Lucia Politi

ESI-MS/MS library of 1,253 compounds for application in forensic and clinical toxicology.

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) library which contains over 5,600 spectra of 1,253 compounds relevant in clinical and forensic toxicology has been developed using a hybrid tandem mass spectrometer with a linear ion trap. Pure compound solutions—in some cases solutions made of tablets—were prepared and 1 to 2,000xa0ng of each compound were injected into the system using standard reversed-phase analytical columns with gradient elution. To obtain maximum mass spectral information enhanced product ion spectra were acquired with positive and/or negative ionization at low, medium, and high collision energies and additionally applying collision energy spread. In this mode, all product ions generated by the different collision energies are trapped in the linear ion trap prior to their detection. The applicability of the library for other types of hybrid tandem mass spectrometers with a linear ion trap of the same manufacturer as well as a standard triple-quadrupole tandem mass spectrometer has been investigated with a selection of compounds. The spectra of the developed library can be used to create methods for target analysis, either screening methods or quantitative procedures by generating transitions for multiple reaction monitoring. For those procedures, suitable transitions and convenient collision energies are selected from the library. It also has been utilized to identify compounds with a multi target screening approach for clinical and forensic toxicology with a standardized and automated system. The novel aspects compared to our former library produced with a standard triple-quadrupole mass spectrometer are the enlargement of the ESI-MS/MS library and the additional acquisition of spectra with collision energy spread.

Ethyl glucuronide in hair. A sensitive and specific marker of chronic heavy drinking

AIMSnThis study aims to define a cut-off concentration for ethyl glucuronide in hair to determine if there was a history of heavy drinking.nnnSETTINGSnPavia, Italy.nnnPARTICIPANTSnWe analysed hair samples from 98 volunteers among teetotallers, social drinkers and heavy drinkers, whose ethanol daily intake (EDI) was estimated by means of a written questionnaire.nnnMEASUREMENTSnEthyl glucuronide hair concentration (HEtG) was measured by liquid chromatography-tandem mass spectrometry (lower limit of quantification: 3 pg/mg) using a fully validated method.nnnFINDINGSnThe HEtG level providing the best compromise between sensitivity (0.92) and specificity (0.96) at detecting an EDI of 60 g or higher during the last 3 months was 27 pg/mg. None of the factors examined among those known to affect ethanol metabolism and/or the diagnostic power of other markers of ethanol use or hair analyses, including age, gender, body mass index, tobacco smoke, prevalent beverage, hair colour, cosmetic treatments and hygienic habits was found to influence marker performance significantly. However, the slight differences in HEtG performance observed for some factors (e.g. body mass index, smoke and hair treatments) require further studies on larger groups of individuals in order to assess their influence more precisely.nnnCONCLUSIONSnOur results confirm further that HEtG is a sensitive and specific marker of chronic heavy drinking.

Effect of bleaching on ethyl glucuronide in hair: An in vitro experiment

INTRODUCTIONnEthyl 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.nnnAIMnTo set up an in vitro study in order to evaluate the ability of bleaching of modifying HEtG test results.nnnMETHODSnThirty 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 manufacturers 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).nnnRESULTSnHEtG 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.nnnCONCLUSIONSnThis 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.

Cocaine and heroin in waste water plants: a 1-year study in the city of Florence, Italy

The diffusion and trends in use of each substance is a basic information in policy planning of strategies aiming at deterrence of drug abuse or in the organization of the fight against drug trafficking. The actual diffusion of illicit drugs in a population is hardly measurable, but, among the various measures available, the analysis of waste water plants represents one of the most reliable source of data. We analyzed waste water in order to monitor illicit drug use by local population. We investigated the use of cocaine and heroin in the city of Florence, Italy, over a 1-year (July 2006-June 2007) period using state-of-the-art measuring techniques from waste water samples. Cocaine, benzoylecgonine, and morphine were determined in water samples by gas chromatography-mass spectrometer, and the amount of illicit substance was estimated. Data indicate for cocaine a bimodal distribution (December and March), while heroin showed a main peak in April. The heroin-to-cocaine use ratio in terms of estimated doses per month ranged from 0.11 to 0.76, representing new evidence of wider distribution of cocaine than heroin in Florence. Waste water analysis can become a valuable tool in monitoring use of illicit drugs over time. In particular, it can highlight changes in the magnitude and relative use of illicit drug at a population level thereby becoming useful to develop strategies against drug trafficking and abuse. If routinely performed, it can be part of Epidemiologic Surveillance Programmes on drug abuse.

Determination of aminorex in human urine samples by GC–MS after use of levamisole

The Drug Enforcement Administration (DEA) reports that as of October 2010, 79% of all cocaine seized in the United States contained levamisole. The equine conversion of levamisole to aminorex has been demonstrated. However, the metabolic fate of levamisole in humans is unknown. Nevertheless, as aminorex is amphetamine-like and hallucinogenic, it may be used as an adulterant to increase the effects of cocaine. We report here the results of in vivo studies demonstrating for the first time that not only equine, but also canine and human metabolism all result in aminorex formation. Levamisole and aminorex were extracted from real urine samples by liquid-liquid extraction and identified and quantified by GC-MS (identification by 3 ions per substance, LLOQ at 0.15ng/ml for both).

Comparison of ethyl glucuronide in hair with carbohydrate-deficient transferrin in serum as markers of chronic high levels of alcohol consumption

This study was designed with the aim to compare sensitivity and specificity of ethyl glucuronide in hair (HEtG) and carbohydrate-deficient transferrin (CDT) in serum as markers of heavy drinking. Eighty-six volunteers, including teetotalers, social, and heavy drinkers, were interviewed to evaluate their ethanol daily intake (EDI) during the last 2-week and 3-month periods. HEtG determination was performed by a fully validated LC-MS-MS procedure and ranged from <LOD (2 pg/mg) to 890.5 pg/mg. CDT was measured by immunonephelometry or by HPLC. Sensitivity and specificity of the two markers as indicators of an EDI higher than 60 g/day were calculated, with cut-off at 27 pg/mg (HEtG) and 2.5% (CDT). Considering the EDI of the last 2 weeks, HEtG showed equal selectivity (0.93 for both HEtG and CDT-immunonephelometry; 0.70 for both HEtG and CDT-HPLC) and 2 times the sensitivity of either of the two CDT methods (1.00 vs. 0.44 for CDT-immunonephelometry; 0.96 vs. 0.50 for CDT-HPLC). The same difference in performances but with higher absolute sensitivity and selectivity values for HEtG were observed considering the EDI of the last 3-months (selectivity: 1.00 for both HEtG and CDT-immunonephelometry, 0.89 and 0.78 for HEtG and CDT-HPLC, respectively; sensitivity: 1.00 vs. 0.47 for CDT-immunonephelometry; 0.98 vs. 0.51 for CDT-HPLC). Our results indicate that HEtG, as compared to CDT measured using different methods, is a selective marker of ethanol heavy chronic use providing considerably higher sensitivity.

Ethyl glucuronide and ethyl sulfate in autopsy samples 27 years after death

The unique case of a 50-year-old known alcoholic whose corpse was exhumed 27xa0years 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 27xa0years 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 (0–3xa0cm, 3–6xa0cm, and 6–10xa0cm), 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.

Nails of newborns in monitoring drug exposure during pregnancy.

This project was developed to investigate the usefulness of newborn nails for monitoring in utero drug exposure. Cocaine, benzoylecgonine, morphine, methadone, caffeine, nicotine, and cotinine were determined in nail samples from the first 3 months of life of 25 newborns abandoned immediately after birth (group 1) and of 33 babies born at the local maternity hospital whose families were recruited on a voluntary basis (group 2). All substances were measured by gas chromatography-mass spectrometry (detection limit: 0.025 ng/mg). Moreover, analytical results were compared with mothers self-reported habits when the information was available. In group 1, 12 nails were found positive for caffeine and 13 for both nicotine and cotinine. Six samples tested cocaine- (range, median: 0.14-0.25, 0.175 ng/mg) and benzoylecgonine-positive (range, median: 0.12-0.20, 0.165 ng/mg). Both nicotine and cocaine were always retrieved together with their main metabolite. Morphine was found in four samples (range, median: 0.10-0.15, 0.125 ng/mg), methadone in five samples (range, median: 0.12-0.26, 0.170 ng/mg) that were found negative for all other compounds. In group 2, two samples tested positive for methadone (0.16, 0.17 ng/mg). The mothers self-report of the use of coffee always corresponded to caffeine positivity in the newborn nails (n=6), whereas six samples tested positive for nicotine and/or cotinine with a non-smoking mother. Sixteen out of the 33 samples of group 2 tested negative for all compounds. In conclusion, for the first time, results showed that, once that sample collection problems are solved, nails of the first period of life can be a very interesting indicator of in utero drug exposure.

Determination of fatty acid ethyl esters in hair by GC–MS and application in a population of cocaine users

A gas chromatography-mass spectrometry method for the determination of ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate in hair samples was developed, validated and applied to real samples. Ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate are fatty acid ethyl esters (FAEE) which are known to be direct biotransformation products of ethanol. Their presence in the body fluids and tissue is therefore indicative of alcohol intake and, in particular, FAEE concentration in hair higher than 0.5 ng/mg is indicative of excessive chronic alcohol consumption. The method was applied to 80 hair samples formerly found positive for cocaine and FAEE analytical results were compared with the presence of cocaethylene, a cocaine metabolite formed only when alcohol and cocaine are used together. According to our data the two biomarkers (FAEE and cocaethylene in hair) are tools of great value in the assessment of the diagnosis of use of cocaine and ethanol. In fact, discrepancies were noted and might be related to various factors including differences in consumption habits and thus permitting to distinguish the use of both substances non-concurrently or concurrently. Also, the determination of both markers may, in some cases, discriminate the use of moderate or heavy alcohol amounts when associated with cocaine. Finally, in a population of non-cocaine-users our results support FAEE as valuable means in the assessment of excessive alcohol chronic use.

What constitutes a normal ante-mortem urine GHB concentration?

Gamma-hydroxybutyric acid (GHB) is endogenously produced within the central nervous system, however it is also used as a medication for the treatment of a variety of clinical conditions, sold under the name Zyrem in the United States and Alcover in Europe. It is a very dangerous drug with a very limited safety margin, and is classified as a controlled substance in many countries. The interpretation of post-mortem studies of GHB concentrations is problematic; GHB can be detected in urine and blood from non-GHB users, both before and after death, and concentrations in both matrices may rise with prolonged storage. Because it is produced as a post-mortem artifact, forensically defensible cut-offs for post-mortem blood concentrations have yet to be established. Given the enormous degree of inter and intra-individual variation in GHB production that has been documented, it is unlikely they ever will. The important issue for forensic scientists is whether the detection of GHB in urine, in concentrations above some yet to be determined value, can be used as evidence for drug facilitated assault. In an attempt to see if a cut-off level could be determined we analyzed urine from 39 alcoholics who were being treated with known oral doses of Alcover (group 1), and compared the results with concentrations found in the urine of 30 volunteers who had no exogenous GHB intake (group 2), and 30 urine specimens taken from the alcoholics before they initiated GHB therapy (Alcover treatment group 3). More than one third (36.6%) of subjects being treated with GHB were found to have urinary GHB concentration that fell between 2.75 and 10 microg/mL. The data suggests that caution must be used when applying the currently used cut-off of 10 microg/mL.