The evaluation of bacterial biosensors for screening of water pollutants

Bacterial biosensors incorporating the cyanobacterium Synechoccus as the biocatalyst have been evaluated by three laboratories as potential biomonitors for detecting water pollutants. The biosensors were capable of detecting at low concentrations herbicides that interact with photosynthetic electron transfer chains. Statistical evaluation of the interlaboratory comparison results for linuron and atrazine indicated that these compounds can be detected rapidly at 50 μg/L concentrations.     

Recent trends in development of biosensors for detection of microcystin

Increased cyanobacterial blooms, a source of cyanotoxins are linked with climate change and eutrophication in aquatic bodies, a major concern worldwide. Microcystins are potently hepatotoxic, nephrotoxic as well as carcinogenic. Thus microcystins are threat to tourism, agriculture and animal's health. However, there is a still lacuna in the knowledge of regulation of microcystins production. Presence of toxic and non-toxic cyanobacterial strains together and occurrence of various microcystin variants in aquatic bodies compounded the problem. Although several analytical techniques for microcystin detection such as bioassay, ELISA, HPLC and LC-MS etc. have been already prevalent, the development of biosensors offered rapid and accurate detection, high reproducibility and portability. Sequencing of Microcystis spp., opened the new vistas towards the development of biosensor at molecular and genetic level. This review incorporates the current trends in the development of biosensors for microcystin detection in the light of state-of-the-art techniques.     

Exposure simulation for pharmaceuticals in European surface waters with GREAT-ER

The Geo-referenced Regional Exposure Assessment Tool for European Rivers (GREAT-ER) model was developed as an aquatic chemical exposure prediction tool for use within environmental risk assessment (ERA) schemes and river basin management. The GREAT-ER software calculates the distribution of predicted environmental concentrations (PECs) of consumer chemicals in surface waters, for individual river stretches, as well as representative average PECs for entire catchments. The system uses an ARC/INFO-ArcView (ESRI) based Geographical Information System (GIS) for data storage and visualization, combined with simple mathematical models for prediction of chemical fate. Use of GREAT-ER 1.03 to derive PECs is illustrated for Ethinyl Oestradiol, Paracetamol, Aspirin, Dextropropoxyphene, Clofibrate and Oxytetracycline in three river basins, i.e. Aire (UK), Lambro (Italy) and Rur (Germany). In contrast with household consumer chemicals the transformation of pharmaceuticals in the human body needs to be incorporated in the emission estimation. The "PECinitial" of these pharmaceuticals in surface waters ranges from >1 microg/l (Oxytetracycline and Paracetamol) down to <1 ng/l (Ethinyl Oestradiol). Risk characterization employing PECs or measured environmental concentrations (MECs) and predicted-no-effect-concentrations (PNECs) from available ecotoxicity data is also reported.     

Pharmaco-toxicological screening of commercially available Italian natural mineral waters

The consumption of natural mineral water has increased enormously during the past few years, yet doubts are arising about the real utility of using such water instead of ordinary drinking water (tap water). Mineral water's 'special' mineral composition might have properties favourable to health, which should be assessed by clinical and pharmacological analyses. A comparative pharmaco-toxicological study was performed on 14 commercially available Italian natural mineral waters with a wide range of mineral content. The waters were microbiologically analysed and the Allium cepa test done; in addition, Irwin, charcoal meal and diuresis tests were also performed on laboratory animals. No toxicological signs were recorded at the Allium cepa assay for any but the water with the highest mineral content. The diuretic effect was comparable to that of control tap water. Waters with high magnesium content significantly enhanced intestinal motility while at the Irwin test none of the water samples evoked behavioural changes. All the tested samples were microbiologically pure. In conclusion, mineral water can be an alternative to aqueduct water in places where the latter comes from superficial water and has to be subjected to hard potabilisation techniques; water from the 'Acqua Marcia' aqueduct (control) was found to have characteristics comparable to the mineral waters studied.     

Metabolic profiling of endocrine-disrupting compounds by on-line cytochrome p450 bioreaction coupled to on-line receptor affinity screening

We present a fully automated and hyphenated bioanalytical method for metabolic profiling of potentially harmful xenoestrogens. The system consists of an on-line cytochrome P450 bioreactor coupled to a reversed-phase, gradient high-performance liquid chromatograph. A C18 solid-phase extraction (SPE) unit is used as an interface between the P450 bioreactor and the HPLC column. The HPLC column is linked on-line to a high-resolution screening (HRS)-estrogen receptor alpha affinity detection (ERAD) assay. In effect, the P450 bioreactor produces metabolites that are subsequently trapped on-line by SPE and separated by HPLC. The separated metabolites are then screened on-line, at the moment of elution, for affinity toward estrogen receptor alpha (ERalpha) using the HRS-ERAD assay. The SPE method was optimized with methoxychlor (MXC) and its metabolites mono- and bis-OH-MXC. After optimization, the P450-bioreactor-SPE-HPLC system was made generally applicable to the biocatalysis and trapping of polar to highly apolar compounds. The precision of the P450-bioreactor-SPE-HPLC system is high (relative standard deviation相似文献   

Human health risk assessments for three neuropharmaceutical compounds in surface waters

Enhanced sensitivity of analytical chemistry methods has enabled the detection of low-levels of pharmaceuticals in the environment, resulting in questions about the safety of surface waters used for drinking supplies. Human health risk assessments were performed to evaluate the risks from residues of atomoxetine, duloxetine, and olanzapine, which might be found in surface waters. Preclinical safety studies and human clinical data were used to determine an acceptable daily intake (ADI) for each compound: atomoxetine, 1.4 microg/kg/day; duloxetine, 1.8 microg/kg/day; and olanzapine, 1.4 microg/kg/day. The calculated predicted no-effect concentrations (PNECs) for children were 25.7, 19.1, and 35.9 microg/L for atomoxetine, duloxetine, and olanzapine, respectively. Estimated exposure concentrations determined using United States Food and Drug Administration guidelines and predicted exposure concentrations from the PhATE model were compared with each PNEC to determine margins of safety, which ranged from 147 to 642. Based on currently available data used in this assessment, no appreciable human health risks exist from exposure to the highest 99th percentile of predicted residue levels of atomoxetine, duloxetine or olanzapine in surface waters under low-flow conditions.     

哮喘患者报告临床结局量表的研制和条目筛选

目的研制具有良好信度、效度与反应度,适用于新药临床试验或临床疗效评价的哮喘患者报告临床结局(PRO)量表。方法遵照美国食品药品管理局(FDA)PRO量表研制的程序化方法,建立量表的理论框架,建立条目池,并通过专家咨询、患者访谈等修改量表,得到含有72个条目的初量表。现场调查共收集108例样本,使用离散趋势法、因子分析法、相关系数法、克朗巴赫α系数法及修正条目的总相关系数法和项目反应理论5种方法对初量表进行条目筛选。结果经过条目筛选,调整量表的框架结构,共保留67个条目,分为生理、心理、社会、治疗4个领域及13个方面。结论哮喘PRO量表严格按照国际量表的操作原则和方法进行,科学性强、结论可信,有利于临床研究中推广使用。     

Bone surface and whole bone as biomarkers for acute fluoride exposure

This study compares fluoride concentrations ([F]) in surface and whole bone for up to 27 days following an acute oral dose of F. Four groups of rats received single oral F dose (50 mg/kg body weight), and the control group received deionized water (n = 10/group). Groups were euthanized at 1, 3, 9, or 27 days after F administration. Plasma and femurs were collected. F on the femur surface was removed from a circular area (4.52 mm(2)) by immersion in 0.5M HCl for 15 s. The solution was buffered with total ionic strength adjustment buffer and analyzed with an electrode. The subjacent bone was sectioned and ashed at 600 degrees C. Ash and plasma were analyzed for F with the electrode following hexamethyldisiloxane-facilitated diffusion. Data were analyzed by Kruskall-Wallis and Dunn's test and by linear regression (p < 0.05). Peak plasma and bone surface [F] occurred on day 1 (0.26 +/- 0.14 microg/mL and 1801 +/- 888 microg/g, respectively). Bone surface [F] at 3, 9, and 27 were not statistically different from control. A significant increase in whole bone [F] was observed 3 days after F administration and the [F] remained relatively constant thereafter. The mean (+/- SD) surface/whole bone [F] ratios for the control and F groups were 2.45 +/- 0.98, 3.92 +/- 1.32, 1.61 +/- 0.82, 1.73 +/- 0.39, and 1.09 +/- 0.28, respectively. Plasma and bone surface [F]s were positively correlated (r = 0.74). Thus, bone surface was found to be a suitable biomarker for acute, sublethal F exposure 1 day after F administration. Whole bone [F] were significantly increased at 3, 9, and 27 days after F administration.     

An in vitro screening paradigm for extracts of whole foods for detection of potential toxicants.

The application of organic, conventional and biotechnology techniques can alter the intrinsic levels of natural toxicants in crop foods and methods are needed to screen for unexpected changes in toxicant levels. We evaluated crude, aqueous preparations of 37 foods purchased from a local market in a battery of four in vitro mammalian toxicity screens. The foods were evaluated in one or more of the following tests: (1) cytotoxicity (37 foods) and (2) chromosomal aberration test (nine foods), both in Chinese hamster ovary cells, (3) limb bud micromass assay (nine foods) using 11-day old CD-1 mouse embryos and (4) estrogenicity (MCF-7 cells transfected with estrogen receptor and lucerifase reporter constructs, 12 foods). IC50s for cellular proliferation ranged from < 1% (v/v, garlic) to > 10% (v/v, 18 foods), the maximal concentration tested. Five of nine preparations (soybeans, broccoli, garlic, snow peas and corn) were clastogenic and two (soybeans and snow peas) inhibited chrondrogenesis in the limb bud micromass assay. Five of nine preparations (soybeans, snow peas, cumin, asparagus and bean sprouts) produced significant estrogenic responses. Overall, the 12 foods evaluated in two or more of the tests showed different patterns of response. These preliminary data indicate that screening for potential toxicants is possible with fast, relatively inexpensive in vitro tests. These in vitro tests, while potentially useful to detect unexpected toxicants in plants that may signal the need for further evaluation, are not directly useful to predict human or animal risk from eating these plants.     

Toxicological screening of basic drugs in whole blood using UPLC-TOF-MS

Ultra performance liquid chromatography (UPLC) coupled with time-of-flight (TOF) mass spectrometry (MS) was established for toxicological screening of basic drugs in whole blood and tested on authentic samples. Whole blood samples (0.2 ml) were extracted using a Gilson apparatus equipped with Bond Elut Certify columns. Screening was performed for 175 compounds (psychotropic, cardiovascular, designer, and abused drugs). The drugs were separated in 15 min using a UPLC system (Waters ACQUITY BEH C18, 1.7 μm, 2.1 mm × 100 mm column) coupled to an LCT Premier XE (Waters) instrument. Data were processed by ChromaLynx XS using identification criteria of ± 0.2 min retention time, and ± 5 mDa mass tolerance. Whole blood was spiked with the 175 compounds in concentrations from 5-100 μg/kg to assess approximately the lowest concentrations that could be identified. This method was further applied to 119 samples from forensic investigations, leading to 302 hits, of which 291 (96%) were subsequently verified, in concentrations exceeding the lower limit of quantification (LLOQ), by a liquid chromatography (LC)-MS/MS confirmation method. In conclusion, this UPLC-TOF-MS method is a useful and effective screening method for basic drugs in whole blood.     

Preliminary evaluation of the Abbott TDx for benzoylecgonine and opiate screening in whole blood

A method for the detection of benzoylecgonine (cocaine metabolite) and opiates in whole blood is described. This method employs the Abbott TDx fluorescence polarization immunoassay technique, which was designed for urine analysis. Drug-free whole blood was spiked with varying concentrations of benzoylecgonine, morphine, and codeine. Samples were prepared for analysis by adding 300 microL of 10% trichloroacetic acid to 300 microL of blood. Specimens were vortexed and centrifuged with 50 microL of supernatant required per assay. Precision studies of six replicate samples spiked with benzoylecgonine at 0.5 mg/L gave a within-run CV of 4.7% and a between-run CV of 4.7% with a detection limit of 0.1 mg/L. Within-run CVs for morphine at 0.5 mg/L and 0.1 mg/L were 1.7% and 7.9% respectively. The detection limits for morphine and codeine were 0.05 mg/L. Correlation coefficients for spiked whole blood calibration curves of benzoylecgonine, morphine, and codeine were 0.984, 0.999, and 0.997 respectively. This preliminary evaluation demonstrates a potential application of the TDx fluorescence polarization immunoassay technique to the analysis of drugs in whole blood.     

Flow amperometric determination of pharmaceuticals with on-line electrode surface renewal

In this article, a flow system developed for the amperometric determination of a great variety of pharmaceuticals that are known to lead the rapid poisoning of the working electrode surface is described. The referred system was made up of two parallel flow channels that shared the voltammetric detector of tubular configuration, whose movement in the manifold followed the concept of multi-site location of detector. In this way, after each measurement, the conditioning of the working electrode was possible through the passage by its surface of a regeneration solution without implying the alteration of the carrier that flowed in the analytical channel of the manifold. The methodology proposed was evaluated through the determination of two drugs belonging to two distinct therapeutic groups: an antihypertensive (diltiazem) and a non-steroid anti-inflammatory (nimesulide). The results obtained after evaluation of various pharmaceutical formulations on the Portuguese market were in the case of diltiazem compared with those supplied by the reference US Pharmacopoeia XXIV method, with no statistically significant differences having been observed for a confidence interval of 95%. In the case of nimesulide, since no official reference method exists, a series of recovery experiments were proceeded with and a mean value of 101.1% with a R.S.D. of 0.7% was obtained.     

A single-step extraction for screening whole blood for basic drugs by capillary GC/NPD

Basic drugs were routinely extracted from whole blood under alkaline conditions into n-butyl acetate. An aliquot of the n-butyl acetate was injected into a gas chromatograph equipped with a nitrogen-phosphorous detector and a wide-bore cross-linked 50% phenylmethyl silicone capillary column. Absolute and relative retention times were recorded for more than 100 extracted drug standards. Recovery from whole blood was determined for some of the more frequently encountered drugs. This one-step extraction proved to be reliable for general screening and has been used routinely in forensic and clinical toxicological analyses.     

Preliminary evaluation of the Abbott TDx for screening of D-methamphetamine in whole blood specimens.

The Abbott TDx Urine Amphetamine/Methamphetamine II fluorescence polarization immunoassay technique was applied to the determination of D-methamphetamine in hemolyzed whole blood. The assay was found to have 100% cross-reactivity with D-methamphetamine and only an 8% cross-reactivity with L-methamphetamine. Whole blood was fortified with D-methamphetamine at concentrations ranging from 25 to 1000 ng/mL. These whole blood calibrators were used to evaluate the following sample preparation techniques: direct, diluted and buffer, and precipitated using methanol, acetone, sulfosalicylic acid, trichloroacetic acid, and zinc sulfate. Calibrators and samples were prepared by mixing 200 microL of whole blood and 200 microL precipitation reagent and centrifuging at 10,000 rpm for 5 min (9600 x g). A 50-microL aliquot of the supernatant was used for the assay. Using the zinc sulfate precipitation, blood calibration curves showed a linear range of 25-1000 ng/mL. The within-run precision for the 25-, 60-, and 200-ng/mL D-methamphetamine blood controls showed percent coefficients of variation of 17.8, 17.0, and 5.4, respectively. The TDx results were compared to RIA and GC/MS assays for the methamphetamine controls and for eight positive case specimens and found reliable for the screening of hemolyzed whole blood.     

Radioimmunoassay screening and GC/MS confirmation of whole blood samples for drugs of abuse

From 1981 to 1984, an average of 300 radioimmunoassay screens on whole blood were performed each week in the authors' laboratory. Most samples were screened for opiates, phencyclidine and its analogs, barbiturates, and cocaine or its metabolite benzoylecgonine. A commercially available radioimmunoassay was used with modifications to facilitate screening of whole blood. Increasing sample size increased the sensitivity of the assay. Changing reagent concentration (1:1 dilution), incubation time, sample matrix (water, urine, or blood), or fraction counted (precipitate or supernatant) did not affect the utility of the standard curve or the sensitivity of the assay. All positive results for phencyclidine, opiates, cocaine, and related compounds were confirmed by GC/MS. Barbiturate positives were confirmed by UV spectrophotometry. The rate of confirmation in postmortem bloods from coroner's cases for 1981-84 was: cocaine/benzoylecgonine, 57%; opiates, 79%; phencyclidine, 49%; and barbiturates, 58%.     

Drug screening of whole blood by ultra-performance liquid chromatography-tandem mass spectrometry

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for screening of drugs in whole blood has been developed and validated. Samples were prepared by supported liquid-liquid extraction on ChemElute(?) columns with ethyl acetate/heptane (4:1). LC separation was achieved with an Acquity HSS T3-column (2.1 100 mm, 1.8-μm particle). Mass detection was performed by positive ion mode electrospray MS-MS and included the following drugs/metabolites: morphine, codeine, ethyl morphine, oxycodone, buprenorphine, methadone, cocaine, methylphenidate, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), Δ(9)-tetrahydrocannabinol (THC), fentanyl, alprazolam, bromazepam, clonazepam, diazepam, nordiazepam, 3-OH-diazepam, fenazepam, flunitrazepam, lorazepam, nitrazepam, oxazepam, zopiclone, zolpidem, carisoprodol, and meprobamate. The cycle time was 9 min, and within- and between-day relative coefficients of variation varied from 1% to 33% and 2% to 58%, respectively. Extraction recoveries from whole blood were > 50% except for morphine and THC. The limit of quantitation was 0.1 to 521 ng/mL, depending on the drug.