Quantification of 4 antidepressants and a metabolite by LC-MS for therapeutic drug monitoring.

A liquid chromatography method coupled to mass spectrometry was developed for the quantification of bupropion, its metabolite hydroxy-bupropion, moclobemide, reboxetine and trazodone in human plasma. The validation of the analytical procedure was assessed according to Société Française des Sciences et Techniques Pharmaceutiques and the latest Food and Drug Administration guidelines. The sample preparation was performed with 0.5mL of plasma extracted on a cation-exchange solid phase 96-well plate. The separation was achieved in 14min on a C18 XBridge column (2.1mm×100mm, 3.5μm) using a 50mM ammonium acetate pH 9/acetonitrile mobile phase in gradient mode. The compounds of interest were analysed in the single ion monitoring mode on a single quadrupole mass spectrometer working in positive electrospray ionisation mode. Two ions were selected per molecule to increase the number of identification points and to avoid as much as possible any false positives. Since selectivity is always a critical point for routine therapeutic drug monitoring, more than sixty common comedications for the psychiatric population were tested. For each analyte, the analytical procedure was validated to cover the common range of concentrations measured in plasma samples: 1-400ng/mL for reboxetine and bupropion, 2-2000ng/mL for hydroxy-bupropion, moclobemide, and trazodone. For all investigated compounds, reliable performance in terms of accuracy, precision, trueness, recovery, selectivity and stability was obtained. One year after its implementation in a routine process, this method demonstrated a high robustness with accurate values over the wide concentration range commonly observed among a psychiatric population.

S. Jérôme, sur les psaumes. Ms. fait en 1488 pour Mathias Corvin, enluminé par Attavante.

La Vallière.

Body fluid and tissue analysis using filter paper sampling support prior to LC-MS/MS: application to fatal overdose with colchicine

Because of the various matrices available for forensic investigations, the development of versatile analytical approaches allowing the simultaneous determination of drugs is challenging. The aim of this work was to assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform allowing the rapid quantification of colchicine in body fluids and tissues collected in the context of a fatal overdose. For this purpose, filter paper was used as a sampling support and was associated with an automated 96-well plate extraction performed by the LC autosampler itself. The developed method features a 7-min total run time including automated filter paper extraction (2 min) and chromatographic separation (5 min). The sample preparation was reduced to a minimum regardless of the matrix analyzed. This platform was fully validated for dried blood spots (DBS) in the toxic concentration range of colchicine. The DBS calibration curve was applied successfully to quantification in all other matrices (body fluids and tissues) except for bile, where an excessive matrix effect was found. The distribution of colchicine for a fatal overdose case was reported as follows: peripheral blood, 29 ng/ml; urine, 94 ng/ml; vitreous humour and cerebrospinal fluid, < 5 ng/ml; pericardial fluid, 14 ng/ml; brain, < 5 pg/mg; heart, 121 pg/mg; kidney, 245 pg/mg; and liver, 143 pg/mg. Although filter paper is usually employed for DBS, we report here the extension of this alternative sampling support to the analysis of other body fluids and tissues. The developed platform represents a rapid and versatile approach for drug determination in multiple forensic media.

Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces.

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm(-2)) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.

The future key role of LC-high-resolution-MS analyses in clinical laboratories: a focus on quantification.

For the last decade, high-resolution (HR)-MS has been associated with qualitative analyses while triple quadrupole MS has been associated with routine quantitative analyses. However, a shift of this paradigm is taking place: quantitative and qualitative analyses will be increasingly performed by HR-MS, and it will become the common 'language' for most mass spectrometrists. Most analyses will be performed by full-scan acquisitions recording 'all' ions entering the HR-MS with subsequent construction of narrow-width extracted-ion chromatograms. Ions will be available for absolute quantification, profiling and data mining. In parallel to quantification, metabotyping will be the next step in clinical LC-MS analyses because it should help in personalized medicine. This article is aimed to help analytical chemists who perform targeted quantitative acquisitions with triple quadrupole MS make the transition to quantitative and qualitative analyses using HR-MS. Guidelines for the acceptance criteria of mass accuracy and for the determination of mass extraction windows in quantitative analyses are proposed.

Intrathecal immune responses to EBV in early MS.

EBV has been consistently associated with MS, but its signature in the CNS has rarely been examined. In this study, we assessed EBV-specific humoral and cellular immune responses in the cerebrospinal fluid (CSF) of patients with early MS, other inflammatory neurological diseases (OIND) and non-inflammatory neurological diseases (NIND). The neurotropic herpesvirus CMV served as a control. Virus-specific humoral immune responses were assessed in 123 consecutive patients and the intrathecal recruitment of virus-specific antibodies was expressed as antibody indexes. Cellular immune responses tested in the blood of 55/123 patients were positive in 46/55. The CD8(+) CTL responses of these 46 patients were assessed in the blood and CSF using a CFSE-based CTL assay. We found that viral capsid antigen and EBV-encoded nuclear antigen-1, but not CMV IgG antibody indexes, were increased in early MS as compared with OIND and NIND patients. There was also intrathecal enrichment in EBV-, but not CMV-specific, CD8(+) CTL in early MS patients. By contrast, OIND and NIND patients did not recruit EBV- nor CMV-specific CD8(+) CTL in the CSF. Our data, showing a high EBV-, but not CMV-specific intrathecal immune response, strengthen the association between EBV and MS, in particular at the onset of the disease.

Monarquia vs. República : què costa més?

“El coneixement condueix a la unitat, com la ignorància a la diversitat” Ramakrishna.Fins a quin punt les nostres opinions estan realment ben fonamentades? És la diversitat d’opinió, fruit de la ignorància col·lectiva?Tants punts a tractar sobre la Monarquia o la República, i tan difícils cada un d’ells. En aquest cas, pretenem saber si hi ha o no grans diferències econòmiques entre un sistema i altre, donant un repàs als conceptes i magnituds importants per entendre bé el significat de les dades resultants.Si bé pensàvem que seria difícil arribar a una conclusió clara, i ens sorprèn haver-ho aconseguit, més ens sorprèn el fet d’haver-nos adonat, durant la realització de la investigació, de la carència d’informació que tenim. I no només els ciutadans, sinó els propis governs sobre les seves pròpies dades i sobre el que reflecteixen les mateixes. Conceptes entremesclats, dades poc clares, diferències en les informacions dins llocs oficials d’un mateix país... La República és més cara. Ho sabien? Però...ara que ho saben...és aquest un resultat que podria variar les seves preferències? O necessiten saber més?Tots tenim dret a opinar sobre aquest tema, a triar el que ens agrada més,però, com bé diu Ramakrishna, el resultat de la diversitat d’opinions sobre eltema és, probablement, conseqüència de la poca informació que tenim sobre laglobalitat de factors que influencien en la execució d’una d’aquestes formes d’estat.

L'Envàs : més que una protecció

Aquest és un treball essencialment de camp. Hem volgut investigar la importància de l’envàs mitjançant dues vies d’estudi. Primer des del punt de vista del productor idesprés des del punt de vista del consumidor.Pel primer anàlisi vam realitzar una recol·lecta de dades que ens ha permès demostrar la hipòtesi que els empresaris obtenen una marge de benefici major quan venen un producte presentat en envàs premium (un envàs sofisticat, més ergònomic, de mésqualitat…) en comparació amb el que està en el mercat en envàs estàndard. Nosaltreshem volgut veure si, realment, aquest marge és més gran del que justifica la millorad’envàs estàndard a envàs premium. I pel segon anàlisi vam elaborar unes enquestesque ens han permés donar per vàlida la hipòtesi restant: que existeix una relació entre recta pressupostària i envàs que s’escull i que, per tant, els consumidors, sempre que puguin, escolliran l’envàs premium.

L'Audi Q3: més enllà de Seat i la crisi de L'automòbil

La crisi econòmica global és, sens dubte, el principal motiu de preocupació de la societat. Les dificultats que estant patint empreses i treballadors queden plasmades enl'evolució catastròfica de certes variables macroeconòmiques tals com els índex de(de)creixement del PIB, la taxa d'atur, el dèficit públic o la inversió privada.En aquest context, els grans sectors de l'economia són els que reben major atenciómediàtica i popular.Els problemes en el sistema financer han estat els grans protagonistes d'aquesta crisi, doncs en són l'origen i s'espera que siguin part important en la recuperació.En el context espanyol, el sector immobiliari ha endurit encara més la crisi, deixant a centenars de milers de treballadors a l'atur i donant símptomes de ser un sector que mai podrà recuperar la dimensió de la que havia gaudit els últims anys. Dins d'un entorn econòmic recessiu, el sector automobilístic mundial ha estat colpejat molt durament, en especial a Espanya.A les notícies de caigudes en les matriculacions, s'han succeït retallades de la producció, EROs temporals, reduccions de plantillla.... i l'adjudicació de l'Audi Q3 a la fàbrica que Seat té a Martorell, i que salvarà milers de llocs de treball.Hem volgut doncs, analitzar la crisi de l'automòbil a Espanya, centrant-nos en Seat i el Grup Volkswagen, i tractar de manera profunda el cas de l'Audi Q3.Estudiarem la importància de que el Q3 es fabriqui aquí, els motius pels quals Seat haestat l'elegida, el seguiment que la premsa n'ha fet i l'impacte econòmic i social querepresenta. Finalment, esperem conèixer què necessita el nostre país per aconseguir que el sector automobilístic segueixi sent important en el futur, i s'eviti que poc a poc es vagi deslocalitzant.

VOLUMES RELIES du Cabinet des titres : recherches de noblesse, armoriaux, preuves, histoires généalogiques. Recueils généalogiques de « messire Jean, baron DE LAUNAY et du St-Empire, chevalier de l'ordre militaire de Christo, sr de Montigny et d'Asfelt, vicomte de Zelande » († 1687). XXXV « Copie d'un vieux livre ms. intituté : Recueil genealogicq des plus nobles et anciennes maisons et familles du pays d'Haynaut, faicte par Me François Vinchant, prestre... » (1661).

Contient : Copies de différentes pièces, des XVIe et XVIIe siècles, concernant Namur ; « Tournoy à fer esmolu tenu à Bruxelles, en l'an 1516 » ; Généalogies diverses

An ultra performance liquid chromatography-tandem MS assay for tamoxifen metabolites profiling in plasma: first evidence of 4'-hydroxylated metabolites in breast cancer patients.

There is increasing evidence that the clinical efficacy of tamoxifen, the first and most widely used targeted therapy for estrogen-sensitive breast cancer, depends on the formation of the active metabolites 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). Large inter-individual variability in endoxifen plasma concentrations has been observed and related both to genetic and environmental (i.e. drug-induced) factors altering CYP450s metabolizing enzymes activity. In this context, we have developed an ultra performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) requiring 100 μL of plasma for the quantification of tamoxifen and three of its major metabolites in breast cancer patients. Plasma is purified by a combination of protein precipitation, evaporation at room temperature under nitrogen, and reconstitution in methanol/20 mM ammonium formate 1:1 (v/v), adjusted to pH 2.9 with formic acid. Reverse-phase chromatographic separation of tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen is performed within 13 min using elution with a gradient of 10 mM ammonium formate and acetonitrile, both containing 0.1% formic acid. Analytes quantification, using matrix-matched calibration samples spiked with their respective deuterated internal standards, is performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of relative matrix effects variability, as well as tamoxifen and metabolites short-term stability in plasma and whole blood. The method is precise (inter-day CV%: 2.5-7.8%), accurate (-1.4 to +5.8%) and sensitive (lower limits of quantification comprised between 0.4 and 2.0 ng/mL). Application of this method to patients' samples has made possible the identification of two further metabolites, 4'-hydroxy-tamoxifen and 4'-hydroxy-N-desmethyl-tamoxifen, described for the first time in breast cancer patients. This UPLC-MS/MS assay is currently applied for monitoring plasma levels of tamoxifen and its metabolites in breast cancer patients within the frame of a clinical trial aiming to assess the impact of dose increase on tamoxifen and endoxifen exposure.

Simultaneous determination of antidementia drugs in human plasma: Procedure transfer from HPLC-MS to UPLC-MS/MS.

A previously developed high performance liquid chromatography mass spectrometry (HPLC-MS) procedure for the simultaneous determination of antidementia drugs, including donepezil, galantamine, memantine, rivastigmine and its metabolite NAP 226-90, was transferred to an ultra performance liquid chromatography system coupled to a tandem mass spectrometer (UPLC-MS/MS). The drugs and their internal standards ([(2)H(7)]-donepezil, [(13)C,(2)H(3)]-galantamine, [(13)C(2),(2)H(6)]-memantine, [(2)H(6)]-rivastigmine) were extracted from 250μL human plasma by protein precipitation with acetonitrile. Chromatographic separation was achieved on a reverse phase column (BEH C18 2.1mm×50mm; 1.7μm) with a gradient elution of an ammonium acetate buffer at pH 9.3 and acetonitrile at a flow rate of 0.4mL/min and an overall run time of 4.5min. The analytes were detected on a tandem quadrupole mass spectrometer operated in positive electrospray ionization mode, and quantification was performed using multiple reaction monitoring. The method was validated according to the recommendations of international guidelines over a calibration range of 1-300ng/mL for donepezil, galantamine and memantine, and 0.2-50ng/mL for rivastimgine and NAP 226-90. The trueness (86-108%), repeatability (0.8-8.3%), intermediate precision (2.3-10.9%) and selectivity of the method were found to be satisfactory. Matrix effects variability was inferior to 15% for the analytes and inferior to 5% after correction by internal standards. A method comparison was performed with patients' samples showing similar results between the HPLC-MS and UPLC-MS/MS procedures. Thus, this validated UPLC-MS/MS method allows to reduce the required amount of plasma, to use a simplified sample preparation, and to obtain a higher sensitivity and specificity with a much shortened run-time.

Moscas frugívoras (Diptera, Tephritoidea) coletadas em Aquidauana, MS

No Brasil as moscas frugívoras são pragas importantes de frutas e hortaliças. O conhecimento da flutuação populacional dessas espécies em cada bioma é um importante requisito para a adoção de estratégia de controle de pragas nos agroecossistemas. O objetivo desse trabalho foi avaliar a diversidade de espécies de moscas-das-frutas infestantes de frutas silvestres e cultivadas em Aquidauana, MS. Vinte e uma espécies de frutas foram amostradas de fevereiro de 2003 a janeiro de 2004. As espécies de Tephritidae encontradas foram: Anastrepha striata Schiner, 1868, Anastrepha obliqua (Macquart, 1835) e Ceratitis capitata (Wiedemann, 1824). Os frugívoros Lonchaeidae e Muscidae encontrados foram: Neosilba sp. e Atherigona orientalis (Schiner, 1868), respectivamente. Um total de 2.568 moscas foram coletadas, das quais 2.394 representadas pela mosca-do-Mediterrâneo C. capitata. A associação entre moscas frugívoras e espécies de frutas é discutida.

Natalizumab treatment alters the expression of T-cell trafficking marker LFA-1 α-chain (CD11a) in MS patients.

OBJECTIVE: To determine the long-term effect of natalizumab (NTZ) treatment on the expression of integrins and chemokine receptors involved in the migration of T cells towards the central nervous system (CNS). METHODS: We drew the blood of 23 patients just before starting NTZ therapy and every 12 months thereafter, for up to 48 months of treatment. We assessed the ex-vivo expression of phenotype markers (CCR7 and CD45RA), CNS-addressing integrins (CD11a, CD49d and CD29) and chemokine receptors (CXCR3 and CCR6) in CD4+ or CD8+ T-cell subsets by flow cytometry. RESULTS: As compared to the pre-NTZ values, there was a marked increase in central memory (CCR7+/CD45RA-) CD4+ T cells and in effector memory (CCR7-/CD45RA-) CD8+ T cells at 12 and 24 months. In addition to an expected downregulation of both VLA-4 subunits (CD49d/CD29), we also found decreased T-cell expression of CXCR3 at 12 months, and of CD11a (LFA-1 αL subunit) at 12 months, but mostly at 24 months of NTZ treatment. CONCLUSION: Our data show a nadir of CD11a expression at 2 years of NTZ treatment, at the peak of incidence of progressive multifocal leukoencephalopathy (PML), indirectly suggesting that a lack of these molecules may play a role in the onset of PML in NTZ-treated patients.