1,721,128 research outputs found
Development and validation of analytical methods for the determination of direct ethanol metabolites in biological matrices by liquid chromatography tandem mass spectrometry. Applications in forensic toxicology
No full textL’abuso alcolico è un problema che ha rilevanti implicazioni cliniche, sociali,
economiche e amministrative. La diagnosi specifica e sensibile delle diverse forme
di abuso alcolico acuto e cronico costituisce elemento essenziale per poter
affrontare correttamente questo problema.
Recentemente, il rilievo di metaboliti minori dell’alcol, l’etil glucuronide (EtG) e l’etil
solfato (EtS), in matrici biologiche tradizionali (sangue e derivati, urina) o meno
convenzionali (capello, meconio) è stato prospettato come un approccio
potenzialmente efficace ed efficiente alla diagnosi di abuso alcolico sia per
finalità forensi, sia per finalità cliniche ed epidemiologiche.
Lo scopo di questa tesi è stato quello di (a) sviluppare e sottoporre a validazione
completa metodi analitici per la determinazione di questi metaboliti mediante
cromatografia liquida abbinata alla spettrometria di massa tandem (LC-MS/MS) in
diversi campioni biologici (urina, sangue/siero, capello - limitatamente all’EtG - e
meconio) e (b) applicare questi metodi analitici in diversi contesti diagnostici e, in
particolare: 1) studio della cinetica di eliminazione nel sangue, nonché del
rapporto sangue/siero dell’EtG e dell’EtS in soggetti alcolisti all’inizio della terapia
riabilitativa (studio condotto in collaborazione con il National Insitute of Public
Health Norvegese); 2) studio della stabilità dell’EtG e dell’EtS in campioni
cadaverici a distanza di diversi anni dall’inumazione; 3) valutazione delle
potenzialità dell’EtG nel capello (HEtG) quale marker di abuso alcolico cronico,
anche in rapporto ad altri indicatori correntemente utilizzati nella routine (CDT); 4)
applicazione della determinazione di EtG e di EtS nel meconio quali indicatori di
esposizione intrauterina all’alcol.
I risultati ottenuti nelle sperimentazioni condotte negli ambiti appena descritti
hanno dimostrato, in alcuni casi per la prima volta, ovvero confermato, il notevole
potenziale dell’EtG e dell’EtS quali indicatori specifici e sensibili di abuso alcolico.
Inoltre, hanno permesso di accertare che: l’eliminazione di EtG e EtS in soggetti
abusatori di alcool è sovrapponibile a quella osservata in bevitori moderati (con
l’importante eccezione di soggetti con patologie renali nei quali metabolismo
dell’alcol e conseguente eliminazione dei suoi metaboliti dall’organismo
appaiono rallentati); l’EtG e l’EtS si distribuiscono preferenzialmente nel siero
rispetto alla frazione corpuscolata del sangue, con potenziali implicazioni
sull’interpretazione dei risultati qualora l’analisi sia eseguita su siero/plasma
piuttosto che su sangue intero; l’EtG e l’EtS sono rilevabili in campioni biologici
prelevati da cadaveri esumati anche a distanza di molti anni dal decesso;
l’impiego di un metodo analitico specifico, sensibile (LLOQ di 3 pg/mg) e
sottoposto a validazione completa conferisce all’HEtG elevata sensibilità e
specificità diagnostica; l’HEtG appare correlato qualitativamente con altri marker
(es. il cocaetilene nei capelli di abusatori di cocaina; la CDT) pur manifestando,
rispetto a questi, una maggiore sensibilità (> 90%) nell’evidenziare un abuso
alcolico cronico; l’EtG e L’EtS sono presenti e rilevabili nel meconio (anche se la
loro concentrazione non è correlata con quella di altri indicatori di consumo di
alcol quali gli esteri etilici degli acidi grassi) ciò costituendo la premessa per una
più approfondita valutazione dell’utilità della loro determinazione in questo
substrato biologico nell’evidenziare l’esposizione intrauterina all’alcol.Alcohol abuse and misuse is a growing problem with relevant clinical, social,
economic and administrative implications. A sensitive and specific diagnosis of the
different forms of acute and chronic alcohol abuse is an essential tool to properly
face this social burden. Recently, the determination of two minor non oxidative
products of alcohol metabolism, ethyl glucuronide (EtG) and ethyl sulphate (EtS),
in traditional (blood and derivatives, urine) as well as alternative (hair, meconium)
biological matrices has been proposed as a promising approach to the efficient
and effective diagnosis of alcohol abuse for forensic, clinical, and epidemiological
purposes.
The aim of this thesis was to (a) develop and fully validate analytical methods for
the determination of these two metabolites in different biological samples (urine,
blood/serum, meconium and, for EtG, hair) by liquid chromatography-tandem
mass spectrometry (LC-MS/MS), and b) to apply these methods in several
diagnostic contexts and, in particular: 1) the study of the blood kinetics and
blood/serum ratio of EtG and EtS in samples of heavy drinkers at the beginning of a
withdrawal treatment (study carried out in collaboration with the Norwegian
National Institute of Health); 2) study of the stability of EtG and EtS in post-mortem
samples several years after burial; 3) evaluation of EtG in hair (HEtG) as a potential
marker of chronic alcohol abuse, in comparison with other biomarkers currently
used in the routine (i.e. CDT); 4) application of EtG and EtS in meconium as markers
of gestational alcohol exposure.
Results obtained in the different fields mentioned above showed, in some cases
for the first time, or confirmed the great potential of EtG and EtS as sensitive and
specific markers of alcohol misuse. Moreover they allowed to ascertain that: the
elimination of EtG and EtS in heavy drinkers is similar to that occurring in social
drinkers (with the important exception of subjects suffering from renal pathologies
where the ethanol metabolism and consequently the metabolites elimination
appear to be slowed down); EtG and EtS are to mainly distributed in serum than in
blood cells, with relevant implications when the analysis is carried out on
serum/plasma instead of whole blood; EtG and EtS are detectable in post-mortem
biological samples even when collected several years after death; the use of an
analytical, sensitive (LLOQ of 3 pg/mg), specific and fully validated method gives
to HEtG a high diagnostic sensitivity and specificity; HEtG appears to qualitatively
correlate with other markers ( i.e. cocaethylene in hair of cocaine users; CDT),
although it exhibits better sensitivity (>90%) in ascertaining alcohol chronic abuse;
EtG and EtS are detectable in meconium (even if their concentration doesn’t
correlate with that of other markers of alcohol consumption such as fatty acids
ethyl esters), thus offering the basis for an in-depth evaluation of the usefulness of
their determination in this matrix for the diagnosis of prenatal exposure to alcohol
Prendersi (il) gioco: la prospettiva della co-progettazione contro l’egemonia dei sistemi chiusi nei giochi educativi
No full textL'articolo contestualizza l'adozione, l'uso e la progettazione di Serious Games in contesti educativi, problematizzando alcuni argomenti di senso comune sul loro impiego e affrontando alcune questioni legate all'uso strumentale dei giochi. Come alternativa, l'articolo delinea un paradigma per la concettualizzazione e la co-progettazione di Serious Games, basato sul riconoscimento delle tensioni e delle opportunità inerenti alle dimensioni collegate, ma distinte, del gioco sociale/culturale (play) e formale/sistemico (game), sottolineando il valore della promozione dell'autonomia dello studente attraverso il suo coinvolgimento diretto nella creazione di attività e artefatti ludici.The article contextualises the adoption, use and design of Serious Games in educational contexts, problematising some common sense arguments about their deployment, and articulating some general issues with the instrumental use of games. As an alternative, the article outlines a paradigm for the conceptualisation of and co-design of Serious Games, grounded in acknowledging the tensions and opportunities inherent to the linked but distinct dimensions of play (social/cultural) and game (formal/systemic), emphasising the value of promoting the autonomy of learner through their direct involvement in the creation of playful activities and artefacts
A direct screening procedure for diuretics in human urine by liquid chromatography-tandem mass spectrometry with information dependent acquisition
No full textBackground: Diuretics are a class of compounds largely used for either therapeutic (edemas, hypertension, etc.) or illegal (doping) purposes. Probably owing to the substantial variety of their chemical structures, which makes them hardly extractable from a biological matrix in a single procedure, a quite short list of screening methods can be retrieved in the literature.
Methods: This work presents a screening procedure for 24 diuretics based on the direct injection of urine (after 50 folds dilution) in a LC-ESI-MS/MS system (Applied Biosytems 4000 QTrap). Two information dependent acquisitions (IDA), one in positive, one in negative ionization, allowed the acquisition of one selected reaction monitoring transition for each compound, which, when a significant peak was found, triggered the acquisition of the enhanced product ion (EPI) spectrum.
Results and conclusions: EPI spectra were stored in a library and the procedure was able to recognize by library matching various diuretics in real positive samples. The limits of detection were comprised between 0.002 and 0.25 mg/l and ion suppression was not found to significantly influence the analysis
Determination of 18 cardiovascular drugs in urine and whole blood by LC-MS/MS: application in clinical and forensic toxicology
No full textEthyl glucuronide in hair: sensitivity, specificity, and factors potentially influencing its performance
No full textTreatments against hair loss may hinder cocaine and metabolites detection
No full text: Recently, some of the hair samples that we routinely analyse for drugs of abuse did not produce valid results for cocaine and metabolites. A series of very intense interfering peaks with ion fragments common to cocaine (CO), and benzoylecgonine (BE) were found to cover up the "cocaine" region of the chromatogram. In one of these cases the subject declared he had used a lotion containing Minoxidil in order to prevent hair loss. Starting from this observation we found that the interfering peaks belonged to four different TMS derivatives of Minoxidil. Minoxidil interference was further investigated by applying Tricoxidil(®), a Minoxidil solution, to the hair of CO-free volunteers and to a CO-positive hair strand dipped into Tricoxidil. Hair were analysed before and after treatment. In both cases interfering peaks were absent in the chromatograms of untreated hair and appeared in treated hair. In the CO-positive hair detection of CO, BE and internal standard was completely hindered after treatment with Minoxidil. Attempts to separate interfering peaks from CO and metabolites by modifying the temperature programme failed. None of the hair washing methods tested (methanol; dichloromethane; sodium dodecyl sulphate water solution, 1% w/v followed by methanol; phosphate buffer 0.1 M, pH 6 followed by methanol) succeeded in removing Minoxidil interference. However, a simple solution to partially overcome the problem was to dry up the derivatised extract, reconstitute it in methanol (in order to switch back Minoxidil derivatives to the native molecule), and re-inject it: owing to the higher polarity, underivatised Minoxidil does not interfere any more with the chromatography of CO, at the expense of the disappearance of BE and ecgonine methyl ester both producing TMS derivatives. This strategy was applied to four real cases where Minoxidil interference was recognised: in two of these cases CO was detected. The problem of Minoxidil interference on CO detection may be limited to procedures involving trimethylsilylation, which is probably the most commonly adopted derivatisation in laboratories performing hair analysis for drugs of abuse
Ethyl glucuronide and ethyl sulfate in autopsy samples 27 years after death
No full textThe unique case of a 50-year-old known alcoholic whose corpse was exhumed 27 years after death is reported. The man apparently committed suicide by hanging, but many years later the case was questioned and homicide-linked to a long-lasting serial killer case-was suspected. Thus, the corpse was exhumed, and at the autopsy it was found to be naturally mummified. This fact permitted the analysis of body tissues with the aim to investigate the persistence of ethanol conjugates in the biological material 27 years after death. Fragments of liver and kidney, a blood clot, and a hair strand were collected and submitted to liquid chromatography tandem mass spectrometry analysis. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) were identified and quantified in the liver, the kidney, and the blood clot. Hair analysis was found to be severely affected by ion suppression even after solid phase extraction. Consequently, EtG was identified in all hair segments (0-3 cm, 3-6 cm, and 6-10 cm), but no reliable quantification could be carried out. In summary, our findings demonstrate that, notwithstanding the expected conjugate degradation, EtG and EtS can be indicative of ante-mortem use of alcohol even many years after death
Effect of bleaching on ethyl glucuronide in hair: An in vitro experiment
No full textINTRODUCTION: Ethyl glucuronide in hair (HEtG) has recently gained great attention, because of its high sensitivity and specificity in the diagnosis of chronic alcohol abuse. Due to its high polarity hydrophilicity, a strong hair treatment followed by a shampooing may lead to removal/degradation of this molecule from hair matrix.
AIM: To set up an in vitro study in order to evaluate the ability of bleaching of modifying HEtG test results.
METHODS: Thirty hair samples from teetotalers (n=5), social drinkers (n=4) and heavy drinkers (n=21), after an informed written consent, were collected and divided longitudinally into four aliquots. The first aliquot was kept untreated and was processed following the method routinely used in our lab for the determination of HEtG (double washing with methanol/dichloromethane, overnight incubation in water, and LC-MS/MS analysis, LLOQ: 3pg/mg). To the other three aliquots a commercially available bleaching solution was applied, according to the manufacturer's instructions. One out of the three aliquots was submitted to the analysis by following the same procedure used for the untreated sample. The other two were submitted to a purification step before LC-MS/MS analysis, by using two different SPE cartridges (aminopropyl and dimethyl butylamine).
RESULTS: HEtG levels in the untreated samples from social drinkers and heavy drinkers ranged from 7.7 to 149.0pg/mg. All the samples from teetotalers tested negative. The treated samples processed without any SPE extraction and with aminopropyl cartridges showed a relevant ion suppression for both EtG and D(5)-EtG (IS) signals. Samples treated with the bleaching solution and extracted with dimethyl butylamine cartridge allowed to sensitively reduce ion suppression (less than 35%) and to verify that EtG, after a strong treatment like bleaching, completely disappears.
CONCLUSIONS: This in vitro study showed that HEtG disappears from hair matrix after a strong hair treatment. It is not clear whether the mechanism involved is chemical degradation or physical removal from the damaged keratinic matrix. However, owing to the highly hydrophilic character of the compound, the second mechanism seems more likely to occur. Finally, bleaching solutions could lead to a heavy ion suppression of this metabolite that may be avoided by using an SPE purification before instrumental analysis
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