Development of a two-step screening ESI-TOF-MS method for rapid determination of significant stress-induced metabolome modifications in plant leaf extracts: the wound response in Arabidopsis thaliana as a case study.

To study the stress-induced effects caused by wounding under a new perspective, a metabolomic strategy based on HPLC-MS has been devised for the model plant Arabidopsis thaliana. To detect induced metabolites and precisely localise these compounds among the numerous constitutive metabolites, HPLC-MS analyses were performed in a two-step strategy. In a first step, rapid direct TOF-MS measurements of the crude leaf extract were performed with a ballistic gradient on a short LC-column. The HPLC-MS data were investigated by multivariate analysis as total mass spectra (TMS). Principal components analysis (PCA) and hierarchical cluster analysis (HCA) on principal coordinates were combined for data treatment. PCA and HCA demonstrated a clear clustering of plant specimens selecting the highest discriminating ions given by the complete data analysis, leading to the specific detection of discrete-induced ions (m/z values). Furthermore, pool constitution with plants of homogeneous behaviour was achieved for confirmatory analysis. In this second step, long high-resolution LC profilings on an UPLC-TOF-MS system were used on pooled samples. This allowed to precisely localise the putative biological marker induced by wounding and by specific extraction of accurate m/z values detected in the screening procedure with the TMS spectra.

Development of a Rational Characterization Method for Iowa Fly Ash, HR-286, 1988

This research project was conducted in an attempt to determine the cause of paste strength variability in Iowa fly ashes and to develop test methods to more adequately reflect fly ash physical and chemical characteristics. An extensive three year sampling and testing program was developed and initiated which incorporated fly ash from several Iowa power plants. Power plant design and operating data were collected. The variability was directly linked to power plant maintenance schedules and to sodium carbonate coal pretreatment. Fly ash physical and chemical properties can change drastically immediately before and after a maintenance outage. The concentrations of sulfate bearing minerals in the fly ash increases sharply during shutdown. Chemical, mineralogical, and physical testing indicated that the sodium, sulfate bearing minerals, lime and tricalcium aluminate contents of the fly ashes play important roles in the development of hydration reaction products in fly ash pastes. The weak pastes always contained ettringite as the major reaction product. The strong pastes contained straetlingite and monosulfoaluminate as the major reaction products along with minor amounts of ettringite. Recommendations for testing procedure changes and suggested interim test methods are presented.

Development and validation of a gas chromatography-negative chemical ionization tandem mass spectrometry method for the determination of ethyl glucuronide in hair and its application to forensic toxicology.

Ethyl glucuronide (EtG) is a minor and direct metabolite of ethanol. EtG is incorporated into the growing hair allowing retrospective investigation of chronic alcohol abuse. In this study, we report the development and the validation of a method using gas chromatography-negative chemical ionization tandem mass spectrometry (GC-NCI-MS/MS) for the quantification of EtG in hair. EtG was extracted from about 30 mg of hair by aqueous incubation and purified by solid-phase extraction (SPE) using mixed mode extraction cartridges followed by derivation with perfluoropentanoic anhydride (PFPA). The analysis was performed in the selected reaction monitoring (SRM) mode using the transitions m/z 347-->163 (for the quantification) and m/z 347-->119 (for the identification) for EtG, and m/z 352-->163 for EtG-d(5) used as internal standard. For validation, we prepared quality controls (QC) using hair samples taken post mortem from 2 subjects with a known history of alcoholism. These samples were confirmed by a proficiency test with 7 participating laboratories. The assay linearity of EtG was confirmed over the range from 8.4 to 259.4 pg/mg hair, with a coefficient of determination (r(2)) above 0.999. The limit of detection (LOD) was estimated with 3.0 pg/mg. The lower limit of quantification (LLOQ) of the method was fixed at 8.4 pg/mg. Repeatability and intermediate precision (relative standard deviation, RSD%), tested at 4 QC levels, were less than 13.2%. The analytical method was applied to several hair samples obtained from autopsy cases with a history of alcoholism and/or lesions caused by alcohol. EtG concentrations in hair ranged from 60 to 820 pg/mg hair.

Development of a Testing Method for Convertibility of Paperboard

Työssä tutkittiin muovattujen kartonkivuokien sekä muovattujen kartonkinäytteiden rinnastettavuutta. Puristusvaiheen prosessiolosuhteiden miellettiin vaikuttavan eniten multidimensionaliseen muodonmuutokseen. Multidimensionaalista muodonmuutosta simuloitiin uudella muovaamiseen soveltuvalla muovauslaitteella. Kirjallisuusosassa keskeisiä teemoja ovat kartongin muovaus sekä kuitupohjaisen materiaalin reologinen käyttäytyminen. Kirjallisuusosassa esitellään lisäksi yksi tekninen sovellus, jonka avulla kyetään ennustamaan kuitumateriaalin muovautuvuutta sekä mittaamaan tapahtunutta muodonmuutosta. Prosessiparametrien teoreettista vaikutustakuituihin tarkastellaan myös kirjallisuusosassa. Kokeellisessa osassa toteutettiin kartonkivuokien valmistus puristamalla. Vastaavilla prosessiparametreilla muovattiin myös pienemmät testinäytteet. Perinteiset yksidimensionaliset deformaatiomittaukset toteutettiin lujuusominaisuuksien laboratoriomäärityksinä. Myös kitka, joka toimii tärkeänä muuttujana prässäysprosessissa, mitattiin laboratorio-olosuhteissa. Tämän työn tulokset osoittavat uuden kehitetyn muovausmenetelmän toimivuuden. Asema-voima kuvaajat ovat selkeitä sekä helposti luettavia. Tuloksissa havaittiin materiaalin muovauspotentiaalin sekä asema-voima kuvaajan välillä vallitseva yhteys. Erittäin merkittävä huomio oli myös, että muovipäällystetyllä kartongilla oli yhteys päällystämättömän kartongin asema-voima kuvaajaan. Tämä tulos osoittaa, että muovipäällystetyn kartongin muovautuvuutta voi olla mahdollista ennustaa pohjakartongin muovautuvuustulosten perusteella. Perinteiset yksidimensionaliset laboratoriomittaukset eivät kykene antamaan riittävää informaatiota muovautuvuuden ennustamiseen. Tästä näkökulmasta on tärkeää että kartongin multidimensionalista muotoutuvuutta voidaankin tutkia kehitetyllä muovausmenetelmällä.

L'immunoscintigraphie par les anticorps monoclonaux radiomarqués. Evolution d'une méthode de diagnostic du cancer et espoir d'une nouvelle forme de thérapie [Immunoscintigraphy using radio-labeled monoclonal antibodies. Development of a method of cancer diagnosis and hopes for a new form of therapy].

Personal results are presented to illustrate the development of immunoscintigraphy for the detection of cancer over the last 12 years, from the early experimental results in nude mice grafted with human colon carcinoma to the most modern form of immunoscintigraphy applied to patients, using I123 labeled Fab fragments from monoclonal anti-CEA antibodies detected by single photon emission computerized tomography (SPECT). The first generation of immunoscintigraphy used I131 labeled, immunoadsorbent purified, polyclonal anti-CEA antibodies and planar scintigraphy, as the detection system. The second generation used I131 labeled monoclonal anti-CEA antibodies and SPECT, while the third generation employed I123 labeled fragments of monoclonal antibodies and SPECT. The improvement in the precision of tumor images with the most recent forms of immunoscintigraphy is obvious. However, we think the usefulness of immunoscintigraphy for routine cancer management has not yet been entirely demonstrated. Further prospective trials are still necessary to determine the precise clinical role of immunoscintigraphy. A case report is presented on a patient with two liver metastases from a sigmoid carcinoma, who received through the hepatic artery a therapeutic dose (100 mCi) of I131 coupled to 40 mg of a mixture of two high affinity anti-CEA monoclonal antibodies. Excellent localisation in the metastases of the I131 labeled antibodies was demonstrated by SPECT and the treatment was well tolerated. The irradiation dose to the tumor, however, was too low at 4300 rads (with 1075 rads to the normal liver and 88 rads to the bone marrow), and no evidence of tumor regression was obtained. Different approaches for increasing the irradiation dose delivered to the tumor by the antibodies are considered.

Development of a harmonised method for the profiling of amphetamine

Résumé : Cette thèse de doctorat est le fruit d'un projet de recherche européen financé par le quatrième programme cadre de la Commission Européenne (DG XII, Standards, Measurement and Testing). Ce projet, dénommé SMT-CT98-2277, a été financé pour la partie suisse par l'Office Fédéral de l'Education et de la Science (OFES, Berne, Suisse). Le but de ce projet était de développer une méthode harmonisée et collaborativement testée pour le profilage des impuretés de l'amphétamine illicite par chromatographie capillaire en phase gazeuse. Le travail a été divisé en sept phases majeures qui concernaient la synthèse de l'amphétamine, l'identification d'impuretés, l'optimisation de la préparation de l'échantillon et du système chromatographique, la variabilité des résultats, l'investigation de méthodes mathématiques pour la classification et la comparaison de profils et finalement l'application de la méthode à des réels échantillons illicites. La méthode résultant de ce travail n'a pas seulement montré que les données étaient interchangeables entre laboratoires mais aussi qu'elle était supérieure en de nombreux points aux méthodes préalablement publiées dans la littérature scientifique. Abstract : This Ph.D. thesis was carried out in parallel to an European project funded by the fourth framework program of the European Commission (DG XII, Standards, Measurement and Testing). This project, named SMT-CT98-2277 was funded, for the Swiss part, by the Federal Office of Education and Science (OFES, Bern, Switzerland). The aim of the project was to develop a harmonised, collaboratively tested method for the impurity profiling of illicit amphetamine by capillary gas chromatography. The work was divided into seven main tasks which deal with the synthesis of amphetamine, identification of impurities, optimization of sample preparation and of the chromatographic system, variability of the results, investigation of numerical methods for the classification and comparison of profiles and finally application of the methodology to real illicit samples. The resulting method has not only shown to produce interchangeable data between different laboratories but was also found to be superior in many aspects to previously published methods.

Development of a shelling method to recover whole kernels of the Cutia nut (Couepia edulis)

The kernel of the cutia nut (castanha-de-cutia, Couepia edulis (Prance) Prance) of the western Amazon, which is consumed by the local population, has traditionally been extracted from the nut with a machete, a dangerous procedure that only produces kernels cut in half. A shelling off machine prototype, which produces whole kernels without serious risks to its operator, is described and tested. The machine makes a circular cut in the central part of the fruit shell, perpendicular to its main axis. Three ways of conditioning the fruits before cutting were compared: (1) control; (2) oven drying immediately prior to cutting; (3) oven drying, followed by a 24-hour interval before cutting. The time needed to extract and separate the kernel from the endocarp and testa was measured. Treatment 3 produced the highest output: 63 kernels per hour, the highest percentage of whole kernels (90%), and the best kernel taste. Kernel extraction with treatment 3 required 50% less time than treatment 1, while treatment 2 needed 38% less time than treatment 1. The proportion of kernels attached to the testa was 93%, 47%, and 8% for treatments 1, 2, and 3, respectively, and was the main reason for extraction time differences.

Development of a systematic computer vision-based method to analyse and compare images of false identity documents for forensic intelligence purposes-Part I: acquisition, calibration and validation issues

Following their detection and seizure by police and border guard authorities, false identity and travel documents are usually scanned, producing digital images. This research investigates the potential of these images to classify false identity documents, highlight links between documents produced by a same modus operandi or same source, and thus support forensic intelligence efforts. Inspired by previous research work about digital images of Ecstasy tablets, a systematic and complete method has been developed to acquire, collect, process and compare images of false identity documents. This first part of the article highlights the critical steps of the method and the development of a prototype that processes regions of interest extracted from images. Acquisition conditions have been fine-tuned in order to optimise reproducibility and comparability of images. Different filters and comparison metrics have been evaluated and the performance of the method has been assessed using two calibration and validation sets of documents, made up of 101 Italian driving licenses and 96 Portuguese passports seized in Switzerland, among which some were known to come from common sources. Results indicate that the use of Hue and Edge filters or their combination to extract profiles from images, and then the comparison of profiles with a Canberra distance-based metric provides the most accurate classification of documents. The method appears also to be quick, efficient and inexpensive. It can be easily operated from remote locations and shared amongst different organisations, which makes it very convenient for future operational applications. The method could serve as a first fast triage method that may help target more resource-intensive profiling methods (based on a visual, physical or chemical examination of documents for instance). Its contribution to forensic intelligence and its application to several sets of false identity documents seized by police and border guards will be developed in a forthcoming article (part II).

Development and validation of an alternative titration method for the determination of sulfate ion in indinavir sulfate

A simple and rapid precipitation titration method was developed and validated to determine sulfate ion content in indinavir sulfate raw material. 0.1 mol L-1 lead nitrate volumetric solution was used as titrant employing potentiometric endpoint determination using a lead-specific electrode. The United States Pharmacopoeia Forum indicates a potentiometric method for sulfate ion quantitation using 0.1 mol L-1 lead perchlorate as titrant. Both methods were validated concerning linearity, precision and accuracy, yielding good results. The sulfate ion content found by the two validated methods was compared by the statistical t-student test, indicating that there was no statistically significant difference between the methods.

Development and application of spectrophotometric method for the determination of cefaclor in pharmaceutical formulations

A simple, fast and sensitive spectrophotometric method for the determination of cefaclor in pharmaceutical raw and dosage forms based on reaction with ninhydrin is developed, optimized and validated. The purple color (Ruhemenn's purple) that resulted from the reaction was stabilized and measured at 560 nm. Beer's law is obeyed in the concentration range of 4-80 µg mL-1 with molar absorptivity of 1.42 × 10(5) L mole-1 cm-1. All variables including the reagent concentration, heating time, reaction temperature, color stability period, and cefaclor/ninhydrin ratio were studied in order to optimize the reaction conditions. No interference was observed from common pharmaceutical adjuvant. The developed method is easy to use, accurate and highly cost-effective for routine studies relative to HPLC and other techniques.

Development of dissolution method for benznidazole tablets

The aim of this work was the development of a dissolution method for benznidazole (BNZ) tablets. Three different types of dissolution media, two stirring speeds and apparatus 2 (paddle) were used. The accomplishment of the drug dissolution profiles was compared through the dissolution efficiency. The assay was performed by spectrophotometry at 324 nm. The better conditions were: sodium chloride\hydrochloride acid buffer pH 1.2 with stirring speed of 75 rpm, volume of 900 mL and paddle as apparatus. Ahead of the results it can be concluded that the method developed consists in an efficient alternative for assays of dissolution for benznidazole tablets.

Development of a flotation method for preconcentration-separation of trace amounts of some metal ions in plant tissues prior to their FAAS determinations

An efficient flotation method based on the combination of flame atomic absorption spectrometry (FAAS) and separation and preconcentration step for determination of Cr3+, Cu 2+, Co2+, Ni2+, Zn2+, Cd 2+, Fe3+ and Pb2+ ions in various real samples by the possibility of applying bis(2-hydroxyacetophenone)-1,4-butanediimine (BHABDI) as a new collector was studied. The influence of pH, amount of BHABDI as collector, sample matrix, type and amount of eluting agent, type and amount of surfactant as floating agent, ionic strength and air flow rates i.e. variables affecting the efficiency of the extraction system was evaluated. It is ascertained that metal ions such as iron can be separated simultaneously from matrix in the presence of 0.012 mM ligand, 0.025% (w/v) of CTAB to a test sample of 750 mL at pH 6.5. These ions can be eluted quantitatively with 6 mL of 1.0 mol L-1 HNO3 in methanol which lead to the enrichment factor of 125. The detection limits for analyte ions were in the range of 1.3-2.4 ng mL-1. The method has been successfully applied for determination of trace amounts of ions in various real samples.

Development of new dissolution test and HPLC-RP method for anti-parasitic ornidazole coated tablets

This work aimed the development and validation of a new dissolution test for ornidazole coated tablets. The dissolution conditions were determined after testing Sink conditions, dissolution medium, apparatus, stirring speed, 24 h stability and medium filtration influence. The best conditions were paddle at a stirring speed of 75 rpm and 900 mL of 0.1 M HCl. A new HPLC quantification method was developed and validated. The dissolution test and quantification method showed to be adequate for their purposes and could be applied for quality control of ornidazole coated tablets, since there is no official monograph.

Development of a simple, rapid and validated spectrophotometric method for nitazoxanide in pharmaceutical formulations and comparison with HPLC

A rapid, economical, reproducible, and simple direct spectrophotometric method was developed and validated for the assay of nitazoxanide in pharmaceutical formulations. Nitazoxanide concentration was estimated in water at 345 nm and pH 4.5. The method was suitable and validated for specificity, linearity, precision, and accuracy. There was no interference of the excipients in the determination of the active pharmaceutical ingredient. The proposed method was successfully applied in the determination of nitazoxanide in coated tablets and in powders for oral suspension. This method was compared to a previously developed and validated method for liquid chromatography to the same drug. There was no significative difference between these methods for nitazoxanide quantitation.

Development and validation of UV spectrophotometric method for determination of levofloxacin in pharmaceutical dosage forms

The objective of this research was to develop and validate an alternative analytical method for quantitative determination of levofloxacin in tablets and injection preparations. The calibration curves were linear over a concentration range from 3.0 to 8.0 μg mL-1. The relative standard deviation was below 1.0% for both formulations and average recovery was 101.42 ± 0.45% and 100.34 ± 0.85% for tablets and injection formulations, respectively. The limit of detection and limit of quantitation were 0.08 and 0.25 μg mL-1, respectively. It was concluded that the developed method is suitable for the quality control of levofloxacin in pharmaceuticals formulations.