Precursor Ion Scan Research Articles

High Specificity and Sensitivity Method for Quantitation of Anthocyanins by Precursor Ion Scan Using Liquid Chromatography‐Tandem Mass Spectrometry Technique

ABSTRACTAnthocyanins, primarily found in flowers, vegetables, and fruits, play a critical role in human and plant health. These polyphenolic pigments are predominantly found in plants in the form of glucosides linked to one or more sugar units and exhibit a broad range of structural diversity. This creates a complex spectrum of anthocyanins making it challenging to perform its absolute quantification in different plant matrices due to the lack of availability of all reference standards. More commonly today, anthocyanins are quantified by spectrophotometric methods, which is non‐specific quantification due to a lack of information on the anthocyanin classes present. In this study, a systematic approach has been proposed to address this challenge, utilizing liquid chromatography‐tandem mass spectrometry using precursor ion scan mode for high‐specificity identification and subsequent quantification of the anthocyanin classes. A method was developed for six anthocyanin classes and it was used to quantify anthocyanins in black carrot extract. The initial scan was conducted for all anthocyanin classes, followed by the quantification of the anthocyanin classes detected using the reference standard specific to that class. This approach has demonstrated that precursor ion scan mass spectrometry can serve as a valuable tool for the rapid identification and specific quantification of anthocyanin classes in plants.

Analysis of pentacyclic triterpenoids and phytosterols in cloudberry (Rubus chamaemorus L.) by LC-MS/MS.

Cloudberry (Rubus chamaemorus L.) is a plant rich in various biologically active compounds. In this work, the composition and concentrations of pentacyclic triterpenoids (PCTs) and phytosterols in various parts of cloudberries were studied using high-performance liquid chromatography-tandem mass spectrometry by multiple reaction monitoring mode (MRM, targeted analysis) and precursor ion scan mode (non-targeted analysis and semi-quantitative determination). Moderately polar fractions of cloudberry leaves, sepals and berries extracts are rich in β-sitosterol glycoside and various triterpenoid acids, among which tormentic acid (∼190-1800 μg/g dw part of cloudberries) and its hydroxyl derivatives (∼3.1-470 μg/g dw part of cloudberries) predominate. Non-polar fractions are characterised by the presence of amyrins (0.76-110 μg/g dw part of cloudberries), β-sitosterol (62-750 µg/g dw part of cloudberries) and β-sitosterol glycoside (∼92-360 μg/g dw sepals).

Comprehensive profiling and evaluation of free/conjugated Phytosterols in crops using chemical derivatization coupled with UHPLC-ESI-QTOF-MS/MS

Phytosterols are naturally existed in crops but their detection is constrained by sensitivity and accuracy due to the inefficient analytical approaches. This study hypothesizes that an untargeted analytical method combining chemical derivatization with ultrahigh performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry can identify the various composition and contents of phytosterols in different crops. The results showed that chemical derivatization significantly enhanced intensity of phytosterols compared with non-derivatized samples. Using precursor ion scanning (PIS) of m/z 252.0690, dansyl chloride-labeled phytosterols were identified, demonstrating that rapeseeds had the highest content of total phytosterol (3981.2 ± 95.3 mg/kg), followed by sunflower seeds, flaxseeds, corn and rice, respectively. Principal component analysis revealed significant variations in phytosterol distribution among 15 crop samples, suggesting the applicability of phytosterol profile as a marker for phytosterols-contained crops. Hence, the proposed analytic approach proves high efficiency and accuracy in determining phytosterols and advances the study for phytosterol-enriched crops.

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.

Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of N-Linked Glycopeptides.

Glycosylation is a common modification across living organisms and plays a central role in understanding biological systems and disease. Our ability to probe the gylcome has grown exponentially in the past several decades. However, further improvements to the analytical toolbox available to researchers would allow for increased capabilities to probe structure and function of biological systems and to improve disease treatment. This article applies the developing technique of two-dimensional Fourier transform ion cyclotron resonance mass spectrometry to a glycoproteomic workflow for the standard glycoproteins coral tree lectin (CTL) and bovine ribonuclease B (BRB) to demonstrate its feasibility as a tool for glycoproteomic workflows. 2D infrared multiphoton dissociation and electron capture dissociation spectra of CTL reveal comparable structural information to their 1D counterparts, confirming the site of glycosylation and monosaccharide composition of the glycan. Spectra collected in 2D of BRB reveal correlation lines of fragment ion scans and vertical precursor ion scans for data collected using infrared multiphoton dissociation and diagonal cleavage lines for data collected by electron capture dissociation. The use of similar techniques for glycoproteomic analysis may prove valuable in instances where chromatographic separation is undesirable or quadrupole isolation is insufficient.

Unveiling distinct influencing mechanisms of Br- on NDMA formation during ozonation of actual water Matrixes: Identification of precursors and brominated substance

Bromide ion (Br-) in water sources has been found to affect the formation of carcinogenic non-brominated N-nitrosodimethylamine (NDMA) during ozonation of typical amines. The increasing bromide levels bring more concern due to climate change and industrial discharge, but its effects on actual water matrixes were still unclear. In this study, NDMA formation from three actual water matrixes during ozonation as the Br- concentration increased from 0 to 100 µM were investigated (promotion (100.4 ∼ 231.6 ng/L), promotion then inhibition (399.5 ∼ 461.2 ∼ 428.2 ng/L), and subtle influence (51.9 ± 6.9 ng/L)); from which the NDMA precursors were screened and 8 precursors were selected for exploring the influencing mechanisms. The results indicated that the roles of Br- on NDMA formation significantly depended on the water characteristics of actual matrixes. A total of 16 typical NDMA precursors, which were mainly derived from industrial, agricultural and pharmaceutical sources, were identified. Moreover, 6 brominated substance (Br-DMA, Br-UDMH, Br-DMSC, Br-DMS, Br-TMDS, Br2-TMDS), which were thought to play a crucial role in promoting NDMA formation, were also identified in actual water by precursor ion scan (PIS) method. Hypobromous acid (HOBr), which has been proved to be the culprit to the generation of brominated substances, tend to attack the electron-donating groups of amines, such as acylamino (–NHCO-R) or amidogen (–NH2) groups. However, the reactive bromine species (∙Br and ∙Br2-) was inclined to attack the aromatic and anti-oxidant moieties then result in Br- release, as no bromine substitution but free radical transfer reaction occurs. Therefore, decreasing HOBr formation is the crucial step to reduce NDMA generation. This work would provide technical guide for NDMA risks control during ozonation of Br--amines co-containing water matrixes.

Insights into the transformation of natural organic matter during UV/peroxydisulfate treatment by FT-ICR MS and machine learning: Non-negligible formation of organosulfates

Natural organic matter (NOM) is a major sink of radicals in advanced oxidation processes (AOPs) and understanding the transformation of NOM is important in water treatment. By using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in conjunction with machine learning, we comprehensively investigated the reactivity and transformation of NOM, and the formation of organosulfates during the UV/peroxydisulfate (PDS) process. After 60 min UV/PDS treatment, the CHO formula number and dissolved organic carbon concentration significantly decreased by 83.4 % and 74.8 %, respectively. Concurrently, the CHOS formula number increased substantially from 0.7 % to 20.5 %. Machine learning identifies DBE and AImod as the critical characteristics determining the reactivity of NOM during UV/PDS treatment. Furthermore, linkage analysis suggests that decarboxylation and dealkylation reactions are dominant transformation pathways, while the additions of SO3 and SO4 are also non-negligible. According to SHAP analysis, the m/z, number of oxygens, DBE and O/C of NOM were positively correlated with the formation of organosulfates in UV/PDS process. 92 organosulfates were screened out by precursor ion scan of HPLC-MS/MS and verified by UPLC-Q-TOF-MS, among which, 7 organosufates were quantified by authentic standards with the highest concentrations ranging from 2.1 to 203.0 ng L‒1. In addition, the cytotoxicity of NOM to Chinese Hamster Ovary (CHO) cells increased by 13.8 % after 30 min UV/PDS treatment, likely responsible for the formation of organosulfates. This is the first study to employ FT-ICR MS combined with machine learning to identify the dominant NOM properties affecting its reactivity and confirmed the formation of organosulfates from sulfate radical oxidation of NOM.

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).

Effects of ozone dose on brominated DBPs in subsequent chlor(am)ination: A comprehensive study of aliphatic, alicyclic and aromatic DBPs

Ozone‒chlor(am)ine is a commonly used combination of disinfectants in drinking water treatment. Although there are quite a few studies on the formation of some individual DBPs in the ozone‒chlor(am)ine disinfection, an overall picture of the DBP formation in the combined disinfection is largely unavailable. In this study, the effects of ozone dose on the formation and speciation of organic brominated disinfection byproducts (DBPs) in subsequent chlorination, chloramination, or chlorination‒chloramination of simulated drinking water were investigated. High-molecular-weight, aliphatic, alicyclic and aromatic brominated DBPs were selectively detected and studied using a powerful precursor ion scan method with ultra performance liquid chromatography/electrospray ionization triple quadrupole mass spectrometry (UPLC/ESI-tqMS). Two groups of unregulated yet relatively toxic DBPs, dihalonitromethanes and dihaloacetaldehydes, were detected by the UPLC/ESI-tqMS for the first time. With increasing ozone dose, the levels of high-molecular-weight (m/z 300–500) and alicyclic and aromatic brominated DBPs generally decreased, the levels of brominated aliphatic acids were slightly affected, and the levels of dihalonitromethanes and dihaloacetaldehydes generally increased in the subsequent disinfection processes. Despite different molecular compositions of the detected DBPs, increasing ozone dose generally shifted the formation of DBPs from chlorinated ones to brominated analogues in the subsequent disinfection processes. This study provided a comprehensive analysis of the impact of ozone dose on the DBP formation and speciation in subsequent chlor(am)ine disinfection.

Intramolecular interactions and the neutral loss of ammonia from collisionally activated, protonated ω-aminoalkyl-3-hydroxyfurazans.

Gas phase fragmentation reactions of monoprotonated 4-(3-aminopropyl)- and 4-(4-aminobutyl)-3-hydroxyfurazan were investigated to examine potential interactions between functional groups. The two heterocyclic alkyl amines were ionized by electrospray ionization (ESI, positive mode) and fragmented using tandem mass spectrometry (MS/MS). The fragmentation pathways were characterized using pseudo MS3 experiments, precursor-ion scans, and density functional computations. For both heterocyclic ions, loss of ammonia was the only fragmentation process observed at low collision energies. Computational analysis indicated that the most feasible mechanism was intramolecular nucleophilic displacement of ammonia from the protonated ω-aminoalkyl side chain by N5 of the furazan ring. The alkylated nitrogen in the resulting bicyclic product ion facilitated N-O bond cleavage; subsequent neutral losses of nitric oxide (NO) and carbon monoxide (CO) occurred by homolytic bond cleavages. Next in the multistep sequence, neutral loss of ethylene from a radical cation was observed. A less favorable, competing fragmentation pathway of protonated 4-(3-aminopropyl)-3-hydroxyfurazan was consistent with cleavage of the 3-hydroxyfurazan ring and losses of NO and CO. Overall, the similar fragmentation behavior found for protonated 4-(3-aminopropyl)- and 4-(4-aminobutyl)-3-hydroxyfurazan differed from that previously characterized for furazan analogs with shorter alkyl chains. These observations demonstrate that a small change in the structure of multifunctional, heterocyclic alkyl amines may significantly influence interactions between distinct functional groups and the nature of the fragmentation process.

Toward Machine Learning-Driven Mass Spectrometric Identification of Trichothecenes in the Absence of Standard Reference Materials.

While a significant body of work exists on the detection of commonly known trichothecene toxins, biological, environmental, and other transformational processes can generate many under-characterized and unknown modified trichothecenes. Lacking both analytical reference standards and associated mass spectral databases, identification of these modified compounds reflects both a challenge and a critical gap from forensic and public health perspectives. We report here the application of machine learning (ML) techniques toward identification of discriminative fragment ions from mass spectrometric data that can be exploited to detect evidence of type A and B trichothecenes. The goal of this work is to establish a new method for the identification of unknown, though structurally similar trichothecenes, by leveraging objective ML techniques. Discriminative fragments derived from a series of gradient-boosted machine learners are then used to develop ML-driven precursor ion scan (PIS) methods on a triple quadrupole mass spectrometer (QQQ) for screening of "unknown unknown" trichothecenes. Specifically, we apply the PIS method to a laboratory-synthesized trichothecene, a first step in demonstrating the power of alternative, machine learning-driven mass spectrometric methods.

A comprehensive analytical strategy based on characteristic fragments to detect synthetic cannabinoid analogs in seized products and hair samples

Synthetic cannabinoids, one of the most widely abused new psychoactive substances (NPS), are now placed under national control generally in China. Due to continuous modification of synthetic cannabinoid structure, an ongoing dilemma in the forensic laboratory is that newly emerging substances cannot be detected by established methods. Thus, the screening methods for simultaneous detection of known or unknown substances have become research hotspots. In this study, the ultra high performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-QqQ-MS) with precursor ion scan (PIS) as acquisition mode was used for prescreening purposes of all possible synthetic cannabinoids-related substances. In detail, four common characteristic fragments, m/z of 144.0, 145.0, 135.1, and 109.0 corresponding to acylium-indole, acylium-indazole, adamantyl, and fluorobenzyl cation respectively, were selected for PIS mode, and their collision energies were optimized by 97 available synthetic cannabinoids standards with relevant structures. Those suspicious signals observed in the screening experiment were confirmed by ultra high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) via high-resolution MS and MS2 data obtained by full scan (TOF MS) and product ion scan mode. After methodological validation, the integrated strategy established above was applied to the screening and identification of the seized e-liquids, herbal blends and hair samples, confirming the presence of multiple synthetic cannabinoids in these samples. In particular, a novel synthetic cannabinoid was identified as 4 F-ABUTINACA, for which no relevant high-resolution mass spectrometry (HRMS) data has been retrieved until now, making this study the first to report the cleavage pattern of this compound in electrospray ionization (ESI) mass spectrometry. In addition, four other suspected by-products of the synthetic cannabinoids were found in the herbal blends and e-liquids, and their possible structures were also deduced via the information from high-resolution mass spectra.

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.

Recent advances of data‐independent acquisition mass spectrometry‐based proteomics

Recent advances of data‐independent acquisition mass spectrometry‐based proteomics

Suspect Screening and Semi-Quantification of Macrolide Antibiotics in Municipal Wastewater by High-Performance Liquid Chromatography—Precursor Ion Scan Tandem Mass Spectrometry

Macrolides are widely used in medicine and veterinary medicine, and are the leading antibiotics in terms of consumption. The release of macrolides and their metabolites into the environment through municipal wastewater can have an adverse effect on aquatic ecosystems and human health. In the present study, a method for the non-targeted screening and semi-quantitative determination of macrolide antibiotics and their derivatives in wastewater based on a combination of chromatographic separation and tandem mass spectrometric detection in precursor ion scan (PrecIS) mode has been proposed. Product ions with m/z 158 and 174 related to specific desosamine fragments were used as diagnostic ions for the PrecIS detection of the macrolide structures without (14- and 15-membered macrocycles) and with a (16-membered macrocycle) glycosylated desosamine moiety, respectively. The combination of the optimized solid phase extraction procedure and HPLC-MS/MS analysis in PrecIS mode allowed for the suspect screening of macrolides in municipal wastewater with limits of detection in the range of 4–150 ng L−1. The developed approach made it possible to detect and tentatively identify in municipal wastewater 17 compounds belonging to the macrolide class, including azithromycin, clarithromycin, josamycin and 14 metabolites with a total concentration of 1450 ng L−1.

Chemical constituents in different parts of Ixeris sonchifolia based on UPLC-LTQ-Orbitrap-MS~n

The chemical constituents in stem leaf, root, and flower of Ixeris sonchifolia were identified by the ultra performance li-quid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS~n). The separation was performed on an Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) with a mobile phase of water(containing 0.1% formic acid, A)-acetonitrile(B) with gradient elution. With electrospray ionization source, the data of 70% methanol extract from stem leaf, root and flower of I. sonchifolia were collected by high-resolution full-scan Fourier transform spectroscopy, data dependent acquisition, precursor ion scan, and selected ion monitoring in the negative and positive ion modes. The compounds were identified based on accurate molecular weight, retention time, fragment ions, comparison with reference standard, Clog P and references. A total of 131 compounds were identified from the 70% methanol extract of I. sonchifolia, including nucleosides, flavonoids, organic acids, terpenoids, and phenylpropanoids, and 119, 110, and 126 compounds were identified from the stem leaf, root and flower of I. sonchifolia, respectively. In addition, isorhamnetin, isorhamnetin-7-O-sambubioside and caffeylshikimic acid were discovered from I. sonchifolia for the first time. This study comprehensively analyzed and compared the chemical constituents in different parts of I. sonchifolia, which facilitated the discovery of effective substances and the development and application of medicinal material resources of I. sonchifolia.

Efficient optimization and development of two methods for the determination of acrylamide in deep-frying oil by liquid chromatography-tandem mass spectrometry: Application of multifactor analysis assessment strategy.

A new multifactor analysis assessment strategy was developed for evaluating, optimizing, and comparing analytical techniques for acrylamide in frying oils. Based on five indices (absolute recovery, absolute matrix effect, the intensity of the full ion scan, and the precursor ion scan to m/z 184 and m/z 241), the proposed strategy was performed with radar analysis, relative contribution analysis, and the entropy-weighted technique for order performance by similarity to ideal solution analysis. Two novel methods based on quick, easy, cheap, effective, rugged, and safe extraction methodology and gel permeation chromatography-liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry have been developed for the analysis of acrylamide in frying oils. Two methods were suitable for rapid and sensitive analysis of acrylamide in oils in different laboratories, with a limit of quantitation at 2μg/kg, and the average recovery ranging from 92.5% to 107.8%, with relative standard deviations below 10%. When considering automation efficiency and matrix effects, gel permeation chromatography is the most efficient method, whereas the other method has an advantage when analyzing large samples. The developedmethods were used in a pilot study to analyze frying oils with acrylamide content below 9.82μg/kg, showing that the repeated frying process did not produce significant content of acrylamide in oils.

Differentiating root and rhizome of panax notoginseng based on precursor ion scanning and multi heart-cutting two-dimensional liquid chromatography.

Owing to increasing demand for Panax notoginseng-based medicines and health products, establishing a fast, simple, and reliable assay to analyze the chemical differences between its root and rhizome is important. Although previous studies showed that the chemical and biological differences between the root and rhizome of P. notoginseng seem to be small, efforts should be taken to investigate such differences to ensure the safety and efficacy of the products. This work describes a holistic approach that combines characteristic fingerprinting using ultra-high performance liquid chromatography-tandem mass spectrometry parent ion scanning with charged aerosol detection and targeted separation by online heart-cutting two-dimensional liquid chromatography, to identify and evaluate characteristic markers allowing differentiation of the root and rhizome. A total of five potential markers chikusetsusaponin L5 , ginsenoside Rb2 , stipuleanoside R2, malonyl-ginsenoside Rb1 , and malonyl-ginsenoside Rd, were identified and confirmed by comparing chromatographic retention time, the accurate mass of molecular weight, and the fragments of secondary MS with the available reference materials. The results showed that all five markers were 2.8-7 times higher in content in the rhizome than in the root.

Characterization of long-chain fatty acid-linked bile acids: a major conjugation form of 3β-hydroxy bile acids in feces

Although most bile acids (BAs) in feces are present in noncovalent forms that can be extracted with ethanol, non-negligible amounts of saponifiable BAs are also present. It is a major concern that such saponifiable BAs are routinely omitted from fecal BA measurements. We compared the BA profiles of healthy stools that were obtained with/without alkaline hydrolysis and found that as much as 29.7% (2.1–67.7%) of total BAs were saponifiable. Specifically, alkaline treatment led to significant elevations of isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) concentrations, suggesting that considerable proportions of isoDCA and isoLCA were esterified. Precursor ion scan data from LC/MS suggested the presence of long-chain FA-linked BAs. We chemically synthesized a series of fatty acid 3β-acyl conjugates of isoDCA and isoLCA as analytical standards and analyzed their fecal profiles from newborns to adults (n = 64) by LC/MS. FA-conjugated isobile acids (FA-isoBAs) were constantly present from 2 years of age to adulthood. C16- and C18-chain FA-isoBA esters were predominantly found regardless of age, but small amounts of acetic acid esters were also found. FA-isoBA concentrations were not correlated to fecal FA concentrations. Interestingly, there were some adults who did not have FA-isoBAs. Gut bacteria involved in the production of FA-isoBAs have not been identified yet. The present study provides insight into the establishment of early gut microbiota and the interactive development of esterified BAs.The contribution of FA-isoBAs to gut physiology and their role in pathophysiologic conditions such as inflammatory bowel disease are currently under investigation.

Chain-locked precursor ion scanning based HPLC–MS/MS for in-depth molecular analysis of lipase-catalyzed transesterification of structured phospholipids containing ω-3 fatty acyl chains

Lipase-catalyzed transesterification of structured phospholipids (sPLs) is a hot topic, but the structural variation of the fatty acyl chains in intact phospholipids at the molecular level remains unclear to date. The present study explored the detailed characteristics of synthesized phospholipids through high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) in precursor ion scan mode. The optimal conditions were in-depth inspected and determined for the reaction system, including phospholipase A1 as catalyst, 15% lipase loading, and 1% water content. The sPLs enriched with EPA/DHA were structurally and quantitatively characterized by focusing on the fragments of m/z 301.6 (eicosapentaenoic acid, EPA) and m/z 327.6 (docosahexaenoic acid, DHA), and the results were statistically analyzed using partial least squares discriminant analysis and clustered heatmap hierarchical clustering analysis. PC 38:6 (18:1/20:5), PC 38:7 (18:2/20:5), PC o-40:6 (o-18:0/22:6), and PE 40:8 (18:2/22:6) etc. were revealed as the main variables that were active in the reaction.