Potentially Toxic Planktic and Benthic Cyanobacteria in Slovenian Freshwater Bodies: Detection by Quantitative PCR

Due to increased frequency of cyanobacterial blooms and emerging evidence of cyanotoxicity in biofilm, reliable methods for early cyanotoxin threat detection are of major importance for protection of human, animal and environmental health. To complement the current methods of risk assessment, this study aimed to evaluate selected qPCR assays for detection of potentially toxic cyanobacteria in environmental samples. In the course of one year, 25 plankton and 23 biofilm samples were collected from 15 water bodies in Slovenia. Three different analyses were performed and compared to each other; qPCR targeting mcyE, cyrJ and sxtA genes involved in cyanotoxin production, LC-MS/MS quantifying microcystin, cylindrospermopsin and saxitoxin concentration, and microscopic analyses identifying potentially toxic cyanobacterial taxa. qPCR analyses detected potentially toxic Microcystis in 10 lake plankton samples, and potentially toxic Planktothrix cells in 12 lake plankton and one lake biofilm sample. A positive correlation was observed between numbers of mcyE gene copies and microcystin concentrations. Potential cylindrospermopsin- and saxitoxin-producers were detected in three and seven lake biofilm samples, respectively. The study demonstrated a potential for cyanotoxin production that was left undetected by traditional methods in both plankton and biofilm samples. Thus, the qPCR method could be useful in regular monitoring of water bodies to improve risk assessment and enable timely measures.     

First Report of Cylindrospermopsin Production by Two Cyanobacteria (Dolichospermum mendotae and Chrysosporum ovalisporum) in Lake Iznik,Turkey

Cylindrospermopsin (CYN) is a cytotoxic alkaloid produced by cyanobacteria. The distribution of this toxin is expanding around the world and the number of cyanobacteria species producing this toxin is also increasing. CYN was detected for the first time in Turkey during the summer months of 2013. The responsible species were identified as Dolichospermum (Anabaena) mendotae and Chrysosporum (Aphanizomenon) ovalisporum. The D. mendotae increased in May, however, C. ovalisporum formed a prolonged bloom in August. CYN concentrations were measured by LC-MS/MS and ranged from 0.12 µg·mg⁻¹to 4.92 µg·mg⁻¹ as dry weight, respectively. Both species were the only cyanobacteria actively growing and CYN production was attributed solely to these species. Despite CYN production by C. ovalisporum being a well-known phenomenon, to our knowledge, this is the first report of CYN found in D. mendotae bloom.     

基于多种微生物鉴定技术的制药企业环境微生物鉴定结果分析和应用研究

目的 运用多种微生物鉴定技术建立制药企业无菌制剂高风险生产区环境微生物鉴定信息库。方法 连续4个季度对中间体、制药用水、洁净空间、人员设备表面等收集微生物,采用VITEK生化鉴定、MALDI-TOF MS蛋白鉴定和16S rRNA/ITS基因鉴定技术进行鉴定,结合微生物形态学和来源信息进行分析,建立相应的微生物鉴定信息库。结果 共收集获得89株细菌和5株真菌的鉴定结果,开展相关形态学分析：在收集的94株菌中,革兰氏阳性球菌和革兰氏阳性芽孢杆菌共占比60.6%,是洁净区常见污染菌;B级环境收集到46株微生物,葡萄球菌是主要优势菌属,占比45.7%,其次是芽孢杆菌(17.4%)、微球菌(6.5%)和酵母菌(4.3%),B级还分离到1株洋葱伯克霍尔德菌;制药用水收集到29株微生物,主要为革兰氏阴性菌(58.6%)。结论 本研究建立了融合生化、蛋白、基因鉴定结果的微生物鉴定信息库并开展相关分析,为制药企业开展风险控制,加强对不可接受微生物的管理,开展微生物数据偏差调查和溯源分析提供依据;企业还可利用鉴定结果建立环境微生物地图,有针对性地控制并消除微生物污染,提高无菌保障水平,确保药品质量安全。     

放线菌1161代谢产物分离纯化及其菌种鉴定

本文对放线菌1161粗提物进行抗菌活性检测,并利用超高效液相色谱-串联质谱(UPLC-MS/MS)分析该菌株代谢产物,然后对其代谢产物1161-S8和1161-S9进行分离,最后对该菌株进行分类鉴定。结果表明,该菌株代谢产物对Bacillus subtilis、Sarcina lutea、Staphylococcus aureus、methicillin-resistant Staphylo-coccus aureus(MRSA)、vancomycin-resistant Enterococcus faecalis(VRE)均显示出较强的抗菌活性;UPLC-MS/MS分析其粗提物显示可能为结构相似的一系列的化合物,通过质谱氢谱和碳谱数据判断1161-S8和1161-S9分别为阿扎霉素F4和阿扎霉素F5。同时结合该菌株培养特性、生理生化特性、16SrRNA序列比对与系统进化分析,将其初步鉴定为Streptomyces malaysiensis,是阿扎霉素新的产生菌。     

Identification of a benthic microcystin-producing filamentous cyanobacterium (Oscillatoriales) associated with a dog poisoning in New Zealand

In November 2008 a dog died soon after ingesting benthic “algal” mat material from the Waitaki River, New Zealand. Based on a morphological examination of environmental material, the causative organism was putatively identified as the filamentous cyanobacterium Phormidium sp. Two strains (VUW25 and CYN61) were isolated and cultured to enable further taxonomic and cyanotoxin characterisation. Phylogenetic analyses based on a region of the 16S rRNA gene sequence, intergenic spacer (ITS) region and the mcyE gene demonstrated that the species was likely to be a new Planktothrix species that is either benthic or has a biphasic life cycle. Using liquid chromatography-mass spectrometry (LC-MS), microcystin-LR, [D-Asp³, Dha⁷] microcystin-LR, [D-Asp³] microcystin-LR, and minor proportions of [D-Asp³, ADMAdda⁵] microcystin-LhR were identified. This is the first report of [D-Asp³] microcystin-LR, [D-Asp³, Dha⁷] microcystin-LR and an ADMAadda variant in New Zealand. No cylindrospermopsins, saxitoxins or anatoxins were detected. Dog deaths caused by the consumption of cyanobacterial mats containing anatoxins have previously been reported in New Zealand. To our knowledge, however, this is the first instance of a benthic microcystin-producing species causing an animal death in New Zealand.     

Response of Fecal Bacterial Flora to the Exposure of Fumonisin B1 in BALB/c Mice

Fumonisins are a kind of mycotoxin that has harmful influence on the health of humans and animals. Although some research studies associated with fumonisins have been reported, the regulatory limits of fumonisins are imperfect, and the effects of fumonisins on fecal bacterial flora of mice have not been suggested. In this study, in order to investigate the effects of fumonisin B1 (FB1) on fecal bacterial flora, BALB/c mice were randomly divided into seven groups, which were fed intragastrically with 0 mg/kg, 0.018 mg/kg, 0.054 mg/kg, 0.162 mg/kg, 0.486 mg/kg, 1.458 mg/kg and 4.374 mg/kg of FB1 solutions, once a day for 8 weeks. Subsequently, feces were collected for analysis of microflora. The V3-V4 16S rRNA of fecal bacterial flora was sequenced using the Illumina MiSeq platform. The results revealed that fecal bacterial flora of mice treated with FB1 presented high diversity. Additionally, the composition of fecal bacterial flora of FB1 exposure groups showed marked differences from that of the control group, especially for the genus types including Alloprevotella, Prevotellaceae_NK3B31_group, Rikenellaceae_RC9_gut_group, Parabacteroides and phylum types including Cyanobacteria. In conclusion, our data indicate that FB1 alters the diversity and composition of fecal microbiota in mice. Moreover, the minimum dose of FB1 exposure also causes changes in fecal microbiota to some extent. This study is the first to focus on the dose-related effect of FB1 exposure on fecal microbiota in rodent animals and gives references to the regulatory doses of fumonisins for better protection of human and animal health.     

Screening method for stimulants in urine by UHPLC‐MS/MS: identification of isomeric compounds

A fast screening method for the detection of more than 60 stimulants in urine was developed. The method consisted of a dilution of the urine (1:5 v/v) and analysis by ultra high performance liquid chromatography coupled to tandem mass spectrometry, using a C18 column (1.7 µm particle size), a mobile phase containing deionized water and acetonitrile with formic acid, and gradient elution. The chromatographic run time was 5 min. The detection was performed in positive mode electrospray ionization, monitoring one or two specific ion transitions for each analyte. Appropriate repeatability was obtained, with relative standard deviation (RSD) values below 25% for most of the analytes. Regarding intermediate precision, estimated during routine work, higher RSDs were obtained, probably due to between‐day differences in the status of the mass spectrometer and in the chromatographic system. Matrix effect ranged from 60 to 255% with RSD lower than 35% for the majority of compounds. Despite the matrix effect observed, the signal/noise ratio of the analytes spiked at 50 ng/mL was greater than three in all tested samples, allowing a correct detection of all substances at the minimum required performance levels required by the World Anti‐Doping Agency and demonstrating the suitability of the method. The method was tested in administration study samples and satisfactorily in operation for more than one year with routine doping samples. The presence of isomeric stimulants with closely similar chromatographic and/or mass spectrometric properties did not allow the unequivocal identification of these compounds after the first analysis. Different possibilities for separation and identification of isomeric compounds are presented. Copyright © 2015 John Wiley & Sons, Ltd.     

First report of homoanatoxin-a and associated dog neurotoxicosis in New Zealand.

In November 2005, at least five dogs died rapidly after contact with water from the Hutt River (lower North Island, New Zealand). Necropsy performed 24h later on one of the dogs (a 20-month-old Labrador) revealed few findings of interest, except for copious amounts of froth in the respiratory tract down to the bifurcation of the trachea and large quantities of algal material in the dog's stomach. Low and relatively stable flows in the Hutt River during spring had resulted in the proliferation of benthic cyanobacteria that formed large black/brown mats along the river edge. Samples from the Labrador's stomach contents and cyanobacterial mats were analysed microscopically and screened using chemical and biochemical assays for cyanotoxins: anatoxin-a, homoanatoxin-a, cylindrospermopsins, saxitoxins and microcystins. Liquid chromatography-mass spectrometry (LC-MS) confirmed the presence of the neurotoxic cyanotoxins anatoxin-a and homoanatoxin-a and their degradation products, dihydro-anatoxin-a and dihydro-homoanatoxin-a. This is the first report of homoanatoxin-a and associated degradation product in New Zealand. Based on morphology, the causative species was identified as Phormidium sp. Subsequent phylogenetic analysis of 16S rRNA gene sequences demonstrated that the causative organism was most similar to Phormidium autumnale. Further investigations led to the detection of homoanatoxin-a and anatoxin-a in cyanobacterial mats from four other rivers in the Wellington region (lower North Island, New Zealand). Access restrictions were placed on over 60% of river catchments in the western Wellington region, severely affecting recreational users.     

甘草饮片中抗氧化活性成分的快速筛选及鉴定

目的:建立在线检测甘草饮片中抗氧化活性成分的方法,并对其进行鉴定.方法:通过检测1,1-二苯基-2-三硝基苯肼(DPPH)清除率来评价甘草饮片的抗氧化活性;采用在线高效液相色谱-紫外-1,1-二苯基-2-三硝基苯肼法(HPLC-UV-DPPH)筛选甘草饮片中的抗氧化活性成分;采用高效液相色谱-飞行时间高分辨质谱法(HP...     

Chemical Screening for Bioactive Substances in Culture Media of Microalgae and Cyanobacteria from Marine and Brackish Water Habitats: First Results

Abstract

Culture media of 24 commonly occurring microalgal and cyanobacteria species were subjected to a screening for total alkaloids, saponins, and phenol compounds, especially flavonoids and tannins. Both Wagner's and Mayer's reagents were employed in the alkaloid screening. Ferric chloride solution was used to detect phenolic compounds. Tannins were identified by the gelatine-salt block test. Shinoda's color reaction was used for flavonoids. Saponins were detected by the froth method. The species investigated were isolated from different marine and brackish water habitats. Media of five species gave positive alkaloid reactions with both reagents, whereas those of seven others were reactive only in one test. For the remaining 23 species, the presence of alkaloids in the culture medium can be excluded. In the media of 16 species, phenolic compounds were present. Five species produced ferric chloride–positive phenolic compounds. In six cases, positive gelatine-salt block tests were recorded. Four media gave positive reactions to the flavonoid test. It can therefore be assumed that at least two species excreted phenolic compounds other than flavonoids or tannins. Two media gave a positive saponin test.     

保健食品中新型二硫代卡地那非类似物的分析鉴定

目的 鉴定保健食品中非法添加的一个未知结构的二硫代卡地那非类似物。方法 采用高效液相色谱-二极管阵列检测器联用（HPLC-DAD）技术进行补肾壮阳类保健食品非法添加筛查时发现一个未知结构的二硫代卡地那非类似物,经过正相硅胶薄层色谱分离纯化得到目标化合物后,用超高效液相色谱-二级质谱联用（UPLC-MS/MS）技术获得其准分子离子和二级质谱图,用核磁共振得到碳谱和氢谱数据,结合文献分析,最终鉴定该化合物的结构。结果 在保健食品中发现了一个新型二硫代卡地那非类似物,结构为3,5-二甲基哌嗪基二硫代去甲卡地那非。结论 该化合物不在现有补肾壮阳类中成药检验标准的13种目标化合物范围内,是一种新的非法添加化学物质。     

UR‐144 in products sold via the Internet: Identification of related compounds and characterization of pyrolysis products

The synthetic cannabinoid, UR‐144 ((1‐pentyl‐1H‐indol‐3‐yl)(2,2,3,3‐tetramethylcyclopropyl)methanone), was identified in commercial ‘legal high’ products (herbal, resin, and powder). Along with this, six related compounds were detected. The most abundant one (2.1) was identified as 4‐hydroxy‐3,3,4‐trimethyl‐1‐(1‐pentyl‐1H‐indol‐3‐yl)pentan‐1‐one, a product of the electrophilic addition of water to the cyclopropane moiety in UR‐144. Compound 2.1 was found to be undergo cyclisation which leads to the formation of two additional interconvertable compounds (2.3, tentatively identified as 1‐pentyl‐3‐(4,4,5,5‐tetramethyl‐4,5‐dihydrofuran‐2‐yl)‐1H‐indole which is stable only in absence of water and also observed as GC artifact) and 2.2, a protonated derivative of 2.3 which is formed in acidic solutions. The remaining compounds were identified as possible degradation products of the group 2 compounds (4,4,5,5‐tetramethyldihydrofuran‐2(3H)‐one and 1‐pentylindoline‐2,3‐dione) and intermediates or by‐products from the synthesis of UR‐144 ((1H‐indol‐3‐yl)(2,2,3,3‐tetramethylcyclopropyl)methanone, 1‐pentyl‐1H‐indole and 1‐(1‐pentyl‐1H‐indol‐3‐yl)hexan‐1‐one). Pyrolysis of herbal products containing the group 2 compounds or UR‐144 resulted in the formation of 3,3,4‐trimethyl‐1‐(1‐pentyl‐1H‐indol‐3‐yl)pent‐4‐en‐1‐one (3). This was confirmed by separate pyrolysis of 2.1 and UR‐144. Also, the two additional minor compounds, 1‐(1‐pentyl‐1H‐indol‐3‐yl)ethanone and 1‐(1‐pentyl‐1H‐indol‐3‐yl)propan‐1‐one, were detected. Pathways for these transformations are presented. Copyright © 2013 John Wiley & Sons, Ltd.     

A Summer of Cyanobacterial Blooms in Belgian Waterbodies: Microcystin Quantification and Molecular Characterizations

In the context of increasing occurrences of toxic cyanobacterial blooms worldwide, their monitoring in Belgium is currently performed by regional environmental agencies (in two of three regions) using different protocols and is restricted to some selected recreational ponds and lakes. Therefore, a global assessment based on the comparison of existing datasets is not possible. For this study, 79 water samples from a monitoring of five lakes in Wallonia and occasional blooms in Flanders and Brussels, including a canal, were analyzed. A Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method allowed to detect and quantify eight microcystin congeners. The mcyE gene was detected using PCR, while dominant cyanobacterial species were identified using 16S RNA amplification and direct sequencing. The cyanobacterial diversity for two water samples was characterized with amplicon sequencing. Microcystins were detected above limit of quantification (LOQ) in 68 water samples, and the World Health Organization (WHO) recommended guideline value for microcystins in recreational water (24 µg L⁻¹) was surpassed in 18 samples. The microcystin concentrations ranged from 0.11 µg L⁻¹ to 2798.81 µg L⁻¹ total microcystin. For 45 samples, the dominance of the genera Microcystis sp., Dolichospermum sp., Aphanizomenon sp., Cyanobium/Synechococcus sp., Planktothrix sp., Romeria sp., Cyanodictyon sp., and Phormidium sp. was shown. Moreover, the mcyE gene was detected in 75.71% of all the water samples.     

Quantitation of escitalopram and its metabolites by liquid chromatography‐tandem mass spectrometry in psychiatric patients: New metabolic ratio establishment

Therapeutic drug monitoring (TDM) is used to determine the concentration of drug in plasma/serum to adjust the dose of the therapeutic drug. Selective and sensitive analytical methods are used to determine drug and metabolite levels for the successful application of TDM. The aim of the study was to develop and validate using LC‐MS/MS to analyse quantitative assay of escitalopram (S‐CT) and metabolites in human plasma samples. In order to provide a convenient and safe treatment dose, it was aimed to determine the levels of S‐CT and its metabolites in the patients’ plasma. A new method with short sample preparation and analysis time was developed and validated using LC‐MS/MS to analyse quantitative assay of S‐CT and its metabolites in plasma. Also, plasma samples of 30 patients using 20 mg S‐CT between the ages of 18 and 65 years were analysed by the validated method. The mean values of S‐CT, demethyl escitalopram and didemethyl escitalopram in plasma of patients were 27.59, 85.52 and 44.30 ng/mL, respectively. At the end of the analysis, the metabolic ratio of S‐CT and metabolites was calculated. It is considered that the method for the quantitative analysis of S‐CT and its metabolites in human plasma samples may contribute to the literature on account of its sensitive and easy application. Additionally, the use of our data by physicians will contribute to the effective drug treatment for their patients who take S‐CT.     

New perspectives in bio-analytical techniques for preclinical characterization of a drug candidate: UPLC-MS/MS in in vitro metabolism and pharmacokinetic studies

Lead optimization requires rapid bio-analytical turnover for the generation of early absorption, distribution, metabolism, excretion (ADME) and pharmacokinetics (PK) data maintaining a high quality level. Therefore, one of the major challenges in the bio-analytical field is to achieve faster and more sensitive quantification protocols. In the present communication, a comparison between HPLC and ultra performance liquid chromatography (UPLC) performances in terms of sensitivity and resolution is shown using a pharmakokinetic study and a metabolism study as models. The studies highlight the features of the new technology and the resulting impact in the PK throughput and in the characterization of isomeric metabolites using UPLC/MS/MS technique.     

Identification of ginkgolic acid (15:1) metabolites in rats following oral administration by high-performance liquid chromatography coupled to tandem mass spectrometry

1.?In this article, metabolites of ginkgolic acid (GA) (15:1) in rats plasma, bile, urine and faeces after oral administration have been investigated for the first time by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) with the aid of on-line hydrogen/deuterium (H/D) exchange technique and β-glucuronidase hydrolysis experiments.

2.?After oral administration of GA (15:1, M0) to rats at a dose of 10?mg/kg, it was found that metabolites M1-M5 together with parent compound (M0) existed in rat plasma; parent compound (M0) and metabolites M2–M5 were observed in rat bile, and parent compound (M0) with metabolites M1 and M2 were discovered in rat faeces, and there was no parent compound and metabolite detectable in rat urine.

3.?Two oxidative metabolites of GA (15:1, M0) were identified as 2-hydroxy-6-(pentadec-8-enyl-10-hydroxy) benzoic acid (M1) and 2-hydroxy-6-(pentadec-8-enyl-11-hydroxy-13-carbonyl) benzoic acid (M2), respectively. Metabolites M3, M4 and M5 were identified as the mono-glucuronic acid conjugates of parent compound (M0), M1 and M2, respectively.

4.?The results indicated that M1 and M2 with parent compound (M0) were mainly eliminated in faeces and three glucuronide metabolites (M3, M4 and M5) excreted in bile as the predominant forms after oral administration of GA (15:1) to rats.     

Fisetin disposition and metabolism in mice: Identification of geraldol as an active metabolite

Although the natural flavonoid fisetin (3,3′,4′,7-tetrahydroxyflavone) has been recently identified as an anticancer agent with antiangiogenic properties in mice, its in vivo pharmacokinetics and metabolism are presently not characterized. Our purpose was to determine the pharmacokinetics and metabolism of fisetin in mice and determine the biological activity of a detected fisetin metabolite. After fisetin administration of an efficacious dose of 223 mg/kg i.p. in mice, the maximum fisetin concentration reached 2.5 μg/ml at 15 min and the plasma concentration declined biphasically with a rapid half-life of 0.09 h and a terminal half-life of 3.1 h. Three metabolites were detected, one of which was a glucuronide of fisetin (M1), whereas another glucuronide (M2) was a glucuronide of a previously unknown fisetin metabolite (M3). HPLC–MS/MS analysis indicated that M3 was a methoxylated metabolite of fisetin (MW = 300 Da). The UV spectrum of M3 was identical to that of fisetin and standard 3,4′,7-trihydroxy-3′-methoxyflavone (geraldol). In addition, because M3 co-eluted with standard geraldol in 4 different chromatographic ternary gradient conditions, M3 was therefore assigned to geraldol. Of interest, this metabolite was shown to achieve higher concentrations than fisetin in Lewis lung tumors. We also compared the cytotoxic and antiangiogenic activities of fisetin and geraldol in vitro and it was found that the latter was more cytotoxic than the parent compound toward tumor cells, and that it could also inhibit endothelial cells migration and proliferation. In conclusion, these results suggest that fisetin metabolism plays an important role in its in vivo anticancer activities.