (m⁶A), uridine, and taurine and decreases in carnitine, nicotinamide, N-acetylputrescine and l -palmitoylcarnitine. By analyzing the correlation among these metabolites and clinical computer-assisted semen analysis reports, we found that m⁶A is significantly correlated with not only the four decreased metabolites but also with sperm count, motility, and curvilinear velocity. Furthermore, nicotinamide was shown to correlate with other identified metabolites, indicating its important role in the metabolic pathway of AS. Current results implied that sensitive untargeted seminal plasma metabolomics could identify distinct metabolic patterns of AS and would help clinicians by offering novel cues for discovering the pathogenesis of male infertility.     

Analysis of a series of chlorogenic acid isomers using differential ion mobility and tandem mass spectrometry

Chlorogenic acids are among the most abundant phenolics found in the human diet. Of these, the mono-caffeoylquinic acids are the predominant phenolics found in fruits, such as apples and pears, and products derived from them. In this research, a comprehensive study of the electrospray ionization (ESI) tandem mass spectrometric (MS/MS) dissociation behavior of the three most common mono-caffeoylquinic acids, namely 5-O-caffeoylquinic acid (5-CQA), 3-O-caffeoylquinic acid (3-CQA) and 4-O-caffeoylquinic acid (4-CQA), were determined using both positive and negative ionization. All proposed structures of the observed product ions were confirmed with second-generation MS³ experiments. Similarities and differences between the dissociation pathways in the positive and negative ion modes are discussed, confirming the proposed structures and the established MS/MS fingerprints. MS/MS dissociation was primarily driven via the cleavage of the ester bond linking the quinic acid moiety to the caffeic acid moiety within tested molecules. Despite being structural isomers with the same m/z values and dissociation behaviors, the MS/MS data in the negative ion mode was able to differentiate the three isomers based on ion intensity for the major product ions, observed at m/z 191, 179 and 173. This differentiation was consistent among various MS instruments. In addition, ESI coupled with high-field asymmetric waveform ion mobility spectrometry-mass spectrometry (ESI-FAIMS-MS) was employed for the separation of these compounds for the first time. By combining MS/MS data and differential ion mobility, a method for the separation and identification of mono-caffeoylquinic in apple/pear juice samples was developed with a run time of less than 1 min. It is envisaged that this methodology could be used to identify pure juices based on their chlorogenic acid profile (i.e., metabolomics), and could also be used to detect juice-to-juice adulteration (e.g., apple juice addition to pear juice).     

NMF-Based Approach for Missing Values Imputation of Mass Spectrometry Metabolomics Data

In mass spectrometry (MS)-based metabolomics, missing values (NAs) may be due to different causes, including sample heterogeneity, ion suppression, spectral overlap, inappropriate data processing, and instrumental errors. Although a number of methodologies have been applied to handle NAs, NA imputation remains a challenging problem. Here, we propose a non-negative matrix factorization (NMF)-based method for NA imputation in MS-based metabolomics data, which makes use of both global and local information of the data. The proposed method was compared with three commonly used methods: k-nearest neighbors (kNN), random forest (RF), and outlier-robust (ORI) missing values imputation. These methods were evaluated from the perspectives of accuracy of imputation, retrieval of data structures, and rank of imputation superiority. The experimental results showed that the NMF-based method is well-adapted to various cases of data missingness and the presence of outliers in MS-based metabolic profiles. It outperformed kNN and ORI and showed results comparable with the RF method. Furthermore, the NMF method is more robust and less susceptible to outliers as compared with the RF method. The proposed NMF-based scheme may serve as an alternative NA imputation method which may facilitate biological interpretations of metabolomics data.     

超高效液相色谱-四极杆-飞行时间质谱定性研究茶叶籽中的酚类化合物

采用超高效液相色谱(UHPLC)-四极杆-飞行时间质谱(Q-TOF/MS)技术定性分析茶叶籽中的酚类化合物。茶叶籽样品经乙醇水溶液提取后经反相色谱分离,通过Q-TOF/MS进行化合物的鉴定。基于山茶属及相关植物化学组成的文献,建立了一个含有106种酚类化合物的数据库。对UHPLC-Q-TOF/MS采集得到的一级质谱数据进行数据库检索,然后对检索到的化合物色谱峰进行二级质谱扫描,根据得到的碎片离子推断化合物的结构。初步推断出茶叶籽提取物中的24种酚类化合物,包括13种酚酸类、4种儿茶素类和7种黄酮类化合物,并通过与标准品比对,进一步确证了这些化合物。结果表明UHPLC-Q-TOF/MS技术可以用于对茶叶籽中酚类化合物进行快速、准确、可靠的定性分析,促进新化合物的发现与鉴别。     

Development and validation of a sensitive UHPLC–MS/MS method for quantitative analysis of farrerol in rat plasma: Application to pharmacokinetic and bioavailability studies

Farrerol is a 2,3‐dihydro‐flavonoid isolated from rhododendron. In this study, a sensitive and selective ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed for the determination of farrerol in rat plasma. Liquid–liquid extraction by ethyl ether was used for sample preparation. Chromatographic separation was achieved on an Agilent UHPLC XDB‐C₁₈ column (2.1 × 100 mm, 1.8 μm) with water and methanol (30:70, v /v) as the mobile phase. An electrospray source was applied and operated in negative ion mode; selection reaction monitoring was used for quantification using target fragment ions m/z 299 → 179 for farrerol and m/z 267 → 252 for internal standard. Calibration plots were linear in the range of 2.88–1440 ng/mL for farrerol in rat plasma. Intra‐ and inter‐day precisions were <11.6%, and the accuracy ranged from −13.9 to 11.9%. The UHPLC–MS/MS method was successfully applied in pharmacokinetics and bioavailability studies of farrerol in rats.     

Instrument dependence of electrospray ionization and tandem mass spectrometric fragmentation of the gingerols

The gingerols, including [6]-, [8]-, and [10]-gingerols, a series of chemical homologs differentiated by the length of their unbranched alkyl chains, have been identified as major active components in fresh ginger rhizome. The purpose of this study was to investigate the utility of ion trap liquid chromatography/tandem mass spectrometry (LC/MS/MS) as an online tool to identify and quantify these compounds in raw or processed ginger rhizome samples. Negative mode electrospray ionization (ESI) was used in MS, MS/MS and MS(n) experiments in quadrupole ion trap instruments from two different manufacturers and in high-resolution and accurate mass MS and MS/MS experiments in a Fourier transform ion cyclotron resonance mass spectrometer to elucidate the ionization and fragmentation mechanisms of these compounds in these instruments. Positive mode ESI, which generated many more fragment ions in full scan MS even under gentle ionization conditions, was also used in LC/MS and MS/MS experiments and in direct infusion MS and MS/MS experiments. Consistent and predictable ionization and fragmentation behaviors were observed for all gingerols when analyzed in the same instrument. Instruments from different manufacturers, however, had different ionization mechanisms. The major difference between instruments was their ability to form covalent dimer adducts of the gingerols. Subsequent fragmentation patterns of the precursor ions were essentially identical. These results clearly demonstrate that LC/MS instruments produce data that cannot necessarily be replicated in other laboratories, especially if those laboratories do not have the same instrument model from the same manufacturer. This presents major problems for metabolite target analysis, metabolic profiling and metabolomics investigations, which would benefit from LC/MS mass spectrum libraries as they do from GC/MS mass spectrum libraries, because such libraries may not be valid across platforms.     

Semi‐quantitative and structural metabolic phenotyping by direct infusion ion trap mass spectrometry and its application in genetical metabolomics

The identification of quantitative trait loci (QTL) for plant metabolites requires the quantitation of these metabolites across a large range of progeny. We developed a rapid metabolic profiling method using both untargeted and targeted direct infusion tandem mass spectrometry (DIMSMS) with a linear ion trap mass spectrometer yielding sufficient precision and accuracy for the quantification of a large number of metabolites in a high‐throughput environment. The untargeted DIMSMS method uses top‐down data‐dependent fragmentation yielding MS² and MS³ spectra. We have developed software tools to assess the structural homogeneity of the MS² and MS³ spectra hence their utility for phenotyping and genetical metabolomics. In addition we used a targeted DIMS(MS) method for rapid quantitation of specific compounds. This method was compared with targeted LC/MS/MS methods for these compounds. The DIMSMS methods showed sufficient precision and accuracy for QTL discovery. We phenotyped 200 individual Lolium perenne genotypes from a mapping population harvested in two consecutive years. Computational and statistical analyses identified 246 nominal m/z bins with sufficient precision and homogeneity for QTL discovery. Comparison of the data for specific metabolites obtained by DIMSMS with the results from targeted LC/MS/MS analysis showed that quantitation by this metabolic profiling method is reasonably accurate. Of the top 100 MS¹ bins, 22 ions gave one or more reproducible QTL across the 2 years. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid screening and characterization of drug metabolites using a multiple ion monitoring-dependent MS/MS acquisition method on a hybrid triple quadrupole-linear ion trap mass spectrometer

A novel LC/MS/MS method that uses multiple ion monitoring (MIM) as a survey scan to trigger the acquisition of enhanced product ions (EPI) on a hybrid quadrupole-linear ion trap mass spectrometer (Q TRAP) was developed for drug metabolite identification. In the MIM experiment, multiple predicted metabolite ions were monitored in both Q1 and Q3. The collision energy in Q2 was set to a low value to minimize fragmentation. Results from analyzing ritonavir metabolites in rat hepatocytes demonstrate that MIM-EPI was capable of targeting a larger number of metabolites regardless of their fragmentation and retained sensitivity and duty cycle similar to multiple reaction monitoring (MRM)-EPI. MIM-based scanning methods were shown to be particularly useful in several applications. First, MIM-EPI enabled the sensitive detection and MS/MS acquisition of up to 100 predicted metabolites. Second, MIM-MRM-EPI was better than MRM-EPI in the analysis of metabolites that undergo either predictable or unpredictable fragmentation pathways. Finally, a combination of MIM-EPI and full-scan MS (EMS), as an alternative to EMS-EPI, was well suited for routine in vitro metabolite profiling. Overall, MIM-EPI significantly enhanced the metabolite identification capability of the hybrid triple quadrupole-linear ion trap LC/MS.