Development and Validation of a Method for the Detection and Quantification of Antidepressants and Antipsychotics in Bone Samples by GC-MS.

Bone marrow is the tissue contained inside the bones and can be considered one of the potential alternative tissues in forensic toxicology. This matrix could be particularly useful in those cases where the routine sample is not available due to an advanced state of decomposition or skeletonization of the corpse. The aim of this study was to develop, validate and apply an analytical method of extraction and analysis of different antidepressants and antipsychotics, commonly used in therapy, from spiked pig ribs. Pig ribs, each of 5 g and 5 cm long, were spiked at three concentration levels (100, 200 and 500 ng/g). For each concentration, 10 pig ribs were prepared. The method involves the leaching by ethanol at different pHs of nine drugs from the inside of ribs, in particular from the bone marrow, without the fragmentation of bone tissue. Following a liquid-liquid purification and extraction, analysis was performed by gas chromatography coupled with mass spectrometry in selected ion monitoring mode. For method validation was assessed linearity, sensitivity, precision and accuracy, matrix interferences and, finally, carryover. Analytical method performance was acceptable respect to acceptance criteria for validation. No matrix interferences were detected; for this reason, it is possible to affirm that this method has a good selectivity. Moreover, the method was not affected by carryover. Considering that the study conducted on pig ribs has given encouraging results, it can be assumed that this method can be used in forensic toxicological protocols (human autopsy cases) as alternative to classic procedures.

Toxicological investigations in a fatal and non-fatal accident due to hydrogen sulphide (H2S) poisoning.

Hydrogen sulphide (H2S) is one of the most toxic natural gas and represents a not rare cause of fatal events in workplaces. We report here a serious accidental poisoning by hydrogen sulphide inhalation involving six sailors. Three of them died while the other three survived and were transported to the emergency room. No greenish discolouration of the body, that could be a feature of these type of deaths, was observed at autopsy. Given that blood and/or urine H2S detection does not allow to discriminate if it is related to inhalation or to putrefactive processes, the determination of thiosulphate, H2S main metabolite, is decisive. The succession of fatal events reported here can be rebuilt by toxicological data interpretation: the subject 1 died after a longer interval of time as demonstrated by the highest blood and urine thiosulfate concentrations; the subject 2 died after a short interval of time as showed by a lower blood and urine thiosulfate concentrations than subject 1; the subject 3 died almost immediately after H2S inhalation since he showed the lowest blood thiosulfate concentration, and no trace of sulphide and thiosulfate was found in the urine.

Evaluation of the Role of Toxicological Data in Discriminating Between H2 S Femoral Blood Concentration Secondary to Lethal poisoning and Endogenous H2 S Putrefactive Production.

Hydrogen sulfide is a colorless gas and has a strong odor of rotten eggs. It is absorbed by the upper respiratory tract mucosa, and it causes histotoxic hypoxemia and respiratory depression by exerting an inhibitory effect on cytochrome oxidase. To evaluate the role of toxicological data in distinguishing between the H2 S blood concentration secondary to lethal poisoning and the endogenous H2 S produced during putrefaction, we compared the postmortem H2 S concentrations of six fatal H2 S poisoning cases (8.7-28.6 mg/L) with the postmortem concentrations of endogenous H2 S of 12 subjects who died from other causes (traffic-related deaths) (2.2-32.7 mg/L). These results will be of interest to the forensic community as it underlines the importance of considering circumstantial evidence along with the toxicological and pathological findings in the identification of H2 S lethal poisoning.

The therapeutic use of cannabinoids: Forensic aspects.

UNLABELLED: Since 2013 in the Italian market has been introduced the Nabiximols, a drug containing two of the main active cannabinoids: Δ(9)-tetrahydrocannabinol (Δ(9)-THC) and cannabidiol (CBD). This drug has been approved in Italy in the treatment of Multiple Sclerosis (MS). It is an oral spray formulation and each puff of 100µl contains 2.7mg of Δ(9)-THC and 2.5mg of CBD. In the present study we analyzed urine and blood samples collected from a group of 20 patients treated with Nabiximols in order to evaluate: blood Δ(9)-THC concentrations in relation to the dose administered and the duration of treatment and the potentiality of this medication to be used for drug habit. METHODS: The study was conducted on a sample group of patients affected by MS, of both sexes, age: 49-61 years, treated with Nabiximols for short (28 days) or long-term. The results of our study allow affirming that it is unlikely to use this medication for drug habit or to sale it in the black market because of the low blood concentrations available and of its high costs. These statements were confirmed by: (a) the low Δ(9)-THC concentrations in the pharmaceutical formulation; (b) the low blood concentrations produced by Nabiximols administration, more than 10 times smaller than the blood concentrations known to produce psychotropic effects; (c) the presence of CBD (Δ(9)-THC natural antagonist); (d) the route of administration (inhaled, not smoked).

Domino effect: An unusual case of six fatal hydrogen sulfide poisonings in quick succession.

Hydrogen sulfide (H2S) is one of the most serious toxic gases encountered in forensic practice. Aside from being a by-product of many industrial processes, this gas is naturally produced during the putrefaction of organic substances. We report six autopsy cases of fatal H2S poisonings from inhalation of H2S gas after an occupational accident. These six men died during the unblocking of a wastewater cistern. The first worker died shortly after clearing the obstruction, the other five died, one by one, as they attempted to help their colleagues. The macroscopic and histological findings are discussed here to provide useful information for future cases. Greenish discoloration of the skin and of internal organs (liver, trachea, esophagus, stomach) was observed, and one case showed signs typical of drowning. We present a very unusual incident, complete with rare photographs and toxicological analysis. In these cases, based on both macroscopic and microscopic findings, the cause of death was most likely an inhibitory effect on cellular cytochrome oxidase causing respiratory failure.

Levamisole-adulterated cocaine: Two fatal case reports and evaluation of possible cocaine toxicity potentiation.

Levamisole has been identified as a cocaine adulterant in the United States since 2002. Although there is a variation in the percentage of levamisole in cocaine samples between European countries, measurement of levamisole in human samples of cocaine users has become increasingly important. To our best knowledge, only five deaths are reported (one twice) as a result of complications secondary to levamisole-tainted cocaine and none of these cases reports the post-mortem levamisole concentration. In this article, we present the post-mortem levamisole concentrations in fluids and tissues in two young cocaine users, dead after levamisole-adulterated cocaine intake. With the dearth of levamisole reported concentrations in literature, this particular report is of interest to the forensic toxicological and pathological communities. This article aims to be a supplementary alert to aware the risk that may occur using levamisole-adulterated cocaine and an incentive to publication of toxicity reports and new researches involving the combination of levamisole and cocaine.

A fatal accidental subarachnoid injection of lidocaine and levobupivacaine during a lumbar paravertebral block.

Paravertebral block (PVB) is the technique of injecting a local anesthetic solution alongside the vertebral column, close to where the spinal nerves emerge, resulting in unilateral somatic and sympathetic nerve blockade. Here is reported a fatal case involving a 60-year-old woman with spondylitis arthropathy, who developed cardiac and respiratory arrest 40min after receiving an accidental subarachnoid injection (L5-S1 bilaterally) of depomedrol lidocaine and levobupivacaine. A complete autopsy including histological and toxicological analyses was performed in order to establish the cause of death. Liquid/liquid extraction (LLE) and GC-MS analysis were performed according to a previously published method. Lidocaine and bupivacaine were detected both in blood, at concentrations of 14.8mg/L and 13.3mg/L respectively, and in cerebrospinal fluid (CSF) at concentrations of 287.1mg/L and 464.2mg/L respectively. Both lidocaine and bupivacaine were also detected in the urine. The toxicological findings along with the autopsy allowed us to establish that the accidental subarachnoid injection of lidocaine and levobupivacaine had led to a progressive hypotension and normovolaemic shock caused by a severe ganglionic block, determining the patient's death.

Determination of GHB in human hair by HPLC-MS/MS: Development and validation of a method and application to a study group and three possible single exposure cases.

Gamma-hydroxybutyrate (GHB) over the last two decades has generated increased notoriety as a euphoric and disinhibiting drug of abuse in cases of drug-related sexual assault and for this reason it is considered a 'date rape' drug. The first aim of this paper was to develop and fully validate a method for the detection of GHB in human hair by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after liquid-liquid extraction (LLE). The second aim was the application of the method to hair samples of 30 GHB-free users in order to determine the basal level. The results obtained showed no significant differences in endogenous concentrations (p = 0.556) between hair samples of the three groups (black, blonde, and dyed hair) and the age and sex of the subjects did not affect the endogenous levels. Another 12 healthy volunteers, with no previous history of GHB use, were selected and a single dose (25 mg/Kg) was orally administered to all of them and hair samples were collected before the administration of the single dose and other two samples were collected one month and two months later, respectively. The segmental analysis of the latter two samples allowed us to calculate two ratios: 4.45:1 (95% C.I. 3.52-5.63) and 3.35:1 (95% C.I. 2.14-5.18), respectively, which can be recommended as reasonable values for a positive identification of GHB intake. Finally the method was applied to three real cases where a GHB single exposure probably occurred.

A singular case of survival after acute methanol poisoning: toxicological and neuroimaging findings.

Acute methanol poisoning is a relatively uncommon and dangerous form of intoxication. It generally occurs after suicidal or accidental events and can be potentially fatal if not diagnosed and treated promptly. Here reported is the case of a 52-year-old Romanian man who survived acute methanol intoxication. Therefore, it was possible to monitor the clinical evolution, the arterial blood gas assay and toxicological research of methanol in blood and urine, as well as the brain damage by computed tomography and magnetic resonance imaging during a period of 20 days after the intake.

Post mortem concentrations of endogenous gamma hydroxybutyric acid (GHB) and in vitro formation in stored blood and urine samples.

Gamma-hydroxybutyrate (GHB) is a central nervous system depressant, primarily used as a recreational drug of abuse with numerous names. It has also been involved in various instances of drug-facilitated sexual assault due to its potential incapacitating effects. The first aim of this paper is to measure the post-mortem concentration of endogenous GHB in whole blood and urine samples of 30 GHB free-users, who have been divided according to the post-mortem interval (PMI) in three groups (first group: 24-36h; second group: 37-72h; third group: 73-192h), trying to evaluate the role of PMI in affecting post mortem levels. Second, the Authors have evaluated the new formation of GHB in vitro in blood and urine samples of the three groups, which have been stored at -20°C, 4°C and 20°C over a period of one month. The concentrations were measured by GC-MS after liquid-liquid extraction according to the method validated and published by Elliot (For. Sci. Int., 2003). For urine samples, GHB concentrations were creatinine-normalized. In the first group the GHB mean concentration measured after autopsy was: 2.14mg/L (range 0.54-3.21mg/L) in blood and 3.90mg/g (range 0.60-4.81mg/g) in urine; in the second group it was: 5.13mg/L (range 1.11-9.60mg/L) in blood and 3.93mg/g (range 0.91-7.25mg/g) in urine; in the third group it was: 11.8mg/L (range 3.95-24.12mg/L) in blood and 9.83mg/g (range 3.67-21.90mg/g) in urine. The results obtained in blood and urine samples showed a statistically significant difference among groups (p<0.001) in the first analysis performed immediately after autopsy. Throughout the period of investigation up to 4 weeks, the comparison of storage temperatures within each group showed in blood and urine samples a mean difference at 20°C compared to -20°C not statistically significant at the 10% level. These findings allow us to affirm that the PMI strongly affects the post mortem production of GHB in blood and urine samples. Regarding the new formation of GHB in vitro both in blood and urine samples of the three groups, which have been stored at -20°C, 4°C and 20°C over a period of one month, although there was no significant increases of GHB levels throughout the period of investigation, the lowest increases were found both in blood and urine at -20°C, therefore we recommend the latter as optimal storage temperature.

A suicidal poisoning due to tramadol. A metabolic approach to death investigation.

Tramadol is a synthetic opioid, widely used for post-surgical and chronic pain. Lethal overdose due only to tramadol is not common; more often the poisoning is due to tramadol in combination with other substances. Reported is a suicidal case of lethal tramadol poisoning in a 48-year-old woman. Tramadol and its metabolites O-desmethyltramadol (M1), N-desmethyltramadol (M2), N,N-didesmethyltramadol (M3), N,O-didesmethyltramadol (M5) were detected by GC/MS in biological fluids (femoral blood, bile, urine, gastric content) and viscera (brain, lung, liver and kidney). The tramadol concentration in femoral blood was 61.83 mcg/ml which is approximately 30 times higher than that believed to be lethal. According with other Authors, a preferential formation of M1 over M2 (M1/M2 ratio >1) is indicative of acute death, while M1/M2 ratio <1 suggests that death occurred after a longer time lapse from ingestion.

The pathogenetic role of adulterants in 5 cases of drug addicts with a fatal outcome.

The purpose of the present study is to determine the role of lidocaine, caffeine and dextromethorphan, used as adulterant substances, in five cases of drug overdose which have come to our attention. Taking into account the pharmacological mechanism, blood concentration and route of administration (intravenous) we evaluated the hypothesis that these substances could act with a synergistic effect - or at least additive - with the illicit drugs on the central nervous system and cardiovascular system.

Determination of drugs of abuse in hair: evaluation of external heroin contamination and risk of false positives.

One of the most controversial point regarding the validity of hair testing is the risk of false positive due to external contamination. The aim of our experience is to verify if a 5 consecutive days contamination with a small amount of a powdered mixture of heroin hydrochloride and acetylcodeine hydrochloride (10:1 w/w) will last sufficiently long to make a contaminated subject indistinguishable from active users, and if normal washing practices together with the decontamination procedure are sufficient to completely remove the external contamination. Our results suggest that decontamination procedures are not sufficient to remove drugs penetrated into hair from external source. In fact, all contaminated subjects were positive for opiates (heroin, 6-MAM, morphine, acetylcodeine and codeine) for at least 3 months. Significant 6-MAM concentrations (>0.5 ng/mg) were found in each subject until 6th week. Further, 6-MAM/morphine ratio were always above 1.3.

Lethal diltiazem poisoning.

A 60-year-old man presented to an emergency department 2 h after the ingestion of 8 g of diltiazem (about 40 slow-release capsules, 200 mg/each) in a suicide attempt. The subject was treated with a gastric lavage and activated charcoal; then, a temporary transvenous pacing was also inserted. Despite emergency pharmacological treatment, the subject died about 20 h after ingestion. Postmortem diltiazem and desacetyl-diltiazem concentrations, measured by gas chromatography-mass spectrometry, were as follows: 31.1 mg/mL diltiazem and 9.7 mg/mL desacetyl-diltiazem in blood; 33.1 mg/g diltiazem and 13.7 mg/g desacetyl-diltiazem in brain; 179.5 mg/g diltiazem and 47.5 mg/g desacetyl-diltiazem in lung; 41.8 mg/g diltiazem and 10.1 mg/g desacetyl-diltiazem in heart; 182.1 mg/g diltiazem and 47.3 mg/g desacetyl-diltiazem in liver; 49.2 mg/g diltiazem and 22.6 mg/g desacetyl-diltiazem in kidney; and 294.9 mg/mL diltiazem and 29.4 mg/mL desacetyl-diltiazem in bile. It is interesting to note that although several cases of acute diltiazem poisoning have been reported in literature, only a few were lethal. Diltiazem concentrations found in our case are notably higher than those reported in other studies, including those in which diltiazem ingestion resulted in the death of the patient. Notably, in many of these latter cases, the doses of diltiazem ingested were higher than those taken by our patient.