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This paper reviews liquid chromatographic-mass spectrometric (LC-MS) procedures for the screening, identification and quantification of doping agents in urine and other biological samples and devoted to drug testing in sports. Reviewed methods published approximately within the last five years and cited in the PubMed database have been divided into groups using the same classification of the 2004 World Anti-Doping Agency (WADA) Prohibited List. Together with procedures specifically developed for anti-doping analysis, LC-MS applications used in other fields (e.g., therapeutic drug monitoring, clinical and forensic toxicology, and detection of drugs illicitly used in livestock production) have been included when considered as potentially extensible to doping control. Information on the reasons for potential abuse by athletes, on the requirements established by WADA for analysis, and on the WADA rules for the interpretation of analytical findings are provided for the different classes of drugs.

Mirtazapine is a noradrenergic and specific serotoninergic antidepressant agent that stimulates norepinephrine and serotonin release while also blocking serotonin receptors (5-HT2 and 5-HT3). Although the drug is used extensively, at present we do not know of any fatal cases due to mirtazapine alone. On the contrary, the published literature describes several fatal poisoning cases related to the intake of mirtazapine together with other drugs. Here we describe a fatal case of mirtazapine self-poisoning, since the other drug detected (lorazepam), was within the therapeutic range. Analyses were performed by LC-MS/MS on body fluids and a hair sample and mirtazapine concentration measured in blood was very high: 9.3mg/L. N-Desmethylmirtazapine was also quantitated. We then compared our results with those of previously published cases. In conclusion, even though mirtazapine can be considered a relatively safe drug, taking a large amount alone or in combination with other drugs, could lead to death. [ABSTRACT FROM AUTHOR]

Introduction: An in vitro study on authentic positive samples was carried out, aiming the evaluation of the effect of chlorinated water on benzodiazepines in hair. Two subjects exposed to chlorinated water for several consecutive times were also investigated.Method: Seven hair samples collected from autopsy cases, previously tested positive for benzodiazepines, were washed with dichloromethane and methanol. They were longitudinally divided in six aliquots of about 30mg. An aliquot was processed without treatment while other five ones were soaked in chlorinated water (0.1% sodium dichloroisocyanurate and 0,1M sulfuric acid at pH 5.5) for 4, 20, 24 and 30h respectively. Hair samples were then processed following a fully validated and previously published method. Briefly hair samples were sonicated in 600 microliters methanol containing halazepam (IS) up to two hours. Ten microliters were injected in a liquid chromatographic tandem mass spectrometric (LC-MS/MS) system. Analytes were eluted from a C18 reversed-phased column. Two transitions on multiple reaction monitoring and positive ionization mode were monitored for each compound.Result and Discussions: Six compounds among benzodiazepines and metabolites were identified and quantified in the seven hair samples: diazepam (575pg/mg), desmethyldiazepam (562pg/mg), chlordesmethyldiazepam (173pg/mg), desalkylflurazepam (320pg/mg), clonazepam (three cases-195, 119 and 111pg/mg respectively), lormetazepam (two cases-182 and 416pg/mg respectively). Traces of 7-aminoclonazepam were identified into 2 samples. Stability of benzodiazepines in water was evaluated by soaking an aliquot of hair for up to 30h in deionized water. No significant degradation was observed. Samples soaked in chlorinated water showed considerable decreasing from the initial concentration even after the 4-h treatment: the fastest degradation was provided by clonazepam that showed a 61% loss. The greatest loss was measured for diazepam (86% loss after 30-h soaking).Conclusion: To the best of our knowledge this is the first in vitro study that evaluated benzodiazepines stability in hair after prolonged exposure to chlorinated water. The results showed that the longer the exposure the higher the degradation. Prolonged exposure to chlorinated water and sunlight must be always taken into account as possible causes of false negative results. [ABSTRACT FROM AUTHOR]

A multi-analyte method for detection and quantification of 16 synthetic cathinones (known also as "bath salts") in human hair has been developed and fully validated using liquid chromatography-tandem mass spectrometry system. About 20 mg of hair samples, previously washed and homogenized, were ultrasonicated with 1 mL HCl 0.1 M solution. Samples were then extracted using a solid phase extraction procedure (SPE), taken to dryness and reconstituted in 100 μL mobile phase. Finally, they were directly injected into a liquid chromatographic system, coupled with tandem mass spectrometer detector. The validation criteria parameters were satisfactory according with the international guidelines. A LOQ of 5 pg/mg was obtained for 4-fluoromethcathinone (4-FMC), buphedrone, ethcathinone, methcathinone, mephedrone and naphyrone, while the method proved to be more sensitive for 4-methylethcathinone (4-MEC), methedrone, alpha-pyrrolidinopentiophenone (α-PVP), alpha-pyrrolidinohexiophenone (α-PHP), methylenedioxypyrovalerone (MDPV), butylone, ethylone, 3,4-dimethylmethcathinone (3,4-DMMC), pentedrone and pentylone, reaching a LOQ of 1 pg/mg. Potential use of bath salts was investigated in postmortem cases of young subjects previously tested positive at least to one traditional drug of abuse. Two samples out of 17 cases analyzed provided positive results for synthetic cathinones. One sample has been divided in two segments of 2.5 cm length each. Both segments were positive for 8 different cathinone derivatives, namely: 3,4-DMMC, 4-FMC, 4-MEC, α-PHP, α-PVP, methcathinone, methedrone and pentedrone. The second case provided positive results for ethcathinone. [ABSTRACT FROM AUTHOR]

Over the last 10 years we have registered in our district (about 500,000 inhabitants) 36 cases of fatal methadone poisoning, involving both patients on treatment and naive subjects: this is a significant increase of deaths due to methadone use, misuse or abuse compared with previous years. Twenty-four patients (66.7%) were on methadone maintenance programs for heroin detoxification, while 12 (33.3%) were taking the drug without a medical prescription. The average blood concentration of methadone in patients undergoing a maintenance program was 1.06 mg/L (0.21-3.37 mg/L), against 0.79 mg/L (0.2-3.15 mg/L) in those taking the non-prescribed drug. Since 111 heroin-related deaths were recorded in our district in the same period, the fact that there appear to be many methadone deaths (about a third of heroin-related deaths) cannot be overlooked. The aim of this work is to understand the possible reasons for such a large number of methadone-related deaths. On this subject, we have noticed that risks associated with methadone intake are often underestimated by clinicians prescribing the drug: sometimes methadone is prescribed without taking into account patient's tolerance to opiates, and a large number of subjects enrolled in methadone maintenance programs in Italy, have also been given take-home doses, thus increasing the risk of abuse and diversion. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: 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 D5-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. [Copyright &y& Elsevier]

A teenager male was found dead in a waterway after he was spotted jumping off into the water stream. The boy looked agitated and confused after a party with friends. At the gathering place, investigators seized packages of blotter papers. A complete autopsy and a histological evaluation of the main tissues were performed; although the death occurred by drowning, the prosecutor requested toxicological exams, in order to evaluate the potential role of drugs of abuse in the episode. Blood (both peripheral and central) and urine samples as well as seized blotter papers were collected and analyzed as follows. The blotter paper, analyzed through a GC-MS method, revealed the presence of 25-NBOMes. A liquid chromatography tandem mass spectrometric (LC-MS/MS) system was used to identify and quantify 5 different 25-NBOMes (namely 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25H-NBOMe, 25I-NBOMe) in blood and urine. 25E-NBOMe was used as internal standard (IS). 1mL of urine and 1mL of blood (both peripheral and cardiac) were diluted in 2mL phosphate buffer at pH 6.0, containing IS and purified on a solid phase extraction (SPE) cartridge. LOD and LOQ for the five 25-NBOMes were calculated at 0.05 and 0.1ng/mL respectively. Linearity, accuracy, precision, ion suppression, carry over and recovery were tested and all parameters fulfilled the acceptance criteria. Blood and urine provided positive results for 25C-NBOMe and 25H-NBOMe. Eventually, the seized blotter papers were analyzed by means of LC-MS/MS and the presence of the two NBOMes was confirmed: 25C-NBOMe and 25H-NBOMe were measured at the concentration of 2.80 and 0.29ng/mL in peripheral blood, of 1.43 and 0.13ng/mL in central blood and of 0.94 and 0.14ng/mL in urine, respectively. THC and THCCOOH were also detected in biological fluids, at the concentration of 15.5 and 56.0ng/mL in peripheral blood, 9.9 and 8.5ng/mL in central blood, respectively. NBOMes can produce severe hallucination even at very low doses, and the 25C-NBOMe levels measured in the subject's blood are considered potentially toxic. [ABSTRACT FROM AUTHOR]