A new clean-up approach for stomach content toxicological analysis.

Stomach content is a matrix often applied in post-mortem cases. It is especially important in oral intoxication cases. The main advantages are the usually high concentration of analytes and the lack of biotransformation process. Still, even with extensive sample preparation, the final extract is not always suitable for analysis. The aim of this study was to develop an alternative method using QuEChERS for the extraction of drugs and pesticides from postmortem stomach content. Sample preparation started acetonitrile with 1% of acetic acid and QuEChERS salts. Later, the initial extract was cleaned-up using the EMR-Lipid sorbent. Residual water was withdrawn with MgSO4/NaCl in the third step and a final step with MgSO4. Vigorous shaken and centrifugation was performed after each step. The final supernatant was evaporated, re-suspended, and injected into GC-MS in full scan mode. This approach was successfully applied to stomach content, resulting in clean extracts, with low lipid levels. The method was able to detected target drugs and pesticides (cocaine, tramadol, diazepam, amytriptiline, phenobarbital, prochloraz, diazinon, heptachlor, permethrin, malathion and carbaryl) at the limit of detection of 0.1 mg/g or 0.1 mg/L. Recovery was over 70% for the majority of analytes, precision and accuracy was within acceptable range. The method was also applied to real forensic cases and carbofuran, terbuphos and fluoxetine was detected likewise. This work demonstrates that this method can provide an effective clean-up in high lipids samples such as stomach content, and can be used to analyze of pesticides and drugs in forensic cases.

Behavioural, biochemical and molecular changes induced by chronic crack-cocaine inhalation in mice: The role of dopaminergic and endocannabinoid systems in the prefrontal cortex.

Crack-cocaine addiction has increasingly become a public health problem worldwide, especially in developing countries. However, no studies have focused on neurobiological mechanisms underlying the severe addiction produced by this drug, which seems to differ from powder cocaine in many aspects. This study investigated behavioural, biochemical and molecular changes in mice inhaling crack-cocaine, focusing on dopaminergic and endocannabinoid systems in the prefrontal cortex. Mice were submitted to two inhalation sessions of crack-cocaine a day (crack-cocaine group) during 11 days, meanwhile the control group had no access to the drug. We found that the crack-cocaine group exhibited hyperlocomotion and a peculiar jumping behaviour ("escape jumping"). Blood collected right after the last inhalation session revealed that the anhydroecgonine methyl ester (AEME), a specific metabolite of cocaine pyrolysis, was much more concentrated than cocaine itself in the crack-cocaine group. Most genes related to the endocannabinoid system, CB1 receptor and cannabinoid degradation enzymes were downregulated after 11-day crack-cocaine exposition. These changes may have decreased dopamine and its metabolites levels, which in turn may be related with the extreme upregulation of dopamine receptors and tyrosine hydroxylase observed in the prefrontal cortex of these animals. Our data suggest that after 11 days of crack-cocaine exposure, neuroadaptive changes towards downregulation of reinforcing mechanisms may have taken place as a result of neurochemical changes observed on dopaminergic and endocannabinoid systems. Successive changes like these have never been described in cocaine hydrochloride models before, probably because AEME is only produced by cocaine pyrolysis and this metabolite may underlie the more aggressive pattern of addiction induced by crack-cocaine.