Stability of benzodiazepines in hair after prolonged exposure to chlorinated water.

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.

Methadone-related deaths. A ten year overview.

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.

A case of suicide by ingestion of caffeine.

Intentional or unintentional caffeine abuse due to excessive intake of beverages or energy drinks containing caffeine is relatively frequent. However, death due to caffeine intoxication is rare and case reports of fatalities from caffeine toxicity are relatively infrequent. In this report, we describe an autopsy case involving a 31 year-old man who intentionally took a large amount of caffeine tablets in the form of a weight loss supplement as part of a suicide plan. Caffeine femoral blood concentration (170 mg/l) was within the toxic and potentially lethal ranges reported in the literature in similar cases. Postmortem biochemistry results suggested depressed glomerular filtration rate and pre-renal failure at the time of death but failed to reveal myoglobinuria, glycosuria, ketonuria or ketonemia. Based on the absence of pathological findings at autopsy and the high blood caffeine level, death was attributed to acute caffeine toxicity. The case emphasizes the usefulness of performing exhaustive toxicology and searching for all potentially relevant information in order to formulate appropriate hypotheses concerning the cause and manner of death.

Fatal metformin overdose: case report and postmortem biochemistry contribution.

Metformin is an oral antihyperglycemic agent used in the management of type 2 diabetes mellitus. Lactic acidosis from metformin overdose is a rare complication of metformin therapy and occurs infrequently with therapeutic use. Fatal cases, both accidental and intentional, are extremely rare in clinical practice. Metformin is eliminated by the kidneys, and impaired renal function can result in an increased plasma concentration of the drug. In this report, we describe an autopsy case involving a 70-year-old woman suffering from diabetes mellitus and impaired renal function who received metformin treatment. Metformin concentrations in the peripheral blood collected during hospitalization and femoral blood collected during autopsy were 42 and 47.3 µg/ml, respectively. Lactic acidosis (29.10 mmol/l) was objectified during hospitalization. Furthermore, postmortem biochemistry allowed ketoacidosis to be diagnosed (blood ß-hydroxybutyrate, 10,500 µmol/l). Death was attributed to lactic acidosis due to metformin intoxication. Increased plasma concentrations of the drug were attributed to severely impaired renal function. The case emphasizes the usefulness of performing exhaustive toxicology and postmortem biochemistry towards the more complete understanding of the pathophysiological mechanisms that may be involved in the death process.

Workplace drug testing in Italy - critical considerations.

Workplace drug testing (WDT) was established in Italy on 30 October 2007. Two tiers of survey are required: the first tier concerns drug testing on urine samples, the second involves both urine and hair analysis. Between July 2008 and December 2011, 10 598 workers' urine samples and 72 hair samples for opiates, cocaine, cannabinoids, amphetamines, methylenedioxyamphetamines, methadone, and buprenorphine were tested in our laboratory. Urine analyses were performed by immunological screening (EMIT); hair analysis and confirmation tests in urine were performed by gas chromatography-mass spectrometry (GC-MS). Employees tested positive in urine for drugs of abuse numbered 2.8% in 2008, 2.03% in 2009, 1.62% in 2010, and 1.43% in 2011. As regards the second level of analysis, we observed that only one-third of the workers who had been tested positive for drugs of abuse were referred to an Addiction Treatment Unit in order to verify drug addiction. Our experience shows that, four years after approval of the law on WDT, the percentage of workers positive for drugs of abuse in urine has reduced in comparison to the first year. Moreover, our data show that most of the times employees who tested positive are tardily referred or not referred at all to a Public Addiction Treatment Unit to verify drug addiction. This makes us believe that the legal provisions are widely disregarded not paying the right tribute to the fact that Italy is one of few European countries with legislation on WDT.

Direct determination of the ethanol metabolites ethyl glucuronide and ethyl sulfate in urine by liquid chromatography/electrospray tandem mass spectrometry.

A liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) method for the determination in urine samples of two ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), was developed and validated. Pentadeuterated EtG was used as internal standard for both EtG and EtS. In addition to the surviving ions, two MS/MS reactions were monitored for each analyte, with the deprotonated molecule as precursor ion: m/z 221 --> 75, m/z 221 --> 85 (EtG), and m/z 125 --> 97, m/z 125 --> 80 (EtS). Sample pretreatment, though very simple and rapid (1:50 water dilution and centrifugation of 50 muL of urine), was found to contain the occurrence of matrix effects. The method was accurate and precise over the linear dynamic range (0.05-10 mg/L). The analytes were stable in frozen urine for at least 1 month. The assay was applied to several authentic urine samples from social drinkers and to alcoholic beverages.