Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 mug kg (-1) in liver and 5, 15 and 50 mug kg (-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 mug kg (-1), respectively. (C) 2003 Elsevier Science B.V. All rights reserved.

Development and validation of dry reagent time-resolved fluoroimmuno assays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 961 23\EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin a-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TR-FIAs, the results being available within 1 h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.

Validation and application of reporter gene assays for the determination of estrogenic and androgenic endocrine disruptor activity in sport supplements.

Previously developed estrogen and androgen mammalian reporter gene assays (RGAs) were assessed for their potential use as a quantitative screening method in the detection of estrogenic and androgenic endocrine disruptors (EDs) in sport supplements. The validation of both RGAs coupled with dispersive solid phase extraction (dSPE) was performed in accordance with European Commission Decision EC/2002/6579 for biological screening methods. Decision limits (CCa) and detection capabilities (CCß) were established for both the estrogen and androgen RGAs. All samples were compliant with CCa and CCß in both bioassays. Recovery rates were 96 % for 17ß-estradiol and 115 % for dihydrotestosterone as obtained in their corresponding RGA. Both estrogens and androgens were stable in samples for more than 3 weeks, when stored at -20 °C. Specificity, good repeatability (coefficients of variation (CV), 12–25 %), reproducibility and robustness of both bioassays were also observed. Four different ED modes of action were determined for estrogens and androgens in 53 sport supplements, using the validated RGAs. This study revealed that 89 % of the investigated sport supplements contained estrogenic EDs and 51 % contained androgenic compounds. In conclusion, both bioassays are suitable for sport supplement screening of estrogenic and androgenic EDs.

Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a ? coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.

Development and validation of a method for the confirmation of nicarbazin in chicken liver and eggs using LC-electrospray MS-MS according to the revised EU criteria for veterinary drug residue analysis

A method is described for the quantitative confirmation of 4,4'-dinitrocarbanilide (DNC), the marker residue for nicarbazin in chicken liver and eggs. The method is based on LC coupled to negative ion electrospray MS-MS of tissue extracts prepared by liquid-liquid extraction. The [M-H](-) ion at m/z 301 is monitored along with two transition ions at m/z 137 and 107 for DNC and the [M-H](-) ion at m/z 309 for the internal standard, d(8)-DNC. The method has been validated according to the new EU criteria for the analysis of veterinary drug residues at 100, 200 and 300 mug kg(-1) in liver and at 10, 30 and 100 mug kg(-1) in eggs. Difficulties concerning the application of the new analytical limits, namely the decision limit (CC) and the detection capability (CC) to the determination of DNC in both liver and eggs are discussed.

How Organizations Engage with External Complexity: A Political Action Perspective

This paper offers a new insight into how organizations engage with external complexity. It applies a political action perspective that draws attention to the hitherto neglected question of how the relative power organizational leaders enjoy within their environments is significant for the actions they can take on behalf of their organizations when faced with external complexity. It identifies cognitive and relational complexity as two dimensions of the environment with which organizations have to engage. It proposes three modes whereby organizations may engage with environmental complexity that are conditioned by an organization's power within its environment. It also considers the intention associated with each mode, as well as the implications of these modes of engagement for how an organization can learn about its environment and for the use of rationality and intuition in its strategic decision-making. The closing discussion considers how this analysis integrates complexity and political action perspectives in a way that contributes to theoretical development and provides the basis for a dynamic political co-evolutionary approach. © The Author(s) 2011.

Validation of membrane protein topology models by oxidative labeling and mass spectrometry

Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ·OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ·OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

Do smokers with chronic obstructive pulmonary disease report their smoking status reliably?:A comparison of self-report and bio-chemical validation

Chronic obstructive pulmonary disease (COPD) is predominantly caused by cigarette smoking and is considered a worldwide preventable chronic illness. Smoking cessation is considered the primary intervention for disease management and nurses should play a major role in assisting patients to stop smoking. Currently there is a lack of professional consensus on how cessation interventions should be evaluated. The vast array of biochemical markers reported in the literature can be confusing and can make the comparisons of results difficult.

The technical merits of turbogenerating shown through the design, validation and implementation of a 1 dimensional engine model

Turbocompounding is the process of recovering a proportion of an engine’s fuel energy that would otherwise be lost in the exhaust process and adding it to the output power. This was first seen in the 1930s and is carried out by coupling an exhaust gas turbine to the crankshaft of a reciprocating engine. It has since been recognised that coupling the power turbine to an electrical generator instead of the crankshaft has the potential to reduce the fuel consumption further with the added flexibility of being able to decide how this recovered energy is used. The electricity generated can be used in automotive applications to assist the crankshaft using a flywheel motor generator or to power ancillaries that would otherwise have run off the crankshaft. In the case of stationary power plants, it can assist the electrical power output. Decoupling the power turbine from the crankshaft and coupling it to a generator allows the power electronics to control the turbine speed independently in order to optimise the specific fuel consumption for different engine operating conditions. This method of energy recapture is termed ‘turbogenerating’.

This paper gives a brief history of turbocompounding and its thermodynamic merits. It then moves on to give an account of the validation of a turbogenerated engine model. The model is then used to investigate what needs to be done to an engine when a turbogenerator is installed. The engine being modelled is used for stationary power generation and is fuelled by an induced biogas with a small portion of palm oil being injected into the cylinder to initiate combustion by compression ignition. From these investigations, optimum settings were found that result in a 10.90% improvement in overall efficiency. These savings relate to the same engine without a turbogenerator installed operating with fixed fuelling.