Brain/blood ratios of methadone and ABCB1 polymorphisms in methadone-related deaths.

Methadone is an opioid that often leads to fatalities. Interpretation of toxicological findings can be challenging if no further information about the case history is available. The aims of this study were (1) to determine whether brain/blood ratios can assist in the interpretation of methadone findings in fatalities; (2) to examine whether polymorphisms in the gene encoding the P-glycoprotein (also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1)), which functions as a multispecific efflux pump in the blood-brain barrier, affect brain/blood ratios of methadone. Femoral venous blood and brain tissue (medulla oblongata and cerebellum) from 107 methadone-related deaths were analysed for methadone by gas chromatography-mass spectrometry. In addition, all the samples were genotyped for three common ABCB1 single nucleotide polymorphisms (SNPs rs1045642, rs1128503, and rs2032582) using ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS). In nearly all cases, methadone concentrations were higher in the brain than in the blood. Inter-individual brain/blood ratios varied (0.6-11.6); the mean ratio was 2.85 (standard deviation 1.83, median 2.35). Moreover, significant differences in mean brain/blood ratios were detected among the synonymous genotypes of rs1045642 in ABCB1 (p = 0.001). Cases with the T/T genotype had significantly higher brain/blood ratios than cases with the other genotypes (T/T vs. T/C difference (d) = 1.54, 95% CI [1.14, 2.05], p = 0.002; T/T vs. C/C d = 1.60, 95% CI [1.13, 2.29], p = 0.004). Our results suggest that the rs1045642 polymorphisms in ABCB1 may affect methadone concentrations in the brain and its site of action and may be an additional factor influencing methadone toxicity.

The challenge of post-mortem GHB analysis: storage conditions and specimen types are both important.

BACKGROUND: For the interpretation of concentrations of gamma-hydroxybutyrate (GHB) in post-mortem specimens, a possible increase due to post-mortem generation in the body and in vitro has to be considered. The influence of different storage conditions and the specimen type was investigated. METHOD AND MATERIAL: Post-mortem GHB concentrations in femoral venous blood (VB), heart blood (HB), serum (S) from VB, urine (U), cerebrospinal fluid (CSF) and vitreous humour (VH) were determined by gas chromatography-mass spectrometry after derivatisation. Various storage conditions, that is 4 °C or room temperature (RT) and the addition of sodium fluoride (NaF), were compared during storage up to 30 days. Additionally, bacterial colonisation was determined by mass spectrometry fingerprinting. RESULTS: Twenty-six cases without involvement of exogenous GHB were examined. GHB concentrations (by specimen) at day 0 were 3.9-22.1 mg/L (VB), 6.6-33.3 mg/L (HB), < 0.5-18.1 mg/L (U), 1.1-10.4 mg/L (CSF) and 1.7-22.0 mg/L (VH). At 4 °C, concentrations increased at day 30 to 5.6-74.5 mg/L (VB), 4.6-76.5 mg/L (HB) and < 0.5-21.3 mg/L (U). At RT, concentrations rose to < 0.5-38.5 mg/L (VB), 1.2-94.6 mg/L (HB) and < 0.5-37.5 mg/L (U) at day 30. In CSF, at RT, an increase up to < 0.5-21.2 mg/L was measured, and at 4 °C, a decrease occurred (< 0.5-6.5 mg/L). GHB concentrations in VH remained stable at both temperatures (1.2-20.9 mg/L and < 0.5-26.2 mg/L). The increase of GHB in HB samples with NaF was significantly lower than that without preservation. No correlation was found between the bacterial colonisation and extent of GHB concentration changes. CONCLUSION: GHB concentrations can significantly increase in post-mortem HB, VB and U samples, depending on storage time, temperature and inter-individual differences. Results in CSF, VH, S and/or specimens with NaF are less affected.

Post-mortem analysis of prescription opioids-A follow-up examination by LC-MS/MS with focus on fentanyl.

Our aim was to investigate the reason for relatively low detection rates for opioids and fentanyl in particular in post-mortem cases in the State of Hamburg. We re-analysed 822 blood samples from two different time periods, 2011/12 and 2016. These samples had been previously analysed in accordance with post-mortem routine by a case selected strategy. All samples were re-analysed with an LC-MS/MS method specific for prescription opioids. The main point in the evaluation was to determine whether the previous analysis strategy had led to underreporting of drug-related deaths (DRD), especially with regard to fentanyl. Another aim was to evaluate changes in prescribing prevalence of opiates and opioids. We compared pharmacy claims data in Hamburg with Germany. The analyses showed that the number of DRD remained unaffected by the new analytical strategy. Detection rates in DRD, however, increased for fentanyl 3.4-fold from 1.2% to 4.1%, buprenorphine from 5.9% to 7.6%, oxycodone from 0% to 1.8%, tilidine from 1.8% to 2.4%. The most frequently detected opioids in DRD cases were methadone (39.4%) and heroin (20%). Prescription rates between 2011-2017 decreased in Hamburg for nearly all opioids, morphine by - 43.5%, buprenorphine - 43%, codeine - 57%, fentanyl - 25%, tilidine -17%, tramadol - 31%, and hydromorphone -6%. Oxycodone, tapentadol, and piritramide prescription rates increased. For Germany, a decrease in the prescription rates for fentanyl was also found during this period (-12.9 %), although not as pronounced as in Hamburg. Prescription rates for methadone were three to greater than five times higher in Hamburg as compared to the German average due to the higher number of substituted persons per inhabitant. Conclusion: Despite the global problem of opioid abuse, there are significant regional differences in the nature and extent of opioid abuse. It is necessary to collect data at the national level to develop appropriate prevention strategies.

Helium poisoning: new procedure for sampling and analysis.

An increasing number of suicidal asphyxiation with a plastic bag with inert gases, and in particular helium (He), have been reported from numerous countries over the last decade. These cases are differently managed and lead to different and variable interpretations. Based on the 12 last cases analysed in the laboratory and on the review of the most recent literature about this topic, updated autopsy guidelines for sampling have been proposed regarding to the samples choice and analytical challenges required by the gaseous state of this substance. Biological samples from airways (lungs lobe) followed by brain and cardiac blood are the best matrices to take during the autopsy to diagnose He exposure. Gaseous samples from trachea, pulmonary bronchi, gastric and cardiac areas are also recommended as alternative samples. The anatomical site of sampling must be carefully detailed, and to this end, forensic imaging constitutes a beneficial tool. Even if He detection is sufficient to conclude to He exposure, He concentrations in samples may be related to He exposure conditions (duration, breathing rate, etc.). A quantification in biological samples could be helpful to document more precisely the case. He concentrations in gaseous samples are reported up to 6.0 µmol/mL (tracheal gas), 2.4 µmol/mL (pulmonary gas), 0.64 µmol/mL (cardiac gas) and 12 µmol/mL (gastric gas). He concentrations in solid/liquid samples are reported up to 28 µmol/g (lungs) and 0.03 µmol/g (cardiac blood). The other matrices usually sampled during autopsy such as urine, peripheral blood, liver, fat matter and kidney appear as not relevant.

Mass poisoning with NPS: 2C-E and Bromo-DragonFly.

BACKGROUND: Reports of intoxications with new psychoactive substances (NPS) mostly involve young people, as they are the main consumers of these types of drugs. This report centers on a case that was unusual due to it being a mass-poisoning event involving middle-aged individuals who had consumed a combination of the two different new psychoactive drugs 2,5-dimethoxy-4-ethylphenethylamine (2C-E) and 1-(8-bromofuro[2,3-f][1]benzofuran-4-yl)-2-propanamine (Bromo-DragonFly, BDF). CASE HISTORY: The mass poisoning of 29 individuals (24-56 years old) resulted in their admission to six different hospitals with severe symptoms of intoxication. All symptoms manifested after consumption of an unknown drug formulation around lunchtime during an esoteric weekend seminar. INVESTIGATION: Urine (n = 11) and blood samples (n = 29), collected from the 29 individuals for police investigation, were analyzed with immunochemical techniques, GC/MS and LC-MS/MS. 2C-E was confirmed in seven urine samples, but not in blood. BDF was confirmed in all urine samples, and in 17 blood samples. The blood samples exhibited BDF concentrations between ca. 0.6 and ca. 2.0 µg/L, while urine concentrations of BDF ranged from ca. 1.6 to 35 µg/L. The concentration of 2C-E in urine was found to be between ca. 1.5 and 183 µg/L. All patients made a complete recovery, although some had required mechanical ventilation. CONCLUSION: The investigation and the presentation of this case illustrates not only mass intoxication with 2C-E and BDF, with corresponding blood and urine concentrations, but also the necessity of collecting urine samples in cases where NPS-consumption is suspected, in order to improve the chances of analytical detection.

Correction to: Mass poisoning with NPS: 2C-E and Bromo-DragonFly.

The original version of this article contains an error. The Author S. Lehmann incorrectly listed as S. Lehman. The correct spelling is presented above. The original article has been corrected.

Endogenous gamma-hydroxybutyric acid (GHB) concentrations in post-mortem specimens and further recommendation for interpretative cut-offs.

When interpreting gamma-hydroxybutyric acid (GHB) concentrations in post-mortem specimens, a possible increase in GHB concentrations because of post-mortem generation must be considered. In this study, endogenous GHB concentrations in post-mortem biological fluids were investigated. Additionally, we review post-mortem GHB concentrations already published in the literature. Heart and peripheral blood samples, cerebrospinal fluid, urine, and vitreous humor were collected from 64 autopsies in subjects where the cause of death excluded GHB exposure. Sample analysis was carried out either on the day of autopsy or later after immediate freezing and storage at -20 °C. GHB concentrations in venous blood samples (n = 61) were <0.6-28.7 mg/L (mean 11.9 mg/L; median 10.6 mg/L), <0.6-65.3 mg/L (mean 15.2 mg/L; median 12.8 mg/L) in heart blood (n = 56), <0.6-25.1 mg/L (mean 6.0 mg/L; median 3.8 mg/L) in urine (n = 50), <0.6-39.0 mg/L (mean 9.6 mg/L; median 7.5 mg/L), in vitreous humor (n = 54), and <0.6-24.0 mg/L (mean 4.2 mg/L; median 3.2 mg/L) in cerebrospinal fluid (n = 52). There was no significant difference between GHB concentrations in cases where there were signs of beginning putrefaction at the time of autopsy (n = 9) and cases without obvious signs of putrefaction. In one case with advanced putrefaction, the GHB concentration in venous blood was 32.7 mg/L. In conclusion, for post-mortem venous blood, urine, and cerebrospinal fluid, an interpretative cut-off of 30 mg/L for GHB concentrations is suggested in cases where GHB analysis is conducted on the day of sample collection at autopsy or if samples have been stored at -20 °C immediately after collection.

Uptake of gamma-valerolactone--detection of gamma-hydroxyvaleric acid in human urine samples.

Gamma-valerolactone (GVL) is reported to be a substance that can be used as a legal substitute for gamma-hydroxybutyric acid (GHB), which is currently a controlled substance in several countries. Unlike gamma-butyrolactone and 1,4-butanediol, GVL is not metabolized to GHB, which causes the effects after uptake of these two chemicals. In the case of GVL, the lactone ring is split to gamma-hydroxyvaleric acid (GHV or 4-methyl-GHB) by a lactonase. Because of its affinity for the GHB receptor, GHV reveals similar effects to GHB, although it is less potent. Intoxications with GVL, or its use as a date rape drug, are conceivable. Despite these facts, there are no publications in the literature regarding detections of GHV in human samples. This study reports three cases, including five urine samples, in which GHV could be detected in concentrations between 3 and 5.8 mg/L. In one of these cases, a drug-facilitated sexual assault (DFSA) was assumed; four of these samples were from two people suspected of abusing GHB. The results indicate that GVL is used as an alternative to GHB and its precursors and should be taken seriously. GVL or GHV should be included in toxicological analysis, particularly in DFSA cases. More information is needed regarding the pharmacokinetics of GVL/GHV for the meaningful interpretation of positive or negative results.

An overview of gamma-hydroxybutyric acid: pharmacodynamics, pharmacokinetics, toxic effects, addiction, analytical methods, and interpretation of results.

Abuse of gamma-hydroxybutyric acid (GHB) has been known since the early 1990's, but is not as widespread as the consumption of other illegal drugs. However, the number of severe intoxications with fatal outcomes is comparatively high; not the least of which is brought about by the consumption of the currently legal precursor substances gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD). In regards to previous assumptions, addiction to GHB or its analogues can occur with severe symptoms of withdrawal. Moreover, GHB can be used for drug-facilitated sexual assaults. Its pharmacological effects are generated mainly by interaction with both GABA(B) and GHB receptors, as well as its influence on other transmitter systems in the human brain. Numerous analytical methods for determining GHB using chromatographic techniques were published in recent years, and an enzymatic screening method was established. However, the short window of GHB detection in blood or urine due to its rapid metabolism is a challenge. Furthermore, despite several studies addressing this problem, evaluation of analytical results can be difficult: GHB is a metabolite of GABA (gamma-aminobutyric acid); a differentiation between endogenous and exogenous concentrations has to be made. Apart from this, in samples with a longer storage interval and especially in postmortem specimens, higher levels can be measured due to GHB generation during this postmortem interval or storage time.

Gamma-hydroxybutyrate in urine and serum: additional data supporting current cut-off recommendations.

Besides the use of Gamma-Hydroxybutyrate (GHB) as a recreational drug, use of GHB as an agent in drug-facilitated crime should also be considered. In these cases, there is often a delay of several hours from the incident to collection of the samples. As GHB has a very short plasma half-life, the window of detection is small and in the majority of these specimens, levels of GHB are low. Because GHB is naturally occurring in humans, discrimination between endogenous and exogenous GHB is complicated, particularly in those samples with low concentrations. In this study, endogenous GHB levels of 50 serum and 50 urine samples were determined by GC-MS after conversion to trimethyl-silyl-derivatives. Concentrations in serum ranged from 0.62 to 3.24 mg/L (mean=1.14 mg/L; median=0.97 mg/L) and from 0.64 to 4.20mg/L (mean=1.21 mg/L; median=0.96 mg/L) in urine. Based on this substantial data, the current suggested lower cut-off of 4 mg/L in ante mortem serum samples could be confirmed. For urine, we propose the lower cut-off of 6 mg/L instead of 10mg/L to avoid false negative interpretation.

[Use of psychotropic substances by the elderly and driving accidents]. / Altere Menschen und psychotrope Substanzen im Strassenverkehr.

In the context of a study for the Federal Highway Research Institute (Bundesanstalt für Strassenwesen), a database containing almost 40,000 toxicological blood analysis results from drivers with various traffic offences (time frame 1998-2001) from throughout Germany was evaluated. The database contains the results of 25 laboratories in Germany performing toxicological blood analysis of driving offences. Of these laboratories, 23 gave information about their methodology for toxicological analysis by questionnaire. This database and the results from 10,000 toxicological blood analyses of traffic offences in Hamburg (time frame 2003-2008) were evaluated. The number of elderly driver cases, the spectrum of detectable substances, the type of offence and the medical findings were compared to the results of the total sample in the corresponding period. The number of traffic offences with elderly drivers has risen in recent years but to a much smaller degree than the total number of traffic offences. The relative frequency of detection decreased from 2% in 1998 to 1.1% in 2001 (Germany wide) and from 2.3% (2003) to 1.4% (2008) in Hamburg. In the group of elderly drivers, only 39% (43% in Hamburg) of the sample showed positive results for medical drugs or drugs of abuse (73.9% of all samples). The medical diagnosis on the symptomatic level of intoxication, which had been assigned by medical doctors after blood sampling, did not correlate with the actual presence of therapeutic drugs in the blood. This demonstrates the interactions of senso-motor and physical frailties with potential additive, substance-related effects when driving skills appear to be compromised in the elderly. Drugs of abuse were detected in 5.6% (10.6% in Hamburg) among the senior drivers. Benzodiazepines were detected in 24.5% (23% in Hamburg) of the cases with a high frequency of detection in traffic accidents compared to traffic offences without accident. The type of offence was given in 87 of benzodiazepine-positive cases (20 in Hamburg), 59 of them (11 in Hamburg) were traffic accidents. Keeping the increasing percentage of senior drivers in mind, the results show that most of the elderly drivers are very careful with medical therapy and driving. However traffic-related safety advice including pharmaceutical side effects on driving ability given by general practitioners is important and necessary.

Death after excessive propofol abuse.

Abuse of the anaesthetic agent propofol (2,6-diisopropylphenol) is rare, but we report a case of a 26-year-old male nurse in which the autopsy showed unspecific signs of intoxication and criminological evidence pointed towards propofol abuse and/or overdose. Intravenously administered propofol is a fast and short-acting narcotic agent, therefore it seemed questionable whether the deceased would have been able to self-administer a lethal overdose before losing consciousness. The blood and brain concentrations corresponded to those found 1-2 min after bolus administration of a narcotic standard dose of 2.5 mg propofol/kg body weight. Extremely high propofol concentrations were found in the urine indicating excessive abuse before death. However, due to the short half-life of propofol, the cumulative effects of repeated injections should not be relevant for toxicity, since this would result in a blood level increase of only 1-2 micrograms/ml. Furthermore, the detection and quantitation of propofol in three different hair segments indicated chronic propofol abuse by the deceased. The results of the investigation suggest that death was not caused by a propofol overdose but by respiratory depression resulting from overly rapid injection.

Methadone-related fatalities in Hamburg 1990-1999: implications for quality standards in maintenance treatment?

Drug-related fatal poisonings were analysed in Hamburg from 1990 to 30th June 1999 with special attention to the role of methadone. The first methadone-related fatalities were observed in Hamburg three years after methadone maintenance treatment (MMT) was introduced in 1990. Meanwhile more than half of all fatal poisonings among drug addicts are monovalent or polydrug intoxications with evidence for methadone. From January 1997 until June 1999 methadone was the predominant cause of death in about 39% of all drug-related fatal poisonings while the proportion of mixed heroin/methadone intoxications was about 10%. The rising problem of methadone-related fatalities goes with a decline of monovalent heroin intoxications which decreased in the last 9 years from 60% to 11%. Sixty-five per cent of those who died of fatal methadone-related poisonings had no history of MMT (60% of those with methadone as predominant cause of death). Since take-home doses for up to 7 days are prescribed to the patient due to a change in the German Narcotics Act in 1998, the diversion of methadone into illegal markets may have been accelerated. This results in rising numbers of non-intentional methadone-related fatalities among addicts who have never been in MMT. The prerequisites for the prescription of take-home doses should be taken more serious. There is no doubt that MMT reduced the mortality rate among the great majority of patients in Hamburg but supreme efforts should be made to prevent or reduce fatal intoxications by methadone in the non-treatment group.

Acute intoxication with aniline: detection of acetaminophen as aniline metabolite.

A 47-year-old woman unwittingly ingested an unknown substance together with her breakfast coffee. She suffered effects such as strong headache, generalized cyanosis, and a burning sensation of the lips and collapsed some minutes later. After admission into hospital a methemoglobin level of 35% was determined in the blood. Treatment by administration of tolonium chloride (toluidine blue) resulted in complete recovery of the patient. The toxic agent was identified as aniline by GC with mass selective detection after organic solvent extraction and 11 h after ingestion the plasma aniline level was 0.13 mg/l. Acetanilide (0.79 mg/ml) and acetaminophen (2.3 mg/ml) were identified in plasma as metabolites of aniline. It was assumed that a high metabolic capacity for acetylation protected the victim from more severe reactions. Her husband confessed later that he had tried to poison her.

Direct semiquantitative screening of drugs of abuse in serum and whole blood by means of CEDIA DAU urine immunoassays.

The purpose of this study was to test the direct applicability of CEDIA DAU urine immunoassays to serum or whole blood. The performance of the urine assays for sensitive screening of amphetamines (AMP), benzoylecgonine (BZE), benzodiazepines (BENZ), methadone (MET), opiates (OPI), and tetrahydrocannabinol carboxylic acid (THCCOOH) was evaluated on the BM/Hitachi 911 analyzer with unpretreated serum and whole blood. The limit of detection was 0 ng/mL for all tests. Cutoff values were set from 10 to 40 ng/mL for the different assays. The assays were found to be linear between the following concentrations: AMP 0-2500 ng/mL, BZE 0-1200 ng/mL, BENZ 0-1600 ng/mL, MET 0-600 ng/mL, OPI 0-720 ng/mL, and THCCOOH 24-60 ng/mL. Precision results (within run) for different concentrations were as follows: AMP 3.1-5.7%, BZE 2.4-6.6%, BENZ 4.3-8.0%, MET 2.0-5.5%, OPI 2.8-7.6%, and THCCOOH 1.4-2.4%. Between-run results were as follows: AMP 8.7-15.5%, BZE 6.4-7.5%, BENZ 8.2-15.8%, MET 2.7-5.1%, OPI 4.3-11.2%, and THCCOOH 2.6-7.4%. Sensitivity, specificity, and comparison of CEDIA semiquantitation with GC-MS quantitative results were performed on 500 original serum and whole blood samples. The data provided sufficient documentation to use the CEDIA urine-screening technique without any adaptation as a sensitive serum/whole blood screening for BZE, BENZ, MET, OPI, and THCCOOH. Serum screening for amphetamines is not sensitive enough in the unchanged urine mode. It will require some adaptation to a serum mode (probably a higher sample volume [BM/Hitachi 911] combined with protein precipitation of the sample).