High Performance Liquid Chromatography And Mass Spectrometry Research Articles

Bioactive compounds of spent coffee grounds and their potential use as functional food

Hot brewed coffee is the most popular hot beverage in the world, and its health properties have been published in the literature(1). Conversely, over the past decade, cold-brewed coffee has gained popularity, but its eventual nutritional properties are unclear. Both hot and cold brewed coffee produces over 6 million tons of spent coffee grounds (SCG) yearly disposed in landfills(1). Interestingly, studies have shown that SCG can improve several metabolic parameters via changes in the gut microbiome in obese and diabetic rats(2), and reduce energy consumption in overweight humans(3). However, studies investigating the nutritional properties of SCG are lacking in the literature. Hence, in this study, we aimed to identify, quantify and compare two main bioactive compounds in hot- and cold-brewed coffee as a beverage, as well as in the SCG. Samples from hot and cold coffee beverages and SCG were obtained from a local coffee shop (n = 3 per group). The coffee beans were composed of Coffea arabica from Papa New Guinea, Brazil, Ethiopia, and Colombia (in order from highest to lowest proportion). All samples were analysed by high-performance liquid chromatography and mass spectrometry (HPLC-MS). The analyses focused on two main bioactive compounds; trigonelline and chlorogenic acid (CGA). Statistical analyses were performed using an unpaired t-test with Welch’s correction and two-way ANOVA with Tukey’s post-hoc test (p<0.05). When compared to hot-brewed coffee beverages, cold-brewed coffee beverages have shown lower (p<0.05) levels of trigonelline (17.26 mg/g + 1.305 vs. 8.46 mg/g + 0.74, respectively) and CGA (9.82 mg/g + 0.93 vs. 5.31 mg/g + 0.48, respectively). In SCG obtained from hot-brewed coffee, a higher concentration of CGA was found (0.12 mg/g + 0.006), when compared to SCG obtained from cold-brewed coffee (0.10 mg/g + 0.03). However, trigonelline in cold-brewed SCG was found in higher (p<0.05) concentration, when compared to hot-brewed SCG (0.11 mg/g + 0.03 vs. 0.09 mg/g + 0.017, respectively). Moreover, hot-brewed coffee beverages showed higher (p<0.05) concentrations of trigonelline and CGA, when compared to hot-brewed SCG. Similarly, cold-brewed coffee beverages showed higher (p<0.05) concentrations of both bioactive compounds, when compared to cold-brewed SCG. Our results indicated that hot brewed coffee beverage contains high concentrations of bioactive compounds (CGA and trigonelline), which possibly explain its health properties. Although SCG obtained from hot and cold-brewed coffee showed lower concentrations of both bioactive compounds than coffee beverages, our results shed light on the possible health benefits of SCG consumption. In a world seeking more sustainable solutions, further studies investigating the potential use of SCG as a functional food are required.

Antioxidant and Antifungal Activities and Characterization of Phenolic Compounds Using High-Performance Liquid Chromatography and Mass Spectrometry (HPLC-MS) in Empetrum rubrum Vahl ex Willd.

In searching for compounds with antioxidant and antifungal activity, our study focused on the subshrub species Empetrum rubrum Vahl ex Willd. (Ericaceae). We measured the antioxidant activity of its methanolic extract (MEE) obtained from the aerial parts (leaves and stems) and of its methanolic extract (MEF) obtained from the lyophilized fruits. The antioxidant activity of the MEE and MEF was evaluated in vitro via a 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical and 2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) cationic radical. The results were expressed in gallic acid and Trolox equivalents for the DPPH and ABTS assays, respectively. The antioxidant activities, for the DPPH and ABTS assays, were also evaluated by considering the IC50 values. Concerning the antioxidant activity, the total phenolic content (TPC) in the MEE and MEF was determined using the Folin-Ciocalteu method. Polyphenols contained in the leaves, stems, and fruits of E. rubrum were determined qualitatively by employing high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analysis. The antifungal activity of the MEE obtained from the aerial parts of E. rubrum was tested against Rhizoctonia solani. The results of IC50 values measured by the DPPH and ABTS methods with MEE were 0.4145 ± 0.0068 mg mL-1 and 0.1088 ± 0.0023 mg mL-1, respectively, and the IC50 values for MEF were 6.4768 ± 0.0218 mg mL-1 and 0.7666 ± 0.0089 mg mL-1 measured by the DPPH and ABTS methods, respectively. The HPLC-MS analysis revealed the presence of anthocyanins, phenolic acids derivatives, and flavonols. In vitro, mycelial growth of this fungus was reduced from 90% to nearly 100% in the presence of MEE. The observed antifungal effect is related to the presence of the abovementioned phenols, detected in the MEE.

Network Pharmacology and in vitro Experimental Verification on Intervention of Quercetin, Present in Chinese Medicine Yishen Qutong Granules, on Esophageal Cancer.

To explore the potential mechanism of Yishen Qutong Granules (YSQTG) for the treatment of esophageal cancer using network pharmacology and experimental research. The effective components and molecular mechanism of YSQTG in treating esophageal cancer were expounded based on network pharmacology and molecular docking. The key compound was identified by high-performance liquid chromatography and mass spectrometry (HPLC-MS) to verify the malignant phenotype of the key compounds in the treatment of esophageal cancer. Then, the interaction proteins of key compounds were screened by pull-down assay combined with mass spectrometry. RNA-seq was used to screen the differential genes in the treatment of esophageal cancer by key compounds, and the potential mechanism of key compounds on the main therapeutic targets was verified. Totally 76 effective compounds of YSQTG were found, as well as 309 related targets, and 102 drug and disease interaction targets. The drug-compound-target network of YSQTG was constructed, suggesting that quercetin, luteolin, wogonin, kaempferol and baicalein may be the most important compounds, while quercetin had higher degree value and degree centrality, which might be the key compound in YSQTG. The HPLC-MS results also showed the stable presence of quercetin in YSQTG. By establishing a protein interaction network, the main therapeutic targets of YSQTG in treating esophageal cancer were Jun proto-oncogene, interleukin-6, tumor necrosis factor, and RELA proto-oncogene. The results of cell function experiments in vitro showed that quercetin could inhibit proliferation, invasion, and clonal formation of esophageal carcinoma cells. Quercetin mainly affected the biological processes of esophageal cancer cells, such as proliferation, cell cycle, and cell metastasis. A total of 357 quercetin interacting proteins were screened, and 531 genes were significantly changed. Further pathway enrichment analysis showed that quercetin mainly affects the metabolic pathway, MAPK signaling pathway, and nuclear factor kappa B (NF- κ B) signaling pathway, etc. Quercetin, the key compound of YSQTG, had stronger binding activity by molecular docking. Pull-down assay confirmed that NF- κ B was a quercetin-specific interaction protein, and quercetin could significantly reduce the protein level of NF- κ B, the main therapeutic target. YSQTG can be multi-component, multi-target, multi-channel treatment of esophageal cancer, it is a potential drug for the treatment of esophageal cancer.

Chemical composition, antioxidant and gastrointestinal properties of Sedum dendroideum Moc & Sessé ex DC leaves tea infusion

Ethnopharmacological relevanceSedum dendroideum Moc & Sessé ex DC (Crassulaceae) is a medicinal plant employed in Mexican and Brasilian folk medicine as juice or infusion, as remedy for the treatment of different diseases, including gastric disorders. Aim of the studyAlthough some studies carried out with Sedum dendroideum have demonstrated its gastroprotective effect, the purpose of this study was to elucidate the chemical constituents, antioxidant, cytotoxic and mechanisms underlying the gastrointestinal properties of Sedum dendroideum accordingly its traditional use, as fresh leaves tea infusion (SDI). Materials and methodsChemical constituents were analyzed using high performance liquid chromatography and mass spectrometry (HPLC-MS). Antioxidant and cytotoxicity were evaluated in in vitro assays. The efficacy of the SDI on macroscopic ulcer appearance, mucus and GSH maintenance on ethanol- and indomethacin-induced ulcer models, gastric acid secretion and gastrointestinal motility were investigated. ResultsPhytochemical analysis by HPLC–MS revealed the presence of different flavonol glycosides, containing myricetin and quercetin, along with the kaempferol as aglycones. In vitro pharmacological investigation of SDI demonstrated potent antioxidant activity in DPPH assay (IC50: 13.25 ± 3.37 µg/mL) and absence of cytotoxicity in Caco-2 cells by MTT method. Oral administration of SDI (ED50 of 191.00 ± 0.08 mg/kg) in rats promoted gastroprotection against ethanol or indomethacin in rats through reinforcement of gastric wall mucus, GSH content and nitric oxide release, without present antisecretory properties. The gastroprotective effect was maintained when SDI (19 mg/kg) was administrated by intraperitoneal route. Furthermore, SDI (150 mg/kg) unchanged the gastric emptying but increase small bowel transit in mice through cholinergic pathways. ConclusionsCollectively, this study confirmed that Sedum dendroideum promotes gastroprotection through preventing of endogenous defense mechanisms, represented by mucus and GSH without changes gastric acid secretion. Sedum dendroideum tea infusion features a chemical profile that contributes to the antioxidant and gastric health-promoting effects, supporting the use in folk medicine for the treatment of gastrointestinal disorders.

Hydroalcoholic Extract and Ethyl Acetate Fraction of Bixa orellana Leaves Decrease the Inflammatory Response to Mycobacterium abscessus Subsp. massiliense.

The incidence of infections caused by rapidly growing mycobacteria (RGM), especially Mycobacterium abscessus subsp. massiliense (Mabs), is increasing worldwide. Severe infections are associated with abscess formation and strong inflammatory response. This study evaluated the antimicrobial and anti-inflammatory activities of a hydroalcoholic extract (BoHE) and ethyl acetate fraction (BoEA) of Bixa orellana leaves. Antimicrobial activity was evaluated by broth microdilution to determine the minimum inhibitory (MIC) and the minimum bactericidal (MBC) concentrations. Cytotoxicity was evaluated using erythrocytes and RAW 264.7 cells. Nitric oxide (NO) was assayed in stimulated RAW 264.7 cells, and inflammatory cell migration and acute toxicity were evaluated in a Mabs-induced peritonitis mouse model. The compounds present in BoEA were identified by high performance liquid chromatography and mass spectrometry (HPLC-MS). The MIC and MBC values were 2.34 mg/mL and 37.5 mg/mL for BoHE and 0.39 mg/mL and 6.25 mg/mL for BoEA. The extracts did not induce significant toxicity in erythrocytes and RAW 264.7 cells. High levels of NO induced by Mabs were decreased by treatment with both extracts. The anti-inflammatory activity was confirmed in vivo by significant reduction of the cell migration to the peritoneum following BoHE and BoEA pretreatment. Animals treated with BoHE or BoEA did not show signs of acute toxicity in stomach, liver, and kidney. The chemical characterization of BoEA (the most active extract) revealed that kaempferol-3-O-coumaroyl glucose is its major component. The extract of B. orellana may be effective for treating infections caused by Mabs.

Rapidly screening of α-glucosidase inhibitors from Dioscorea opposita Thunb. peel based on rGO@Fe3O4 nanocomposites microreactor

Present study aimed to immobilise the α-glucosidase on suitable supports to construct enzymatic microreactors and their subsequent applicability in efficient inhibitor screening from the Chinese Yam (Dioscorea opposita Thunb.) peel. A type of lamellar and porous composites (rGO@Fe3O4) were synthesised with a facile one-step solvothermal method and employed as carriers to construct enzymatic microreactors for screening α-glucosidase ligand from the Chinese Yam peel in league with the high performance liquid chromatography and mass spectrometry (HPLC-MS). The immobilisation amount of α-glucosidase on rGO@Fe3O4 under the optimised conditions was about 40 μg α-glucosidase/mg carriers. Furthermore, the binding capacities of screened inhibitors, 2,4-dimethoxy-6,7-dihydroxyphenanthrene and batatasin I, were 35.6 and 68.2%, respectively. Hence, considering their high screening efficiency and excellent magnetic separation ability, these as-prepared nanocomposite consisting of rGO and Fe3O4 may be potential supports for the enzyme (such as α-glucosidase) immobilisation for rapid α-glucosidase inhibitors screening from the diverse nature resources.

Effects of Trimetaphosphate on Abiotic Formation and Hydrolysis of Peptides.

The primordial Earth probably had most of the factors needed for the emergence and development of life. It is believed that it had not only water, but also simple inorganic and organic materials. While studies since the 1950s on the origins of organic matter have established key roles for amino acids, conditions that would have promoted their condensation to make polymers, such as peptides or proteins, have yet to be fully defined. The condensation of amino acids in a water-rich environment is not thermodynamically favored. Therefore, the efficient formation of peptides requires the presence of a catalyst or the activation of a substrate. In living cells, the biosynthesis of proteins is assisted by enzymes and requires adenosine triphosphate (ATP), a relatively complex organic polyphosphate, which serves as an energy source. Outside the living organism, simpler inorganic polyphosphates can form active aminoacyl–phosphate anhydrides, which suggests the broader potential of phosphorus for enabling the polymerization of amino acids. However, this has yet to be demonstrated. To address this gap, aqueous solutions containing a simple dipeptide, diglycine, and a simple polyphosphate, trimetaphosphate, were dried, and reaction products were analyzed by high performance liquid chromatography and mass spectrometry (HPLC-MS). Different reaction environments, which were defined by the initial solution composition, pH, temperature, and incubation time, were found to affect the distribution and yield of products. Our results collectively provide strong evidence for reactions that both condense and hydrolyze peptides. It is noteworthy that the co-occurrence of reactions that form and cleave peptides are a central feature of Kauffman’s theory for the emergence of autocatalytic sets, which is a key step in the chemical origins of life.

Overproduction of Ristomycin A by activation of a silent gene cluster in Amycolatopsis japonicum MG417-CF17.

The emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products is Amycolatopsis. However, Amycolatopsis japonicum does not produce an antibiotic under standard laboratory conditions. In contrast to most Amycolatopsis strains, A. japonicum is genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, the bbr gene from Amycolatopsis balhimycina (bbrAba), into A. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing of A. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed the in silico prediction that the recombinant A. japonicum/pRM4-bbrAba synthesizes ristomycin A.

A Unique Leukemia Mouse Model Established From AML Patient with IDH2 R140Q and FLT3-ITD Mutations Among Other Common AML Mutations

Abstract 3579Somatic mutations in isocitrate dehydrogenase 1 (IDH1, cytosolic) and IDH2 (mitochondrial homolog) have been associated with acute myeloblast leukemia (AML, 7% and 15% respectively), as well as other malignancies, e.g. gliomas, etc1,2. These mutations lead to 2-hydroxyglutarate (2HG) production in leukemic cells, which can be detected both in leukemic cells and plasma of AML patients. This association has led to speculation that IDH mutations are important in AML leukemogenesis, or oncogenic drivers, and could potentially be used as drug targets for AML treatment. However, these assumptions have never been verified for lack of experimental models. We recently successfully engrafted leukemic cells from bone marrow of an AML patient into immunocompromised NOD/SCID mice. This patient has aggressive disease of poor prognosis, with initial response to standard of care of chemotherapy and subsequent fast recurrence, followed by death. The patient was diagnosed as M5 subtype AML with heterozygous IDH2 R140Q mutation, and also mutations of FLT3-ITD, DNMT3A R882H and NPM1. The transplanted mice (AM7577) developed AML leukemia with typical symptoms (BW loss, hunched, inactivity, labored breathing, ruffled coat and eventual mortality) and with leukemic cells in bone and peripheral organs (e.g. spleen, blood, etc) (gradual detection of leukemic cells in peripheral blood after 30 days). The leukemic cells can serially be passed in mice with 100% take-rate and cause 100% leukemia induced mortality (even with < 1e5 cells), thus creating a renewable and potentially unlimited source of leukemia cells. The leukemic cells in mice are identical to those of the original patient leukemic cells (CD45+, CD33+, CD13+, CD123+, and CD19−, heterozygous IDH2 R140Q mutation, along with FLT3-ITD; DNMT3A R882H and NPM1. This mutation pattern is somewhat in contrast to the previous belief that IDH mutations are mutually exclusive to other common AML mutation3. The engrafted animals produce 2HG in leukemic cells and in mouse plasma, both of which can be readily detected and quantified by high-performance liquid chromatography and mass spectrometry (HPLC/MS). 2HG and cytokines could be useful biomarkers to monitor the disease progression as well as treatment response. The treatment leukemic models in vivo with standard of care (SOC: Ara-C, 5day-on/2day-off dosing scheme) resulted in significant response, manifested by reduction of leukemic load to undetectable in peripheral blood and serum biomarkers for both tested doses (2 and 10mg/kg), as well as extended survivals. Leukemia, however, rapidly relapse after the treatment withdrawal, suggesting that AraC has effect only on the leukemic blasts, but not on leukemic stem cells. The appearance of leukemic blasts in peripheral blood after araC withdrawal can be effectively re-suppressed by araC. Further tests on Flt3 inhibitor for anti-leukemic activities are currently ongoing. In summary, the engrafted mice could serve as a useful experimental model to investigate the role of IDH mutation in leukemogenesis and help to identify new treatment strategies for AML, including compounds targeting IDH mutations and FLT3, etc. This is, to our knowledge, the first reported AML experimental model of featuring IDH mutation. Disclosures:Liu:Crown Bioscience, Inc.: Employment. Wu:Crown Bioscience, Inc.: Employment. Cai:Crown Bioscience: Employment. Wang:Crown Bioscience: Employment. Chen:Crown Bioscience: Employment. Wery:Crown Bioscience: Employment. Chen:Crown Bioscience: Employment. Li:Crown Bioscience, Inc.: Employment.

Use of ion pairing reagents for sensitive detection and separation of phospholipids in the positive ion mode LC-ESI-MS

Phospholipids make up one of the more important classes of biological molecules. Because of their amphipathic nature and their charge state (e.g., negatively charged or zwitterionic) detection of trace levels of these compounds can be problematic. Electrospray ionization mass spectrometry (ESI-MS) is used in this study to detect very small amounts of these analytes by using the positive ion mode and pairing them with fifteen different cationic ion pairing reagents. The phospholipids used in this analysis were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), cardiolipin (CA) and sphingosyl phosphoethanolamine (SPE). The analysis of these molecules was carried out in the single ion monitoring (SIM) positive mode. In addition to their detection, a high performance liquid chromatography and mass spectrometry (HPLC-MS) method was developed in which the phospholipids were separated and detected simultaneously within a very short period of time. Separation of phospholipids was developed in the reverse phase mode and in the hydrophilic interaction liquid chromatography (HILIC) mode HPLC. Their differences and impact on the sensitivity of the analytes are compared and discussed further in the paper. With this technique, limits of detection (LODs) were very easily recorded at low ppt (ng L(-1)) levels with many of the cationic ion pairing reagents used in this study.

A combined cell membrane chromatography and online HPLC/MS method for screening compounds from Radix Caulophylli acting on the human α 1A-adrenoceptor

We have developed an online analytical method that combines α 1A-adrenoceptor (α 1AAR) cell membrane chromatography (α 1AAR-CMC) with high performance liquid chromatography and mass spectrometry (HPLC/MS) for the identification of active components from Radix Caulophylli acting on the human α 1AAR. Fractions retained by the α 1AAR-CMC column were captured into a loop and the components were directly analyzed by combining an 8 port column switcher with an HPLC/MS system for separation and preliminary identification. Using methoxamine as a positive control drug, magnoflorine and caulophine from Radix Caulophylli were identified as the active molecules acting on the α 1AAR. This new α 1AAR-CMC-online-HPLC/MS method can be applied for screening active components acting on α 1AAR from traditional Chinese medicines exemplified by Radix Caulophylli. This method will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.

Characterization of glucosinolate—myrosinase system in developing salt cress Thellungiella halophila

Glucosinolates are specialized plant metabolites derived from amino acids. They can be hydrolyzed by myrosinases into different degradation products, which have a variety of biological activities. In this study, the compositions and contents of glucosinolates in salt cress (Thellungiella halophila) at different developmental stages were analyzed by high performance liquid chromatography and mass spectrometry (HPLC-MS). Myrosinase activities were also measured. Seven glucosinolates were identified in T. halophila throughout its life cycle. The glucosinolate profiles varied significantly among different tissues. The roots at stage 4 contained the highest concentrations of total, aromatic and indole glucosinolates among all tissues. Whereas roots, flowers and siliques contained all seven glucosinolates, seeds contained only four aliphatic glucosinolates. During development, the concentrations also displayed significant changes. From seeds to cotyledons and from stage 4 roots to stage 5 roots, there were dramatic declines of glucosinolates, which correlated well with changes in myrosinase activities. In other tissues, myrosinase activity alone could not explain the glucosinolate concentration changes. Certain tissues of T. halophila contained Arabidopsis myrosinase TGG1 and TGG2 orthologs. The molecular basis and functional significance of our findings are discussed here.

Toxin characterisation and identification of a Microcystis flos-aquae strain from a Spanish drinking-water reservoir

The Santillana reservoir, a drinking-water supply in the central part of Spain, was monitored weekly from June to November of 1997 for cyanobacteria and microcystins (MCs) in plankton biomass. Several blooms appeared, some of them with a high MC content. Large changes in both phytoplankton biomass and MCs were observed in a short time period, but not in a concomitant way. We have characterised one Microcystis strain, UAM97, that seemed to dominate a bloom, and with an unusually high MC content. The results obtained strongly indicate that Microcystis UAM97 is a M. flos-aquae strain, containing 4 major MCs: MC-LR, MC-LF, MC-LW, and MC-LY. Species assignment is supported by morphology and molecular criteria. Molecular identification was based on comparison of 16S-23S rDNA internal transcribed spacer (ITS) sequences from Microcystis UAM97 and three other collection Microcystis strains. MCs were characterised by amino acid analysis and combined high performance liquid chromatography and mass spectrometry (HPLC/MS) techniques. This report is the first on a M. flos-aquae strain dominating a toxic bloom in Spanish waterbodies.

Decolorization and biodegradation of dye wastewaters by a facultative-aerobic process

Dye wastewater is one of the main pollution sources of water bodies in China. Conventional biological processes are relatively ineffective for color removal, the development of alternative treatment methods will become important. Our subjective was that of introducing a new biotreatment technology which combined a facultative biofilm reactor (FBR) with an aerobic reactor (AR) to treat a dye wastewater. The efficiencies of color and chemical oxygen demand (COD) removal and the mechanism of dye degradation were investigated. The anthraquinone acid dye (acid blue BRLL) concentration, organic loading rate (OLR) and hydraulic retention time (HRT) were varied in the experiments to evaluate the treatment efficiency and process stability. The biodegradation products were detected by infrared (IR) and high performance liquid chromatography and mass spectrometry (HPLC-MS). The results demonstrated that the facultative biofilm process was more effective for decolorization than the anaerobic stage of an anaerobic-aerobic process. Most color removal occurred in the facultative reaction (maximum to 88.5%) and the BOD (biochemical oxygen demand): COD of the FBR effluent increased by 82.2%, thus improving the biodegradability of dyes for further aerobic treatment. The dye concentration, OLR and HRT will be the factors affecting decolorization. Color removal efficiency falls as the influent dye concentration increases, but rises with increased HRT. The infrared and HPLC-MS analyses of the effluents of FBR and AR reveal that the dye parent compound was degraded in each reactor during the process. The Facultative-aerobic (F-A) system can effectively remove both color and COD from the dye wastewater. The FBR played an essential role in the process. The average overall color and COD in the system were removed by more than 93.9% and 97.1%, respectively, at an OLR of 1.1 kg COD m(-3) d(-1) and at the HRT of 18-20 hours in the FBR and 4-5 hours in the AR. The color removal mechanism in each reactor was not only a sort of biosorption on the floc materials, but even more an effect of biodegradation, especially in the facultative process. Recommendation and Outlook. In applying the F-A system to treat a dye wastewater, the control of facultative processes and the set up of appropriate operation conditions appear to be critical factors. Also, it is suggested a moderate COD loading rate and about a 24-hour HRT will favor the F-A system.

Sequencing of new beauverolides by high‐performance liquid chromatography and mass spectrometry

Mass spectrometry (MS) and tandem mass spectrometry (MS(n)) were used for the identification of beauverolides in the fermentation broth of Beauveria bassiana and for evaluation of the purified fraction obtained by sublimation of beauverolides. Besides being a new efficient route for purification of beauverolides, sublimation provided an enrichment of new minor lipophilic beauverolides of lower molecular weight from the original complex mycelial extract. The product ion collision-induced dissociation (CID) spectra obtained on an ion trap (electrospray ionization), the in-source CID mass spectra on a sector instrument (atmospheric-pressure chemical ionization) and the post-source decay matrix-assisted laser desorption/ionization mass spectra of beauverolides were compared and evaluated. All MS(n) experiments started with singly charged precursor ions. The following two new representatives of this group of compounds were identified by high-performance liquid chromatography and MS (HPLC/MS): cyclo-(3-hydroxy-4-methyloctanoyl-valyl-alanyl-leucyl) and cyclo-(3-hydroxy-4-methyloctanoyl-tyrosyl-alanyl-leucyl). Individual structures were confirmed by preparative isolation and nuclear magnetic resonance spectroscopy. The structure of a third novel and minor beauverolide was tentatively assigned by HPLC/MS only as cyclo-(3-hydroxy-4-methyldecanoyl-valyl-alanyl-Lxx), Lxx = leucyl, isoleucyl, or allo-isoleucyl.