Neutral Loss Scan Research Articles

Screening and identification of reactive metabolic compounds of Cortex Periplocae based on glutathione capture-mass spectrometry.

As a traditional Chinese medicine (TCM), Cortex Periplocae (CP) has a wide range of pharmacological effects, as well as toxic side effects. The main toxic components of it are cardiac glycosides, which tend to cause cardiotoxicity. Currently, it has also been reported in studies to cause hepatotoxicity, but it is not clear whether the hepatotoxicity is related to the toxicity caused by the reactive metabolites. This study aims to investigate the target components of CP that generate reactive metabolic toxicity. The fluorescent probe method was used to detect glutathione (GSH)-trapped reactive metabolites in a co-incubation system of CP extract with rat liver microsomes. Identification of GSH conjugates was performed by LC-MS/MS and that of the possible precursor components that produce reactive metabolites was conducted by UPLC-Q-TOF/MS. Cell viability assays were performed onHepG2 and L02 cells to determine the cytotoxicity of the target components. The findings of our study demonstrate that the extract derived from CP has the ability to generate metabolites that exhaust the intracellular GSH levels, resulting in the formation of GSH conjugates and subsequent cytotoxic effects. Through the utilization of the UPLC-Q-TOF/MS technique, we were able to accurately determine the molecular weight of the precursor compound in CP to be 355.1023. The primary evidence to determining the GSH conjugetes relies on the appearance of characteristic product ions resulting from central neutral loss (CNL) scanning of 129Da and product scanning of m/z 660 in the positive MS/MS spectrum. Through analysis, it was ultimately ascertained that the presence of chlorogenic acid (CGA) and its isomers, namely neochlorogenic acid (NCGA) and cryptochlorogenic acid (CCGA), could lead to the production of GSH conjugates, resulting in cytotoxicity at elevated levels. Taking these findings into consideration, the underlying cause for the potential hepatotoxicity of CP was initially determined.

An integrated LC-MS/MS platform for noninvasive urinary nucleus acid adductomics: A pilot study for tobacco exposure

Tobacco smoke exposure significantly increases the level of global nucleoside damage. To evaluate all aspects of nucleic acid (NA) modifications, NA adductomics analyzes DNA, RNA and nucleobase adducts and provides comprehensive data. Liquid chromatography-tandem triple quadrupole mass spectrometry (LC-QQQ-MS/MS) and LC-Zeno-TOF-MS/MS were employed to screen for DNA, RNA and nucleobase adducts, as part of the analytical platform that was designed to combine high sensitivity and high resolution detection. We identified and distinguished urine nucleoside adducts via precursor ion and neutral loss scanning. A total of 245 potential adducts were detected, of which 28 were known adducts. The smoking group had significantly higher concentrations of nucleoside adducts in rat urine than the control group, based on MRM scanning, which was then used to perform relative quantitative analysis of these adducts. Urine nucleoside adducts were further confirmed using LC-Zeno-TOF-MS/MS. This highlights the potential of untargeted detection methods to provide comprehensive data on both known and unknown adducts. These approaches can be used to investigate the interactions among oxidative and alkylation stresses, and epigenetic modifications caused by exposure to tobacco smoke.

Determination of Triacylglycerol Composition in Mealworm Oil (Tenebrio molitor) via Electrospray Ionization Tandem Mass Spectrometry with Multiple Neutral Loss Scans.

Mealworms (Tenebrio molitor) have been used as an alternative source of proteins and lipids. Triacylglycerols (TAGs) are major sources of energy and have been used to provide essential fatty acids. They are also the main components of mealworm oil, and their composition and content are extensively linked to its physical and chemical properties. However, because of the complexity of TAG molecules, their identification and quantitation are challenging. This study employed electrospray ionization tandem mass spectrometry (ESI-MS/MS) with multiple neutral loss scans (NLS) to analyze the TAG composition and content in mealworm oil. Identifying and quantifying TAGs using ESI-MS/MS in combination with multiple NLS was an efficient way to improve accuracy and timeliness. For the accurate quantification of TAGs, isotopic deconvolution and correlation factors were applied. A total of 57 TAGs were identified and quantified: C52:2 (16:0/18:1/18:1) (1549.4 nmol/g, 18.20%), C52:3 (16:0/18:1/18:2) (1488.1 nmol/g, 17.48%), C54:4 (18:1/18:1/18:2) (870.1 nmol/g, 10.23%), C54:6 (18:1/18:2/18:2) (659.8 nmol/g, 7.76%) and C52:4 (16:0/18:2/18:2) (600.5 nmol/g, 7.06%), which were the most abundant TAGs present in the mealworm oil. The fundamental properties of mealworm oil, including its degree of oxidation, nutritional effect and physical properties, were elucidated.

Investigating the discriminatory potential of urinary DNA adductomics in smokeless tobacco-treated rats and head-neck cancer patients

The genotoxicity of smokeless tobacco (ST) remains an essential public health issue. We conducted a comprehensive study of rats exposed to ST and head and neck cancer patients to elucidate the DNA adduct profiles. ST exposure assessment used alkaloids and tobacco-specific nitrosamines in rat urine. High ST doses led to significantly elevated nicotine, cotinine, N-nitrosonornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) levels, indicating substantial exposure. DNA adductomics identified 17 significant DNA adducts using a neutral loss scan in liquid chromatography-mass spectrometry in rat urine samples upon ST exposure, highlighting the intricate landscape of DNA modifications associated with ST using univariate and multivariate statistical analyses. LC-ESI-QTOF-MS/MS analysis and accurate mass measurements of DNA adducts unveiled specific adducts upregulated in response to ST exposure, particularly those associated with methylation and NNK release. Intriguing correlations emerged between DNA adducts and alkaloids-TSNA in ST-exposed rat urine. Nicotine correlated positively with N2-paraldol-dG and O2/O4-[4-(3-Pyridyl)-4-oxobut-1-yl] thymidine in high-dose rats. NNK correlated with 6O-methylgaunine, while NNN correlated with 6-hydroxy-1-N2-propanodeoxyguanosine and 7-methylguanine. With this aim, we also applied analysis to human samples, where we observed specific DNA adducts for ST users, controls, and subjects with HNC. Two-way ANOVA showed that ST users and cancer patients had higher DNA adduct levels in human urine, which aids cancer risk assessment, early detection, and environmental exposure evaluation. This comprehensive study explores DNA adducts caused by ST exposure across rats and head and neck cancer patients, illuminating genotoxic mechanisms and health risks and providing valuable insights for future preventive and therapeutic strategies.

Applications of Liquid Chromatography-Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Fast and reliable characterization of egg triacylglycerol profiles using atmospheric solids analysis probe tandem mass spectrometry (ASAP-MS/MS) combined with chemometric tools

In this study, the potential of atmospheric solid analysis probe tandem mass spectrometry (ASAP-MS/MS) was assessed as a green and rapid method combined with gas chromatography with flame ionization detection (GC-FID) and chemometrics to determine the triacylglycerol (TAG) profile of eggs from various housing systems, including indoor, cage-free, free-range, organic, and enriched systems. The most abundant ions were identified using the product ion and neutral loss scan. The fatty acid composition, mainly unsaturated, palmitic, and stearic acids of eggs was influenced by the housing systems. The optimal conditions of ASAP-MS for cone voltage, desolvation gas temperature, and desolvation gas flow were determined to be 40 V, 600 °C, and 1100 L h−1, respectively. The main TAGs (50:2, 50:1, 52:4, 52:3, 52:2, 52:1, 54:4, 54:3, 54:2, 54:1) were observed in the mass range m/z 830–890. Notable differences were observed for TAGs 54:1 and 52:4, which were found in significantly higher levels on WE and CE. The proposed methodology enables rapid and efficient TAG profiling of egg yolks, providing a solid foundation for future research in this field.

A miniaturized comprehensive approach for total lipidome analysis and vitamin D metabolite quantification in human serum.

The balance between the different lipid molecules present in biological fluids accurately reflects the health state of the organism and can be used by medical personnel to finely tune therapy to a single patient, a process known as precision medicine. In this work, we developed a miniaturized workflow for the analysis of different lipid classes at theintact level, as well as their fatty acid constituents, starting from human serum. Fatty acids were identified by using flow-modulated comprehensive gas chromatography coupled to mass spectrometry (FM-GC × GC-MS), and their relative amount as well as the ratio of specific FA classes was determined by using FM-GC × GC with a flame ionization detector. Ultra-high-performance liquid chromatography coupled to tandem mass spectrometry was used for the simultaneous quantification of vitamin D metabolites and assessment of different intact lipid classes. An MRM method was developed for the quantification of five vitamin D metabolites (vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3), and validated in terms of LoD, LoQ, accuracy, and precision, also using a certified reference material. At the same time, a combination of SCAN, precursor ion scan, and neutral loss scan, in both positive and negative modes, was used for the identification of 81 intact lipid species, such as phospholipids, cholesteryl esters, and triacylglycerols, in less than 25min. In order to easily monitor the lipid composition and speed up the identification process, a two-dimensional map of the lipidome was generated, by plotting the molecular weight of the identified molecules versus their retention time. Moreover, a relative quantification was performed within each lipid class identified. The combination of untargeted and targeted data could provide useful information about the pathophysiological condition of the organism and evaluate, in a tailored manner, an efficient action.

Isotope-coded derivatization with designed Girard-type reagent as charged isobaric mass tags for non-targeted profiling and discovery of natural aldehydes by liquid chromatography-tandem mass spectrometry

Aldehyde-containing metabolites are reactive electrophiles that have attracted extensive attention due to their widespread occurrence in organisms and natural foods. Herein we described a newly-designed Girard's reagent, 1-(4-hydrazinyl-4-oxobutyl)pyridin-1-ium bromide (HBP), as charged tandem mass (MS/MS) tags to facilitate selective capture, sensitive detection and semi-targeted discovery of aldehyde metabolites via hydrazone formation. After HBP labeling, the detection signals of the test aldehydes were increased by 21–2856 times, with the limits of detection were 2.5–7 nM. Upon isotope-coded derivatization with a pair of labeling reagents, HBP-d0 and its deuterium-labeled counterpart HBP-d5, the aldehyde analytes were converted to hydrazone derivatives, which generated characteristic neutral fragments of 79 Da and 84 Da, respectively. The isobaric HBP-d0/HBP-d5 labeling based LC-MS/MS method was validated by relative quantification of human urinary aldehydes (slope=0.999, R2 > 0.99, RSDs ≤ 8.5%) and discrimination analysis between diabetic and control samples. The unique isotopic doubles (Δm/z = 5 Da) by dual neutral loss scanning (dNLS) provided a generic reactivity-based screening strategy that allowed non-targeted profiling and identification of endogenous aldehydes even amidst noisy data. The LC-dNLS-MS/MS screening of cinnamon extracts led to finding 61 possible natural aldehydes and guided discovery of 10 previously undetected congeners in this medicinal plant.

Free Amino Acids and Biogenic Amines Profiling and Variation in Wild and Sub-Endemic Cardueae Species from Sardinia and Corse.

The cardueae are a common species in the Mediterranean area where they grow spontaneously and are traditionally employed as food and for health purposes. In this work, five Cardueae, including two sub-endemic species (four Carduus and three Ptilostemon casabonae (L.) Greuter samples from different locations) were collected from Sardinia and the Corse islands. All the considered plants are characteristic of the area, in particular the sub-endemic species C. cephalanthus and P. casabonae. This work aims to obtain, for the first time, the amino compounds profile (primary metabolites) of these little-studied species to detect for any similarities and differences among the different samples using statistical analyses. A recently developed method was employed, where diethyl ethoxymethylenemalonate (DEEMM) derivatives are detected in a neutral loss scan mode using high performance liquid chromatography in tandem with a mass spectrometry technique. In total, 42 amino compounds were detected, of which 33 were fully identified and semi-quantified. Overall, the results show that DEEMM-derivatized amino compounds are qualitatively similar among the considered samples. Nonetheless, a discrimination at the genus level is possible. This work adds more information regarding the phytochemical composition regarding the primary metabolites of the considered samples, their discriminations and the search for compounds with potential health benefits.

Comparison of two azobenzene-based amino acid derivatization reagents for LC-MS/MS analysis in positive and negative ESI modes

Derivatization reagents based on azobenzene containing different N-hydroxysuccinimidyl moieties– AzoB (carbamate) and AzoC (ester) – are proposed for the LC-ESI-MS analysis of free amino acids in fermented beverages and juices. A dual comparison between LC-MS/MS in positive and negative ESI modes in dynamic Multiple Reaction Monitoring (dMRM) and Neutral Loss Scan was investigated. The results indicate that the studied carbamate derivatization reagent, AzoB, can be employed for targeted analysis (MS/MS) but also for non-targeted analysis of derivatized amino acids thanks to its constant neutral loss (223 Da) that is the same in both ionisation modes. For amines, precursor ion scan can be used as identification tool. The derivatization properties of AzoB and AzoC were compared against other derivatization reagents, and they showed advantages such as fast derivatization reaction and good reactivity with secondary amines. AzoC also displayed a disadvantage –side products were formed that affect the quantitation. Free amino acids profile of Kvass (a fermented beverage from eastern Europe) was determined for first time, proline was found to be the most abundant amino acid.

Metabolite Identification of a Novel Anti-Leishmanial Agent OJT007 in Rat Liver Microsomes Using LC-MS/MS.

The purpose of this study was to identify potential metabolic pathways and metabolites of OJT007, a methionine aminopeptidase 1 (MetAP1) inhibitor. OJT007 is a novel drug with potent antiproliferative effects against Leishmania Major. We conducted in vitro Phase I oxidation and Phase II glucuronidation assays on OJT007 using rat liver microsomes. Four unknown metabolites were initially identified using a UPLC-UV system from microsomal incubated samples. LC-MS/MS analysis was then used to identify the structural characteristics of these metabolites via precursor ion scan, neutral loss scan, and product ion scan. A glucuronide metabolite was further confirmed by β-glucuronidase hydrolysis. The kinetic parameters of OJT007 glucuronidation demonstrated that OJT007 undergoes rapid metabolism. These results demonstrate the liver’s microsomal ability to mediate three mono-oxidated metabolites and one mono-glucuronide metabolite. This suggests hepatic glucuronidation metabolism of OJT007 may be the cause of its poor oral bioavailability.

Instantaneous characterization of crude vegetable oils via triacylglycerols fingerprint by atmospheric solids analysis probe tandem mass spectrometry with multiple neutral loss scans

Vegetable oils have many applications, including in food and cosmetics industries. In this study, triacylglycerols (TAG), the main components of vegetable oils, were investigated via Fourier transform infrared with attenuated total reflection analysis (FTIR-ATR), gas chromatography with flame ionization detection (GC-FID), and atmospheric solids analysis probe tandem mass spectrometry (ASAP-MS/MS). Instantaneous characterization of 20 vegetable oils extracted according to the Bligh and Dyer method, including soybean, sunflower, sweet almond, and coconut oils, via TAGs fingerprint was provided by ASAP-MS/MS, directly from a droplet of the crude oil, without sample preparation and solvent use. It was found that each vegetable oil has a characteristic fingerprint of TAG and results were according to those obtained by GC-FID and FTIR-ATR analysis. In addition, the difference in the relative abundance of TAG composition could be used as the target in differentiating vegetable oil types from each other. As coconut oil was differentiated from other oils only by its mass spectrum, it was not applied to principal component analysis (PCA), which effectively differentiate sweet almond oil from soybean and sunflower oils. Therefore, the results demonstrate the potentiality of ASAP-MS/MS with chemometrics analysis as powerful, fast, clean, and non-destructive tools for characterization and quality control of vegetable oils.

Neutral loss scan in complement with high-resolution MS/MS: Combination of detection methods for flavonoid and limonoid glycosides analysis.

In this study, neutral loss scan and high-resolution MS/MS were used in combination to detect and tentatively identify various flavonoid and limonoid glycosides in navel orange albedo, juice, peel and pulp. These compound classes are of research interest due to their flavour and bioactive properties, and although flavonoid glycosides have been previously studied in other food matrices, to the best of our knowledge, neutral loss scans have not been used for the elucidation of limonoid glycosides. Neutral loss masses of 120, 162 and 308 Da were selected for the detection of hexose, rutinose and neohesperidose-substituted flavonoids, whereas 197 Da was explored for limonoid glycosides due to their tendency to form ammonium adducts. Fragmentation patterns obtained from targeted MS/MS were then used to differentiate rutinose and neohesperidose substituents as well as flavonoid subclasses of flavones, flavanones and flavonols. Additionally, high-resolution MS/MS was also used for the identification of aglycones by accurate mass (to four decimal places), allowing for the differentiation of aglycones with similar unit masses but different chemical formulas. In total, 19 flavonoid glycosides and six limonoid glycosides were detected. This workflow allows for a rapid screening of flavonoid and limonoid glycosides in citrus, which can be further extended to other food products such as tea.

Derivatization-targeted analysis of amino compounds in plant extracts in neutral loss acquisition mode by liquid chromatography-tandem mass spectrometry

Amino compounds, such as amino acids and biogenic amines, are important metabolites that can be found in diverse natural matrices. The most common method for amino compound analysis nowadays is reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS). However, due to the polar and the basic nature of amines, their RPLC retention is often insufficient or peaks are tailing. Derivatization is a way to overcome the issue and in the present work amino compounds are derivatized with diethyl ethoxymethylenemalonate (DEEMM) and analyzed by a RPLC triple quadrupole MS system in neutral loss scan (NLS) mode (loss of 46). This allows to target all compounds in the sample that undergo derivatization with DEEMM, so that the amino compound profile of the sample is obtained. To the best of our knowledge, the NLS acquisition mode has never been employed to target amino compounds after DEEMM derivatization. In the first part of the study, eight amino acids (arginine, aspartic acid, threonine, proline, tyrosine, tryptophan, phenylalanine and isoleucine) were employed as model compounds for method optimization, with good results in terms of DEEMM derivatives detection and repeatability. The developed method was successfully applied to a complex extract from the plant species Carduus nutans subsp. macrocephalus (Desf.) Nyman, with 18 amino acids and 3 other amines being identified. The proposed approach could be employed for straightforward identification of known and unknown amino compounds in different types of matrices.

Isotope-Coding Derivatization for Quantitative Profiling of Reactive α-Dicarbonyl Species in Processed Botanicals by Liquid Chromatography-Tandem Mass Spectrometry.

α-Dicarbonyls (α-DCs) are key reactive Maillard intermediates with structural diversity and are widely found in foods and in vivo, but little is known regarding the complete molecular profiles of these potentially harmful electrophiles. Herein, we reported a novel isotope-coding derivatization (ICD) strategy for the broad-spectrum, quantitative profiling of (non)target α-DC species in natural foodstuffs. It utilized differential isotope labeling (DIOL) with a reagent pair o-phenylenediamine (OPD)/OPD-d4 (deuterated) to form stable quinoxalines for class-specific fragmentation-dependent acquisition using liquid chromatography-hybrid quadrupole linear ion trap mass spectrometry (LC-QqLIT). A combination of facile one-pot quantitative labeling and convenient cleanup protocol afforded satisfactory sensitivity, linearity, accuracy (81-116%), and process recovery (86-109% with RSDs < 10%) by matrix-matched ICD-internal standard calibration, without significant matrix interference (-9 to 5%), isotopic effect (<0.5%), and cocktail effect. A more generic DIOL-based LC-QqLIT algorithm integrated double precursor ion and neutral loss scan to trigger enhanced product ions with the unique isobaric doublet tags (4 Da shift), enabling simultaneous screening and relative quantitation of nontarget α-DC analogues in a single analysis. This study has widened the vision on complex α-DC profiles in traditional botanicals, which revealed a wide occurrence of α-DCs in such processed sugar-rich products, yet their abundance varied greatly among different samples.

Atmospheric Pressure Drift Tube Ion Mobility Spectrometry Coupled with Two-Dimensional Tandem Mass Spectrometry.

Atmospheric pressure drift tube ion mobility was coupled with two-dimensional tandem mass spectrometry (2D MS/MS) in a linear ion trap to simultaneously collect ion mobility and the entire MS/MS data domain. Utilizing ion intensities from precursor ion and neutral loss scan lines, ion mobility spectra of multiple compounds with particular functional groups were acquired in a single experiment. Functional group-specific ion mobility spectra were demonstrated for a standard mixture of lipids. Additionally, ion mobility was used to separate isobaric ions prior to 2D MS/MS. The combination of these two methods offers improvements for the analysis of complex mixtures.

Efficient Matrix Cleanup of Soft-Gel-Type Dietary Supplements for Rapid Screening of 92 Illegal Adulterants Using EMR-Lipid dSPE and UHPLC-Q/TOF-MS.

An efficient matrix cleanup method was developed for the rapid screening of 92 illegal adulterants (25 erectile dysfunction drugs, 15 steroids, seven anabolic steroids, 12 antihistamines, 12 nonsteroidal anti-inflammatory drugs (NSAIDs), four diuretics, and 17 weight-loss drugs) in soft-gel-type supplements by ultra-high performance liquid chromatography-quadrupole/time of flight-mass spectrometry (UHPLC-Q/TOF-MS). As representative green chemistry methods, three sample preparation methods (dispersive liquid-liquid microextraction (DLLME), “quick, easy, cheap, effective, rugged, and safe” dispersive solid-phase extraction (QuEChERS-dSPE), and enhanced matrix removal-lipid (EMR-Lipid) dSPE) were evaluated for matrix removal efficiency, recovery rate, and matrix effect. In this study, EMR-Lipid dSPE was shown to effectively remove complicated matrix contents in soft-gels, compared to DLLME and QuEChERS-dSPE. For the rapid screening of a wide range of adulterants, extracted common ion chromatogram (ECIC) and neutral loss scan (NLS) based on specific common MS/MS fragments were applied to randomly collected soft-gel-type dietary supplement samples using UHPLC-Q/TOF-MS. Both ECICs and NLSs enabled rapid and simple screening of multi-class adulterants and could be an alternative to the multiple reaction monitoring (MRM) method. The developed method was validated in terms of limit of detection (LOD), precision, accuracy, recovery, and matrix effects. The range of LODs was 0.1–16 ng/g. The overall precision values were within 0.09–14.65%. The accuracy ranged from 81.6% to 116.6%. The recoveries and matrix effects of 92 illegal adulterants ranged within 16.9–119.4% and 69.8–114.8%, respectively. The established method was successfully applied to screen and identify 92 illegal adulterants in soft-gels. This method can be a promising tool for the high-throughput screening of various adulterants in dietary supplements and could be used as a more environmentally friendly routine analytical method for screening dietary supplements illegally adulterated with multi-class drug substances.

A global profiling strategy using comprehensive two-dimensional liquid chromatography coupled with dual-mass spectrometry platforms: Chemical analysis of a multi-herb Chinese medicine formula as a case study

Although ultraviolet detector or mass spectrometer could be coupled with two-dimensional liquid chromatography (2DLC) to analyze complex constituents, full detection and identification of the compounds are difficult. Suffering from biased UV detection and insufficient MS data interpretation, a number of minor compounds are neglected though they are separated. In this study, we report a global chemical profiling strategy using comprehensive 2DLC coupled with dual-MS platforms, including Orbitrap-MS and QqQ-MS. It was exemplified by an 11-herb Chinese medicine formula Xiaoer-Feire-Kechuan (XFK). Firstly, constituents in XFK were separated on a CSH C18 × Phenyl-Hexyl 2DLC system with a practical peak capacity of 990.5 and an orthogonality of 90.3%. Secondly, untargeted mass spectral data was collected using dd-MS2 scan on an Orbitrap-MS. In total 542 peaks were detected, which was 4 times of that detected by 2DLC/UV (131 peaks). A total of 108 compounds were tentatively identified. Thirdly, targeted mass spectral data was collected for 8 characteristic substructures using neutral loss and precursor ion (NL/PRE) scan on a QqQ-MS. Extracted ion chromatogram was used to recognize minor constituents. An additional of 151 compounds were detected. Our study indicated that comprehensive 2DLC coupled with dd-MS2 and NL/PRE-MS is a powerful technique for the global profiling of multi-component systems.

Expanding the Coverage of Metabolic Landscape in Cultivated Rice with Integrated Computational Approaches

Genome-scale metabolomics analysis is increasingly used for pathway and function discovery in the post-genomics era. The great potential offered by developed mass spectrometry (MS)-based technologies has been hindered, since only a small portion of detected metabolites were identifiable so far. To address the critical issue of low identification coverage in metabolomics, we adopted a deep metabolomics analysis strategy by integrating advanced algorithms and expanded reference databases. The experimental reference spectra and in silico reference spectra were adopted to facilitate the structural annotation. To further characterize the structure of metabolites, two approaches were incorporated into our strategy, i.e., structural motif search combined with neutral loss scanning and metabolite association network. Untargeted metabolomics analysis was performed on 150 rice cultivars using ultra-performance liquid chromatography coupled with quadrupole-Orbitrap MS. Consequently, a total of 1939 out of 4491 metabolite features in the MS/MS spectral tag (MS2T) library were annotated, representing an extension of annotation coverage by an order of magnitude in rice. The differential accumulation patterns of flavonoids between indica and japonica cultivars were revealed, especially O-sulfated flavonoids. A series of closely-related flavonolignans were characterized, adding further evidence for the crucial role of tricin-oligolignols in lignification. Our study provides an important protocol for exploring phytochemical diversity in other plant species.

Multiple reaction monitoring profiling (MRM profiling): Small molecule exploratory analysis guided by chemical functionality

Small molecules, including metabolites and lipids, provide information on metabolic pathways and active biological processes in living organisms. They are often diagnostic of disease. Current exploratory methods for metabolomics and lipidomics mostly rely on separation using liquid or gas chromatography (LC or GC) coupled with mass spectrometers capable of acquiring high resolution data to generate an enormous data, but at the cost of lengthy processing and data acquisition. Even though many molecules can be identified and quantified by these methods, the laborious protocols for purification, identification, and validation limit the accessible sample chemical information. To improve the speed and efficiency of exploratory metabolomics and lipidomics, multiple reaction monitoring profiling (MRM profiling) has been developed. This strategy involves a three-stage workflow which starts by considering the metabolome as a collection of functional groups. The Discovery Stage interrogates a representative sample mixture for functional groups using the functional group specific precursor ion (Prec) scans and neutral loss (NL) scans. This experiment usually uses a triple quadrupole mass spectrometer without chromatography, i.e. by direct sample infusion. In the second Screening Stage, the main features seen in the Prec and NL scans are organized into lists of precursor ion/product ion transitions (MRMs) which are then used for the fast, specific, and sensitive interrogation of each individual sample. Data analysis by univariate and multivariate statistical methods is used to identify the most informative MRMs and so classify the individual samples. The compounds (biomarkers) which are responsible for the most informative MRMs in particular sample classes can be investigated in an optional third Identification Stage i.e. in a structural identification study. MRM profiling benefits from the much smaller number of functional groups compared to the number of individual metabolites existing in biological samples (where most metabolites are still unknown), resulting in acquisition of a much smaller data set and a shorter analysis time. The application of MRM Profiling to several biological and clinical problems is used to illustrate its features.