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A simple and rapid method is presented for the trace-level analysis of 10 polar pharmaceutical residues in various types of water samples from the aquatic environment. Using this method, the pharmaceuticals and several drug metabolites can be analyzed in drinking and surface waters and in wastewater (treated and untreated sewage) at concentrations down to 0.01 [micro]g/L. Samples are prepared by a simple in situ derivatization enabling the preconcentration of very polar metabolites by automated solid-phase extraction. The analytes were separated by liquid chromatography with tandem mass spectrometric detection and quantified by comparison with an internal standard. Limits of quantification were between 0.01 and 0.02 [micro]g/L for three phenazone-type pharmaceuticals, six of their metabolites, and the antiepileptic drug carbamazepine. Except for dimethylaminophenazone, recoveries for all analytes were between 87 and 117% for raw and purified sewage, groundwater, and surface and drinking water. Investigations of some environmental samples revealed that sewage and surface water treatment causes a slight reduction of the concentrations of some analytes whereas other compounds were persistent during water treatment. Thus, some compounds were detected at the low-microgram per liter level in sewage effluents of wastewater treatment plants in Berlin (Germany) and were also found at high-nanogram per liter concentrations in Berlin surface water samples.

Mycotoxin contamination of wheat kernels and diversity of fungal pathogens were monitored in a survey of 26 fields in Nakuru district, Kenya, in 2006. Sampling was performed twice; at mid-anthesis in July for leaves, stems and spikelets and at harvest in September for spikelets and kernels. Kernels were analysed for the occurrence of 32 mycotoxins using a high performance liquid chromatography – electrospray tandem mass spectrometry (LC-ESI-MS/MS) method. Fungi were isolated from surface-sterilized tissues and differentiated morphologically to the genus level. Fusarium isolates were identified based on the sequence of translation elongation factor 1-alpha gene. Eleven Fusarium-related mycotoxins were quantified with deoxynivalenol being detected at highest frequency (69%) and highest concentrations. Occurrence of enniatins in wheat (50%) is reported for the first time in Kenya. Non- Fusarium mycotoxins detected included aflatoxin G2, ochratoxin A, alternariol and alternariol monomethyl ether. Prevalence of Fusarium species on different wheat parts was 100% at both growth stages. Nineteen Fusarium species were identified with seven species – Fusarium chlamydosporum, Fusarium boothi, Fusarium poae, Fusarium scirpi, Fusarium arthrosporioides, Fusarium oxysporum and Fusarium graminearum– accounting for 80% of infections. At anthesis, spikelets and leaves were the most and least susceptible tissue, respectively. At harvest, infection of spikelets was 59% higher than that of kernels. [ABSTRACT FROM AUTHOR]

Abstract: The degradation of a chiral nonylphenol isomer, 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112), in two agricultural soils from Monheim and Dortmund, Germany has been studied. The degradation of NP112 and the formation of a nitro-nonylphenol metabolite were determined by means of GC–MS analysis. The degradation followed bi-exponential order kinetics, with half-life of less than 5 days in both soils. The nitro-metabolite was found at different concentration levels in the two soils. The nitro-metabolite of NP112 was more persistent than its parent compound. After 150 days about 13% of the initially applied NP112 remained in the Monheim soil as its nitro-metabolite. Results of the E-screen assay revealed that the nitro-NP112 has oestrogenic potency of 85% of that of NP112. Furthermore, the results of chiral GC–MS analysis revealed that no chiral degradation of NP112 occurred in this study. [Copyright &y& Elsevier]

Abstract: The occurrence and fate of residues from the therapeutic use of the non-steroidal anti-inflammatory drug metamizole have been studied in investigations of sewage effluents from a military hospital, municipal sewers and a sewage treatment plant (STP) in Berlin, Germany. The loads of the metabolites aminoantipyrin (AA), 4-acetylaminoantipyrin (AAA) and 4-formyl-aminoantipyrin (FAA), rapidly formed after the application of metamizole, were predicted from pharmacokinetic data and based on the evaluation of extensive data sets of on the administration in hospitals and private households. In parallel, the actual concentrations were measured within three field trials. For the military hospital, the estimated average annual discharges of AA/AAA and FAA were 10.5 and 3.2kg, respectively. For the STP, annual loads of 333 and 133 kg were determined for AA/AAA and FAA, respectively. During sewage treatment, an average decrease of 26% of the loads was measured for AA/AAA whereas no changes were observed for FAA. Generally, the prediction of the loads resulted in an overestimation of the residue levels compared to those measured in the respective sewers. Thus, modeling of predicted loads or concentrations alone will not be sufficient for a realistic assessment. Concerns for human or other mammals’ health are not expected from the occurrence of metamizole residues in the aquatic system measured at concentrations up to 7μgl−1 in STP effluents. However, a rest of uncertainty remains as it was not possible to derive a no observed effect level for the induction of rare but potentially fatal toxicological side effects reported for metamizole. [Copyright &y& Elsevier]

Residues of phenazone-type pharmaceuticals originating from spills of a former pharmaceutical production plant have recently been detected in ground water in Berlin, Germany. The degradation pathways of phenazone, propyphenazone, and dimethylaminophenazone (DMAA) during water purification were enlightened in batch experiments with groundwater and filter material obtained from operating waterworks. For phenazone and propyphenazone a complete biological transformation into their respective metabolites 1,5-dimethyl-1,2-dehydro-3-pyrazolone (DP) and 4-(2-methylethyl)-1,5-dimethyl-1,2-dehydro-3-pyrazolone (PDP) was observed. Generally, removal of phenazone-type pharmaceutical residues during rapid sand filtration was almost exclusively caused by microorganisms only present in polluted raw water. DMAA applied to fresh filter materials was rapidly degraded into its metabolites 1-acetyl-1-methyl-2-phenylhydrazide (AMPH), acetoaminoantipyrine (AAA), formylaminoantipyrine (FAA), and 1-acetyl-1-methyl-2-dimethyloxamoyl-2-phenylhydrazide (AMDOPH). DMAA, AAA, and FAA were, however, only detected at low levels in a few samples of purified water from an operating water works. Whereas, the metabolites AMDOPH and DP were detected up to 1μgl−1. Propyphenazone was rapidly removed and AMPH, phenazone, and PDP were only measured with concentrations in the low ngl−1 range. The concentrations of the metabolites DP and PDP are even higher in the purified water than in the raw water caused by their formation during degradation of phenazone and propyphenazone. Reduction of filtration velocity on an experimental filter from 5mh−1 down to 2mh−1 resulted in improved removal of phenazone, propyphenazone and their metabolites DP and PDP, respectively. AMDOPH, however, was highly persistent in all experiments independent from filtration velocities and contact times. [Copyright &y& Elsevier]

The elimination has been studied of racemic and enantioenriched metalaxyl applied as an emulsifiable concentrate and wettable powder combined with copper in a Cameroonian field site. The kinetics of the degradation/dissipation of metalaxyl and its acid metabolite were investigated using reversed phase HPLC-MS/MS, while the enantiomeric ratios were measured by HPLC-MS/MS using a Chiralcel OD-H HPLC column. Some soil enzymes activities were determined concurrently for 120d. The elimination of racemic metalaxyl was shown to be enantioselective, with the R-enantiomer being degraded more slowly than the S-enantiomer. Dissipation followed approximate square root first-order kinetics (R >0.98) without lag phases. The enantiomers of metalaxyl have different elimination rates, with half-lives ranging from only 0.8 to 1.5 days. After application to soil, the elimination of metalaxyl in the copper containing formulation was slower. The activities of acid phosphatase, alkaline phosphatase, and alkaline glucosidase were monitored throughout the experiments. No significant influence of metalaxyl and copper could be observed on these parameters. The significantly shorter half-life values of all forms of metalaxyl under field conditions, compared to the previously reported laboratory derived ones, may have implications for the plant disease control with these fungicides in tropical rainforest areas. [Copyright &y& Elsevier]

Abstract: It has recently become clear that an isomer specific view of technical 4-nonylphenol (NP) is absolutely necessary for the evaluation of the biological behavior of NP, raising additional questions concerning enantiomer specific effects. For the first time, in this study enantioselective HPLC was applied to enantiomeric separation of chiral NP isomers. A semipreparative separation of two NP isomers could be achieved. A GC–MS method has been developed for the simultaneous detection of three chiral NP isomers in water samples. Investigation of influent and effluent samples from a wastewater treatment plant in Germany indicated that enantioselective degradation could occur in the environment. In one examined influent, an enantiomer ratio of 1.7 for two different isomers was determined. [Copyright &y& Elsevier]

A reliable and sensitive analytical method for the determination of estrone (E1), 17β- estradiol (E2), and the synthetic hormone 17α-ethinylestradiol (EE2) has been established. Samples are concentrated using automated solid-phase extraction and analysis is performed by liquid chromatography with detection by tandem mass spectrometry. Recoveries of all analytes were between 93% and 107%, and limits of quantification (LOQs) between 0.1 and 0.4 ng/L for purified sewage, and surface, ground, and drinking water, and between 1 and 2 ng/L in case of raw sewage. For the investigation of estrogen release into the environment and its behavior during sewage and surface water treatment, and during ground water recharge, samples from municipal waste water treatment plants (WWTPs), a surface water treatment plant, a bank filtration site, and a ground water enrichment (GWE) pond were analyzed. E1 was found in the raw waste water with an average concentration of 157 ng/L, whereas E2 and EE2 were found with mean concentrations of only 18 and 9 ng/L, respectively. Sewage treatment by municipal WWTPs affected a removal of EE2 (76%), E1 (92%), and E2 (94%). In the investigated surface water of Berlin, only E1 could be detected at concentrations around or below 1 ng/L. E2 and EE2 were not present in the Berlin surface water above the LOQ of 0.2 ng/L, respectively. Surface water treatment also leads to a significant removal of E1 (> 80%). In the ground water samples from the GWE site near Lake Tegel, only a few samples contained detectable concentrations of E1. These samples were collected from a shallow monitoring well located very close to the bank of the pond. Even a short distance between the bank and observation wells led to concentrations of E1 below the LOQ showing the potential of ground water recharge systems for the retention of estrogenic steroids depending on the environmental setting. [ABSTRACT FROM AUTHOR]

Research on the fate and transport of endocrine-disrupting compounds and other organic wastewater compounds released into the environment and their potential presence in drinking water is in its infancy. Studies conducted during the last decade in Lincoln, Nebraska, and Berlin, Germany, indicate that removal of less polar compounds probably can be obtained through bank filtration, ground-water enrichment, and additional drinking-water and wastewater treatment processes. Polar compounds, such as atrazine and some metabolites, occur in drinking water obtained from contaminated surface water or ground water, but at concentrations generally lower than those occurring in wastewater and surface water. The results of the studies also suggest that concentrations of nonpolar estrogenic compounds decrease during drinking-water pretreatment processes such as bank filtration and ground-water enrichment. [ABSTRACT FROM AUTHOR]

During production of rivaroxaban, an orally administrated anticoagulant, process waste water is generated at several stages of manufacturing. Due to low biodegradability in conventional waste water treatment plants, it is currently incinerated. Thus, new pre-treatment methods for rivaroxaban-containing waste water could facilitate its subsequent biological processing. In this study, ozonation was investigated as the pre-treatment method, with special emphasis on the elimination of the target compound and the generation of its main transformation products. These were determined by high performance liquid chromatography – high resolution mass spectrometry (HPLC-HRMS). Our results show that the concentration of rivaroxaban in the ozonated waste water can be effectively reduced (below detection limit) under acidic conditions, whereas neutral or basic reaction environment proved less suitable. Four main transformation products were identified and their concentrations determined. Rivaroxaban and its most prominent transformation product acetoxamide were elucidated in multiple-stage mass spectrometry (MSn) experiments. Transformation products derived from the degradation of rivaroxaban were then compared to its natural (animal and human) metabolites as well as other known derivatives. In contrast to previously published rivaroxaban-derived metabolites, the presented ozonation experiments resulted in new transformation products. Unlabelled Image • Ozonation of original waste water from production of rivaroxaban • Identification of new rivaroxaban transformation products (incl. acetoxamide and oxazolinone acetamide) • Calculated pseudo first order rate constants for the degradation of rivaroxaban • Comparison of rivaroxaban ozonation products and its known metabolites • Improvement of the ecotoxicological parameters of rivaroxaban containing process waste water after ozonation [ABSTRACT FROM AUTHOR]

During production of active pharmaceutical ingredients, process waste water is generated at several stages of manufacturing. Whenever possible, the resulting waste water will be processed by conventional waste water treatment plants. Currently, incineration of the process waste water is the method to eliminate compounds with high biological activity. Thus, ozone treatment followed by biological waste water treatment was tested as an alternative method. Two prominent representatives of the large group of fluoroquinolone antibiotics (ciprofloxacin and moxifloxacin) were investigated, focussing on waste water of the bulk production. Elimination of the target compounds and generation of their main transformation products were determined by liquid chromatography – high resolution mass spectrometry (LC-HRMS). The obtained results demonstrated, that the concentration of moxifloxacin and its metabolites can be effectively reduced (>99.7%) prior entering the receiving water. On the contrary, the concentration of ciprofloxacin and its metabolites remained too high for safe discharge, necessitating application of prolonged ozonation for its further degradation. The required ozonation time can be estimated based on the determined kinetics. To assure a low biological activity the ecotoxicity of the ozonated waste water was investigated using three trophic levels. By means of multiple-stage mass spectrometry (MS n ) experiments several new transformation products of the fluoroquinolones were identified. Thus, previously published proposed structures could be corrected or confirmed. [ABSTRACT FROM AUTHOR]

Abstract: The phenolic constituents and antioxidant activity of ethanol and water extract of the flowers of Hypericum venustum, collected in Turkey, were investigated. The extracts possess strong reducing power, free radicals and hydrogen peroxide scavenging activity, as well as metal chelating ability. [Copyright &y& Elsevier]

The extent of phenotypic variation of St. John's wort (Hypericum perforatum L.) plants transformed with wild agropine ATCC 15834 Agrobacterium rhizogenes plasmid was evaluated with respect to the number of rol genes integrations. The transfer of T(L)-DNA to plant explants during each transformation event was incomplete with different rolA, rolB, and rolC copy numbers. Along with typical features representing the hairy root syndrome, an altered size, number and density of dark and translucent glands, changes in ability to synthesize secondary metabolites, and reduced fertility were observed. The highest copy number of transferred rol genes resulted in weak expression of transgenic character and comparable quantitative parameters with the controls. Only 1 out of 11 transgenic clones was able to produce seed progeny and not more than 4 out of its 35 offsprings were positive for rolC gene integration. Sterility of the clones was due to retarded development of both gametophytes.

Biosynthesis of the hypericins that accumulate in the dark glands of some members of the genus Hypericum is poorly understood. The gene named hyp-1, isolated from Hypericum perforatum L. has been proposed as playing an important role in the final steps of hypericin biosynthesis. To study the role of this candidate gene in relation to the production of hypericins, the expression of this gene was studied in 15 Hypericum species with varying ability to synthesise hypericin. While the accumulation of hypericins and emodin, an intermediate in the respective pathway, was associated with the dark glands in the hypericin-producing species, the hyp-1 gene was expressed in all studied species regardless of whether hypericins and emodin were detected in the plants. The coding sequences of hyp-1 cDNA were isolated from all species and showed more than 86% similarity to each other. Although, in general, an increased level of the hyp-1 gene transcript was detected in hypericin-producing species, several of the hypericin-lacking species expressed comparable levels as well. Our results question the role of the hyp-1 gene product as a key enzyme responsible for biosynthesis of hypericins in the genus Hypericum. The function of the hyp-1 gene may not be restricted to hypericin biosynthesis only, or some additional factors are necessary for completion of hypericin biosynthesis.

Enantiomers of 4-(1,1,2-trimethylhexyl)phenol, a chiral isomer of the endocrine disrupting chemical nonylphenol, have been resolved and isolated by preparative chiral HPLC. The absolute configurations of the enantiomers were then determined by an X-ray crystallographic study of the (-)-camphanoyl derivative of the first eluted enantiomer NP(35)E1. The first enantiomer (NP(35)E1) and the second enantiomer (NP(35)E2) eluted were found to have the S and R absolute configurations, respectively. The estrogenic potencies of the S and R enantiomers were tested by the E-screen assay. A slight difference was observed in the relative proliferative effect between the S enantiomer and R enantiomer in the E-screen assay., ((c) 2008 Wiley-Liss, Inc.)

A study has been carried out on the chemical composition and in vivo anti-oedematogenic activity of several extracts from Hypericum grandifolium Choisy (Hypericaceae) collected in Tenerife (Canary Islands). The HPLC-MS analysis (HPLC-ESI/MS and HPLC-FT/MS) revealed the presence of hyperforin, flavonoids and probably benzophenone derivatives, while naphthodianthrones were absent. Pharmacological results revealed that the methanol extracts and the aqueous, butanol and chloroform fractions obtained therefrom possess anti-oedematogenic activity against TPA-induced ear oedema and carrageenan-induced paw oedema, the chloroform fraction being the most active. Subfractions derived from the chloroform fraction also showed anti-oedematogenic activity. From these results, it can be suggested that different constituents, such as the flavonoids and the benzophenone derivatives, could be responsible, at least in part, for the anti-oedematogenic effects observed for this species.

A multimethod based on liquid-liquid extraction and solid-liquid extraction for the analysis of persistent organic pollutants in water and sludge from sewage treatment plants has been established. Traces of 22 organic compounds used in industry and personal care products (PCPs) were analyzed by GC/MS. The LODs for the analytes were less than 2.3 ng/L for wastewater and 31 microg/kg (dry weight matter) for sewage sludge. Satisfactory recoveries (70-130%) were achieved. The validated method permits the analysis of water and sludge samples at various stages of the treatment from different sewage treatment plants. Thus, the distribution between water and sludge as well as the dissipation of the compounds analyzed were balanced. By this means, the efficiency of different wastewater treatment plants (WWTPs) can be evaluated and measures can be taken to optimize the treatment process at different stages.

Renewed interest in plant-derived drugs has led to an increased need for efficient extraction methods. Hypericum perforatum L. contains several groups of bioactive compounds with noteworthy pharmacological activities. Direct sonication of H. perforatum was investigated and compared with conventional maceration, indirect sonication, Soxhlet extraction, and accelerated solvent extraction (ASE). Highly selective liquid chromatography/tandem mass spectrometry analysis showed that the content of six investigated active compounds (hypericin, pseudohypericin, hyperoside, rutin, quercitrin, and hyperforin) in extracts obtained by direct sonication was significantly higher than in extracts obtained by the other methods. The active compound contents increased on increasing the ultrasonic power from 40 to 60 W when using direct sonication. Conventional maceration gave the lowest amount of analyzed active compounds. Soxhlet extraction gave better results than ASE or indirect sonication.

An analytical method is presented which permits trace level determination of 17alpha-ethynylestradiol (EE2), 17beta-estradiol (E2), and estrone (E1). Using this method, the estrogenic steroids were analyzed in drinking water, surface water, and wastewater (sewage influents and effluents) at concentrations down to 0.1 ng/L. Sample volumes between 100 and 500 mL are concentrated using automated solid-phase extraction. Analysis is performed by liquid chromatography with detection by tandem mass spectrometry. Applying simple clean-up procedures and internal standard calibration, recovery losses resulting from matrix-dependent ion suppression during electrospray ionization could be compensated for all of the investigated compounds. Recoveries around 100% were obtained for all analytes after correction using the internal standards. Limits of quantification (LOQ) were between 0.1 and 0.4 ng/L for purified sewage, surface, ground, and drinking water and between 1 and 2 ng/L in the case of raw sewage. Water treatment by wastewater treatment plants (WWTPs) or by a surface water treatment plant affected the removal of all estrogenic steroids. Thus, E1, E2, and EE2 were removed in the municipal WWTPs to the extent of 93%, 93%, and 80%, respectively. In the effluents of the WWTP in Ruhleben (Berlin, Germany), E1, E2, and EE2 were detected at the low ng/L level. E2 and EE2 were, however, not present in the Berlin surface water above the LOQ (0.2 ng/L). E1 was the only compound that could be detected in surface water samples. After additional surface water treatment it was still detectable but only at trace-level concentrations with a mean value of 0.16 ng/L.