[Show abstract][Hide abstract] ABSTRACT: The increased use of cathinone-type designer drugs, known as legal highs, has led to concerns about their potential neurotoxicity due to their similarity to methamphetamine (METH). Therefore, closer investigations of their toxic effects are needed. We investigated the effects of the cathinones 4-methylmethcathinone (4-MMC) and 3,4-methylenedioxymethcathinone (MDMC) and the amphetamine METH on cytotoxicity and mitochondrial respiration in SH-SY5Y neuroblastoma cells. We also investigated the contribution of reactive species, dopamine, Bcl-2 and tumor necrosis factor α (TNFα) on toxicity. Finally, we investigated the effect of cathinone breakdown products using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry and studied their involvement in toxicity. We observed dose-dependent increases in cytotoxicity and decreases in mitochondrial respiration following treatment with all cathinones and amphetamines. Glutathione depletion increases amphetamine, but not cathinone toxicity. Bcl-2 and TNFα pathways are involved in toxicity but dopamine levels are not. We also show that cathinones, but not amphetamines, spontaneously produce reactive species and cytotoxic methylbenzamide breakdown products when in aqueous solution. These results provide an important first insight into the mechanisms of cathinone cytotoxicity and pave the way for further studies on cathinone toxicity in vivo.
[Show abstract][Hide abstract] ABSTRACT: Lactate is produced in carbohydrate metabolism under anaerobic conditions. Lactic acidosis occurs when the production of lactate exceeds its removal. In post-mortem (PM) context, the lactic acidosis is difficult to interpret due to unknown pathophysiological factors prior to death and PM changes that may affect the lactate levels. We evaluated 1865 medico-legal autopsy cases where the quantitation of glucose, lactate, and ketone bodies was performed as a part of the cause of death (CoD) investigation. Lactate was shown to ascend in a logarithmic manner as the PM interval increased until a plateau was achieved approximately after 8–10 days PM, and the elevation was caused mainly by PM changes. The lactate level was higher than the mean in cases where the CoD was diabetes mellitus type 2 (DM2) or metformin poisoning. Although there was a correlation between metformin and lactate levels, our findings suggest the DM2 and its complications were the cause for elevated lactate levels rather than metformin, since the lactate levels were similar in DM2-associated deaths where no metformin was detected. Elevated lactate levels in PM samples rather referred to metabolic disturbances often caused by DM2. An assay to detect D-lactate in PM samples was described.
Full-text · Article · Sep 2015 · Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin
[Show abstract][Hide abstract] ABSTRACT: In this study, we sought to determine what impact the banning of 3, 4- methylenedioxypyrovalerone (MDPV) had on the incidence of MDPV-positive findings and on user profiles in driving under the influence of drugs (DUID) and postmortem (PM) investigations in Finland. All MDPV-positive cases and a selection of corresponding court cases between 2009 and 2012 were examined. The median serum concentration of MDPV in DUID cases was 0.030 mg/L and in PM blood 0.12 mg/L. The number of MDPV-positive cases decreased both in DUID and PM investigations after the drug was banned. The decrease in the mean monthly numbers of MDPV-positive DUID cases was 51.1%. In court cases, MDPV was rarely mentioned until banned and frequently mentioned thereafter. Of the convicted, 37% were without a fixed abode, 98% had other charges besides that of DUID, and 13% appeared in the study material more than once. In MDPV-positive PM cases, the proportion of suicides was very high (24%). Research on new psychoactive substances is required not only to support banning decisions but more importantly to be able to provide a scientific assessment of the risks of these new substances to the public and potential users.
No preview · Article · Apr 2015 · Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin
[Show abstract][Hide abstract] ABSTRACT: This report is a follow-up to a study on fatal poisoning in drug addicts conducted in 2012 by a Nordic working group. Here we analyse data from the five Nordic countries: Denmark, Finland, Iceland, Norway and Sweden. Data on sex, number of deaths, places of death, age, main intoxicants and other drugs detected in the blood were recorded. National data are presented and compared between the Nordic countries and with data from similar studies conducted in 1991, 1997, 2002 and 2007.
Full-text · Article · Jan 2015 · Forensic Science International
[Show abstract][Hide abstract] ABSTRACT: Opioids are important medications for pain and opioid maintenance treatment. Increasing use and abuse of prescription opioids has, however, caused worldwide concern. Our aim was to estimate the ratio between prescription opioid abuse and total use, based on representative postmortem toxicology. Our material included all the medico-legally examined deaths in Finland during 2010–2011 involving positive findings involving buprenorphine, codeine, fentanyl, methadone, oxycodone, or tramadol. We studied drug abuse by age group, with “abuse” meaning licit opioids used illicitly as narcotics. Drug-abuse history, drug injecting, or laboratory findings of illicit drugs defined an abuser case. We then compared abuser cases and other opioid-related cases between the opioids with the number of fatal poisonings, accidents, suicides, alcohol findings, concomitant opioid use, and median postmortem blood opioid concentrations. Opioid findings numbered 2499 in 2088 cases. Drug abuse involved 545 opioid-positive cases, which in Finland represented 0.5% of those deceased. The proportion of abuser cases among all opioid-related cases for buprenorphine was 85.5%, for methadone 82.4%, for tramadol 29.4%, for codeine 16.3%, for fentanyl 14.5%, and for oxycodone 6.9%. Abuse in age-groups >60 was rare. Concomitant other opioid findings were more frequent in abuser- than in other cases for codeine, oxycodone, and tramadol, whereas alcohol findings were more frequent in buprenorphine, codeine, and fentanyl abuse. Buprenorphine and methadone were most often related to drug abuse. Every other opioid studied involved some abuse, and especially tramadol. Abuse and fatal poisonings were concentrated in men aged 20–49.
No preview · Article · Oct 2014 · Forensic Science International
[Show abstract][Hide abstract] ABSTRACT: Background:
Sedative-hypnotics are commonly encountered in drivers apprehended for driving under the influence of drugs (DUID). Previous research has mainly concentrated on the residual effects of the drugs.
In this study, the extent of sleep medication use and abuse among drivers apprehended on suspicion of DUID was assessed. Additionally, the prevalence and concentrations of the drugs, concomitant use of other drugs of abuse, and the age and sex of the drivers positive for the most commonly prescribed sedative-hypnotics (temazepam, midazolam, nitrazepam, zopiclone, and zolpidem) in DUID cases in Finland in 2009 to 2011 were examined.
Sedative-hypnotics were found in 3155 samples of the 13,248 that were analyzed. Temazepam was present in over half of the cases (57.9%), along with other benzodiazepines such as midazolam (13.1%) and nitrazepam (7.0%) and the non-benzodiazepine hypnotics zopiclone (12.2%) and zolpidem (9.8%). The mean age of the drivers using the studied sedative-hypnotics was 33.5 years. Many of the drivers were polydrug users; concomitant stimulant use was found in nearly half of the cases. Cannabis and alcohol were also very common co-findings. In nearly 20% of the cases, the driver had taken more than 1 of the studied sedative-hypnotics; only 2.5% had no findings other than a single sedative-hypnotic in their blood. The drug use pattern of those positive for zopiclone and zolpidem was somewhat different from that of users of benzodiazepine sedative-hypnotics; their age was higher and the concomitant use of illegal stimulants was markedly less prevalent than among the users of temazepam, midazolam, and nitrazepam.
There were very few cases in our study population where the positive sedative-hypnotic finding could have been due to appropriate medical use. The extremely prevalent concomitant use of other psychoactive drugs and the high median serum concentrations of the studied sedative-hypnotics suggest their widespread abuse among apprehended drivers.
Full-text · Article · Sep 2014 · Therapeutic Drug Monitoring
[Show abstract][Hide abstract] ABSTRACT: Pregabalin is a medicinal drug used mainly for the treatment of epilepsy and neuropathic pain. It has
been shown to possess an abuse potential and in recent years some reports of illegal use have been
published. In order to further evaluate the extent and nature of pregabalin abuse, serum pregabalin
levels of drivers apprehended for driving under the influence of drugs (DUID) in Finland in 2012 were
assessed. The samples were analysed by an LC–MS/MS system and the results were evaluated in relation
to the typical therapeutic range of pregabalin as well as the age and gender of the driver.
Pregabalin was detected in 206 samples in the study period. The median (range) serum concentration
was 6.2 (0.68–111.6) mg/L. In nearly 50% of the cases the serum concentration was above the typical
therapeutic range. In most of the cases the driver had also taken other drugs besides pregabalin, the
mean number of concomitantly taken drugs being four.
Our data indicate that pregabalin is being used at high doses, probably for recreational purposes. The
vast majority of the drivers positive for pregabalin in our study material had used pregabalin as a part of a
spectrum of psycho-active drugs and thus qualified as probable drug abusers. In these cases pregabalin
probably contributed to their driving impairment but to what extent remained unclear in this study.
No preview · Article · Jul 2014 · Forensic Science International
[Show abstract][Hide abstract] ABSTRACT: The β-keto amphetamine (cathinone, β-KA) designer drugs such as mephedrone (4-methylmethcathinone, 4-MMC) show a large degree of structural similarity to amphetamines like methamphetamine (METH). However, little is currently known about whether these substances also share the potential neurotoxic properties of their non-keto amphetamine counterparts, or what mechanisms could be involved. Here, we evaluate the cytotoxicity of β-KAs in SH-SY5Y cells using lactate dehydrogenase (LDH) assays, assess the redox potential of a range of β-KAs and non-keto amphetamines using the sensitive redox indicator 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) and explore the effect of 4-MMC on the formation of protein adducts using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) and on the mitochondrial respiratory chain using high resolution respirometry. We show that treatment with β-KAs increases LDH release. Further, we demonstrate that even under physiological pH, β-KAs are effective and selective-as compared to their non-keto analogues-reductants in the presence of electron acceptors. Increased pH (range 7.6-8.0) greatly enhanced the reactivity up to six-fold. We found no evidence of protein adduct formation, suggesting the reactivity is due to direct electron transfer by the β-KAs. Finally, we show that 4-MMC and METH produce dissimilar effects on the respiratory chain. Our results indicate that β-KAs such as 4-MMC possess cytotoxic properties in vitro. Furthermore, in the presence of an electron-accepting redox partner, the ketone moiety of β-KAs is vital for pH-dependent redox reactivity. Further work is needed to establish the importance of β-KA redox properties and its potential toxicological importance in vivo.
Full-text · Article · Jun 2014 · Toxicological Sciences
[Show abstract][Hide abstract] ABSTRACT: Use and abuse of alcohol are common findings when unnatural deaths are investigated as evidenced by high blood- and urine- alcohol concentrations (BAC and UAC) at autopsy. Because ethanol is metabolized in the liver until the time of death, the autopsy BAC or UAC might be negative even though the deceased had consumed alcohol in the immediate ante-mortem period. Analysis of the non-oxidative metabolite of ethanol [ethyl glucuronide (EtG)] offers a more sensitive test of recent drinking. In this paper, we determined the concentrations of ethanol and EtG in urine samples from 972 consecutive forensic autopsies. In 425 cases (44%) both EtG and ethanol were positive, which supports ante-mortem drinking. In 342 cases (35%), both EtG and ethanol was negative, which speaks against any consumption of alcohol just before death. In 181cases, ethanol was negative in urine (<0.2g/kg), whereas EtG was positive (>0.5mg/L), which points towards ingestion of alcohol some time before death. In these cases, mean and median concentrations of EtG were 53.2mg/L and 23.7mg/L, respectively, although there was no mention of alcohol on 131 of the death certificates. Alcohol was mentioned on death certificates as an underlying or immediate cause of death or a contributing factor in 435 (45%) cases, which rose to 566 (58%) cases when positive EtG results were included. This article demonstrates the usefulness of EtG analysis in routine post-mortem toxicology when ante-mortem drinking and alcohol-related deaths are investigated.
No preview · Article · Jun 2014 · Forensic Science International
[Show abstract][Hide abstract] ABSTRACT: Introduction
Mass spectrometric (MS) methods are today widely used in analytical toxicology, but in comprehensive quantitative drug screening it is laborious to maintain quantitative calibration and use of historic calibration is not feasible. In non-MS gas chromatographic (GC) and liquid chromatographic (LC) methods, historic calibration is widely used. However, GC is not amenable to analysis of current polar drugs and traditional LC-UV methods do not have high enough identification power to meet the requirements of screening. Ultra-performance LC (UPLC) with two consecutive detectors appears to possess high potential for simultaneous multi-component screening and quantification. In this study, a comprehensive quantitative screening method for 170 basic drugs in blood samples was developed and validated using UPLC coupled with photodiode array (PDA) and corona charged aerosol detector (CAD).
Blood samples were extracted with organic solvent (ethyl acetate: butyl acetate, 25:75) in basic conditions. After extraction, the organic solvent was evaporated to dryness and samples were reconstituted with the UPLC mobile phase. Dibenzepine was used as an internal standard. Chromatographic separation was performed at 60 °C using a HSS C18 column (150 mm × 2,1 mm, particle size 1,8 μm), and the mobile phase consisted of 0.1% trifluoroacetic acid and methanol. After a three minute isocratic phase, a linear gradient from 5% to 95% methanol in 15 minutes followed. The flow rate was 0.4 mL/min. UV spectra were collected in the range of 210–400 nm, and wavelength of 230 nm was used for quantification. Substance identification was based on the UV spectrum, retention times on both detectors, and the response ratio of CAD and PDA at wavelength 230 nm. Calibration was carried out using a single calibration point at the vicinity of the upper limit of therapeutic range of each drug. Individual calibration curves were created for both detectors and the average result was used for quantification.
The retention times were found be very stable, the relative standard deviation of retention times being <0.03% intraday and <0.3% interday. In order to simplify data processing and interpretation of the reports, the analytes were divided into five data processing methods, each containing every fifth analyte based the retention time. This approach provided a maximum of five candidates for each peak, thus simplifying manual interpretation even with more complicated samples. Linearity of one-point calibration was found to be acceptable within the therapeutic and toxic ranges of the drugs of interest, with linear ranges generally being 0.05–5,0 mg/L or 0.1–5,0 mg/L. Calibration was found to be stable over one month, the bias between old and new calibration being <15%.
The developed method allows the detection and quantification of 170 basic drugs in therapeutic and toxic concentrations in a single run. Due to the stability of calibration and good linearity, historic one-point calibration can be utilized. Adding the CAD detector with universal response after the PDA detector increases the reliability of both identification and quantification. In addition, drugs with poor UV absorption can be detected and quantitated. This method provides a tool for comprehensive quantitative screening for ordinary basic drugs in blood and leaves LC-MS target analysis to be applied to low-dose compounds.
[Show abstract][Hide abstract] ABSTRACT: Introduction
Immunoassay screening tests are widely used in clinical and forensic toxicology. However, the emergence of new psychoactive substances (NPS) has challenged the scope and sensitivity of these methods. Consequently, specialized analytical toxicology laboratories are today relying more and more on mass spectrometry based techniques. Ultra-high performance liquid chromatography/ high resolution time-of-flight mass spectrometry (UHPLCHR- TOFMS) is an especially attractive technique for comprehensive drug screening in terms of scope, sensitivity and reliability of identification. The objective of the present study was to compare the performance of these two analytical approaches in urine screening for conventional drugs of abuse and NPS.
A total of 279 post-mortem urine samples were analyzed by both an immunoassay (Emit II Plus) and an UHPLC-HRTOFMS screening method. Sample preparation prior to immunoassay was only a centrifugation step, while UHPLC-HR-TOFMS analysis was preceded by solid-phase extraction, including both acidic/neutral and basic fractions. The instrumentation consisted of a Dionex Ultimate 3000 series UHPLC coupled with a Bruker Daltonics maXis Impact HR-TOFMS. Simultaneous acquisition of MS and broad band collision induced fragmentation (bbCID) data was applied. Compound identification was based on post-targeted database search with preset reporting criteria for mass accuracy, isotopic pattern match, retention time, and abundance criteria for qualifier ions. The immunoassay drug panel included amphetamines, benzodiazepines, buprenorphine, cocaine, opiates, and cannabis. The UHPLC-HR-TOFMS method comprised conventional drugs of abuse, various classes of NPS, such as synthetic cannabinoids and cathinones, and commonly abused prescription drugs such as tramadol and pregabalin.
The UHPLC-HR-TOFMS and immunoassay results were compared with quantitative confirmation analyses by established GC-MS and LC-MS/MS methods. The UHPLC-HR-TOFMS results for amphetamines (n=11), buprenorphine (n=27), cannabis (n=28), opiates (n=31), and benzodiazepines (n=99) showed excellent correlation with confirmation analyses, whereas many false negative immunoassay results existed obviously due to high cut-off concentrations and interfering matrix. Moreover, the wider scope of UHPLC-HR-TOFMS method allowed detection of NPS (n=4) and prescription drugs (n=88). A total of 22 false positive (FP) results were produced by immunoassay. For example, the immunoassay gave ten FP buprenorphine results, for which both UHPLC-HRTOFMS and quantification analyses were negative. The UHPLC-HRTOFMS FP results were mainly due to the interference from early eluting endogenous compounds or due to the ion source cleavage of reduced oxycodone resulting in a structure corresponding to codeine. UHPLC-HR-TOFMS revealed additional low-concentration drug findings. The correspondence between symmetric peaks of precursor and qualifier ions supported these results, which were, however, undetectable by confirmation methods. Simultaneous detection of parent compound and metabolites enhances the reliability of compound identification.
Due to the superior results from UHPLC-HR-TOFMS screening, this method has replaced immunoassay in the authors’ laboratory. The possibility to collect both MS and bbCID data simultaneously in a single run increases the reliability of identification and reduces the need for confirmation analyses. However, the data processing parameters of the software should provide more options for individual reporting criteria, which would notably reduce the number of FP results without compromising sensitivity.
[Show abstract][Hide abstract] ABSTRACT: Pregabalin (PRG) and gabapentin (GBP) are used in the treatment of neuropathic pain and epilepsy, and PRG also in generalized anxiety disorder. There is increasing evidence that PRG possesses considerable abuse potential. PRG may have a higher addiction potential than GBP due to its rapid absorption and faster onset of action. Our objective is to estimate the proportion of all PRG- and GBP-related fatalities attributable to PRG and GBP abuse. We investigated all medico-legal death cases in Finland in which PRG or GBP was found in postmortem toxicology during 2010-2011. PRG was found in 316 cases and GBP in 43 cases. Drug abuse was associated with 48.1% of the PRG and 18.6% of the GBP findings. PRG poisoning accounted for 10.1% of all PRG cases and GBP poisoning for 4.7% of all GBP cases. In the drug abuser cases, PRG poisoning represented 19.1%, and GBP poisoning 12.5%. The median blood concentration of PRG was 15mg/L in the abuser group and 5.8mg/L in the other cases. For GBP, these concentrations were 12mg/L and 8.3mg/L, respectively. In the PRG abuser group, 91.4% of cases showed concomitant opioid use, while in the rest of these cases neither alcohol nor opioids were detected, but other central nervous system acting drugs were found in each abuser case. In the GBP abuser group, 87.5% of cases showed concomitant opioid use. PRG abuse with high doses is increasingly common and can be fatal when combined with opioids.
No preview · Article · May 2014 · Forensic Science International