High-performance Liquid Chromatography Coupled To High-resolution Mass Spectrometry Research Articles

Analysis of phytocannabinoids in hemp seeds, sprouts and microgreens

Hemp-sprouts are emerging as a new class of attractive functional food due to their numerous health benefits when compared to other sprout species. Indeed, the high content of beneficial components including polyphenols and flavonoids makes this type of food a promising and successful market. However, the available literature on this topic is limited and often conflicting as regards to the content of phytocannabinoids. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was applied in an untargeted metabolomics fashion to extracts of hemp seeds, sprouts and microgreens of nine different genotypes. Both unsupervised and supervised multivariate statistical analysis was performed to reveal variety-specific profiles of phytocannabinoids with surprisingly remarkable levels of phytocannabinoids even in chemotype V samples. Furthermore, a targeted HPLC-HRMS analysis was carried out for the quantitative determination of the major phytocannabinoids including CBDA, CBD, CBGA, CBG, CBCA, CBC, THCA, and trans-Δ9-THC. The last part of the study was focused on the evaluation of the enantiomeric composition of CBCA in hemp seeds, sprouts and microgreens in the different varieties by HPLC-CD (HPLC with online circular dichroism). Chiral analysis of CBCA showed a wide variability of its enantiomeric composition in the different varieties, thus contributing to the understanding of the intriguing stereochemical behavior of this compound in an early growth stage. However, further investigation is needed to determine the genetic factors responsible for the low enantiopurity of this compound.

Profiling and optimized extraction of bioactive polyphenolic compounds from young, red-fleshed apple using eco-friendly deep eutectic solvents

Red-fleshed apple cultivars with an enhanced content of polyphenolic compounds have attracted increasing interest due to their promising health benefits. Here, we have analysed the polyphenolic content of young, red-fleshed apples (RFA) and optimised extraction conditions of phenolics by utilising natural deep eutectic solvents (NDES). We also compare the antioxidant, neuroprotective and antimicrobial activities of NDES- and methanol-extracted phenolics from young RFA. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was used for phenolics identification and quantification. Besides young RFA, ripe red-fleshed, young and ripe white-fleshed apples were analysed, revealing that young RFA possess the highest phenolic content (2078.4 ± 4.0 mg gallic acid equivalent/100 g), and that ripe white-fleshed apples contain the least amount of phenolics (545.0 ± 32.0 mg gallic acid equivalent/100 g). The NDES choline chloride-glycerol containing 40 % w/w H2O gave similar yields at 40 °C as methanol. In addition, the polyphenolics profile, and bioactivities of the NDES extract from young RFA were comparable that of methanol extracts. Altogether, our data show that NDES extracts of young RFA are a promising source of bioactive polyphenolics with potential applications in diverse sectors, e.g., for functional food production, smart material engineering and natural therapies.

Exposure to micro(nano)plastics polymers in water stored in single-use plastic bottles

Human exposure to micro (nano)plastics (MNPLs) has become a significant concern as a potential health threat. Exposure routes include ingestion, inhalation, and dermal contact, being food and drinking water the primary sources of oral exposure. Here we present the quantification of polymers of MNPLs particles from 700 nm to 20 μm in bottled water commercialised in Spain, including an estimation of the potential risk for daily consumers.We evaluated samples from 20 popular brands in 0.5 and 1.5 L plastic bottles. A double-suspect screening approach developed and validated in our research group for drinking water was adapted for bottled water samples. The identification and quantification of MNPLs-polymers in mass units and the tentative identification of plastic additives (PA) until the second level of confidence was carried out based on high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS).The results showed the presence of polypropylene (PP), polyethylene (PE) and polypropylene terephthalate (PET) in the samples. Among them, PE was the most frequently detected and quantified polymer (55% of samples) followed by PET which was detected in 33% of the samples and showing the highest concentration (4700 ng L−1). The median value of the sum of polymer concentrations was 359 ng L−1.In addition, 28 plastic additives were detected, where at least one of them was present in 100% of the samples. Stabilizers and plasticisers were the most frequently identified. A prioritisation study was performed using a multi-QSAR modelling software, where bis(2-ethylhexyl) adipate and bis(2-ethylhexyl) phthalate were estimated as the most potentially harmful compounds for human health.Overall, findings suggest that bottled water is a non-negligible route to exposure to MNPLs.

Biodegradation mechanism of perfluorooctane sulfonic acid (PFOS) in domestic sewage: Specific methanogenic activity, molecular biology, and ecotoxicological aspects

Among the perfluorinated compounds (PFCs), perfluorooctane sulfonic acid (PFOS) is one of the most used ones worldwide, specially in the textile industry as a surfactant. In addition, this contaminant is present in several products, such as: fire extinguisher foams, insecticides formulations, electronic devices, and kitchen products. PFOS is an endocrine disruptor, undergoes bioaccumulation, resists to biological degradation, and has been detected in human blood, animals, dusts, sediments, surface waters, and groundwater as well. Therefore, this research aimed at assessing the degradation of PFOS (100 μg L−1) in anaerobic conditions using simulated domestic sewage as co-substrate. After 10 days, at pH 6 and 35 °C, a 24 % PFOS degradation was achieved. In this period of time, inhibition of specific methanogenic activity (SMA) and changes to the microbial consortium (Archaea and Bacteria Domains) were not observed. The biodegradation products (BPs) were identified by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-MS). One of them is reported here for the first time: C4F9CHO (2,2,3,3,4,4,5,5,5-nonafluoropentanal). BPs acute and chronic ecotoxicities for three aquatic organisms (fishes, daphnids, and green algae) and lipophilicity (log D, pH 7.4) were estimated using the ECOSAR 1.11 and Chemicalize software, respectively. Taken as a whole, they presented increased lipophilicities and ecotoxicities compared to PFOS. The present study showed, for the first time, that PFOS degradation is possible by a biological process in anaerobic conditions (although low efficiencies were achieved). Three possible degradation mechanisms were proposed.

Non targeted screening of nitrogen containing disinfection by-products in formation potential tests of river water and subsequent monitoring in tap water samples.

The generation of disinfection by-products during water chlorination is a major concern in water treatment, given the potential health risks that these substances may pose. In particular, nitrogen-containing DBPs are believed to have greater toxicological significance than carbon-based DBPs. Hence, high performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) in positive mode was employed to identify new non-volatile nitrogen containing disinfection by-products (DBPs) and to assess their presence in potable water. Nine water samples were taken in the Llobregat river, in the context of a water reuse trial, near the catchment of a drinking water treatment plant (DWTP) in 2019. River samples were disinfected with chlorine under controlled formation potential tests conditions and analysed with a non-target approach. The peak lists of raw and chlorinated samples were compared exhaustively, resulting in an extensive list of 495 DBPs that include bromine and/or chlorine atoms. 172 of these species were found frequently, in three or more chlorinated samples. The empirical formulae of these DBPs were unambiguously annotated on the basis of accurate m/z measurements, isotopic patterns and common heuristic rules. Most of the annotated species (310) contained bromide, which is consistent with the relatively high bromide content of the Llobregat basin (>0.3mg/l). Drinking water samples were taken at the outlet of the DWTP during the same sampling period. According to their analysis, a large portion of the DBPs detected after the formation potential tests do not reach real-life drinking water, which suggests that the treatment train successfully removes a significant fraction of DBP precursors. However, 131 DBPs could still be detected in the final product water. A larger sampling was carried in the Barcelona water distribution network, during six consecutive weeks, and it revealed the presence of 78 halogenated DBPs in end-consumer water, most of which were nitrogen-containing. MS/MS fragmentation and retention times were employed to tentatively suggest molecular structure for these recalcitrant DBPs.

Suicide of a young woman using a kit containing sodium nitrite ordered on the internet

We report the case of a 13-year-old girl, found dead by her father, with a glass containing 15 g of white powder and a bottle containing 5 g of white powder with a moistened appearance. The autopsy was performed four days after the death. The anatomopathological study of the specimens was included in formaldehyde and prepared according to the Saffron Eosin Hematein technique. First of all, a post-mortem toxicological screening was performed on cardiac blood, peripheral blood, urine and gastric contents by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-HRMS). Ethanol testing was performed by gas chromatography coupled to a flame ionization detector (GC-FID), and drug testing by liquid chromatography coupled to a mass spectrometer (LC-MS/MS, opiates, cocaine, amphetamine) or gas chromatography coupled to a mass spectrometer (GC-MS, cannabis). A rapid analysis of powders found near the body was carried out by RAMAN spectrometry, and confirmed by ion chromatography coupled with mass spectrometry and X-ray diffractometry. In order to establish the concentration of the substance found by those analyses in peripheral blood and gastric contents, we later used the saltzman reaction after addition of potassium ferrocyanide and zinc sulfate. External examination and autopsy revealed a non-specific asphyxia syndrome. This was confirmed by the anatomopathological study which showed signs of non-specific acute heart failure: diffuse visceral congestion (heart, lungs, spleen, liver, pancreas, kidneys, thyroid, stomach) and pulmonary haemorrhage. First of all, toxicological analyses revealed the presence of metoclopramide, in cardiac blood (0.37 mg/L), urines (0.20 mg/L), gastric contents (0.24 mg/L) and in peripheral blood at a concentration of 0.40 mg/L. However, no ethanol and presence of drugs of abuse (cannabinoids, opiates, cocaine, and amphetamines) were detected. Secondly, powders found close to the body were analysed by three different techniques, and revealed the presence of sodium nitrite in pure form. Finally, the analysis of gastric contents confirmed the presence of nitrites at a concentration of 30.9 mg/L. This analysis could not be performed on the blood samples due to colour interference. This young girl died of anoxia secondary to the ingestion of a suicide kit containing sodium nitrite. Diverted from their original use as euphoriants (poppers), nitrites are now used for suicidal purposes. Specific kits have emerged on the internet. They contain sodium nitrite, metoclopramide and antacid. NaN02 is presented in the form of a powder, similar to salt, which dissolves easily in a liquid to reconstitute a toxic drink (Durão C, J Forensic Leg Med, 2020;73:101989). The lethal dose of nitrites is estimated for an adult at 2.6 g (Hwang C, Forensic Sci Med Pathol, 2021;17(3):475–80). This dose seems variable since a fatal case is described in the literature for an ingestion of 1 g (Gowans WJ, Br J Gen Pract J R Coll Gen Pract, 1990;40(340):470–1) and one non-fatal case following ingestion of 6 g (Chui JSW, Anaesthesia, 2005;60(5):496–500). In our victim's case, metoclopramide was also detected, at a concentration of 0.40 mg/L in the peripheral blood. The metoclopramide's concentration is therapeutic between 0.05 and 0.15 mg/L and toxic from 0.2 mg/L (Schulz M, Crit Care,2012;16(4):R136). Such a concentration of metoclopramide is therefore potentially toxic and can cause extrapyramidal symptoms (tremors), drowsiness, impaired consciousness, confusion, hallucinations, even cardiorespiratory arrest (Vidal) and may have contributed to death. The alarming increase in the number of cases and the access facility to these suicide kits on the internet shows that it is essential to implement measures to regulate this substance to prevent its diversion and use.

Perfluorooctane sulfonic acid (PFOS) degradation by optimized heterogeneous photocatalysis (TiO2/UV) using the response surface methodology (RSM)

Perfluorinated compounds (PFCs) are chemical substances widely used in industry, especially in textile coatings, often detected in waters all around the world. Those are persistent, bioaccumulative, and endocrine disruptors, causing damage to human health and the environment. Thus, this work aimed at evaluating the degradation of aqueous perfluorooctane sulfonic acid (PFOS), one of the most used PFCs, at near environmental concentrations (initially 100 μg L−1) by heterogeneous photocatalysis (TiO2/UV). At optimized conditions (CTiO2= 1.45 g L−1 and pH= 4.0), which were determined by the response surface methodology (RSM), 83 % PFOS removal was achieved in 8 h at 25 °C. A pseudo-first-order kinetic model was adjusted to the experimental data and the initial rate constant (k) was estimated as 0.64 h−1 with maximum oxidation capacity (MOC) of 86 %. No significant degradation was observed by direct UV-photolysis. Furthermore, the degradation products (DPs) were identified by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-MS), acute and chronic ecotoxicities for three classes of organisms (fishes, daphnids, and green algae) and lipophilicity (log D, pH 7.4) of the DPs were estimated using the ECOSAR 1.11 and Chemicalize software, respectively. The main finding was that PFOS could be degraded by an advanced oxidation process under mild conditions and reasonable times, and that the lipophilicity of the formed DPs affects acute and chronic ecotoxicities of the organisms studied.

Liquid Chromatography-High-Resolution Mass Spectrometry-Based In Vitro Toxicometabolomics of the Synthetic Cathinones 4-MPD and 4-MEAP in Pooled Human Liver Microsomes.

Synthetic cathinones belong to the most often seized new psychoactive substances on an international level. This study investigated the toxicometabolomics, particularly the in vitro metabolism of 2-(methylamino)-1-(4-methylphenyl)-1-pentanone (4-MPD) and 2-(ethylamino)-1-(4-methylphenyl)-1-pentanone (4-MEAP) in pooled human liver microsomes (pHLM) using untargeted metabolomics techniques. Incubations were performed with the substrates in concentrations ranging from 0, 12.5, and 25 µM. Analysis was done by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS/MS) in full scan only and the obtained data was evaluated using XCMS Online and MetaboAnalyst. Significant features were putatively identified using a separate parallel reaction monitoring method. Statistical analysis was performed using Kruskal-Wallis test for prefiltering significant features and subsequent hierarchical clustering, as well as principal component analysis (PCA). Hierarchical clustering or PCA showed a distinct clustering of all concentrations with most of the features z-scores rising with the concentration of the investigated substances. Identification of significant features left many of them unidentified but revealed metabolites of both 4-MPD and 4-MEAP. Both substances formed carboxylic acids, were hydroxylated at the alkyl chain, and formed metabolites after combined hydroxylation and reduction of the cathinone oxo group. 4-MPD additionally formed a dihydroxy metabolite and a hydroxylamine. 4-MEAP was additionally found reduced at the cathinone oxo group, N-dealkylated, and formed an oxo metabolite. These findings are the first to describe the metabolic pathways of 4-MPD and to extend our knowledge about the metabolism of 4-MEAP. Findings, particularly the MS data of the metabolites, are essential for setting up metabolite-based toxicological (urine) screening procedures.

Abstract 5496: A predictive model for selective targeting of the Warburg effect through GAPDH inhibition with a natural product

Abstract Cancer cells undergo numerous adaptive processes to sustain growth and survival. One notable mechanism is by rewiring metabolism, most prominently through a phenomenon known as the Warburg effect (WE). The WE is defined by increased glucose consumption and lactate excretion in the presence or absence of oxygen. Although the WE has been extensively studied, efforts to develop successful glycolytic inhibitors have been largely unsuccessful. Targeting cancer metabolism has remained a challenge due to the lack of obvious metabolic biomarkers and difficulties achieving full enzyme inhibition without inducing toxicity in normal tissue. Although targeted cancer therapies that use genetics have been largely successful, principles for selectively targeting tumor metabolism that also depend on the environment remain unknown. In the present study, we employ metabolic control analysis to reveal that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the sixth enzyme in glycolysis, exhibits differential control properties during the WE and can be used to predict response to targeting glucose metabolism. Using high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS), we conducted comparative metabolomics to establish a natural product produced by Trichoderma fungi, koningic acid (KA), as a selective inhibitor of GAPDH. We expressed a fungal-derived resistant-GAPDH allele in human cells to show that KA is highly specific for GAPDH. With machine learning, integrated pharmacogenomics, and metabolomics, we demonstrate that KA efficacy is not determined by the status of individual genes, but by the quantitative extent of the WE, leading to a therapeutic window in vivo. Thus, the basis of targeting the WE can be encoded by molecular principles that extend beyond genetic status. Current work focuses on elucidating acquired resistance mechanisms of KA in cancer cells undergoing the WE. Together, these data importantly demonstrate that a complete understanding of pharmacogenomics for cancer therapy likely requires information encoded at the metabolic level. Citation Format: Maria V. Liberti, Ziwei Dai, Suzanne E. Wardell, Joshua A. Baccile, Xiaojing Liu, Xia Gao, Robert Baldi, Mahya Mehrmohamadi, Marc O. Johnson, Neel S. Madhukar, Alexander Shestov, Iok I. C. Chio, Olivier Elemento, Jeffrey C. Rathmell, Frank C. Schroeder, Donald P. McDonnell, Jason W. Locasale. A predictive model for selective targeting of the Warburg effect through GAPDH inhibition with a natural product [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5496.

Metabolic Responses of Mytilus galloprovincialis to Fullerenes in Mesocosm Exposure Experiments.

In this study, Mediterranean mussels (Mytilus galloprovincialis) were exposed through the diet to fullerene soot at three concentrations in parallel to a control group. Their metabolomics response was assessed by high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS). The experiments were conducted in marine mesocosms, during 35 days (7 days of acclimatization, 21 days of exposure, and 7 days of depuration). Real conditions were emulated in terms of physicochemical conditions of the habitat. Results confirmed the bioaccumulation of fullerenes, and the metabolome of the exposed organisms revealed significant differences in the concentrations of seven free amino acids in comparison to the control group. An increase in small nonpolar amino acids (e.g., alanine) and branched chain amino acids (leucine and isoleucine) were observed. Also, glutamine concentrations decreased significantly, suggesting the activation of facultative anaerobic energy metabolism. Branched chain amino acids, such as leucine and isoleucine, followed the opposite trend after the highest level of exposure, which can imply hormesis effects. Other significant differences were observed on lipids content, such as the general increase of free fatty acids, i.e., long-chain fatty acids (lauric, myristic, and palmitic acids) when the concentration of exposure was increased. These results were consistent with hypoxia and oxidative stress.

Characterization of enalapril and ranitidine chlorination by-products by liquid chromatography/high-resolution mass spectrometry and their toxicity evaluation

Due to its low cost, its capability for disinfection and oxidation, chlorination using gaseous chlorine or hypochlorite salts, has also been commonly applied in water treatment plants for oxidation and disinfection purposes. Little is known about the identity and toxicity of by-products resulting from the chlorination of pharmaceutical micropollutants, such as enalapril (ENA) and ranitidine (RAN). ENA and RAN chlorination by-products were characterized in this study by high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC/HRMS) and their toxicity were assessed by MTT assay. Chlorination experiments with ENA and RAN solutions (10 mg L-1) indicate degradation efficiencies of 100% for both compounds after only 5 min of exposure to chlorine at concentration of 9.53 mg Cl2 L-1. On the other hand mineralization rates were lower than 3%, thereby indicating there was accumulation of degradation by-products in all experiments. Mass spectrometric analysis revealed, at all times of reaction after the addition of hypochlorite, the presence of 1-(2-((4-(chlorophenyl)-1-ethoxy-1-oxobutan-2-yl)amino)propanoyl)pyrrolidine-2-carboxylic acid (enalapril by-product) and N-chloro-N-(2-(((chloro-5-((dimethylamino)methyl)furan-2-yl)methyl)sulfinyl)ethyl)-N-methyl-2-nitroethene 1,1-diamine (ranitidine by-product). Despite the formation of oxidized chlorinated by-products in all chlorination assays, the treated solutions were nontoxic to HepG2 cells by the MTT assay. It has been observed that chlorination (10 mg L-1, 5 min) of ENA and RAN solutions exhibited high degradation efficiencies of the target compounds and low mineralization rates. Based on the mass spectrometry data, the routes for ENA and RAN successive oxidation by chlorine has been proposed.

Characterisation of captopril photolysis and photocatalysis by-products in water by direct infusion, electrospray ionisation, high-resolution mass spectrometry and the assessment of their toxicities

ABSTRACTPharmaceuticals of different therapeutic classes are found in the environment. Captopril is used worldwide as an antihypertensive drug, and it has been found in the influent, effluent and secondary sludge of wastewater treatment plants. Advanced oxidation processes, such as direct photolysis (UV-C) and heterogeneous photocatalysis (TiO2/UV-C), are alternatives to enhance mineralisation of pharmaceuticals and their removal during water treatment. In this article, it was evaluated the degradation of captopril in aqueous solution induced by UV-C and TiO2/UV-C systems. The process focused on the identification and monitoring of the by-products formed under these conditions by applying direct-infusion electrospray ionisation high-resolution mass spectrometry in the negative ion mode (ESI(-)-HRMS) and high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC/HRMS). To evaluate the by-products toxicity, acute ecotoxicity tests were performed with the crustacean Artemia salina, and the cytotoxicity was evaluated with (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using HepG2 cells. It was observed by ESI(-)-HRMS that after 120 min of light exposure, there was almost complete removal of captopril, with 93.5% removal efficiency during photolysis and 99.9% during photocatalysis. At these conditions, the rate of mineralisation, by total organic carbon (TOC), was only 2.92% for photolysis and 9.09% for photocatalysis, evidencing the formation of degradation by-products. Nine by-products of captopril photodegradation were identified, and their respective chemical structure elucidations were proposed. The treated samples were nontoxic to A. salina and HepG2 cells, indicating that both oxidative treatments (photocatalytic or photolytic processes) can be conveniently employed to remove captopril from aqueous media.

Toxicologically Relevant Aldehydes Produced during the Frying Process Are Trapped by Food Phenolics.

The lipid-derived carbonyl trapping ability of phenolic compounds under common food processing conditions was studied by determining the presence of carbonyl-phenol adducts in both onions fried in the laboratory and commercially crispy fried onions. Four carbonyl-phenol adducts produced between quercetin and acrolein, crotonaldehyde, or (E)-2-pentenal were prepared and characterized by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-HRMS). The synthesized compounds were 2-(3,4-dihydroxyphenyl)-3,5,8-trihydroxy-9,10-dihydro-4H,8H-pyrano[2,3-f]chromen-4-one (4), 2-(3,4-dihydroxyphenyl)-3,5,8-trihydroxy-10-methyl-9,10-dihydro-4H,8H-pyrano[2,3-f]chromen-4-one (5), 2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-8-methyl-4H,8H-pyrano[2,3-f]chromen-4-one (9), and 2-(3,4-dihydroxyphenyl)-8-ethyl-3,5-dihydroxy-4H,8H-pyrano[2,3-f]chromen-4-one (10). When onions were fried in fresh rapeseed oil spiked with acrolein, crotonaldehyde, and (E)-2-pentenal (2.7 μmol/g of oil), adduct 10 was the major compound produced, and trace amounts of adducts 4 and 5, but not of adduct 9, were also detected. In contrast, compound 4 was the major adduct present in commercially crispy fried onions. Compound 10 was also present to a lower extent, and trace amounts of compound 5, but not of compound 9, were also detected. These data suggested that lipid-derived carbonyl-phenol adducts are formed in food products under standard cooking conditions. They also pointed to a possible protective role of food polyphenols, which might contribute to the removal of toxicologically relevant aldehydes produced during deep-frying, assuming that the formed products are stable during food consumption in the human organism.

Discovery of the Rhizopodin Biosynthetic Gene Cluster in Stigmatella aurantiaca Sg a15 by Genome Mining

The field of bacterial natural product research is currently undergoing a paradigm change concerning the discovery of natural products. Previously most efforts were based on isolation of the most abundant compound in an extract, or on tracking bioactivity. However, traditional activity-guided approaches are limited by the available test panels and frequently lead to the rediscovery of already known compounds. The constantly increasing availability of bacterial genome sequences provides the potential for the discovery of a huge number of new natural compounds by in silico identification of biosynthetic gene clusters. Examination of the information on the biosynthetic machinery can further prevent rediscovery of known compounds, and can help identify so far unknown biosynthetic pathways of known compounds. By in silico screening of the genome of the myxobacterium Stigmatella aurantiaca Sg a15, a trans-AT polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene cluster was identified that could not be correlated to any secondary metabolite known to be produced by this strain. Targeted gene inactivation and analysis of extracts from the resulting mutants by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-HRMS), in combination with the use of statistical tools resulted in the identification of a compound that was absent in the mutants extracts. By matching with our in-house database of myxobacterial secondary metabolites, this compound was identified as rhizopodin. A detailed analysis of the rhizopodin biosynthetic machinery is presented in this manuscript.