Recently Discovered Secondary Metabolites from Streptomyces Species

The Streptomyces genus has been a rich source of bioactive natural products, medicinal chemicals, and novel drug leads for three-quarters of a century. Yet studies suggest that the genus is capable of making some 150,000 more bioactive compounds than all Streptomyces secondary metabolites reported to date. Researchers around the world continue to explore this enormous potential using a range of strategies including modification of culture conditions, bioinformatics and genome mining, heterologous expression, and other approaches to cryptic biosynthetic gene cluster activation. Our survey of the recent literature, with a particular focus on the year 2020, brings together more than 70 novel secondary metabolites from Streptomyces species, which are discussed in this review. This diverse array includes cyclic and linear peptides, peptide derivatives, polyketides, terpenoids, polyaromatics, macrocycles, and furans, the isolation, chemical structures, and bioactivity of which are appraised. The discovery of these many different compounds demonstrates the continued potential of Streptomyces as a source of new and interesting natural products and contributes further important pieces to the mostly unfinished puzzle of Earth’s myriad microbes and their multifaceted chemical output.     

Plant cell culture technologies: A promising alternatives to produce high-value secondary metabolites

Plants have been used for its medicinal values since ancient time. The medicinal properties of plants are based on their phytochemical constituent particularly secondary metabolites which are produced in low amounts by plants. Secondary metabolites have been used as medicines, flavors, colors, and fragrances. In recent time, these natural compounds are gaining enormous attention in pharmaceutical, cosmetics, and nutraceutical industries and are regarded economically valuable products. The production of plant secondary metabolites in plant is largely dependent on the plant species, environmental factors and geographical regions. In addition, the main challenges in their mass production is reported to be the quality and quantity issues during their synthesis. Therefore, enthusiasm has grown for increasing the production of secondary metabolites by employing in vitro plant cell culture technology and bioengineering methods. Such technological advancement, has led to production of a huge number of medicinal herbs and high-value secondary metabolites that are mostly used in pharmaceuticals, cosmetics and nutraceuticals industries. The current mini-review article focuses on applications of plant cell culture system for the production secondary metabolites and recent techniques used to improve metabolite contents. Furthermore, our review emphasizes safety issues of plant cell culture derived products.     

Expanding the Myxochelin Natural Product Family by Nicotinic Acid Containing Congeners

Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1–N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.     

Two New Triterpenes from Basidiomata of the Medicinal and Edible Mushroom,Laetiporus sulphureus

In the search for novel anti-infectives from natural sources, fungi, in particular basidiomycetes, have proven to still harbor so much potential in terms of secondary metabolites diversity. There have been numerous reports on isolating numerous secondary metabolites from genus Laetiporus. This study reports on two new triterpenoids, laetiporins C and D, and four known triterpenes from the fruiting body of L. sulphureus. The structures of the isolated compounds were elucidated based on their 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data in combination with high-resolution electrospray mass spectrometric (HR-ESIMS) data. Laetiporin C exhibited weak antifungal activity against Mucor hiemalis. Furthermore, the compounds showed weak antiproliferative activity against the mouse fibroblast L929 and human cancer cell lines, including KB-3-1, A431, MCF-7, PC-3 and A549.     

An approach to identifying sequential metabolites of a typical phenylethanoid glycoside, echinacoside, based on liquid chromatography-ion trap-time of flight mass spectrometry analysis

Metabolite identification for the compounds that undergo multiple and sequential metabolism is still a great challenge. Echinacoside (ECH), a typical phenylethanoid glycoside, contains multiple unstable chemical bonds and high reactive functional groups which are susceptible to multiple pathways of degradation and metabolism, leading great difficulties for its metabolite identification. This study proposed a novel approach for rapidly identifying the complicated and unpredictable metabolites of ECH, based on the powerful liquid chromatography hybrid ion trap and time of flight mass spectrometry (LC/MS-IT-TOF) analysis. Four degradation products were rapidly identified via the “fragmentation-degradation” comparisons. Five phase I and phase II metabolites of the degradation products were rapidly characterized via the crossover mass differences comparisons of their quasi-molecular ions with the potential precursors. Four direct phase I and phase II metabolites of the parent compound were identified by the mass differences analysis of the molecular ions between metabolites and the parent compound. Multiple stages of fragmentation patterns were used to confirm the metabolites characterizations. This study provides a novel approach to characterizing the complicated metabolites, and would be widely applicable for the metabolite identification of natural products.     

Pharmacological Insights into Halophyte Bioactive Extract Action on Anti-Inflammatory,Pain Relief and Antibiotics-Type Mechanisms

The pharmacological activities in bioactive plant extracts play an increasing role in sustainable resources for valorization and biomedical applications. Bioactive phytochemicals, including natural compounds, secondary metabolites and their derivatives, have attracted significant attention for use in both medicinal products and cosmetic products. Our review highlights the pharmacological mode-of-action and current biomedical applications of key bioactive compounds applied as anti-inflammatory, bactericidal with antibiotics effects, and pain relief purposes in controlled clinical studies or preclinical studies. In this systematic review, the availability of bioactive compounds from several salt-tolerant plant species, mainly focusing on the three promising species Aster tripolium, Crithmum maritimum and Salicornia europaea, are summarized and discussed. All three of them have been widely used in natural folk medicines and are now in the focus for future nutraceutical and pharmacological applications.     

Histone deacetylase inhibitor induced the production of three novel prenylated tryptophan analogs in the entomopathogenic fungus, Torrubiella luteorostrata

Suberoyl bis-hydroxamic acid (SBHA), a histone deacetylase (HDAC) inhibitor, led to significant changes in the secondary metabolism of an entomopathogenic fungus, Torrubiella luteorostrata, and induced the production of three new prenylated tryptophan analogs, luteorides A-C (1-3). The structures are characterized by the presence of an (E)-oxime group, which is an unusual functional group in natural products, and a 3-methylbuta-1,3-dienyl unit as a common substituent. The method of culturing entomopathogenic fungi in the presence of HDAC inhibitors, such as SBHA, is convenient and attractive for obtaining novel secondary metabolites.     

Neuronal metabolomics by ion mobility mass spectrometry: cocaine effects on glucose and selected biogenic amine metabolites in the frontal cortex, striatum, and thalamus of the rat

We report results of studies of global and targeted neuronal metabolomes by ambient pressure ion mobility mass spectrometry. The rat frontal cortex, striatum, and thalamus were sampled from control nontreated rats and those treated with acute cocaine or pargyline. Quantitative evaluations were made by standard additions or isotopic dilution. The mass detection limit was ～100 pmol varying with the analyte. Targeted metabolites of dopamine, serotonin, and glucose followed the rank order of distribution expected between the anatomical areas. Data was evaluated by principal component analysis on 764 common metabolites (identified by m/z and reduced mobility). Differences between anatomical areas and treatment groups were observed for 53 % of these metabolites using principal component analysis. Global and targeted metabolic differences were observed between the three anatomical areas with contralateral differences between some areas. Following drug treatments, global and targeted metabolomes were found to shift relative to controls and still maintained anatomical differences. Pargyline reduced 3,4-dihydroxyphenylacetic acid below detection limits, and 5-HIAA varied between anatomical regions. Notable findings were: (1) global metabolomes were different between anatomical areas and were altered by acute cocaine providing a broad but targeted window of discovery for metabolic changes produced by drugs of abuse; (2) quantitative analysis was demonstrated using isotope dilution and standard addition; (3) cocaine changed glucose and biogenic amine metabolism in the anatomical areas tested; and (4) the largest effect of cocaine was on the glycolysis metabolome in the thalamus confirming inferences from previous positron emission tomography studies using 2-deoxyglucose.

Myxobacterial natural product assembly lines: fascinating examples of curious biochemistry

Over the last 20 years myxobacteria have made their way from highly exotic organisms to one of the major sources of microbial natural products with interesting biological activities. Recent progress towards achieving a better understanding of the genetics and the biochemistry of myxobacterial secondary metabolism, revealed the involvement of numerous exceptional combinations of polyketide synthases and nonribosomal peptide synthetases operating far from textbook biosynthetic logic. In this Highlight, selected examples of recently described systems are discussed in comparison to all myxobacterial natural product assembly lines known to date.     

Chromatography-bioluminescence coupling reveals surprising bioactivity of inthomycin A

By employing a novel technique for the direct coupling of a bacterial bioassay with chromatography, we discovered a gyrA promotor active compound in myxobacterial extracts and elucidated the structure directly without any isolation step. As a result, we identified inthomycin A as the bioactive substance. Our method is based on a whole-cell bioluminescent reporter gene assay coupled with thin-layer chromatography for primary hit detection and with liquid chromatography (LC)/mass spectrometry or LC/NMR for dereplication and structure elucidation. Previously, inthomycin A has been isolated from Streptomycetes and was associated with the inhibition of cellulose biosynthesis and herbicidal activity. Thus, our findings support the basic principle that completely different microbial phyla are able to synthesize the same natural product. Moreover, our results indicate that inthomycin A affects bacterial DNA supercoiling, which reveals an unexpected bioactivity for this compound. These results can possibly promote further investigation of the biosynthesis as well as the biological activity of inthomycins and related natural products.

Cytosporin-related compounds from the marine-derived fungus Eutypella scoparia

Chemical investigation of the culture broth of the fungus Eutypella scoparia ICB-OBX, isolated from the marine pulmonate mollusc Onchidium sp., led to the finding of novel compounds 1 and 2, structurally related to angiotensin II binding inhibitors cytosporins, along with unrelated known nitrogen metabolites (compounds 3-5). The structure and the relative stereochemistry of the novel metabolites were assigned mainly by a detailed analysis of two-dimensional NMR techniques whereas the absolute stereochemistry was proposed by modified Mosher's method. Compound 2 contains an unusual cyclic carbonate functionality that is rare among natural products.     

Synergizing the potential of bacterial genomics and metabolomics to find novel antibiotics

Antibiotic development based on natural products has faced a long lasting decline since the 1970s, while both the speed and the extent of antimicrobial resistance (AMR) development have been severely underestimated. The discovery of antimicrobial natural products of bacterial and fungal origin featuring new chemistry and previously unknown mode of actions is increasingly challenged by rediscovery issues. Natural products that are abundantly produced by the corresponding wild type organisms often featuring strong UV signals have been extensively characterized, especially the ones produced by extensively screened microbial genera such as streptomycetes. Purely synthetic chemistry approaches aiming to replace the declining supply from natural products as starting materials to develop novel antibiotics largely failed to provide significant numbers of antibiotic drug leads. To cope with this fundamental issue, microbial natural products science is being transformed from a ‘grind-and-find’ study to an integrated approach based on bacterial genomics and metabolomics. Novel technologies in instrumental analytics are increasingly employed to lower detection limits and expand the space of detectable substance classes, while broadening the scope of accessible and potentially bioactive natural products. Furthermore, the almost exponential increase in publicly available bacterial genome data has shown that the biosynthetic potential of the investigated strains by far exceeds the amount of detected metabolites. This can be judged by the discrepancy between the number of biosynthetic gene clusters (BGC) encoded in the genome of each microbial strain and the number of secondary metabolites actually detected, even when considering the increased sensitivity provided by novel analytical instrumentation. In silico annotation tools for biosynthetic gene cluster classification and analysis allow fast prioritization in BGC-to-compound workflows, which is highly important to be able to process the enormous underlying data volumes. BGC prioritization is currently accompanied by novel molecular biology-based approaches to access the so-called orphan BGCs not yet correlated with a secondary metabolite. Integration of metabolomics, in silico genomics and molecular biology approaches into the mainstream of natural product research will critically influence future success and impact the natural product field in pharmaceutical, nutritional and agrochemical applications and especially in anti-infective research.

Antimicrobial resistance is a major public concern and novel antibiotics are largely based on natural products. We summarize recent analytical and genome based technological developments that gain increasing importance in the natural products field.     

Metabolomics data exploration guided by prior knowledge

In metabolomics research, it is often important to focus the data analysis to specific areas of interest within the metabolome. In this paper, we describe the application of consensus principal component analysis (CPCA) and canonical correlation analysis (CCA) as a means to explore the relation between metabolome data and (i) biochemically related metabolites and (ii) an amino acid biosynthesis pathway. CPCA searches for major trends in the behavior of metabolite concentrations that are in common for the metabolites of interest and the remainder of the metabolome. CCA identifies the strongest correlations between the metabolites of interest and the remainder of the metabolome.CPCA and CCA were applied to two different microbial metabolomics data sets. The first data set, derived from Pseudomonas putida S12, was relatively simple as it contained metabolomes obtained under four environmental conditions only. The second data set, obtained from Escherichia coli, was much more complex as it consisted of metabolomes obtained under 28 different environmental conditions. In case of the simple and coherent P. putida S12 data set, CCA and CPCA gave similar results as the variation in the subset of the selected metabolites and the remainder of the metabolome was similar.In contrast, CCA and CPCA yielded different results in case of the E. coli data set. With CPCA the trends in the selected subset - the phenylalanine biosynthesis pathway - dominated the results. The main trends were related to high and low phenylalanine productivity, and the metabolites showing a similar behavior in concentration were metabolites regulating the phenylalanine biosynthesis route in the subset and metabolites related to general amino acid metabolism in the remainder of the metabolome. With CCA, neither subset truly dominated the data analysis. CCA described the differences between the wild type and the overproducing strain and the differences between the succinate and glucose grown cells. For the difference between the wild type and the overproducing strain, metabolites from the beginning and the end of aromatic amino acid pathways like erythrose-4-phosphate, tryptophan, and phenylalanine were important for the selected metabolites.CCA and CPCA proved to be complementary data analysis tools that enable the focusing of the data analysis on groups of metabolites that are of specific interest in relation to the remainder of the metabolome. Compared to an ordinary PCA, focusing the data analysis on biologically relevant metabolites lead especially for the complex E. coli data to a better biological interpretation of the data.     

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

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

Excretion,Metabolism, and Tissue Distribution of Gelsemium elegans (Gardn. & Champ.) Benth in Pigs

Gelsemium elegans (Gardn. & Champ.) Benth is a toxic flowering plant in the family Loganiaceae used to treat skin diseases, neuralgia and acute pain. The high toxicity of G. elegans restricts its development and clinical applications, but in veterinary applications, G. elegans has been fed to pigs as a feed additive without poisoning. However, until now, the in vivo processes of the multiple components of G. elegans have not been studied. This study investigates the excretion, metabolism and tissue distribution of the multiple components of G. elegans after feeding it to pigs in medicated feed. Pigs were fed 2% G. elegans powder in feed for 45 days. The plasma, urine, bile, feces and tissues (heart, liver, lung, spleen, brain, spinal cord, adrenal gland, testis, thigh muscle, abdominal muscle and back muscle) were collected 6 h after the last feeding and analyzed using high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Five natural products in plasma, twelve natural products and five metabolites in urine, and three natural products in feces were characterized, suggesting that multiple components from G. elegans were excreted in the urine. However, ten natural products and four metabolites were detected in bile samples, which suggested that G. elegans is involved in enterohepatic circulation in pigs. A total of seven of these metabolites were characterized, and four metabolites were glucuronidated metabolites. Ten natural products and six metabolites were detected in the tissues, which indicates that G. elegans is widely distributed in tissues and can cross the blood-brain barrier. Among the characterized compounds, a highly toxic gelsedine-type alkaloid from G. elegans was the main compound detected in all biological samples. This is the first study of the excretion, metabolism and tissue distribution of multiple components from G. elegans in pigs. These data can provide an important reference to explain the efficacy and toxicity of G. elegans. Additionally, the results of the tissue distribution of G. elegans are of great value for further residue depletion studies and safety evaluations of products of animals fed G. elegans.     

Biodiversity of Secondary Metabolites Compounds Isolated from Phylum Actinobacteria and Its Therapeutic Applications

The current review aims to summarise the biodiversity and biosynthesis of novel secondary metabolites compounds, of the phylum Actinobacteria and the diverse range of secondary metabolites produced that vary depending on its ecological environments they inhabit. Actinobacteria creates a wide range of bioactive substances that can be of great value to public health and the pharmaceutical industry. The literature analysis process for this review was conducted using the VOSviewer software tool to visualise the bibliometric networks of the most relevant databases from the Scopus database in the period between 2010 and 22 March 2021. Screening and exploring the available literature relating to the extreme environments and ecosystems that Actinobacteria inhabit aims to identify new strains of this major microorganism class, producing unique novel bioactive compounds. The knowledge gained from these studies is intended to encourage scientists in the natural product discovery field to identify and characterise novel strains containing various bioactive gene clusters with potential clinical applications. It is evident that Actinobacteria adapted to survive in extreme environments represent an important source of a wide range of bioactive compounds. Actinobacteria have a large number of secondary metabolite biosynthetic gene clusters. They can synthesise thousands of subordinate metabolites with different biological actions such as anti-bacterial, anti-parasitic, anti-fungal, anti-virus, anti-cancer and growth-promoting compounds. These are highly significant economically due to their potential applications in the food, nutrition and health industries and thus support our communities’ well-being.     

Total synthesis approaches to natural product derivatives based on the combination of chemical synthesis and metabolic engineering

Secondary metabolites are an extremely diverse and important group of natural products with industrial and biomedical implications. Advances in metabolic engineering of both native and heterologous secondary metabolite producing organisms have allowed the directed synthesis of desired novel products by exploiting their biosynthetic potentials. Metabolic engineering utilises knowledge of cellular metabolism to alter biosynthetic pathways. An important technique that combines chemical synthesis with metabolic engineering is mutasynthesis (mutational biosynthesis; MBS), which advanced from precursor-directed biosynthesis (PDB). Both techniques are based on the cellular uptake of modified biosynthetic intermediates and their incorporation into complex secondary metabolites. Mutasynthesis utilises genetically engineered organisms in conjunction with feeding of chemically modified intermediates. From a synthetic chemist's point of view the concept of mutasynthesis is highly attractive, as the method combines chemical expertise with Nature's synthetic machinery and thus can be exploited to rapidly create small libraries of secondary metabolites. However, in each case, the method has to be critically compared with semi- and total synthesis in terms of practicability and efficiency. Recent developments in metabolic engineering promise to further broaden the scope of outsourcing chemically demanding steps to biological systems.     

Lentzeacins A-E,New Bacterial-Derived 2,5- and 2,6-Disubstituted Pyrazines from a BGC-Rich Soil Bacterium Lentzea sp. GA3-008

Pyrazines (1,4-diazirines) are an important group of natural products that have tremendous monetary value in the food and fragrance industries and can exhibit a wide range of biological effects including antineoplastic, antidiabetic and antibiotic activities. As part of a project investigating the secondary metabolites present in understudied and chemically rich Actinomycetes, we isolated a series of six pyrazines from a soil-derived Lentzea sp. GA3-008, four of which are new. Here we describe the structures of lentzeacins A-E (1, 3, 5 and 6) along with two known analogues (2 and 4) and the porphyrin zincphyrin. The structures were determined by NMR spectroscopy and HR-ESI-MS. The suite of compounds present in Lentzea sp. includes 2,5-disubstituted pyrazines (compounds 2, 4, and 6) together with the new 2,6-disubstituted isomers (compounds 1, 3 and 5), a chemical class that is uncommon. We used long-read Nanopore sequencing to assemble a draft genome sequence of Lentzea sp. which revealed the presence of 40 biosynthetic gene clusters. Analysis of classical di-modular and single module non-ribosomal peptide synthase genes, and cyclic dipeptide synthases narrows down the possibilities for the biosynthesis of the pyrazines present in this strain.     

Novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga15

BACKGROUND: Myxobacteria have been well established as a potent source for natural products with biological activity. They produce a considerable variety of compounds which represent typical polyketide structures with incorporated amino acids (e.g. the epothilons, the myxothiazols and the myxalamids). Several of these secondary metabolites are effective inhibitors of the electron transport via the respiratory chain and have been widely used. Molecular cloning and characterization of the genes governing the biosynthesis of these structures is of considerable interest, because such information adds to the limited knowledge as to how polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) interact and how they might be manipulated in order to form novel antibiotics. RESULTS: A DNA region of approximately 50000 base pairs from Stigmatella aurantiaca Sga15 was sequenced and shown by gene disruption to be involved in myxalamid biosynthesis. Sequence analysis reveals that the myxalamids are formed by a combined PKS/NRPS system. The terminal NRPS MxaA extends the assembled polyketide chain of the myxalamids with alanine. MxaA contains an N-terminal domain with homology to NAD binding proteins, which is responsible during the biogenesis for a novel type of reductive chain release giving rise to the 2-amino-propanol moiety of the myxalamids. The last module of the PKS reveals an unprecedented genetic organization; it is encoded on two genes (mxaB1 and mxaB2), subdividing the domains of one module from each other. A sequence comparison of myxobacterial acyl-transferase domains with known systems from streptomycetes and bacilli reveals that consensus sequences proposed to be specific for methylmalonyl-CoA and malonyl-CoA are not always reliable. CONCLUSIONS: The complete biosynthetic gene cluster of the myxalamid-type electron transport inhibitor from S. aurantiaca Sga15 has been cloned and analyzed. It represents one of the few examples of combined PKS/NRPS systems, the analysis and manipulation of which has the potential to generate novel hybrid structures via combinatorial biosynthesis (e.g. via module-swapping techniques). Additionally, a new type of reductive release from PKS/NRPS systems is described.     

In vitro stable isotope labeling for discovery of novel metabolites by liquid chromatography–mass spectrometry: Confirmation of γ-tocopherol metabolism in human A549 cell

A general approach for discovering novel catabolic metabolites from a parent biocompound was developed and validated on the metabolism of γ-tocopherol in human A549 cell. The method is based on LC–MS analysis of in vitro stable isotope-labeled metabolites and assumes that a parent compound and its metabolites share a common functional group that can be derivatized by well-documented reagents. In this method, two equal aliquots of extracted metabolites are separately derivatized with isotope-coded (heavy) and non-isotope-coded (light) form of derivatizing reagent, mixed at 1:1 ratio and analyzed using LC–MS. The metabolites with common functional group are then easily recognized by determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to the mass difference between heavy and light form of derivatization reagent. The feasibility of this approach was demonstrated and validated by the identification of products of γ-tocopherol catabolism in human A549 cell culture media using N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NANHS) and N-methyl-d3-nicotinic acid N-hydroxysuccinimide ester (C1-d3-NANHS) derivatizing reagent. Overall four γ-tocopherol metabolites were identified including 9′-COOH, 11′-COOH, 13′-COOH and 13′-OH. In addition, the developed LC–MS method can also be used for the fast and sensitive quantitative analysis of γ-tocopherol and other forms of vitamin E related compounds.