Ink dating using thermal desorption and gas chromatography / mass spectrometry: comparison of results obtained in two laboratories

Recent ink dating methods focused mainly on changes in solvent amounts occurring over time. A promising method was developed at the Landeskriminalamt of Munich using thermal desorption (TD) followed by gas chromatography / mass spectrometry (GC/MS) analysis. Sequential extractions of the phenoxyethanol present in ballpoint pen ink entries were carried out at two different temperatures. This method is applied in forensic practice and is currently implemented in several laboratories participating to the InCID group (International Collaboration on Ink Dating). However, harmonization of the method between the laboratories proved to be a particularly sensitive and time consuming task. The main aim of this work was therefore to implement the TD-GC/MS method at the Bundeskriminalamt (Wiesbaden, Germany) in order to evaluate if results were comparable to those obtained in Munich. At first validation criteria such as limits of reliable measurements, linearity and repeatability were determined. Samples were prepared in three different laboratories using the same inks and analyzed using two TDS-GC/MS instruments (one in Munich and one in Wiesbaden). The inter- and intra-laboratory variability of the ageing parameter was determined and ageing curves were compared. While inks stored in similar conditions yielded comparable ageing curves, it was observed that significantly different storage conditions had an influence on the resulting ageing curves. Finally, interpretation models, such as thresholds and trend tests, were evaluated and discussed in view of the obtained results. Trend tests were considered more suitable than threshold models. As both approaches showed limitations, an alternative model, based on the slopes of the ageing curves, was also proposed.

Investigation phytochimique d'une plante médicinale malgache Hypoestes serpens (Vahl) R. Br. (Acanthaceae) variété glabre et étude comparative avec la variété poilue

Résumé A Madagascar, comme dans plusieurs pays en développement, une grande partie de la population utilise les plantes pour se soigner. Plusieurs espèces des plantes sont ainsi utilisées en médecine traditionnelle malgache. Par ailleurs, la plupart de ces plantes ne font l'objet que de très peu d'étude scientifique. En effet, dans le cadre de l'investigation phytochimique de plantes utilisées en médecine traditionnelle malgache et dans la recherche des nouvelles substances biologiquement actives, Hypoestes serpens (Vahl) R. Br. (Acanthaceae) a été étudiée. Elle se présente sous deux variétés (glabre et poilue) qui sont tous utilisées dans la région sud-centre de Madagascar pour traiter la blennorragie. De l'extrait dichlorométhanique des feuilles de H. serpens (Vahl) R. Br. variété glabre, 12 diterpénoides dont 8 nouveaux ont été isolés. Ils ont tous montré une activité antifongique contre un champignon pathogène des plantes, Cladosporium cucumerinum, dans la bioautographie directe sur CCM. Quelques-uns ont également présenté une activité contre une levure saprophyte chez l'homme, Candida albicans et une activité inhibitrice de l'enzyme acétylcholinesterase. Les diterpènoïdes sont déjà considérés comme les principaux métabolites secondaires du genre Hypoestes. Le fractionnement de l'extrait méthanolique a conduit à l'isolement de 5 glycosides des flavonoïdes dont 4 sous formes C-g,lycosides qui n'ont jamais été identifiés dans la famille Acanthaceae. Ces flavonoïdes ont présenté une activité antiradicalaire contre le DPPH. Le fractionnement et la purification des extraits ont été effectués à l'aide des différentes techniques chromatographiques telles que la chromatographie sur colonne ouverte, la filtration sur gel, la chromatographie liquide à haute pression, la chromatographie liquide à moyenne pression et la chromatographie liquide à basse pression. Par ailleurs, les structures des composés isolés ont été élucidées par des techniques spectroscopiques (UV, MS, RMN) et de méthode chimique (hydrolyse acide). En plus de ces techniques, certaines méthodes physiques (cristallographie par rayons-X, mesure de rotation optique) ont été réalisées pour confirmer certaines structures. Comme l'espèce Hypoestes serpens (Vahl) R. Br. se présente en deux variétés, une étude comparative a été effectuée. Cette étude avait montré que ces deux variétés ont une activité biologique similaire. Finalement, une technique analytique couplée, HPLC-UV-APC1-MS a permis de montrer la présence de toutes les substances isolées de la variété glabre dans la variété poilue. Second résumé Depuis des milliers d'almées, l'homme utilise les plantes pour se soigner. De nos jours, même avec le développement de la médecine moderne, la phytothérapie reste toujours la forme des soins de santé abordable et accessible pour la majorité des populations rurales des pays en développement. En outre, les plantes médicinales constituent une source potentielle de molécules biologiquement actives pour les industries pharmaceutiques et actuellement, on estime que 25% des médicaments commercialisés dans le monde sont à base de plantes Dans le cadre de la recherche des nouvelles molécules à intérêt thérapeutique qui pourraient devenir un médicament ou un modèle de structure ("lead compound") pour le développement de nouveaux médicaments, nous avons fait une étude sur l'espèce, Hypoestes serpens (Vahl) R. Br, plante utilisée en médecine traditionnelle malgache. Cette espèce existe en deux variétés, une glabre et une autre poilue qui sont tous utilisées dans la région sud-centre de Madagascar pour traiter la blennorragie. Par ailleurs, les tradipraticiens utilisent de préférence la variété poilue. Dans la première partie de ce présent travail, une investigation phytochimique de H serpens, variété glabre (variété moins utilisée) a d'abord été effectuée afm d'isoler et d'identifier le maximum des molécules biologiquement actives qu'elle contient. De ce fait, 17 composés dont 8 nouveaux ont été isolés. Les potentiels d'activités thérapeutiques des substances isolées ont ensuite été dépistés sur les différents cibles suivants.: deux souches de champignons (Cladosporium cucumerinum et Candida albicans), l'enzyme acétylcholinesterase et le radical DPPH. La deuxième partie de ce travail a été consacrée sur l'étude comparative des deux variétés (glabre et poilue) de H. serpens à la fois sur le plan biologique et sur le plan phytochimique. A l'issue de cette comparaison, nous avons constaté que l'utilisation de ces deux variétés en médecine traditionnelle malgache n'est pas un hasard ; les deux variétés avaient présenté une activité biologique très remarquable et contiennent les mêmes substances actives. Ces résultats démontrent les potentiels thérapeutiques de H serpens en médecine traditionnelle malgache et pourraient également encourager les tradipraticiens à utiliser la variété glabre tout en protégeant la variété poilue qui est en voie de disparition actuellement. En bref, l'investigation phytochimique de H. serpens justifiée par l'isolement et l'identification de certains de ses principes actifs ouvre la voie aux recherches des médicaments d'origine naturelle. Abstract In Madagascar, as in many developing countries, most people use plants to cure. A large number of plant species are employed in Malagasy traditional medicine. Moreover, most of these plants have been subject only very little scientific study. As part of a phytochemical investigation of plants used in Malagasy traditional medicine and in the search for new biologically active substances, Hypoestes serpens (Vah1) R.Br. (Acanthaceae) was investigated. This species exists in two varieties (glabrous and hairy) which are used in the south-center part of Madagascar to treat gonorrhoea. From the dichloromethane extract of the leaves of H. serpens (Vah1) R. Br. glabrous variety, 12 diterpenoids 8 of which were new, were isolated. They showed antifungal activity against the plant pathogen Cladosporium cucumerinum, in the direct TLC bioautography. Some of them also had activity against the yeast Candida athicans and inhibited acetylcholinesterase. The diterpenes are considered as the principal secondary metabolites of the genus Hypoestes. Fractionation of the methanol extract led to the isolation of 5 flavonoid glycosides, 4 of which were C-glycosides, never before identified in the Acanthaceae family. These flavonoids showed radical scavenging activity against DPPH. The fractionation and the purification of the extracts were achieved by different chromatographic techniques such as open-column chromatography, gel filtration, high- pressure liquid chromatography, medium-pressure liquid chromatography and low-pressure liquid chromatography. Moreover, the structures of the isolated compounds were elucidated by spectroscopic techniques (UV, MS, NMR) and chemical technique (acid hydrolysis). In addition, some physical methods (X-ray crystallography, measurement of optical rotation) were performed to confirm some structures. As the species Hypoestes serpens (Vah1) R. Br. is present in two varieties, a comparative study was carried out. This study showed that these two varieties had similar biological activity. Finally, a coupled analytical technique HPLC-UV-APCI-MS showed the presence of the same compounds in both the glabrous and hairy varieties.

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism.

BACKGROUND: The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo. METHODS: We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. RESULTS: In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining. CONCLUSIONS: These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. ABBREVIATIONS: Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50).

Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods, [and] Voriconazole therapeutic drug monitoring in patients with invasive mycoses improves efficacy and safety outcomes

Rapport de synthèse1. Partie de laboratoireCette première étude décrit le développement et la validation, selon les standards internationaux, de deux techniques de mesure des concentrations sanguines de voriconazole, un nouvel agent antifongique à large spectre: 1) la chromatographic en phase liquide à haute pression et 2) le bio-essai utilisant une souche mutante de Candida hypersensible au voriconazole. Ce travail a aussi permis de mettre en évidence une importante et imprévisible variabilité inter- et intra-individuelle des concentrations sanguines de voriconazole malgré l'utilisation des doses recommandées par le fabriquant. Ce travail a été publié dans un journal avec "peer-review": "Variability of voriconazole plasma levels measured by new high- performance liquid chromatography and bioassay methods" by A. Pascual, V. Nieth, T. Calandra, J. Bille, S. Bolay, L.A. Decosterd, T. Buclin, P.A. Majcherczyk, D. Sanglard, 0. Marchetti. Antimicrobial Agents Chemotherapy, 2007; 51:137-432. Partie CliniqueCette deuxième étude a évalué de façon prospective l'impact clinique des concentrations sanguines de voriconazole sur l'efficacité et sécurité thérapeutique chez des patients atteints d'infections fongiques. Des concentrations sanguines élevées étaient significativement associés à la survenue d'une toxicité neurologique (encéphalopathie avec confusion, hallucinations et myoclonies) et des concentrations sanguines basses à une réponse insuffisante au traitement antifongique (persistance ou progression des signes cliniques et radiologiques de l'infection). Dans la majorité des cas, un ajustement de la dose de voriconazole, sur la base des concentrations mesurées, a abouti à une récupération neurologique complète ou à une résolution de l'infection, respectivement. Ce travail a été publié dans un journal avec "peer-review": " Voriconazole Therapeutic Drug Monitoring in Patients with Invasive Mycoses Improves Efficacy and Safety Outcomes" by A. Pascual, T. Calandra, S. Bolay, T. Buclin, J. Bille, and O. Marchetti. Clinical Infectious Diseases, 2008 January 15; 46(2): 201-11.Ces deux études, financées de façon conjointe par un "grant" international de la Société suisse d'infectiologie et la Société internationale de maladies infectieuses et par la Fondation pour le progrès en microbiologie médicale et maladies infectieuses (FAMMID, Lausanne), ont été réalisées au sein du Service des Maladies Infectieuses, Département de Médecine, au CHUV, en étroite collaboration avec la Division de Pharmacologie Clinique, Département de Médecine, au CHUV et l'Institut de Microbiologie du CHUV et de l'Université de Lausanne.

Detection of metabolite induction in fungal co-cultures on solid media by high-throughput differential ultra-high pressure liquid chromatography-time-of-flight mass spectrometry fingerprinting.

Access to new biological sources is a key element of natural product research. A particularly large number of biologically active molecules have been found to originate from microorganisms. Very recently, the use of fungal co-culture to activate the silent genes involved in metabolite biosynthesis was found to be a successful method for the induction of new compounds. However, the detection and identification of the induced metabolites in the confrontation zone where fungi interact remain very challenging. To tackle this issue, a high-throughput UHPLC-TOF-MS-based metabolomic approach has been developed for the screening of fungal co-cultures in solid media at the petri dish level. The metabolites that were overexpressed because of fungal interactions were highlighted by comparing the LC-MS data obtained from the co-cultures and their corresponding mono-cultures. This comparison was achieved by subjecting automatically generated peak lists to statistical treatments. This strategy has been applied to more than 600 co-culture experiments that mainly involved fungal strains from the Fusarium genera, although experiments were also completed with a selection of several other filamentous fungi. This strategy was found to provide satisfactory repeatability and was used to detect the biomarkers of fungal induction in a large panel of filamentous fungi. This study demonstrates that co-culture results in consistent induction of potentially new metabolites.

Analysis of cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry

A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These cannabinoids include a dagger(9)-tetrahydrocannabinol (THC), 11-hydroxy-a dagger(9)-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), a dagger(9)-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and a dagger(9)-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal (TM) device. The advantages of performing a liquid-liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5 min on a Kinetex (TM) column packed with 2.6-mu m core-shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000 (TM) triple quadrupole LC-MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50 pg/ml and from 500 to 80 pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method's LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collection

Biochemical characterization of FANCD2 and FANCM in the Fanconi Anaemia pathway

Abstract : The maintenance of genome stability is a challenge for all living organisms. DNA is regularly subjected to chemical alterations by both endogenous and exogenous DNA damaging agents. If left unrepaired, these lesions will create mutations or lead to chromosomal instability. DNA crosslinking agents probably bring about the most toxic lesions. By linking covalently the two strands of DNA, crosslinking agents will impede essential cellular processes such as replication and transcription. Cells from Fanconi anaemia patients are extremely sensitive to these agents. Fanconi anaemia (FA) is a rare chromosomal instability disorder that leads to developmental defects, pancytopenia and cancer susceptibility. FA is a genetically heterogeneous disease with thirteen complementation groups identified. Proteins encoded by the FA genes work together in the FA pathway. Eight of these proteins form the FA core complex (FANC-A, B, C,E, F, G, L and -M), whose integrity is required to monoubiquitinate FANCD2 and FANCI in response to DNA damage. The hypersensitivity of FA cells to crosslinking agents, which perturb the progression of replication forks, has led to the hypothesis that FA proteins play a crucial role in the response to replication stress. However, at the molecular level, the functions of the FA pathway remain largely unknown. Our efforts were first focused on the characterization of FANCD2, "the key effector of the FA pathway". Using different substrates, we found that in vitro, purified hFANCD2 preferentially binds single strand DNA and double strand DNA extremities. Concomitantly, FANCM was identified as a new component of the FA core complex. Moreover FANCM was shown to have specific branch migration activities and probably a role as a "landing platform" on DNA for the other components of the core complex. By using FANCM mutants carrying deletions within the internal domain, we investigated the role of FANCM as a DNA anchor protein for the core complex. We observed that indeed, a specific part of the internal domain of FANCM interacts with components of the core complex. Finally, in collaboration with Weidong Wang's lab we characterized two new components of the FA pathway: FAAP10 and FAAP16. As a heterodimer these two proteins show affinity for dsDNA, and anneal complementary oligonucleotides in vitro. Moreover these proteins can associate with FANCM via a part of its internal domain. We find that FANCM, FAAP 10 and FAAP 16 can co-exist on the branch point of replication and recombination intermediates, and that FAAP10 and FAAP16 stimulate replication fork reversal by FANCM. These results suggest that FANCM may function as a landing platform for the core complex. After loading on DNA, the core complex can activate FANCD2 through monoubiquitination leading to its recruitment to the site of damage. Since ssDNA and double strand breaks are intermediates that are generated as a consequence of collapsed replication forks, FANCD2 by binding to ds DNA ends and ssDNA could protect such structures from the recombination repair machinery and prevent unscheduled recombination events. Alternatively, FANCD2 could avoid nucleases from gaining access to collapsed forks, preserving the DNA in state that can be used as a starting point for resumption of DNA synthesis. The overall comprehension of the FA pathway is far from been complete. Our results unravel new aspects of Fanconi Anaemia, which hopefully in the near future will address keys questions leading to a better understanding of the fascinating Fanconi Anaemia. Résumé : Le maintien de l'intégrité du génome est fondamentale chez tous les organismes vivants. L'ADN est constamment altéré par des composés aussi bien endogènes qu'exogènes. Si ces altérations ne sont pas réparées, elles peuvent conduire à l'apparition de mutations, ainsi qu'à une instabilité génomique accrue. Les lésions les plus sévères qui peuvent survenir sur l'ADN, sont les pontages inter caténaires. Des agents pontants en liant de façon covalente les deux brins d'ADN, vont empêcher le déroulement normal de processus cellulaires essentiels tels que la réplication ou la transcription. La compréhension des mécanismes permettant à la cellule de tolérer et réparer ces lésions est primordiale, notamment dans le cas des patients atteints de l'anémie de Fanconi qui présentent une très grande sensibilité à ces composés pontants. L'anémie de Fanconi est une maladie génétique rare appartenant à un groupe de pathologies associées à une grande instabilité chromosomique. Les patients atteints de l'anémie de Fanconi présentent des malformations du squelette, une pancytopénie et une forte propension à la survenue de cancer. L'anémie de Fanconi est génétiquement très hétérogène. À ce jour, 13 gènes codant pour 13 protéines FANC différentes ont été identifiés. Huit de ces protéines fonctionnent ensemble au sein d'un complexe (nommé le complexe FANC) ayant pour but de monoubiquitiner FANCD2 et FANCI en réponse à la formation de lésions sur l'ADN. L'extrême sensibilité des cellules de patients atteints de l'anémie de Fanconi à ces agents pontant l'ADN suggère l'implication des protéines FANC dans la réponse cellulaire suite à une stress réplicatif. Cependant, le rôle moléculaire exact de ces protéines demeure encore inconnu. Après purification, nous avons observé que FANCD2 était capable de lier l'ADN simple brin, ainsi que les extrémités d'ADN in vitro. Dans le même temps, FANCM fut identifié comme appartenant au complexe FANC. FANCM est décrit comme une translocase capable de promouvoir le déplacement de point de jonction dans des structures d'ADN spécifiques in vitro. De plus, en se liant à l'ADN, FANCM peut agir comme une plateforme pour les autres protéines FANC, leur permettant ainsi d'être adressées à l'ADN. En créant des protéines FANCM recombinantes ayant des délétions dans le domaine interne, nous avons pu observer que certaines protéines du complexe FANC se fixent à des sites spécifiques sur le domaine interne de FANCM. Enfin, au travers d'une collaboration, nous avons été amenés à caractériser deux nouvelles protéines appartenant au complexe FANC : FAAP 10 et FAAP16. Elles s'associent à FANCM par l'intermédiaire du domaine interne, et forment ainsi un hétérotrimére. La présence de FAAP10 et FAAP16 n'affecte pas la liaison de FANCM à l'ADN, mais semble potentialiser son activité de régression in vitro. FANCM semble donc fonctionner comme une plateforme pour les autres composants du complexe FANC. Ces derniers, une fois liés à l'ADN permettent la monoubiquitination de FANCD2 et son recrutement au site lésé de l'ADN. FANCD2 en se liant de façon préférentielle à l'ADN simple brin et aux extrémités d'ADN qui sont générés lors de l'arrêt et du démantèlement d'une fourche de réplication, pourrait protéger ces même fourches de réplication arrêtées, d'évènements de recombinaison aléatoires. Nos résultats apportent de nouveaux éléments concernant les mécanismes moléculaires de l'anémie de Fanconi. Enfin, l'étude de l'anémie de Fanconi permet aussi de mieux comprendre les mécanismes mis en place par la cellule pour tolérer des lésions survenant lors de la réplication.

Simultaneous determination of human plasma levels of citalopram, paroxetine, sertraline, and their metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry method is presented which allows the simultaneous determination of the plasma concentrations of the selective serotonin reuptake inhibitors citalopram, paroxetine, sertraline, and their pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram, and desmethylsertraline) after derivatization with the reagent N-methyl-bis(trifluoroacetamide). No interferences from endogenous compounds are observed following the extraction of plasma samples from six different human subjects. The standard curves are linear over a working range of 10-500 ng/mL for citalopram, 10-300 ng/mL for desmethylcitalopram, 5-60 ng/mL for didesmethylcitalopram, 20-400 ng/mL for sertraline and desmethylsertraline, and 10-200 ng/mL for paroxetine. Recoveries measured at three concentrations range from 81 to 118% for the tertiary amines (citalopram and the internal standard methylmaprotiline), 73 to 95% for the secondary amines (desmethylcitalopram, paroxetine and sertraline), and 39 to 66% for the primary amines (didesmethylcitalopram and desmethylsertraline). Intra- and interday coefficients of variation determined at three concentrations range from 3 to 11% for citalopram and its metabolites, 4 to 15% for paroxetine, and 5 to 13% for sertraline and desmethylsertraline. The limits of quantitation of the method are 2 ng/mL for citalopram and paroxetine, 1 ng/mL for sertraline, and 0.5 ng/mL for desmethylcitalopram, didesmethylcitalopram, and desmethylsertraline. No interferences are noted from 20 other psychotropic drugs. This sensitive and specific method can be used for single-dose pharmacokinetics. It is also useful for therapeutic drug monitoring of these three drugs and could possibly be adapted for the quantitation of the two other selective serotonin reuptake inhibitors on the market, namely fluoxetine and fluvoxamine.

Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse. The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC-QTOF-MS(E) platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.

Accuracy profile validation of a new analytical method for butane measurement using headspace-gas chromatography-mass spectrometry.

The aim of our study was to provide an innovative HS-GC/MS method applicable to the routine determination of butane concentration in forensic toxicology laboratories. The main drawback of the GC/MS methods discussed in literature concerning butane measurement was the absence of a specific butane internal standard necessary to perform quantification. Because no stable isotope of butane is commercially available, it is essential to develop a new approach by an in situ generation of standards. To avoid the manipulation of a stable isotope-labelled gas, we have chosen to generate in situ an internal labelled standard gas (C(4)H(9)D) following the basis of the stoichiometric formation of butane by the reaction of deuterated water (D(2)O) with Grignard reagent butylmagnesium chloride (C(4)H(9)MgCl). This method allows a precise measurement of butane concentration and therefore, a full validation by accuracy profile was presented.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. I: Investigation of mobile phase and MS conditions.

The conditions for the analysis of selected doping substances by UHPSFC-MS/MS were optimized to ensure suitable peak shapes and maximized MS responses. A representative mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI- modes. The best compromise for all compounds in terms of MS sensitivity and chromatographic performance was obtained when adding 2% water and 10mM ammonium formate in the CO2/MeOH mobile phase. Beside mobile phase, the nature of the make-up solvent added for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the best candidate as it was able to compensate for the negative effect of 2% water addition in ESI- mode and provided a suitable MS response for all doping agents. Sensitivity of the optimized UHPSFC-MS/MS method was finally assessed and compared to the results obtained in conventional UHPLC-MS/MS. Sensitivity was improved by 5-100-fold in UHPSFC-MS/MS vs. UHPLC-MS/MS for 56% of compounds, while only one compound (bumetanide) offered a significantly higher MS response (4-fold) under UHPLC-MS/MS conditions. In the second paper of this series, the optimal conditions for UHPSFC-MS/MS analysis will be employed to screen >100 doping agents in urine matrix and results will be compared to those obtained by conventional UHPLC-MS/MS.

Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry.

A highly sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of buprenorphine and its major metabolite norbuprenorphine in human plasma. In order to speed up the process and decrease costs, sample preparation was performed by simple protein precipitation with acetonitrile. To the best of our knowledge, this is the first application of this extraction technique for the quantification of buprenorphine in plasma. Matrix effects were strongly reduced and selectivity increased by using an efficient chromatographic separation on a sub-2μm column (Acquity UPLC BEH C18 1.7μm, 2.1×50mm) in 5min with a gradient of ammonium formate 20mM pH 3.05 and acetonitrile as mobile phase at a flow rate of 0.4ml/min. Detection was made using a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The procedure was fully validated according to the latest Food and Drug Administration guidelines and the Société Française des Sciences et Techniques Pharmaceutiques. Very good results were obtained by using a stable isotope-labeled internal standard for each analyte, to compensate for the variability due to the extraction and ionization steps. The method was very sensitive with lower limits of quantification of 0.1ng/ml for buprenorphine and 0.25ng/ml for norbuprenorphine. The upper limit of quantification was 250ng/ml for both drugs. Trueness (98.4-113.7%), repeatability (1.9-7.7%), intermediate precision (2.6-7.9%) and internal standard-normalized matrix effects (94-101%) were in accordance with international recommendations. The procedure was successfully used to quantify plasma samples from patients included in a clinical pharmacogenetic study and can be transferred for routine therapeutic drug monitoring in clinical laboratories without further development.

Long synthetic peptides as biologically active proteins: the example of the chemokines.

Chemokines constitute an expanding protein family of over 40 members which exhibit a wide variety of biological activities and are involved in many normal physiological processes, such as cellular migration, differentiation and activation, but also in pathological situations, such as inflammation and metastasis. Over the last few years, we have developed methods to manufacture long synthetic peptides of up to 130 residues, and to achieve the formation of native-like cysteine pairings. This ability prompted us to undertake the total chemical synthesis of chemokines. So far, we have successfully produced over 30 chemokine species, which exhibit biological activities similar to, or greater than, those reported by others. Chemical synthesis offers a clear advantage over recombinant technologies for the introduction of fluorochromes and haptens at molecularly defined positions. In addition, approval of chemically synthesized products for use in humans is straightforward compared with material produced by biological methods.

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.

Graphitized carbon black in quartz tubes for the sampling of indoor air nicotine and analysis by microwave thermal desorption-capillary gas chromatography

Nicotine in a smoky indoor air environment can be determined using graphitized carbon black as a solid sorbent in quartz tubes. The temperature stability, high purity, and heat absorption characteristics of the sorbent, as well as the permeability of the quartz tubes to microwaves, enable the thermal desorption by means of microwaves after active sampling. Permeation and dynamic dilution procedures for the generation of nicotine in the vapor phase at low and high concentrations are used to evaluate the performances of the sampler. Tube preparation is described and the microwave desorption temperature is measured. Breakthrough volume is determined to allow sampling at 0.1-1 L/min for definite periods of time. The procedure is tested for the determination of gas and paticulate phase nicotine in sidestream smoke produced in an experimental chamber.