Dimitri Gerostamoulos

Detection and Quantification of New Designer Drugs in Human Blood: Part 1 - Synthetic Cannabinoids

Synthetic cannabinoids sprayed on herbal mixtures have been abused as a new designer drug all over the world since 2004. In 2008, the first compounds, CP 47,497 and JWH-018, were identified as active ingredients in these mixtures. Most of the compounds have been synthesized for research purposes and are potent CB1 and/or CB2 receptor agonists. To investigate the presence of synthetic cannabinoids in blood samples, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed using only 100 µL of blood. After the addition of 0.2 mL of trizma buffer, the blood was extracted using liquid-liquid extraction with 1 mL of 1-chlorobutane containing 10% of isopropanol for 5 min on a shaker at 1,500 rpm. After centrifugation at 12,000 rpm for 1 min, the separated solvent layer was transferred to an autosampler vial and evaporated to dryness under N₂. The residue was reconstituted in methanol and injected into a Shimadzu 8030 LC-MS-MS system to separate and detect 25 synthetic cannabinoids. The method has been validated according to international guidelines and was found to be selective for all tested compounds. Calibration was satisfactory from 0.5-100 ng/mL, and from 5.0-500 ng/mL. for HU-210, CP 47,497 and the CP 47,497 C-8 homolog, respectively. The extraction efficiencies ranged from 30-101% and the matrix effects from 67-112%. Accuracy data were within the acceptance interval of ±15% (±20% at the lower limit of quantification) of the nominal values for all drugs.

Identification and quantification of 30 antipsychotics in blood using LC-MS/MS

Over the last decade, the prescription rates of antipsychotic (AP) drugs have increased worldwide. Studies have shown that the risk of sudden cardiac death is threefold higher among patients treated with APs. To investigate the presence of APs in postmortem cases, a liquid chromatography (LC)-MS/MS method was developed using only 0.1 ml of blood sample with 10 microl of internal standard (IS) (haloperidol-d(4), 1 microg/ml). After the addition of 0.2 ml of Trizma buffer, the blood sample was extracted using liquid-liquid extraction (LLE) with 1 ml of 1-chlorobutane for 5 min on a shaker at 1500 rpm. After centrifugation at 12,000 rpm for 1 min, the separated solvent layer was transferred to an autosampler vial and evaporated to dryness under N(2). The residue was reconstituted in 0.05 ml acetonitrile containing 0.1% formic acid, vortexed for 30 s and an additional 0.45 ml of 50 mmol/l ammonium formate pH 3.5 was added and the sample vortexed; 0.1 ml of the final extract was injected into a Shimadzu Prominence HPLC system, with detection of drugs achieved using an Applied Biosystems 3200 Q-TRAP LC-MS/MS system equipped with a Turbo V ion source [electron spray ionization (ESI), multiple reaction monitoring (MRM) mode]. The method has been validated according to international guidelines and was found to be selective for all tested compounds. Calibration was satisfactory for all drugs, except olanzapine, from subtherapeutic to toxic concentrations. The lower limits of quantifications (LLOQs) corresponded to the lowest concentrations used for the calibration curves. With the exception of the lowest concentrations of bromperidol, buspirone and perphenazine, accuracy data were within the acceptance interval of +/- 15% (+/- 20% at LLOQ) of the nominal values for all drugs. The method has been proven to be useful for the routine analysis of APs in postmortem blood samples.

The prevalence of drugs in injured drivers

In mid 2009 Victoria introduced compulsory drug testing of blood taken from all injured drivers taken to hospital. Δ(9)-Tetrahydrocannabinol (THC), methylamphetamine (MA) and 3,4-methylenedioxy-methylamphetamine (MDMA) are prohibited and if drivers are positive to any amount an automatic penalty is enforced. Laboratory screens were conducted on preserved blood using ELISA testing for cannabis metabolite and methylamphetamines and a fully validated LC-MS/MS method for 105 drugs including THC, amphetamines, opioids, benzodiazepines, antidepressants and antipsychotics and a number of other psychoactive substances using a minimum of two transitions per drug. Conventional GC-testing for ethanol was used to screen and quantify the presence of alcohol. 1714 drivers were tested and showed alcohol in 29% (≥ 0.01 g/100mL) and drugs in 35%. The positive rate for the three drugs prohibited by legislation was 12.5%. The prevalence of THC, MA and MDMA was 9.8%, 3.1%, and 0.8%, respectively. The range of THC concentrations in blood was 2-42 ng/mL (median 7) of which 70% had a concentration of 10 ng/mL or higher. The range of concentrations for MA and MDMA was 0.02-0.4 and 0.03-0.3mg/L (median for both drugs was 0.05 mg/L). Drugs of any type were detected in 35% of cases. The other drugs were largely prescribed drugs such as the antidepressants (9.3%) and benzodiazepines (8.9%). Neither 6-acetylmorphine nor cocaine (or benzoylecgonine) was detected in these cases.

The incidence of drugs of impairment in oral fluid from random roadside testing

Oral fluid (OF) has become a popular specimen to test for presence of drugs, particularly in regards to road safety. In Victoria, OF specimens from drivers have been used to test for the presence of methylamphetamine (MA) and Δ(9)-tetrahydrocannabinol (THC) since 2003 and 3,4-methylenedioxy-N-methylamphetamine (MDMA) since 2006. LC-MS/MS has been used to test the most recent 853 submitted OF specimens from Victoria Police for 31 drugs of abuse including those listed in the Australian Standard AS4760-2006. At least one proscribed drug was detected in 96% of drivers, of which MA was the most common (77%), followed by THC (42%), MDMA (17%) and the combination of all three (3.9%). Opioids were detected in 14% of drivers of which 4.8% were positive for 6-acetylmorphine and 3.3% for methadone. The incidence of the opioids tramadol (1.2%) and oxycodone (1.1%) were relatively low. Cocaine (8.0%) was as commonly detected as benzodiazepines (8.0%), and was almost always found in combination with MA (7.9%). Samples positive to benzodiazepines were largely due to diazepam (3.5%) and alprazolam (3.4%), with only 0.2% of drivers combining the two. Ketamine was also detected in 1.5% of cases. While the incidences of the proscribed drugs itself are concerning, it is clear that many drivers are also using other drugs capable of causing impairment.

Involvement of Amphetamines in Sudden and Unexpected Death

Abstract:  In the present study, the effects of amphetamine‐class drugs were examined in cases reported to the Victorian coroner from 2001 to 2005 to determine if death can occur from the use of amphetamine‐class drugs alone. A total of 169 cases were reviewed where a forensic autopsy detected amphetamine(s) in the blood. Pathology, toxicology, and police reports were analyzed in all cases to ascertain the involvement of amphetamine‐class drugs in these deaths. In Victoria, methamphetamine (MA) is the principal abused amphetamine‐class followed by methylenedioxymethamphetamine (MDMA). There were six cases in which a cerebral hemorrhage caused death and three cases in which serotonin syndrome was established as being caused by the interaction of MDMA and moclobemide. There were 19 cases in which long‐term use of amphetamines was associated with heart disease. There were three cases where amphetamine‐class drugs alone were regarded as the cause of death, of which two cases exhibited high levels of MDMA and lesser amounts of MA and/or amphetamine. There were no cases in which significant natural disease was absent and death was regarded as caused by the use of MA. There was no correlation between blood concentration of drug and outcome.

Comparison of extraction efficiencies and LC–MS–MS matrix effects using LLE and SPE methods for 19 antipsychotics in human blood

Antipsychotic drugs are frequently associated with sudden death investigations. Detection of these drugs is necessary to establish their use and possible contribution to the death. LC–MS(MS) methods are common; however accurate and precise quantification is assured by using validated methods. This study compared extraction efficiency and matrix effects using common liquid–liquid and solid-phase extraction procedures in both ante-mortem and post-mortem specimen using LC–MS–MS. Extraction efficiencies and matrix effects were determined in five different blank blood specimens of each blood type. The samples were extracted using a number of different liquid–liquid extraction methods and compared with a standard mixed-mode solid-phase extraction method. Matrix effects were determined using a post-extraction addition approach—the blank blood specimens were extracted as described above and the extracts were reconstituted in mobile phase containing a known amount of analytes. The extraction comparison of ante-mortem and post-mortem blood showed considerable differences, in particular the extraction efficiency was quite different between ante-mortem and post-mortem blood. Quantitative methods used for determination of antipsychotic drugs in post-mortem blood should establish that there are no differences in extraction efficiency and matrix effects, particularly if using ante-mortem blood as calibrator.

Review: Pharmacogenetic aspects of the effect of cytochrome P450 polymorphisms on serotonergic drug metabolism, response, interactions, and adverse effects

The field of pharmacogenetics contains a wealth of potential for the enhancement of clinical practice by providing a more effective match between patient and drug, consequently reducing the probability of an adverse drug reaction. Although a relatively novel concept in the forensic context, pharmacogenetics has the capability to assist in the interpretation of drug related deaths, particularly in unintentional drug poisonings where the cause of death remains unclear. However, the complex pharmacology of the drugs when subjected to genetic variations in metabolism makes interpretation of the expected response and adverse events difficult. Many possess multiple metabolic pathways, narrow therapeutic indices and active metabolites or enantiomers which may be eliminated via different pathways to the parent drug. A number of these drugs, which are metabolised primarily by the CYP450 system, are also associated with serotonin syndrome, or serotonin toxicity, especially when used concomitantly with other serotonin active drugs which rely on the same metabolic pathways for drug elimination. A comprehensive understanding of polymorphic drug metabolism and its expected outcomes is therefore essential when interpreting the involvement of drugs in adverse reactions. This review examines the genetically variable CYP450-mediated metabolism of a number of serotonin-active drugs that are often implicated in cases of serotonin toxicity, to assess the impact of pharmacogenetics on drug metabolism, response, interactions and adverse effects.

The time-dependant post-mortem redistribution of antipsychotic drugs

The post mortem redistribution of ten commonly prescribed antipsychotic drugs (APs) was investigated. Femoral blood was collected from 273 cases at admission to mortuary (AD) and at post-mortem (PM). The PM samples were collected at various times up to nine days after admission and the sample pairs analysed using LC-MS/MS. The drugs included in this study were 9OH-risperidone (paliperidone), amisulpride, chlorpromazine, clozapine, haloperidol, olanzapine, promethazine, quetiapine, risperidone, and zuclopenthixol. Haloperidol, quetiapine and risperidone showed minimal changes between AD and PM specimens, whereas the majority of drugs showed significant changes between the sample pairs collected at different time points post mortem (p<0.01) in addition to an average concentration change greater than the uncertainty of measurement of the applied method. Average increases in blood concentrations after admission to the mortuary ranged up to 112% (chlorpromazine and olanzapine) but also decreases up to -43% (9OH-risperidone) were seen. There were large standard deviations between sample pairs and substantial day-to-day unpredictable changes that highlight the difficulty in the interpretation of drug concentrations post-mortem. Based on the presented data, we recommend that specimens for toxicological analysis should to be taken as soon as possible after admission of a deceased person to the mortuary in order to minimise the effects of the PM interval on the drug concentration in blood.

An assessment of the in vivo effects of intravenous lipid emulsion on blood drug concentration and haemodynamics following oro-gastric amitriptyline overdose

Background. Overdose with lipophilic drugs, such as amitriptyline, may cause cardiotoxicity in overdose. Severe poisoning can be resistant to traditional treatments. Intravenous lipid emulsion (ILE) has been recommended as a novel therapy for the treatment of such overdoses; however, a little is known about the effects of ILE-infusion on drug concentration and haemodynamics in the early/absorptive phase after oral poisoning. Method. Thirty minutes after oro-gastric administration of amitriptyline (70 mg/kg), either 20% intravenous lipid emulsion (ILE), 8.4% sodium bicarbonate or Hartmanns solution was infused to anaesthetized and ventilated rodents (n = 10 per group). Heart rate, blood pressure, cutaneous ECG – QRS interval duration (QRS-d), and survival were serially recorded over 120 min. Blood drug concentrations were also collected during this period. Continuous variables were compared using one-way ANOVA. Results. ILE infusion significantly decreased the survival compared to other treatments (10% ILE vs 70% bicarbonate vs 70% Hartmanns solution, p = 0.005). There was a gradual prolongation of QRS-d and fall in blood pressure over time compared to baseline (T0) measurement for both ILE and Hartmanns solution treatments. This was associated with significantly increased blood AMI concentration with ILE treatment at T60, T90 and T120 min to the other treatments (p < 0.02). Conclusion. Administration of ILE early after oral amitriptyline overdose resulted in worse survival and no improvement in haemodynamics. In addition, blood amitriptyline concentrations were higher in the ILE-treated group. This suggests that either drug absorption from the gastrointestinal-tract was facilitated or drug redistribution was retarded when ILE was given early after oral poisoning.

Deaths involving contraindicated and inappropriate combinations of serotonergic drugs

In the Australian state of Victoria, all fatalities that were recorded from 2002 through to 2008 involving the use of certain serotonin active drugs (tramadol, venlafaxine, fluoxetine, sertraline, citalopram and paroxetine), were reviewed to assess the incidence of contraindicated or ill advised drug combinations. More than 1,000 were identified of which 326 cases formed the basis of this study. These cases involved contraindicated or inappropriate drug combinations that can lead to adverse drug reactions (ADRs) and subsequent fatal toxicity. Of these, 46% were drug-related, 35% were a result of natural disease and 13% were classified as external injury cases. The remaining cases were those where the cause of death (COD) was unascertained. Tramadol was the most common drug, usually detected alongside a serotonergic antidepressant (in 20% of cases). Twenty-five (8%) cases involved contraindicated drug combinations while the remainder (301 cases, 92%) involved drug combinations that are associated with adverse interactions ranging from minor to major severity. Of these 326 cases, the Coroner determined 166 cases (51%) to be acts of intentional self-harm or drug misuse, with the remainder unascertained or attributed to natural disease. Very few post-mortem reports and Coroners’ findings made mention of possible ADRs when such combinations were actually present. The majority of cases comprising contraindicated drug combinations involved the combined use of five drugs (24%) at the time of death. A combination of three to five drugs was most common in cases involving inadvisable drug combinations. Combined drug toxicity was the most common COD, with heart disease the most common co-morbidity.