Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity

Expansion of intraocular gas bubbles due to altitude: do meteorological factors play a role?

BACKGROUND: Intraocular gas bubbles expand as patients move up to higher altitude. This may cause an acute intraocular pressure (IOP) rise with associated vascular obstructions and visual loss. MATERIALS AND METHODS: Two pseudophakic patients underwent a pars plana vitrectomy and 23% SF6 gas tamponade for a pseudophakic retinal detachment. During the immediate post-operative phase, the patients travelled daily up to their domicile, which was situated approximately 600 m higher than the level where they had been operated on. These travels were always without any pain or visual loss. However 1 week after surgery both patients developed severe ocular pain, and one patient had complete temporary loss of vision after ascending to altitude levels, which had previously presented no problem. Both episodes occurred in parallel with a change in barometric pressure. RESULTS: Treatment with acetazolamide reduced the increased IOP to normal levels, and visual acuity recovered. CONCLUSIONS: Although the post-operative size of an intraocular gas bubble decreases progressively over time, problems with bubble expansion may still occur even at a late stage if meteorological factors, that may increase the bubble size, change.

Variation in cuticular hydrocarbons among strains of the Anopheles gambiae sensu stricto by analysis of cuticular hydrocarbons using gas liquid chromatography of larvae

Cuticular hydrocarbons of larvae of individual strains of the Anopheles gambiae sensu stricto were investigated using gas liquid chromatography. Biomedical discriminant analysis involving multivariate statistics suggests that there was clear hydrocarbon difference between the Gambian(G3), the Nigerian (16CSS and, its malathion resistant substrain, REFMA) and the Tanzanian (KWA) strains. The high degree of segregation (95%) in hydrocarbons among the four strains investigated indicates that further analysis is needed to enable understanding of hydrocarbon variation in samples of An. gambiae especially from areas where these populations co-exist.

Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure.

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.

Obtenció de capes primes d'alúmina mitjançant "Atomic Layer Deposition"

En aquest projecte s'ha estudiat la posada a punt d’un equip comercial ALD per a l’obtenció de capes primes d'alúmina a escala nanomètrica utilitzant vapor d’aigua i TMA com a precursors. Per tal de comprovar a bondat de les receptes experimentals aportades pel fabricant així com comprovar alguns aspectes de la teoria ALD s’han realitzat una sèrie de mostres variant els diferents paràmetres experimentals, principalment la temperatura de deposició, el nombre de cicles, la durada del cicle i el tipus de substrat. Per a la determinació dels gruixos nanomètrics de les capes i per tant dels ritmes de creixement s’ha utilitzat la el·lipsometria, una de les poques tècniques no destructives capaç de mesurar amb gran precisió gruixos de capes o interfases de pocs àngstroms o nanòmetres. En una primera etapa s'han utilitzat els valors experimentals donats pel fabricant del sistema ALD per determinar el ritme de creixement en funció de la temperatura de dipòsit i del numero de cicles, en ambdós casos sobre diversos substrats. S'ha demostrat que el ritme de creixement augmenta lleugerament en augmentar la temperatura de dipòsit, tot i que amb una variació petita, de l'ordre del 12% en variar 70ºC la temperatura de deposició. Així mateix s'ha demostrat la linealitat del gruix amb el número de cicles, tot i que no s’observa una proporcionalitat exacta. En una segona etapa s'han optimitzat els paràmetres experimentals, bàsicament els temps de purga entre pols i pols per tal de reduir considerablement les durades dels experiments realitzats a relativament baixes temperatures. En aquest cas s’ha comprovat que es mantenien els ritmes de creixement amb una diferencia del 3,6%, 4,8% i 5,5% en optimitzar el cicles en 6,65h, 8,31h, o 8,33h, respectivament. A més, per una d'aquestes condicions s’ha demostrat que es mantenia l’alta conformitat de les capes d’alúmina. A més, s'ha realitzat un estudi de l'homogeneïtat del gruix de les capes en tota la zona de dipòsit del reactor ALD. S’ha demostrat que la variació en gruix de les capes dipositades a 120ºC és com a màxim del 6,2% en una superfície de 110 cm2. Confirmant l’excepcional control de gruixos de la tècnica ALD.

Carbon isotopic ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans.

Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of gamma-butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3 per thousand was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The (13)C/(12)C ratios of GHB in samples of subjects exposed to the drug ranged from -32.1 to -42.1 per thousand, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range -23.5 to -27.0 per thousand. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses.

Memòria del projecte GAS

L'objectiu d'aquest Treball Final de Carrera consisteix en la realització del desenvolupament d'un Gestor d'Administració de Sistemes (GAS). El desenvolupament d'aquest gestor tindrà ús real i vindrà a cobrir una necessitat del departament de Sistemas Informàtic de l'Hospital Americà de la Base Naval de Trencada on treball com a administradora de sistemes.

Human topoisomerase II-DNA interaction study by using atomic force microscopy.

Type II topoisomerases (Topo II) are unique enzymes that change the DNA topology by catalyzing the passage of two double-strands across each other by using the energy from ATP hydrolysis. In vitro, human Topo II relaxes positive supercoiled DNA around 10-fold faster than negative supercoiled DNA. By using atomic force microscopy (AFM) we found that human Topo II binds preferentially to DNA cross-overs. Around 50% of the DNA crossings, where Topo II was bound to, presented an angle in the range of 80-90°, suggesting a favored binding geometry in the chiral discrimination by Topo II. Our studies with AFM also helped us visualize the dynamics of the unknotting action of Topo II in knotted molecules.

Sensitive headspace gas chromatography analysis of free and conjugated 1-methoxy-2-propanol in urine

Glycol ethers still continue to be a workplace hazard due to their important use on an industrial scale. Currently, chronic occupational exposures to low levels of xenobiotics become increasingly relevant. Thus, sensitive analytical methods for detecting biomarkers of exposure are of interest in the field of occupational exposure assessment. 1-Methoxy-2-propanol (1M2P) is one of the dominant glycol ethers and the unmetabolized urinary fraction has been identified to be a good biological indicator of exposure. An existing analytical method including a solid-phase extraction and derivatization before GC/FID analysis is available but presents some disadvantages. We present here an alternative method for the determination of urinary 1M2P based on the headspace gas chromatography technique. We determined the 1M2P values by the direct headspace method for 47 samples that had previously been assayed by the solid-phase extraction and derivatization gas chromatography procedure. An inter-method comparison based on a Bland-Altman analysis showed that both techniques can be used interchangeably. The alternative method showed a tenfold lower limit of detection (0.1 mg/L) as well as good accuracy and precision which were determined by several urinary 1M2P analyses carried out on a series of urine samples obtained from a human volunteer study. The within- and between-run precisions were generally about 10%, which corresponds to the usual injection variability. We observed that the differences between the results obtained with both methods are not clinically relevant in comparison to the current biological exposure index of urinary 1M2P. Accordingly, the headspace gas chromatography technique turned out to be a more sensitive, accurate, and simple method for the determination of urinary 1M2P.[Authors]

Determination of chlorzoxazone and 6-hydroxychlorzoxazone in plasma by gas chromatography--mass spectrometry.

A gas chromatographic-mass spectrometric method is presented which allows the determination of chlorzoxazone and 6-hydroxychlorzoxazone after derivatization with the reagent N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide. No interference was observed from endogenous compounds following the extraction of plasma samples from six different human subjects. The standard curves were linear over a working range of 20 to 4000 ng/ml and of 20 to 1000 ng/ml for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. Recoveries ranged from 65 to 97% for the two compounds and intra- and inter-day coefficients of variation were always less than 9%. The limit of quantitation of the method was found to be 5 ng/ml for the two compounds, hence allowing its use for single low dose pharmacokinetics.

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.