Marta Cippitelli

Unexpected Variation of the Codeine/Morphine Ratio Following Fatal Heroin Overdose

Postmortem samples from 14 cases of suspected heroin overdose were subjected to a preliminary systematic toxicological analysis in order to highlight the presence of unknown exogenous compounds (e.g., drugs of abuse, alcohol) that may have played a role in the mechanism of death. This analysis unveiled histories of poly-drug use in seven of the cases under investigation. Moreover, the concentrations of morphine and codeine in the brain were also investigated, and the results were compared with the data obtained from the blood specimens. The concentration of morphine in blood ranged from 33 to 688 ng/mL, while the concentration of codeine ranged from 0 to 193 ng/mL. However, in the brain, the concentration of morphine was found to be between 85 and 396 ng/g, while the levels of codeine ranged from 11 to 160 ng/g. The codeine/morphine ratio in the blood ranged from 0.043 to 0.619; however, in the brain, the same ratio was found to be between 0.129 and 0.552. In most cases, a significantly higher codeine/morphine ratio was found in the brain, suggesting the accumulation of codeine in brain tissue due its high lipophilicity as compared with morphine.

Hair analysis in order to evaluate drug abuse in driver's license regranting procedures.

In Italy, driving under the influence of drugs determines the suspension of the offenders drivers license. To regain the license the person must be drug free during an observation period. People whose license has been revoked or suspended can obtain, or re-obtain their drivers license subject to the judgment of a medical commission. The exclusion of illicit drug use is determined by means of toxicological analysis, mainly on urine or hair matrices. We reported the results of several years of experience of the forensic toxicology laboratory of the University of Macerata in the use of hair analysis for the assessment of past exposure to drugs in people suspected of driving under the influence of drugs. From 2004 to 2013, 8612 hair samples, were analyzed for opiates, cocaine and delta-9-tetrahydrocannabinol (Δ(9)-THC) using gas chromatography/mass spectrometry (GC/MS) method. We used a cutoff (SoHT or national guidelines) to determine the positive data, regardless of the hair sample concentrations. 1213 samples resulted positive, 71.7% were positive for cocaine and metabolites, 19.8% for morphine and metabolites, 8.5% for Δ(9)-THC. We also studied the timeframe of the abuse, as well as gender and age distribution of positive subjects. Moreover, we analyzed the possible deterrent effect of the hair analysis on driving under the influence of psychoactive substances.

Detection of Cannabinoids by ELISA and GC–MS Methods in a Hair Sample Previously Used to Detect Other Drugs of Abuse

A forensic standard procedure is described that combines enzyme-linked immunoassay for screening and gas chromatography-mass spectrometry (GC-MS) for confirmation to detect drugs of abuse in a sample before used to detect opioids and cocaine. We used two equal aliquots of the same previously selected cannabinoid positive hair samples, one of which was subjected to acid hydrolysis. Afterward, both the aliquots were subjected to basic extraction and then to immunoassay screening. After derivatization, the GC-MS parameters were the same for both the aliquots for the determination of the cannabinoids (Δ(9)-tetrahydrocannabinol, cannabidiol and cannabinol). The results show that there were no statistical differences between the nonpreviously treated and the pretreated hair samples for the quantification of the three cannabis products for immunochemical procedure. No differences between the two groups were shown as for GC-MS confirmation procedures. All substances showed a good linearity between 0.05 and 2 ng/mg. The limit of detection ranged from 0.02 to 0.03 ng/mg, and the limit of quantification was 0.05 ng/mg for all substances. To our knowledge, this is the first time that screening and confirmation procedures have been applied on the same sample of hair to detect more than one drug of abuse.

Determination of 6-Monoacetyl-Morphine (6-MAM) in Brain Samples from Heroin Fatalities

Objective: Post-mortem brain samples from 15 deceased patients whose death was heroin related, were analyzed to determine 6-monoacetyl-morphine (6-MAM) concentrations. The samples belonged to people died between 2008 and 2014. The first eight samples were also analyzed in 2012 to determine only morphine and codeine levels. Method: A GC/MS method was studied in order to enhance sensitivity, thus helping the determination of 6-MAM whose detection is in most cases difficult because of the complexity of the biological matrix. The analytical method was validated using deuterated internal standards (IS-D3, morphine-D3 and codeine-D3) and it showed adequate specificity, linearity, LOD, LOQ precision and accuracy for the determination of the analyte of interest. Results: 6-MAM was evidenced only in the more recent samples, thus pointing out its low stability. Its concentration ranged from 15.6 to 28.9 ng/g. Morphine and codeine was also determined and a comparison was carried out between the blood and the brain levels of the three analytes. Moreover a parallel was established between the concentrations of morphine and codeine found in the brain in 2012 and 2015. Conclusion: 6-MAM determination in the brain is particularly important when discriminating between morphine assumption and heroin abuse. In fact in the cases in which it is not detectable in the blood it can be present in the brain. It was noticed that the concentrations of morphine found in the brain in 2015 are higher respect to the levels of 2012; a possible explanation could be that 6-MAM originally present in the brain has hydrolyzed to morphine, thus increasing its levels.

Driving under the effect of drugs: Hair analysis in order to evaluate recidivism.

Under Italian law drug addiction and regular drug abuse are incompatible with driving ability. One important problem with the enforcement of the impaired driving law is the large number of people that re-offend. To regain their license, offenders must be drug-free for the duration of an observation period, according to the judgement of a medical commission. The exclusion of illicit drug use is determined by toxicological analysis. A few studies exist that have used a hair matrix to monitor recidivism. Hair is an attractive matrix for monitoring drug recidivism, due to the large time window for drug detection, and to the non-alterability of this matrix. We report the results of several years of experience at our forensic toxicology laboratory in the use of hair analysis for the assessment of past exposure to drugs in persons suspected of driving under the influence of drugs. 5592 subjects were analyzed for opiates, cocaine and delta-9-tetrahydrocannabinol (Δ9-THC) using a GC/MS method. 1062 (19.0%) subjects resulted positive. From this group, the individuals that resulted positive at least at the second control were considered recidivists (243, 22.9%). 79.7% of recidivist subjects were positive for cocaine and metabolites, 14.9% for morphine and metabolites, 5.4% for Δ9-THC. We also studied the time frame of the abuse, as well as gender and age distribution of recidivist subjects. Furthermore, we analyzed risk factors associated with recidivist behaviour. Our results show that cocaine consumption was the only factor that showed significance with regard to increased likelihood of being a recidivist.

Toxicological Analysis of Opiates from Alternative Matrices Collected from an Exhumed Body

In this case study, the body of a 45‐year‐old man was exhumed after 1 year at the request of the public prosecutor to assess whether the death was caused by drug consumption. Toxicological analyses were performed on several matrices, including liver, kidney, and the alternative matrices hair and teeth. The systematic toxicological analysis (STA), which consisted of basic and acid liquid/liquid extraction and gas chromatography–mass spectrometry (GC‐MS) analysis, showed the presence of opiates in each of the matrices analyzed. Subsequently, to confirm and quantify the presence of opioids, samples of each of the matrices were subjected to solid‐phase extraction and specific GC‐MS analysis. The case presented demonstrates the possibility of drug detection in an exhumed body that has been buried for 1 year, despite the problems of quantitative interpretation of the data, and that toxicological results could be useful along with other forensic evidence.

Comparison Between Lc/Uv and Gc/Fid Techniques in Determining N,NDimethylacetamide(Dma) in Diacerein

Objectives: The aim of this work was the quantitative determination of N,N-Dimethylacetamide (DMA) as crystallization solvent in samples of Diacerein. DMA is commonly used as a solvent in the chemical, agricultural and pharmaceutical industries. However, in order to ensure product quality and to protect patients from the potentially toxic properties, the substances used as active ingredients in therapeutic drugs should not contain high levels of residual solvents. Methods: LC is commonly used in the pharmaceutical industry to check DMA in pharmaceutical products, but in this work we were interested in validating and comparing LC/UV and GC/FID techniques for determining the presence of DMA in Diacerein Results: Both methods showed good linearity, precision and accuracy with comparable LOD and LOQ. Conclusion: The GC method, however, since it uses DMSO as an internal standard, has higher analytical versatility, thus allowing the qualitative and quantitative determination of DMA at lower levels than those obtained with LC.

Study of the Ratio between Morphine-3-ò-D-Glucuronide and Morphine-6-ò-DGlucuronidein Blood Samples from Heroin Fatalities

In heroin fatalities the diagnosis of the cause of death, based only on chemical and toxicological data, may be particularly difficult; a complete knowledge of the case history is fundamental. Thus, for each case analytical results should be interpreted taking into account also autopsy findings, information from the scene and relevant medical history. In fact wide variability is present in post-mortem blood concentration of morphine (MOR), the main metabolite of heroin, which is usually the most important analytical result for the interpretation of the cause of death. Recently, increasing interest has grown towards the role of the metabolites morphine-3-β-D-glucuronide (M3G) and morphine-6-β-D-glucuronide (M6G) in mediating heroin effects. To this purpose SPE technique was employed to extract MOR, M3G and M6G from autopsy blood samples. Subsequently a LC/MS-MS method for the determination of these analytes was developed, using a gradient elution with a binary mobile phase, the analytes being revealed by means of an ESI-QqQ Mass Spectrometer operating in positive ionization and MRM mode. After validation, the method was applied to twenty-one blood specimens collected from cases of suspected acute narcotism which previously underwent a Systematic Toxicological Analysis (STA) to highlight the presence of ethanol and of the main drugs of abuse and/or their metabolites. The concentration ratios of MOR, M3G and M6G were investigated. The influence of some risk factors, such as the contemporary use of alcohol, methadone or cocaine, was also studied. Some important indications emerged: the ratio M3G/M6G, being quite constant, should be a valid reference value to assess toxicity. Furthermore the value of M3G/M6G ratio detected in heroin addict, in this study, is lower than that found in people who assume morphine for therapeutic purposes. This ratio could be of help to overcome the difficult interpretation of chemical and toxicological data.