A LC-tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir, maraviroc, darunavir, and etravirine.

Raltegravir (RAL), maraviroc (MVC), darunavir (DRV), and etravirine (ETV) are new antiretroviral agents with significant potential for drug interactions. This work describes a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of plasma drug levels. Single-step extraction of RAL, MVC, DRV, ETV and RTV from plasma (100 microl) is performed by protein precipitation using 600 microl of acetonitrile, after the addition of 100 microl darunavir-d(9) (DRV-d(9)) at 1000 ng/ml in MeOH/H(2)O 50/50 as internal standard (I.S.). The mixture is vortexed, sonicated for 10 min, vortex-mixed again and centrifuged. An aliquot of supernatant (150 microl) is diluted 1:1 with a mixture of 20 mM ammonium acetate/MeOH 40/60 and 10 microl is injected onto a 2.1 x 50 mm Waters Atlantis-dC18 3 microm analytical column. Chromatographic separations are performed using a gradient program with 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electrospray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the positive mode. The method has been validated over the clinically relevant concentrations ranging from 12.5 to 5000 ng/ml, 2.5 to 1000 ng/ml, 25 to 10,000 ng/ml, 10 to 4000 ng/ml, and 5 to 2000 ng/ml for RAL, MRV, DRV, ETV and RTV, respectively. The extraction recovery for all antiretroviral drugs is always above 91%. The method is precise, with mean inter-day CV% within 5.1-9.8%, and accurate (range of inter-day deviation from nominal values -3.3 to +5.1%). In addition our method enables the simultaneous assessment of raltegravir-glucuronide. This is the first analytical method allowing the simultaneous assay of antiretroviral agents targeted to four different steps of HIV replication. The proposed method is suitable for the Therapeutic Drug Monitoring Service of these new regimen combinations administered as salvage therapy to patients having experienced treatment failure, and for whom exposure, tolerance and adherence assessments are critical.

Comparative study of the LC-MS/MS and UPLC-MS/MS for the multi-residue analysis of quinolones, penicillins and cephalosporins in cow milk, and validation according to the regulation 2002/657/EC

The aim of this study was to develop and validate an analytical method to simultaneously determine European Union-regulated beta-lactams (penicillins and cephalosporins) and quinolones in cow milk. The procedure involves a new solid phase extraction (SPE) to clean-up and pre-concentrate the three series of antibiotics before analysis by liquid chromatography¿tandem mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography¿tandem mass spectrometry (UPLC-MS/MS). LC-MS/MS and UPLC-MS/MS techniques were also compared. The method was validated according to the Directive 2002/657/EC and subsequently applied to 56 samples of raw cow milk supplied by the Laboratori Interprofessional Lleter de Catalunya (ALLIC) (Laboratori Interprofessional Lleter de Catalunya, Control Laboratory Interprofessional of Milk of Catalunya).

Sensitive bioassay for determination of fluconazole concentrations in plasma using a Candida albicans mutant hypersusceptible to azoles.

The antifungal agent fluconazole (FLC) is widely used in clinical practice. Monitoring FLC levels is useful in complicated clinical settings and in experimental infection models. A bioassay using Candida pseudotropicalis, a simple and cost-effective method, is validated only for FLC levels ranging from 5 to 40 mg/liter. An extension of the analytical range is needed to cover most yeast MICs. A new bioassay in RPMI agar containing methylene blue was developed using C. albicans DSY1024, a mutant rendered hypersusceptible to FLC constructed by the deletion of the multidrug efflux transporter genes CDR1, CDR2, CaMDR1, and FLU1. Reproducible standard curves were obtained with FLC concentrations in plasma ranging from 1 to 100 mg/liter (quadratic regression coefficient > 0.997). The absolute sensitivity was 0.026 microg of FLC. The method was internally validated according to current guidelines for analytical method validation. Both accuracy and precision lied in the required +/-15% range. FLC levels measured by bioassay and by high-performance liquid chromatography (HPLC) performed with 62 plasma samples from humans and rats showed a strong correlation (coefficients, 0.979 and 0.995, respectively; percent deviations of bioassay from HPLC values, 0.44% +/- 15.31% and 2.66% +/- 7.54%, respectively). In summary, this newly developed bioassay is sensitive, simple, rapid, and inexpensive. It allows nonspecialized laboratories to determine FLC levels in plasma to within the clinically relevant concentration range and represents a useful tool for experimental treatment models.

The "handwriting brain": a meta-analysis of neuroimaging studies of motor versus orthographic processes.

INTRODUCTION: Handwriting is a modality of language production whose cerebral substrates remain poorly known although the existence of specific regions is postulated. The description of brain damaged patients with agraphia and, more recently, several neuroimaging studies suggest the involvement of different brain regions. However, results vary with the methodological choices made and may not always discriminate between "writing-specific" and motor or linguistic processes shared with other abilities. METHODS: We used the "Activation Likelihood Estimate" (ALE) meta-analytical method to identify the cerebral network of areas commonly activated during handwriting in 18 neuroimaging studies published in the literature. Included contrasts were also classified according to the control tasks used, whether non-specific motor/output-control or linguistic/input-control. These data were included in two secondary meta-analyses in order to reveal the functional role of the different areas of this network. RESULTS: An extensive, mainly left-hemisphere network of 12 cortical and sub-cortical areas was obtained; three of which were considered as primarily writing-specific (left superior frontal sulcus/middle frontal gyrus area, left intraparietal sulcus/superior parietal area, right cerebellum) while others related rather to non-specific motor (primary motor and sensorimotor cortex, supplementary motor area, thalamus and putamen) or linguistic processes (ventral premotor cortex, posterior/inferior temporal cortex). CONCLUSIONS: This meta-analysis provides a description of the cerebral network of handwriting as revealed by various types of neuroimaging experiments and confirms the crucial involvement of the left frontal and superior parietal regions. These findings provide new insights into cognitive processes involved in handwriting and their cerebral substrates.

Evaluation of respiratory and dermal occupational exposures to monoethanolamine during cleaning

Professional cleaning is a basic service occupation with a wide variety of tasks carried out in all kind of different sectors and workplaces by a large workforce. One important risk for cleaning workers is the exposure to chemical substances that are present in cleaning products.Monoethanolamine was found to be often present in cleaning products such as general purpose cleaners, bathroom cleaners, floor cleaners and kitchen cleaners. Monoethanolamine can injure the skin, and exposure to monoethanolamine was associated to asthma even when the air concentrations were low. It is a strong irritant and known to be involved in sensitizing mechanisms. It is very likely that the use of cleaning products containing monoethanolamine gives rise to respiratory and dermal exposures. Therefore there is a need to further investigate the exposures to monoethanolamine for both, respiratory and dermal exposure.The determination of monoethanolamine has traditionally been difficult and analytical methods available are little adapted for occupational exposure assessments. For monoethanolamine air concentrations, a sampling and analytical method was already available and could be used. However, a method to analyses samples for skin exposure assessments as well as samples of skin permeation experiments was missing. Therefore one main objective of this master thesis was to search an already developed and described analytical method for the measurement of monoethanolamine in water solutions, and to set it up in the laboratory. Monoethanolamine was analyzed after a derivatisation reaction with o-pthtaldialdehyde. The derivated fluorescing monoethanolamine was then separated with high performance liquid chromatography and detection took place with a fluorescent detector. The method was found to be suitable for qualitative and quantitative analysis of monoethanolamine. An exposure assessment was conducted in the cleaning sector to measure the respiratory and dermal exposures to monoethanolamine during floor cleaning. Stationary air samples (n=36) were collected in 8 companies and samples for dermal exposures (n=12) were collected in two companies. Air concentrations (Mean = 0.18 mg/m3, Standard Deviation = 0.23 mg/m3, geometric Mean = 0.09 mg/m3, Geometric Standard Deviation = 3.50) detected were mostly below 1/10 of the Swiss 8h time weighted average occupational exposure limit. Factors that influenced the measured monoethanolamine air concentrations were room size, ventilation system and the concentration of monoethanolamine in the cleaning product and amount of monoethanolamine used. Measured skin exposures ranged from 0.6 to 128.4 mg/sample. Some cleaning workers that participated in the skin exposure assessment did not use gloves and had direct contact with the solutions containing the cleaning product and monoethanolamine. During the entire sampling campaign, cleaning workers mostly did not use gloves. Cleaning workers are at risk to be regularly exposed to low air concentrations of monoethanolamine. This exposure may be problematic if a worker suffers from allergic reactions (e.g. Asthma). In that case a substitution of the cleaning product may be a good prevention measure as several different cleaning products are available for similar cleaning tasks. Currently there are no occupational exposure limits to compare the skin exposures that were found. To prevent skin exposures, adaptations of the cleaning techniques and the use of gloves should be considered. The simultaneous skin and airborne exposures might accelerate adverse health effects. Overall the risks caused by exposures to monoethanolamine are considered as low to moderate when the cleaning products are used correctly. Whenever possible, skin exposures should be avoided. Further research should consider especially the dermal exposure routes, as very high exposures might occur by skin contact with cleaning products. Dermatitis but also sensitization might be caused by skin exposures. In addition, new biomedical insights are needed to better understand the risks of the dermal exposure. Therefore skin permeability experiments should be considered.

Ultra-performance liquid chromatography mass spectrometry and sensitive bioassay methods for quantification of posaconazole plasma concentrations after oral dosing.

Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 ?g/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C(18) analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 ?g/ml) ?cdr1 ?cdr2 ?flu ?mdr1 ?can constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 ?g/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 ?g/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients receiving oral posaconazole.

Omien osakkeidenhankinta - tarkastelu verotuksen näkökulmasta

Tutkielman päätavoitteena on selvittää rajoittaako verolainsäädäntö osakeyhtiön omien osakkeiden hankintaa ja niiden edelleen luovuttamista. Osakkeiden hankinnan verotuskohtelu selvitetään sekä osakkeenomistajan että osakkeita hankkivan yhtiön näkökulmasta. Yhtenä osatavoitteena on selventää omien osakkeiden hankinnan ja luovuttamisen yhtiöoikeudellinen sääntely. Tämän lisäksi selvitetään minkälaisia omien osakkeiden hankinnan motiiveja on aikaisemmissa tutkimuksissa havaittu. Tutkimuksen teoriaosa perustuu käsiteanalyyttiseen tutkimusotteeseen. Empiria osuudessa tutkitaan suomalaisten pörssiyhtiöiden omien osakkeiden hankinnatja Iuovutukset vuosien 1998 - 2000 ajalta. Tutkimus osoittaa, että verotus ei kannusta yhtiöitä hankkimaan omia osakkeita. Tämän lisäksi verotus rajoittaa hankittujen osakkeiden edelleen luovuttamista.

Natural aphrodisiacs: studies of commercially-available herbal recipes, and phytochemical investigation of "Erythroxylum vacciniifolium" Mart. (Erythroxylaceae) from Brazil

De tout temps, hommes et femmes ont cherché par tous les moyens à développer, préserver ou recouvrer leurs propres capacités sexuelles mais également à stimuler le désir du partenaire. L?utilisation d?aphrodisiaques naturels a été l?un des recours les plus répandus. De nos jours, la commercialisation de nouvelles "love drugs" de synthèse, e.g. Viagra®, Cialis®, Levitra®, a remis au goût du jour les aphrodisiaques classiques et à relancer la recherche sur des molécules nouvelles. La pratique croissante de l?automédication, le matraquage publicitaire sur les aphrodisiaques naturels, la prolifération sur le marché de compléments alimentaires non contrôlés et l?absence de véritable législation accroissent les risques qui pèsent sur la santé publique. Dans le but d?évaluer les risques potentiels sur le consommateur de produits aphrodisiaques commercialisés, le développement et la validation d?une méthode rapide d?analyse qualitative et quantitative de la yohimbine dans ces préparations du marché sont exposés dans la première partie de ce travail. La yohimbine est un antagoniste ?2-adrénocepteur du système nerveux central et périphérique, elle est employée depuis plus d?un siècle dans le traitement des dysfonctionnements érectiles. Cette méthode analytique utilise la chromatographie liquide couplée à l?ultraviolet et à la spectrométrie de masse (LC-UV-MS) et au total, vingt préparations aphrodisiaques ont été étudiées. La dose journalière de yohimbine mesurée s?est révélée très variable selon les produits puisqu?elle varie de 1.32 à 23.16 mg. La seconde partie de ce travail concerne l?étude phytochimique et pharmacologique d?Erythroxylum vacciniifolium Mart. (Erythroxylaceae), une plante, appelée localement catuaba, utilisée dans la médecine traditionnelle brésilienne comme tonique et aphrodisiaque. Dans un premier temps, l?extrait alcaloïdique a été analysé par chromatographie liquide haute performance (HPLC) couplée soit à un détecteur UV à barrette d?iode (LC-UV-DAD), soit à un spectromètre de masse (LC-MS), ou soit à un spectromètre de résonance magnétique nucléaire (LC-RMN). L?interprétation de ces données spectrales enregistrées en ligne a permis d?obtenir des informations structurales et d?identifier partiellement près de 24 alcaloïdes appartenant à la classe des tropanes et potentiellement originaux. Par des méthodes classiques de chromatographie liquide sur l?extrait alcaloïdique de la plante, dix sept tropanes nouveaux ont ensuite été isolés dont les catuabines et leurs dérivés, et les vaccinines. Tous ces composés sont des tropane-diols ou triols estérifiés par au moins un groupe acide 1-méthyl-1H-pyrrole-2-carboxylique. Un de ces composés a été identifié comme un tropane N-oxyde. Toutes les structures ont été déterminées par spectrométrie de masse haute résolution et spectroscopie RMN multi-dimensionnelle. Parmi les nombreux tests biologiques réalisés sur ces tropanes, seuls les tests de cytotoxicité se sont révélés faiblement positifs pour certains de ces composés.<br/><br/>Throughout the ages, men and women have incessantly pursued every means to increase, preserve or recapture their sexual capacity, or to stimulate the sexual desire of selected individuals. One of the most recurrent methods has been the use of natural aphrodisiacs. Nowadays, the commercialization of new synthetic "love drugs", e.g. Viagra®, Cialis® and Levitra®, has fascinated the public interest and has led to a reassessment of classical aphrodisiacs and to the search for new ones. The practice of self-medication by an increasing number of patients, the incessant aggressive advertising of these herbal aphrodisiacs, the invasion of the medicinal market with uncontrolled dietary supplements and the absence of real directives amplifies the potential health hazards to the community. In order to evaluate the possible risks of commercialized aphrodisiac products on consumer health, the development and validation of a rapid qualitative and quantitative method for the analysis of yohimbine in these products, is reported in the first part of the present work. Yohimbine, a pharmacologically well-characterized ?2-adrenoceptor antagonist with activity in the central and peripheral nervous system, has been used for over a century in the treatment of erectile dysfunction. The analytical method is based on liquid chromatography coupled with ultraviolet and mass spectrometry (LC-UV-MS) and in total, 20 commercially-available aphrodisiac preparations were analyzed. The amount of yohimbine measured and expressed as the maximal dose per day suggested on product labels ranged from 1.32 to 23.16 mg. The second part of this work involved the phytochemical and pharmacological investigation of Erythroxylum vacciniifolium Mart. (Erythroxylaceae), a plant used in Brazilian traditional medicine as an aphrodisiac and tonic, and locally known as catuaba. With the aim of obtaining preliminary structure information on-line, the alkaloid extract was analyzed by high performance liquid chromatography (HPLC) coupled to diode array UV detection (LC-UVDAD), to mass spectrometry (LC-MS) and to nuclear magnetic resonance spectroscopy (LCNMR). Interpretation of on-line spectroscopic data led to structure elucidation and partial identification of 24 potentially original alkaloids bearing the same tropane skeleton. Seventeen new tropane alkaloids were then isolated from the alkaloid extract of the plant, including catuabines D to I, their derivatives and vaccinines A and B. All compounds were elucidated as tropane-diol or -triol alkaloids esterified by at least one 1-methyl-1H-pyrrole-2-carboxylic acid. One of the isolated compounds was identified as a tropane alkaloid N-oxide. Their structures were determined by high resolution mass spectrometry and multi-dimensional NMR spectroscopy. Among the numerous bioassays undertaken, only the cytotoxicity tests exhibited a weak positive activity of certain compounds.

Contribution à la connaissance du métabolisme secondaire des Thymelaeaceae et investigation phytochimique de l'une d'elles: "Gnidia involucrata" Steud. ex A. Rich

Dans le but de mieux connaître le métabolisme secondaire de la famille des Thymelaeaceae et de découvrir de nouveaux composés naturels à intérêt thérapeutique, 30 extraits provenant de 8 espèces africaines ont été soumis à un criblage chimique et biologique. Les cibles biologiques suivantes ont servi à l?évaluation de l?activité des extraits étudiés : la moisissure phytopathogène Cladosporium cucumerinum, la levure commensale Candida albicans, la bactérie opportuniste Bacillus subtilis, la larve du moustique vecteur de la fièvre jaune Aedes aegypti et l?hôte intermédiaire mollusque de la schistosomiase urinaire Biomphalaria glabrata. Les propriétés antiradicalaires et inhibitrices de l?acétylcholinestérase de ces extraits ont également été dépistées. Des analyses sur CCM avec révélation chimique, ainsi que des expériences LC/DAD-UV, ont permis demettre en évidence la présence de tanins, de flavonoïdes et de xanthones dans les extraits polaires. Sur la base des résultats de ces analyses préliminaires, l?investigation phytochimique des extraits méthanoliques des racines et des parties aériennes de Gnidia involucrata a été entreprise. Cette démarche a permis l?isolement de 8 composés naturels et leur caractérisation complète au moyen de méthodes spectroscopiques (UV, MS, CD, 1H- et 13C-NMR). Les activités de ces produits purs ont été évaluées et il est apparu qu?ils possédaient presque tous des propriétés antiradicalaires intéressantes, supérieures à celles du BHT, un antioxydant de synthèse (E 321) utilisé dans l?industrie alimentaire. Deux benzophénones simples, respectivement O- et C-glucosylées, ont été isolées des parties aériennes de G. involucrata au côté de la mangiférine, une C-glycosylxanthone ubiquitaire. Ces découvertes sont remarquables à plusieurs titres : (1) les benzophénones simples (nonprénylées) sont très rares dans la nature ; (2) c?est la première fois qu?une Oglycosylbenzophénone a été décrite ; (3) aucune xanthone n?avait été mise en évidence auparavant dans la famille et (4) les benzophénones semblent ne pas être que des produits intermédiaires dans la biosynthèse des xanthones. Trois 3,8??-biflavanones du type GB ont été isolées des racines et des parties aériennes de la même plante, dont deux stéréoisomères se trouvant en mélange. Une analyse LC/CD a permis d?attribuer les configurations absolues des quatre carbones asymétriques de chaque molécule. Cette classe de métabolites secondaires est réputée pour ses propriétés analgésiques et sa présence chez les Thymelaeaceae est prometteuse. Des techniques couplées de pointe ont été utilisées dans ce travail et ont montré leur apport inestimable dans le domaine de la recherche phytochimique. Une analyse LC/ MSn a ainsi permis de mettre en évidence on-line trois C-glycosylflavones ? l?isoorientine, l?isovitexine et la vitexine ? dans les extraits méthanoliques bruts de G. involucrata. De plus, les parties aériennes de cette même plante ont servi de matériel pour le développement d?une nouvelle méthode d?analyse d?extraits bruts : la LC/1H-NMR time-slice. Cette approche consiste à « découper » le temps d?analyse par des interruptions régulières du flux LC, durant lesquelles les données NMR nécessaires sont acquises. Le problème de la faible sensibilité relative de la LC/NMR a été partiellement résolu par ce biais et a permis d?envisager l?utilisation de la NMR au sein de systèmes de couplages multiples en série avec d?autres méthodes spectrales (UV, MS, IR, CD,?).<br/><br/>With the aim of acquiring a better knowledge of the secondary metabolism of the family Thymelaeaceae and of the discovering of new natural therapeutics, 30 extracts from 8 African plant species were submitted to chemical and biological screening. The following biological targets were used to estimate the activity of the extracts under study: the phytopathogenic fungus Cladosporium cucumerinum, the commensal yeast Candida albicans, the opportunistic bacteria Bacillus subtilis, larvae of the yellow fever-transmitting mosquito Aedes aegypti and the intermediate snail host of urinary schistosomiasis Biomphalaria glabrata. The antiradical and acetylcholinesterase-inhibiting properties of these extracts were also investigated. TLC analyses followed by chemical detection, together with LC/DAD-UV experiments, showed the presence of tannins, flavonoids and xanthones in the polar extracts. On the basis of these results, a phytochemical investigation of the methanol extracts of the roots and the aerial parts of Gnidia involucrata was undertaken. This procedure led to the isolation of 8 natural products, which were then characterised by spectroscopic means (UV, MS, CD, 1H- and 13C-NMR). The activities of the pure compounds were then further evaluated: almost all of them exhibited very interesting antiradical properties, superior to those of BHT, a synthetic antioxidant (E 321) used in the food industry. Two simple benzophenones, one O- and one C-glycosylated, were isolated from the aerial parts of G. involucrata, together with mangiferin, a ubiquitous C-glycosylxanthone. These findings are of multiple importance: (1) simple (non-prenylated) benzophenones are very rare in nature; (2) it is the first time that an O-glycosylbenzophenone has been described; (3) no xanthones have been previously reported in the family and (4) benzophenones do not seem to be exclusive intermediates in the biosynthesis of xanthones. Three 3,8??-biflavanones of the GB type were isolated from the roots and the aerial parts of the same plant, among them two stereoisomers in mixture. A LC/CD analysis allowed the assignment of the absolute configurations of all four stereocenters in both molecules. This class of secondary metabolite is well known for its analgesic properties and its presence in the Thymelaeaceae is very promising. Advanced hyphenated techniques were used in this work and showed their inestimable contribution to the field of phytochemical research. A LC/MSn analysis, for example, allowed the on-line characterisation of three C-glycosylflavones ? isoorientin, isovitexin and vitexin ? in the crude methanol extracts of G. involucrata. Furthermore, the aerial parts of this plant were used as material for the development of a new analytical method for crude plant extracts: time-slice LC/1H-NMR. This approach consisted in "slicing" the analytical procedure by interrupting the LC flow at given intervals, during which the necessary NMR data were acquired. The relative lack of sensitivity of LC/NMR was partially surmounted by this means, allowing one to envisage the use of NMR in a multiple hyphenated system, together with other spectroscopic methods (UV, MS, IR, CD,?)

A comparative study of ballpoint ink ageing parameters using GC/MS.

For more than a decade scientists tried to develop methods capable of dating ink by monitoring the loss of phenoxyethanol (PE) over time. While many methods were proposed in the literature, few were really used to solve practical cases and they still raise much concern within the scientific community. In fact, due to the complexity of ink drying processes it is particularly difficult to find a reliable ageing parameter to reproducibly follow ink ageing. Moreover, systematic experiments are required in order to evaluate how different factors actually influence the results over time. Therefore, this work aimed at evaluating the capacity of four different ageing parameters to reliably follow ink ageing over time: (1) the quantity of solvent PE in an ink line, (2) the relative peak area (RPA) normalising the PE results using stable volatile compounds present in the ink formulation, (3) the solvent loss ratio (R%) calculated from PE results obtained by the analyses of naturally and artificially aged samples, (4) a modified solvent loss ratio version (R%*) calculated from RPA results. After the determination of the limits of reliable measurements of the analytical method, the repeatability of the different ageing parameters was evaluated over time, as well as the influence of ink composition, writing pressure and storage conditions on the results. Surprisingly, our results showed that R% was not the most reliable parameter, as it showed the highest standard deviation. Discussion of the results in an ink dating perspective suggests that other proposed parameters, such as RPA values, may be more adequate to follow ink ageing over time.

A comparative study of ballpoint ink ageing parameters using GC/MS

For more than a decade scientists tried to develop methods capable of dating ink by monitoring the loss of phenoxyethanol (PE) over time. While many methods were proposed in the literature, few were really used to solve practical cases and they still raise much concern within the scientific community. In fact, due to the complexity of ink drying processes it is particularly difficult to find a reliable ageing parameter to reproducibly follow ink ageing. Moreover, systematic experiments are required in order to evaluate how different factors actually influence the results over time. Therefore, this work aimed at evaluating the capacity of four different ageing parameters to reliably follow ink ageing over time: (1) the quantity of solvent PE in an ink line, (2) the relative peak area (RPA) normalising the PE results using stable volatile compounds present in the ink formulation, (3) the solvent loss ratio (R%) calculated from PE results obtained by the analyses of naturally and artificially aged samples, (4) a modified solvent loss ratio version (R%*) calculated from RPA results. After the determination of the limits of reliable measurements of the analytical method, the repeatability of the different ageing parameters was evaluated over time, as well as the influence of ink composition, writing pressure and storage conditions on the results. Surprisingly, our results showed that R% was not the most reliable parameter, as it showed the highest standard deviation. Discussion of the results in an ink dating perspective suggests that other proposed parameters, such as RPA values, may be more adequate to follow ink ageing over time.

Vitamin D time profile based on the contribution of non-genetic and genetic factors in HIV-infected individuals of European ancestry.

BACKGROUND: Vitamin D deficiency is prevalent in HIV-infected individuals and vitamin D supplementation is proposed according to standard care. This study aimed at characterizing the kinetics of 25(OH)D in a cohort of HIV-infected individuals of European ancestry to better define the influence of genetic and non-genetic factors on 25(OH)D levels. These data were used for the optimization of vitamin D supplementation in order to reach therapeutic targets. METHODS: 1,397 25(OH)D plasma levels and relevant clinical information were collected in 664 participants during medical routine follow-up visits. They were genotyped for 7 SNPs in 4 genes known to be associated with 25(OH)D levels. 25(OH)D concentrations were analysed using a population pharmacokinetic approach. The percentage of individuals with 25(OH)D concentrations within the recommended range of 20-40 ng/ml during 12 months of follow-up and several dosage regimens were evaluated by simulation. RESULTS: A one-compartment model with linear absorption and elimination was used to describe 25(OH)D pharmacokinetics, while integrating endogenous baseline plasma concentrations. Covariate analyses confirmed the effect of seasonality, body mass index, smoking habits, the analytical method, darunavir/ritonavir and the genetic variant in GC (rs2282679) on 25(OH)D concentrations. 11% of the inter-individual variability in 25(OH)D levels was explained by seasonality and other non-genetic covariates, and 1% by genetics. The optimal supplementation for severe vitamin D deficient patients was 300,000 IU two times per year. CONCLUSIONS: This analysis allowed identifying factors associated with 25(OH)D plasma levels in HIV-infected individuals. Improvement of dosage regimen and timing of vitamin D supplementation is proposed based on those results.

Rapid analysis of multiclass antibiotic residues and some of their metabolites in hospital, urban wastewater and river water by ultra-highperformance liquid chromatography coupled to quadrupole-linear ion trap tandem mass spectrometry

The present work describes the development of a fast and robust analytical method for the determination of 53 antibiotic residues, covering various chemical groups and some of their metabolites, in environmental matrices that are considered important sources of antibiotic pollution, namely hospital and urban wastewaters, as well as in river waters. The method is based on automated off-line solid phase extraction (SPE) followed by ultra-high-performance liquid chromatography coupled to quadrupole linear ion trap tandem mass spectrometry (UHPLC–QqLIT). For unequivocal identification and confirmation, and in order to fulfill EU guidelines, two selected reaction monitoring (SRM) transitions per compound are monitored (the most intense one is used for quantification and the second one for confirmation). Quantification of target antibiotics is performed by the internal standard approach, using one isotopically labeled compound for each chemical group, in order to correct matrix effects. The main advantages of the method are automation and speed-up of sample preparation, by the reduction of extraction volumes for all matrices, the fast separation of a wide spectrum of antibiotics by using ultra-high-performance liquid chromatography, its sensitivity (limits of detection in the low ng/L range) and selectivity (due to the use of tandem mass spectrometry) The inclusion of β-lactam antibiotics (penicillins and cephalosporins), which are compounds difficult to analyze in multi-residue methods due to their instability in water matrices, and some antibiotics metabolites are other important benefits of the method developed. As part of the validation procedure, the method developed was applied to the analysis of antibiotics residues in hospital, urban influent and effluent wastewaters as well as in river water samples

Validació del mètode analític per la determinació d'aminoàcids i carbohidrats d'un producte farmacèutic per HPLC

Per poder desenvolupar un producte farmacèutic és necessari establir un mètode d’anàlisis que permeti determinar i quantificar totes aquelles substàncies que conté, ja sigui referent als principis actius; a les impureses i productes de degradació, conservants, antioxidants,... Grans entitats com la ICH remarquen la importància de validar els mètodes analítics ja que és la via per demostrar que aquell producte compleix les garanties de qualitat prèviament establertes. Així doncs, l’objectiu d’aquest Treball Final de Grau és poder desenvolupar i validar dos mètodes analítics per a la determinació d’aminoàcids i carbohidrats respectivament, d’un producte farmacèutic per cromatografia líquida (HPLC). Per tal de poder concloure que aquell mètode és adequat per la determinació per la qual ha estat desenvolupat, és necessari obtenir resultats que compleixin els criteris d’acceptació corresponents als paràmetres que han de ser avaluats en una validació analítica. Aquests paràmetres són: la precisió, la selectivitat, l’exactitud i la linealitat i el rang. Els resultats d’aquest projecte han demostrat que els dos mètodes desenvolupats són adequats per a la determinació de tres dels principis actius (aminoàcid 1, aminoàcid 2 i carbohidrat 1) que conté el producte farmacèutic d’ús veterinari analitzat; i poden ser validats ja que compleixen els criteris d’acceptació dels paràmetres avaluats que proposa la ICH. El mètode per la determinació de carbohidrats no és vàlid per el carbohidrat 2, ja que durant el desenvolupament es va detectar que una bona part d’aquest passava a carbohidrat 1 (desplaçament de l’equilibri ceto-enòlic que hi ha entre el carbohidrat 1 i el carbohidrat 2 a pHs alts). És per aquest motiu, que es pot concloure que aquest mètode no és vàlid i es recomana seguir investigant per a poder desenvolupar un mètode analític adient.

Inférence de source de traces d'essence retrouvées dans des débris d'incendie: Evaluation de la contribution de la chromatographie en phase gazeuse couplée à la spectrométrie de masse à rapport isotopique

Lorsque de l'essence est employée pour allumer et/ou propager un incendie, l'inférence de la source de l'essence peut permettre d'établir un lien entre le sinistre et une source potentielle. Cette inférence de la source constitue une alternative intéressante pour fournir des éléments de preuve dans ce type d'événements où les preuves matérielles laissées par l'auteur sont rares. Le but principal de cette recherche était le développement d'une méthode d'analyse de spécimens d'essence par GC-IRMS, méthode pas routinière et peu étudiée en science forensique, puis l'évaluation de son potentiel à inférer la source de traces d'essence en comparaison aux performances de la GC-MS. Un appareillage permettant d'analyser simultanément les échantillons par MS et par IRMS a été utilisé dans cette recherche. Une méthode d'analyse a été développée, optimisée et validée pour cet appareillage. Par la suite, des prélèvements d'essence provenant d'un échantillonnage conséquent et représentatif du marché de la région lausannoise ont été analysés. Finalement, les données obtenues ont été traitées et interprétées à l'aide de méthodes chimiométriques. Les analyses effectuées ont permis de montrer que la méthodologie mise en place, aussi bien pour la composante MS que pour l'IRMS, permet de différencier des échantillons d'essence non altérée provenant de différentes stations-service. Il a également pu être démontré qu'à chaque nouveau remplissage des cuves d'une station-service, la composition de l'essence distribuée par cette station est quasi unique. La GC-MS permet une meilleure différenciation d'échantillons prélevés dans différentes stations, alors que la GC-IRMS est plus performante lorsqu'il s'agit de comparer des échantillons collectés après chacun des remplissages d'une cuve. Ainsi, ces résultats indiquent que les deux composantes de la méthode peuvent être complémentaires pour l'analyse d'échantillons d'essence non altérée. Les résultats obtenus ont également permis de montrer que l'évaporation des échantillons d'essence ne compromet pas la possibilité de grouper des échantillons de même source par GC-MS. Il est toutefois nécessaire d'effectuer une sélection des variables afin d'éliminer celles qui sont influencées par le phénomène d'évaporation. Par contre, les analyses effectuées ont montré que l'évaporation des échantillons d'essence a une forte influence sur la composition isotopique des échantillons. Cette influence est telle qu'il n'est pas possible, même en effectuant une sélection des variables, de grouper correctement des échantillons évaporés par GC-IRMS. Par conséquent, seule la composante MS de la méthodologie mise en place permet d'inférer la source d'échantillons d'essence évaporée. _________________________________________________________________________________________________ When gasoline is used to start and / or propagate an arson, source inference of gasoline can allow to establish a link between the fire and a potential source. This source inference is an interesting alternative to provide evidence in this type of events where physical evidence left by the author are rare. The main purpose of this research was to develop a GC-IRMS method for the analysis of gasoline samples, a non-routine method and little investigated in forensic science, and to evaluate its potential to infer the source of gasoline traces compared to the GC-MS performances. An instrument allowing to analyze simultaneously samples by MS and IRMS was used in this research. An analytical method was developed, optimized and validated for this instrument. Thereafter, gasoline samples from a large sampling and representative of the Lausanne area market were analyzed. Finally, the obtained data were processed and interpreted using chemometric methods. The analyses have shown that the methodology, both for MS and for IRMS, allow to differentiate unweathered gasoline samples from different service stations. It has also been demonstrated that each new filling of the tanks of a station generates an almost unique composition of gasoline. GC-MS achieves a better differentiation of samples coming from different stations, while GC-IRMS is more efficient to distinguish samples collected after each filling of a tank. Thus, these results indicate that the two components of the method can be complementary to the analysis of unweathered gasoline samples. The results have also shown that the evaporation of gasoline samples does not compromise the possibility to group samples coming from the same source by GC-MS. It is however necessary to make a selection of variables in order to eliminate those which are influenced by the evaporation. On the other hand, the carried out analyses have shown that the evaporation of gasoline samples has such a strong influence on the isotopic composition of the samples that it is not possible, even by performing a selection of variables, to properly group evaporated samples by GC-IRMS. Therefore, only the MS allows to infer the source of evaporated gasoline samples.