Accuracy profile validation of a new method for carbon monoxide measurement in the human blood using headspace-gas chromatography-mass spectrometry (HS-GC-MS).

The aim of our study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable for the routine determination of blood CO concentration in forensic toxicology laboratories. The main drawback of the GC/MS methods discussed in literature for CO measurement is the absence of a specific CO internal standard necessary for performing quantification. Even if stable isotope of CO is commercially available in the gaseous state, it is essential to develop a safer method to limit the manipulation of gaseous CO and to precisely control the injected amount of CO for spiking and calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in a vial in situ, an internal labeled standard gas ((13)CO) formed by the reaction of labeled formic acid formic acid (H(13)COOH) with sulfuric acid. As sulfuric acid can also be employed to liberate the CO reagent from whole blood, the procedure allows for the liberation of CO simultaneously with the generation of (13)CO. This method allows for precise measurement of blood CO concentrations from a small amount of blood (10 μL). Finally, this method was applied to measure the CO concentration of intoxicated human blood samples from autopsies.

Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry: a method comparison and validation.

RATIONALE: The aim of the work was to develop and validate a method for the quantification of vitamin D metabolites in serum using ultra-high-pressure liquid chromatography coupled to mass spectrometry (LC/MS), and to validate a high-resolution mass spectrometry (LC/HRMS) approach against a tandem mass spectrometry (LC/MS/MS) approach using a large clinical sample set. METHODS: A fast, accurate and reliable method for the quantification of the vitamin D metabolites, 25-hydroxyvitamin D2 (25OH-D2) and 25-hydroxyvitamin D3 (25OH-D3), in human serum was developed and validated. The C3 epimer of 25OH-D3 (3-epi-25OH-D3) was also separated from 25OH-D3. The samples were rapidly prepared via a protein precipitation step followed by solid-phase extraction (SPE) using an HLB μelution plate. Quantification was performed using both LC/MS/MS and LC/HRMS systems. RESULTS: Recovery, matrix effect, inter- and intra-day reproducibility were assessed. Lower limits of quantification (LLOQs) were determined for both 25OH-D2 and 25OH-D3 for the LC/MS/MS approach (6.2 and 3.4 µg/L, respectively) and the LC/HRMS approach (2.1 and 1.7 µg/L, respectively). A Passing & Bablok fit was determined between both approaches for 25OH-D3 on 662 clinical samples (1.11 + 1.06x). It was also shown that results can be affected by the inclusion of the isomer 3-epi-25OH-D3. CONCLUSIONS: Quantification of the relevant vitamin D metabolites was successfully developed and validated here. It was shown that LC/HRMS is an accurate, powerful and easy to use approach for quantification within clinical laboratories. Finally, the results here suggest that it is important to separate 3-epi-25OH-D3 from 25OH-D3. Copyright © 2012 John Wiley & Sons, Ltd.

Confirmation of natural gas explosion from methane quantification by headspace gas chromatography-mass spectrometry (HS-GC-MS) in postmortem samples: a case report.

A new analytical approach for measuring methane in tissues is presented. For the first time, the use of in situ-produced, stably labelled CDH(3) provides a reliable and precise methane quantification. This method was applied to postmortem samples obtained from two victims to help determine the explosion origin. There was evidence of methane in the adipose tissue (82 nmol/g) and cardiac blood (1.3 nmol/g) of one victim, which corresponded to a lethal methane outburst. These results are discussed in the context of the available literature to define an analysis protocol for application in the event of a gas explosion.

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.

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.

Santé et styles de vie des adolescents âgés de 16 à 20 ans en Suisse (2002). SMASH 2002 : Swiss multicenter adolescent survey on health 2002

Le présent rapport décrit les grandes tendances qui se dégagent des résultats de l'Enquête SMASH 2002 et propose quelques hypothèses pour éclairer la compréhension et les enjeux de la situation actuelle ainsi que des pistes de réflexion pour l'avenir. Cette étude repose sur deux approches complémentaires de la santé: l'une donne la primauté aux aspects sociologiques des conduites de santé ("lifestyle"); l'autre, de nature épidémiologique, met l'accent sur la prévalence des comportements et procède à des analyses dans divers sous-groupes ou dans le temps. L'étude SMASH 2002 fournit une photographie de la situation des adolescents et de leur état de santé : besoins et comportements de santé, facteurs associés et évaluation des grands changements intervenus depuis une dizaine d'années. Ces données doivent permettre de réfléchir à une planification optimale des services de soins et des programmes de prévention et de promotion de la santé en faveur des adolescents. [extraits du résumé des auteurs]

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.