Detection of pressed hazelnut oil in virgin olive oil by analysis of polar components: improvement and validation of the method

An improved method for the detection of pressed hazelnut oil in admixtures with virgin olive oil by analysis of polar components is described. The method. which is based on the SPE-based isolation of the polar fraction followed by RP-HPLC analysis with UV detection. is able to detect virgin olive oil adulterated with pressed hazelnut oil at levels as low as 5% with accuracy (90.0 +/- 4.2% recovery of internal standard), good reproducibility (4.7% RSD) and linearity (R-2: 0.9982 over the 5-40% adulteration range). An international ring-test of the developed method highlighted its capability as 80% of the samples were, on average, correctly identified despite the fact that no training samples were provided to the participating laboratories. However, the large variability in marker components among the pressed hazelnut oils examined prevents the use of the method for quantification of the level of adulteration. (C) 2003 Elsevier Ltd. All rights reserved.

Validation of the FALL3D ash dispersion model using observations of the 2010 Eyjafjallajökull volcanic ash clouds

During April-May 2010 volcanic ash clouds from the Icelandic Eyjafjallajökull volcano reached Europe causing an unprecedented disruption of the EUR/NAT region airspace. Civil aviation authorities banned all flight operations because of the threat posed by volcanic ash to modern turbine aircraft. New quantitative airborne ash mass concentration thresholds, still under discussion, were adopted for discerning regions contaminated by ash. This has implications for ash dispersal models routinely used to forecast the evolution of ash clouds. In this new context, quantitative model validation and assessment of the accuracies of current state-of-the-art models is of paramount importance. The passage of volcanic ash clouds over central Europe, a territory hosting a dense network of meteorological and air quality observatories, generated a quantity of observations unusual for volcanic clouds. From the ground, the cloud was observed by aerosol lidars, lidar ceilometers, sun photometers, other remote-sensing instru- ments and in-situ collectors. From the air, sondes and multiple aircraft measurements also took extremely valuable in-situ and remote-sensing measurements. These measurements constitute an excellent database for model validation. Here we validate the FALL3D ash dispersal model by comparing model results with ground and airplane-based measurements obtained during the initial 14e23 April 2010 Eyjafjallajökull explosive phase. We run the model at high spatial resolution using as input hourly- averaged observed heights of the eruption column and the total grain size distribution reconstructed from field observations. Model results are then compared against remote ground-based and in-situ aircraft-based measurements, including lidar ceilometers from the German Meteorological Service, aerosol lidars and sun photometers from EARLINET and AERONET networks, and flight missions of the German DLR Falcon aircraft. We find good quantitative agreement, with an error similar to the spread in the observations (however depending on the method used to estimate mass eruption rate) for both airborne and ground mass concentration. Such verification results help us understand and constrain the accuracy and reliability of ash transport models and it is of enormous relevance for designing future operational mitigation strategies at Volcanic Ash Advisory Centers.

Development and validation of a qPCR-based method for quantifying Shiga toxin-encoding and other lambdoid bacteriophages

P>To address whether seasonal variability exists among Shiga toxin-encoding bacteriophage (Stx phage) numbers on a cattle farm, conventional plaque assay was performed on water samples collected over a 17 month period. Distinct seasonal variation in bacteriophage numbers was evident, peaking between June and August. Removal of cattle from the pasture precipitated a reduction in bacteriophage numbers, and during the winter months, no bacteriophage infecting Escherichia coli were detected, a surprising occurrence considering that 1031 tailed-bacteriophages are estimated to populate the globe. To address this discrepancy a culture-independent method based on quantitative PCR was developed. Primers targeting the Q gene and stx genes were designed that accurately and discriminately quantified artificial mixed lambdoid bacteriophage populations. Application of these primer sets to water samples possessing no detectable phages by plaque assay, demonstrated that the number of lambdoid bacteriophage ranged from 4.7 x 104 to 6.5 x 106 ml-1, with one in 103 free lambdoid bacteriophages carrying a Shiga toxin operon (stx). Specific molecular biological tools and discriminatory gene targets have enabled virus populations in the natural environment to be enumerated and similar strategies could replace existing propagation-dependent techniques, which grossly underestimate the abundance of viral entities.

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations. (C) 2008 Published by Elsevier B.V. and the International Society of Chemotherapy.

Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar. The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples. Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive. When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×105 copies per reaction (equals to 1.58×103 to 1.58×107 cfu/ml L. monocytogenes) were detectable. As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels.

Determination of the absolute accuracy of UK chamber facilities used in measuring methane emissions from livestock

Respiration chambers are one of the primary sources of data on methane emissions from livestock. This paper describes the results from a coordinated set of chamber validation experiments which establishes the absolute accuracy of the methane emission rates measured by the chambers, and for the first time provides metrological traceability to international standards, assesses the impact of both analyser and chamber response times on measurement uncertainty and establishes direct comparability between measurements made across different facilities with a wide range of chamber designs. As a result of the validation exercise the estimated combined uncertainty associated with the overall capability across all facilities reduced from 25.7% (k = 2, 95% confidence) before the validation to 2.1% (k = 2, 95% confidence) when the validation results are applied to the facilities’ data.

Design and validation of a sheet metal shearing experimental procedure

Throughout the industrial processes of sheet metal manufacturing and refining, shear cutting is widely used for its speed and cost advantages over competing cutting methods. Industrial shears may include some force measurement possibilities, but the force is most likely influenced by friction losses between shear tool and the point of measurement, and are in general not showing the actual force applied to the sheet. Well defined shears and accurate measurements of force and shear tool position are important for understanding the influence of shear parameters. Accurate experimental data are also necessary for calibration of numerical shear models. Here, a dedicated laboratory set-up with well defined geometry and movement in the shear, and high measurability in terms of force and geometry is designed, built and verified. Parameters important to the shear process are studied with perturbation analysis techniques and requirements on input parameter accuracy are formulated to meet experimental output demands. Input parameters in shearing are mostly geometric parameters, but also material properties and contact conditions. Based on the accuracy requirements, a symmetric experiment with internal balancing of forces is constructed to avoid guides and corresponding friction losses. Finally, the experimental procedure is validated through shearing of a medium grade steel. With the obtained experimental set-up performance, force changes as result of changes in studied input parameters are distinguishable down to a level of 1%.

Development of a novel microextraction by packed sorbent-based approach followed by ultrahigh pressure liquid chromatography as a powerful technique for quantification phenolic constituents of biological interest in wines

A novel analytical approach, based on a miniaturized extraction technique, the microextraction by packed sorbent (MEPS), followed by ultrahigh pressure liquid chromatography (UHPLC) separation combined with a photodiode array (PDA) detection, has been developed and validated for the quantitative determination of sixteen biologically active phenolic constituents of wine. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (linearity, sensitivity, selectivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters on the MEPS performance such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250 μL) in five extraction cycle and in a short time period (about 5 min for the entire sample preparation step). The wine bioactive phenolics were eluted by 250 μL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a HSS T3 analytical column (100 mm × 2.1 mm, 1.8 μm particle size) using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). The method gave satisfactory results in terms of linearity with r2-values > 0.9986 within the established concentration range. The LOD varied from 85 ng mL−1 (ferulic acid) to 0.32 μg mL−1 ((+)-catechin), whereas the LOQ values from 0.028 μg mL−1 (ferulic acid) to 1.08 μg mL−1 ((+)-catechin). Typical recoveries ranged between 81.1 and 99.6% for red wines and between 77.1 and 99.3% for white wines, with relative standard deviations (RSD) no larger than 10%. The extraction yields of the MEPSC8/UHPLC–PDA methodology were found between 78.1 (syringic acid) and 99.6% (o-coumaric acid) for red wines and between 76.2 and 99.1% for white wines. The inter-day precision, expressed as the relative standard deviation (RSD%), varied between 0.2% (p-coumaric and o-coumaric acids) and 7.5% (gentisic acid) while the intra-day precision between 0.2% (o-coumaric and cinnamic acids) and 4.7% (gallic acid and (−)-epicatechin). On the basis of analytical validation, it is shown that the MEPSC8/UHPLC–PDA methodology proves to be an improved, reliable, and ultra-fast approach for wine bioactive phenolics analysis, because of its capability for determining simultaneously in a single chromatographic run several bioactive metabolites with high sensitivity, selectivity and resolving power within only 10 min. Preliminary studies have been carried out on 34 real whole wine samples, in order to assess the performance of the described procedure. The new approach offers decreased sample preparation and analysis time, and moreover is cheaper, more environmentally friendly and easier to perform as compared to traditional methodologies.

Effects of gamma-irradiation on caprolactam level from multilayer PA-6 films for food packaging: Development and validation of a gas chromatographic method

A gas chromatographic method to determine caprolactam in multilayer PA-6 films used for meat foodstuffs and cheese was developed and validated. A wide linear range (0.8-400 mu g/ml), RSD <= 4.1% and recovery higher than 90.0% were obtained for the chromatographic system, while precision and accuracy of the method showed RSD <= 3.8%, recovery from 95.5-100.0% and LOQ of 32 mu g/g. Irradiated (3, 7 and 12 kGy) and non-irradiated commercial films were analyzed. Most of them increased caprolactam levels with the increase of irradiation doses. (C) 2008 Elsevier Ltd. All rights reserved.

EFFECT of GAMMA IRRADIATION on CAPROLACTAM MIGRATION FROM MULTILAYER POLYAMIDE 6 FILMS INTO WATER FOOD SIMULANT and VALIDATION of THE METHOD

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Determination of gatifloxacin in bulk and tablet preparations by high-performance liquid chromatography

A sensitive, precise, and specific high-performance liquid chromatographic (HPLC) method was developed for the assay of gatifloxacin (GATX) in raw material and tablets. The method validation parameters yielded good results and included the range, linearity, precision, accuracy, specificity, and recovery. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. The HPLC separation was carried out by reversed-phase chromatography on a C18 absorbosphere column (250 x 4.6 mm id, 5 pm particle size) with a mobile phase composed of acetic acid 50/o--acetonitrile-methanol (70 + 15 + 15, v/v/v) pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 287 nm. The calibration graph for GATX was linear from 4.0 to 14.0 mu g/mL. The interday and intraday precisions (relative standard deviation) were less than 1.05%.

Microbiological assay for cefoxitin sodium in dosage form

A microbiological assay applying the cylinder-plate method is described for determination of the activity of cefoxitin sodium in injectables. Using a strain of Staphylococcus epidermidis ATCC 12226 as the test organism, cefoxitin sodium was measured in concentrations ranging from 50.0 to 200.0 mu g/mL. The validation showed that the method was linear (r = 0.9998), precise (RSD 0.81%), and accurate. It was concluded that the microbiological assay is satisfactory for quantitation of cefoxitin sodium in injectables.

Determination of lomefloxacin in tablet preparations by liquid chromatography

A sensitive, precise, and specific high-performance liquid chromatography (HPLC) method was developed for the assay of lomefloxacin (LFLX) in raw material and tablet preparations. The method validation parameters yielded good results and included the range, linearity, precision, accuracy, specificity, and recovery. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. The HPLC separation was performed on a reversed-phase Phenomenex C18 column (150 x 4.6 mm id, 5 pm particle size) with a mobile phase composed of 1% acetic acid-acetonitrile-methanol (70 + 15 + 15, v/v/v), pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 280 nm. The calibration graph for LFLX was linear from 2.0 to 7.0 mg/mL. The interday and intraday precisions (relative standard deviation) were less than 1.0%. The method was applied for the quality control of commercial LFLX tablets to quantitate the drug.

Microbiological assay for ceftazidime injection

A simple, sensitive, and specific biodiffusion assay for the! antibacterial ceftazidime was developed using a strain of Staphylococcus epidermidis (ATCC 12228) as the test organism. Ceftazidime was measured in powder for injection at concentrations ranging from 100 to 400 mu g/mL. The calibration graph for ceftazidime was linear (r(2) = 1), and the method validation showed that it was precise (relative standard deviation = 0.415) and accurate. The results obtained by biodiffusion assay were statistically calculated by linear parallel model and by means of regression analysis and were verified using analysis of variance. It was concluded that the microbiological assay is satisfactory for in vitro quantification of the antibacterial activity of ceftazidime in pharmaceuticals.

Microbiological assay for lomefloxacin in coated tablets

The validation of a microbiological assay, applying cylinder plate method for determination of the activity of lomefloxacin in coated tablets is described. Using a strain of Bacillus subtilis ATCC 9372 as the test organism, lomefloxacin was measured in concentrations ranging from 2.0 to 8.0 mu g/mL. The method validation showed that it is linear (r = 0.9999), precise (relative standard deviation 1.15%), and accurate (it measured the added quantities). The excipients did not interfere in the determination. It was concluded that the microbiological assay is satisfactory for quantitation of lomefloxacin in tablets.