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 Soci��t�� Fran��aise 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. J��r��me, sur les psaumes. Ms. fait en 1488 pour Mathias Corvin, enlumin�� par Attavante.

La Valli��re.

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&#8201;min) and chromatographic separation (5&#8201;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&#8201;ng/ml; urine, 94&#8201;ng/ml; vitreous humour and cerebrospinal fluid, < 5&#8201;ng/ml; pericardial fluid, 14&#8201;ng/ml; brain, < 5&#8201;pg/mg; heart, 121&#8201;pg/mg; kidney, 245&#8201;pg/mg; and liver, 143&#8201;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. Rep��blica : qu�� costa m��s?

���El coneixement condueix a la unitat, com la ignor��ncia a la diversitat��� Ramakrishna.Fins a quin punt les nostres opinions estan realment ben fonamentades? ��s la diversitat d���opini��, fruit de la ignor��ncia col��lectiva?Tants punts a tractar sobre la Monarquia o la Rep��blica, i tan dif��cils cada un d���ells. En aquest cas, pretenem saber si hi ha o no grans difer��ncies econ��miques entre un sistema i altre, donant un rep��s als conceptes i magnituds importants per entendre b�� el significat de les dades resultants.Si b�� pens��vem que seria dif��cil arribar a una conclusi�� clara, i ens sorpr��n haver-ho aconseguit, m��s ens sorpr��n el fet d���haver-nos adonat, durant la realitzaci�� de la investigaci��, de la car��ncia d���informaci�� que tenim. I no nom��s els ciutadans, sin�� els propis governs sobre les seves pr��pies dades i sobre el que reflecteixen les mateixes. Conceptes entremesclats, dades poc clares, difer��ncies en les informacions dins llocs oficials d���un mateix pa��s... La Rep��blica ��s m��s cara. Ho sabien? Per��...ara que ho saben...��s aquest un resultat que podria variar les seves prefer��ncies? O necessiten saber m��s?Tots tenim dret a opinar sobre aquest tema, a triar el que ens agrada m��s,per��, com b�� diu Ramakrishna, el resultat de la diversitat d���opinions sobre eltema ��s, probablement, conseq����ncia de la poca informaci�� que tenim sobre laglobalitat de factors que influencien en la execuci�� d���una d���aquestes formes d���estat.

L'Env��s : m��s que una protecci��

Aquest ��s un treball essencialment de camp. Hem volgut investigar la import��ncia de l���env��s mitjan��ant dues vies d���estudi. Primer des del punt de vista del productor idespr��s des del punt de vista del consumidor.Pel primer an��lisi vam realitzar una recol��lecta de dades que ens ha perm��s demostrar la hip��tesi que els empresaris obtenen una marge de benefici major quan venen un producte presentat en env��s premium (un env��s sofisticat, m��s erg��nomic, de m��squalitat���) en comparaci�� amb el que est�� en el mercat en env��s est��ndard. Nosaltreshem volgut veure si, realment, aquest marge ��s m��s gran del que justifica la millorad���env��s est��ndard a env��s premium. I pel segon an��lisi vam elaborar unes enquestesque ens han perm��s donar per v��lida la hip��tesi restant: que existeix una relaci�� entre recta pressupost��ria i env��s que s���escull i que, per tant, els consumidors, sempre que puguin, escolliran l���env��s premium.

L'Audi Q3: m��s enll�� de Seat i la crisi de L'autom��bil

La crisi econ��mica global ��s, sens dubte, el principal motiu de preocupaci�� de la societat. Les dificultats que estant patint empreses i treballadors queden plasmades enl'evoluci�� catastr��fica de certes variables macroecon��miques tals com els ��ndex de(de)creixement del PIB, la taxa d'atur, el d��ficit p��blic o la inversi�� privada.En aquest context, els grans sectors de l'economia s��n els que reben major atenci��medi��tica i popular.Els problemes en el sistema financer han estat els grans protagonistes d'aquesta crisi, doncs en s��n l'origen i s'espera que siguin part important en la recuperaci��.En el context espanyol, el sector immobiliari ha endurit encara m��s la crisi, deixant a centenars de milers de treballadors a l'atur i donant s��mptomes de ser un sector que mai podr�� recuperar la dimensi�� de la que havia gaudit els ��ltims anys. Dins d'un entorn econ��mic recessiu, el sector automobil��stic mundial ha estat colpejat molt durament, en especial a Espanya.A les not��cies de caigudes en les matriculacions, s'han succe��t retallades de la producci��, EROs temporals, reduccions de plantillla.... i l'adjudicaci�� de l'Audi Q3 a la f��brica que Seat t�� a Martorell, i que salvar�� milers de llocs de treball.Hem volgut doncs, analitzar la crisi de l'autom��bil a Espanya, centrant-nos en Seat i el Grup Volkswagen, i tractar de manera profunda el cas de l'Audi Q3.Estudiarem la import��ncia de que el Q3 es fabriqui aqu��, els motius pels quals Seat haestat l'elegida, el seguiment que la premsa n'ha fet i l'impacte econ��mic i social querepresenta. Finalment, esperem con��ixer qu�� necessita el nostre pa��s per aconseguir que el sector automobil��stic 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 g��n��alogiques. Recueils g��n��alogiques 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. intitut�� : Recueil genealogicq des plus nobles et anciennes maisons et familles du pays d'Haynaut, faicte par Me Fran��ois Vinchant, prestre... �� (1661).

Contient : Copies de diff��rentes pi��ces, des XVIe et XVIIe si��cles, concernant Namur ; �� Tournoy �� fer esmolu tenu �� Bruxelles, en l'an 1516 �� ; G��n��alogies 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 &#956;L 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 250&#956;L human plasma by protein precipitation with acetonitrile. Chromatographic separation was achieved on a reverse phase column (BEH C18 2.1mm��50mm; 1.7&#956;m) 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 frug��voras (Diptera, Tephritoidea) coletadas em Aquidauana, MS

No Brasil as moscas frug��voras s��o pragas importantes de frutas e hortali��as. O conhecimento da flutua����o populacional dessas esp��cies em cada bioma �� um importante requisito para a ado����o de estrat��gia de controle de pragas nos agroecossistemas. O objetivo desse trabalho foi avaliar a diversidade de esp��cies de moscas-das-frutas infestantes de frutas silvestres e cultivadas em Aquidauana, MS. Vinte e uma esp��cies de frutas foram amostradas de fevereiro de 2003 a janeiro de 2004. As esp��cies de Tephritidae encontradas foram: Anastrepha striata Schiner, 1868, Anastrepha obliqua (Macquart, 1835) e Ceratitis capitata (Wiedemann, 1824). Os frug��voros 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-Mediterr��neo C. capitata. A associa����o entre moscas frug��voras e esp��cies de frutas �� discutida.

Natalizumab treatment alters the expression of T-cell trafficking marker LFA-1 &#945;-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 &#945;L 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.

Simultaneous determination of antidementia drugs by HPLC-MS: validation data of the method and plasma level determinations in 300 patients

Aims: A rapid and simple HPLC-MS method was developed for the simultaneousdetermination of antidementia drugs, including donepezil, galantamine, rivastigmineand its major metabolite NAP 226 - 90, and memantine, for TherapeuticDrug Monitoring (TDM). In the elderly population treated with antidementiadrugs, the presence of several comorbidities, drug interactions resulting frompolypharmacy, and variations in drug metabolism and elimination, are possiblefactors leading to the observed high interindividual variability in plasma levels.Although evidence for the benefit of TDM for antidementia drugs still remains tobe demonstrated, an individually adapted dosage through TDM might contributeto minimize the risk of adverse reactions and to increase the probability of efficienttherapeutic response. Methods: A solid-phase extraction procedure with amixed-mode cation exchange sorbent was used to isolate the drugs from 0.5 mL ofplasma. The compounds were analyzed on a reverse-phase column with a gradientelution consisting of an ammonium acetate buffer at pH 9.3 and acetonitrile anddetected by mass spectrometry in the single ion monitoring mode. Isotope-labeledinternal standards were used for quantification where possible. The validatedmethod was used to measure the plasma levels of antidementia drugs in 300patients treated with these drugs. Results: The method was validated accordingto international standards of validation, including the assessment of the trueness(-8 - 11 %), the imprecision (repeatability: 1-5%, intermediate imprecision:2 - 9 %), selectivity and matrix effects variability (less than 6 %). Furthermore,short and long-term stability of the analytes in plasma was ascertained. Themethod proved to be robust in the calibrated ranges of 1 - 300 ng/mL for rivastigmineand memantine and 2 - 300 mg/mL for donepezil, galantamine and NAP226 - 90. We recently published a full description of the method (1). We found ahigh interindividual variability in plasma levels of these drugs in a study populationof 300 patients. The plasma level measurements, with some preliminaryclinical and pharmacogenetic results, will be presented. Conclusion: A simpleLC-MS method was developed for plasma level determination of antidementiadrugs which was successfully used in a clinical study with 300 patients.