Findings of illicit drugs in hair of children at different ages.

Hair is a preferred material to detect exposure or use of illegal drugs in children. In the present study, we investigated a total of 387 hair samples for commonly applied illegal drugs of children up to 16 years. Analysis was by liquid chromatography/mass spectrometry with LOQs of 0.01 ng/mg hair for all analytes except tetrahydrocannabinol carboxylic acid with an LOQ of 0.1 pg/mg hair. Results were firstly compared with our in-house statics on results from adults' hair, and secondly to literature data. We started from the assumption that drug concentrations decrease with increasing age.Results were assigned to 4 different age groups (< 1 year, 1-< 6 years, 6-< 14 years, 14-16 years). As expected, higher results were obtained in age groups 1 and 2. The lowest concentrations were present in age group 3, whereas an increase could be observed in group 4 except heroin. In babies, positive results may be due to in utero exposure, breast milk feeding, and a close physical contact. All drugs under investigation such as cannabinoids, cocaine, amphetamines, and opiates have been detected in breast milk as well as in skin excretions such as sebum, sweat and cutaneous cells. For most drugs, average concentrations in children hair were lower than in adult hair when compared with our in-house statistics. Interestingly, the increase of cannabinoids, cocaine, and amphetamines concentrations in adolescents' hair points to a deliberate use of these drugs possibly in addition to passive exposure. This observation shows that age groups 1 and 4 are most vulnerable if caregivers or parents are drug users, even if the sources of positive drug findings differ.

Concentration distribution of more than 100 drugs and metabolites in forensic hair samples.

Drug testing in hair can complement conventional blood and urine analysis as it enlarges the window of detection and may allow a differentiation of heavy from moderate or rare use. Databases of drug concentrations in biological matrices are a valuable support in interpreting analytical results. In forensic toxicology, several databases exist especially for blood/serum samples. In the present paper, the concentration distributions of more than 100 legal and illegal drugs such as narcotic drugs, opioids, antidepressants, antipsychotics, benzodiazepines, and major metabolites detected in authentic Caucasian hair samples in our laboratory are summarized. Depending on availability, the proximal sections of 1-6 cm in length were analyzed by liquid chromatography/tandem mass spectrometry following extraction of the finely chopped specimens by ultrasound in methanol. The data may present a helpful basis also for other laboratories for an initial evaluation of their results. However, these statistical data should not be used uncritically without including the circumstances of the particular case and the analytical procedures used. In addition, each laboratory in charge of interpreting results from hair analysis should balance own results as far as available with this data base.

[Deaths from propofol abuse : Survey of institutes of forensic medicine in Germany, Austria and Switzerland]. / Todesfälle durch Propofolmissbrauch : Befragung in rechtsmedizinischen Instituten in Deutschland, Österreich und der Schweiz.

Previous references suggesting a high mortality of propofol addiction in medical personnel were mostly based on surveys of the heads of medical departments or case reports; therefore, a questionnaire was sent to 48 forensic medicine departments in Germany, Austria and Switzerland concerning the number of autopsies carried out between 2002-2112 on medical personnel with the suspicion of abuse of propofol or other analgesics. The response rate was 67%. In 16 out of the 32 responding departments 39 deaths (27 males) were observed with previous connections to anesthesiology, intensive care or emergency departments of which 22 were physicians, 13 nurses, 2 other personnel and 2 were unknown. Propofol was the major cause of death in 33 cases (85%), in 8 cases including 7 with propofol, an unintentional accident was recorded and 29 were determined to be suicide. In 14 cases chronic abuse was denied but actually excluded by toxicological analysis in only 2 cases. In 11 cases involving suicide the question of abuse was not investigated. This survey confirmed previous data about the central role of propofol for the fatal outcome of addiction and suicide of anesthetists and other medical personnel. A dual prevention strategy with low-threshold offers for persons at risk and strategies for early detection is urgently needed including a stricter control of dispensing, improvement in forensic medical documentation and the use of toxicological investigations in every case of suspected abuse.

First detection of ethylphenidate in human fatalities after ethylphenidate intake.

Methylphenidate, a psychostimulant drug from the group of amphetamines is, among others, established in the treatment of attention deficit hyperactivity disorder and narcolepsy. It is also known to have a certain potential of abuse. In combination with alcohol, the metabolite ethylphenidate was detected in human plasma in small amounts. However, ethylphenidate is sold as "research chemical" via the Internet. It was put under German narcotics law in July 2013. In a recent case, where a deceased person was found in his apartment, the police seized a plastic bag with the inscription "ethylphenidate". An autopsy of the 32-year-old man yielded a mitral valve endocarditis, which must have persisted a while before death, in combination with a pneumonia. At the Forensic Toxicological Centre (FTC) in Munich femoral blood, liver, pericardium fluid, urine, stomach content and hair of the deceased were analyzed for ethylphenidate after sample preparation by an LC-Triple TOF 5600. Calibration curves were spiked with a methanolic 1mg/mL solution of ethylphenidate (substance provided by the State Office of Criminal Investigation in Munich) in whole blood in comparison to liver and femoral blood, in serum in comparison to pericardium fluid and in urine in comparison to urine and stomach content, respectively. Ethylphenidate was detected in all analyzed matrices. The spectrums of the human specimen were compared to those obtained from the calibration curves and identified as ethylphenidate. The measured concentrations were for femoral blood 110ng/mL, for liver 180ng/g, for pericardium fluid 131ng/mL, for urine 987ng/mL and for stomach content 20.7ng/mL, respectively. The stomach contained 200mL of a brownish-coloured liquid, resulting in a total amount of 4000ng ethylphenidate. The lowest calibrator for whole blood and serum was 1ng/mL and for urine 10ng/mL. As far as it is known to the authors, these are the first ethylphenidate levels measured in a case of ethylphenidate intake. Therefore these results can only be compared to methylphenidate concentrations with therapeutic levels ranging from 5 to 60ng/mL in serum. As the toxic levels for methylphenidate start from approximately 500ng/mL serum, we estimate that ethylphenidate in the concentrations mentioned above is not in a directly lethal range. But it has to be considered, that amphetamine-like drugs as methylphenidate are known for their cardiovascular side effects (like tachycardia and arrhythmia) and might therefore have contributed to death, which was attributed to endocarditis in combination with pneumonia.

Determination of hypoglycaemia induced by insulin or its synthetic analogues post mortem.

The determination of human insulin or its synthetic analogues in post-mortem specimens represents a challenge for forensic toxicologists due to its proven instability in post-mortem blood. We present two cases of an insulin-induced hypoglycaemia. In the first case, ante-mortem material was available for the detection of an injection with human insulin. Human insulin was detected by immunopurification with magnetic beads and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses at a concentration of 5180 µU/ml. The molar ratio human insulin:C-peptide was 111. The second case describes a suicide by self-injection of Insulin lispro and determination of the drug after pre-extraction with methanol and immunopurification by LC-MS/MS at the injection site, in vitreous humour and organs. Apart from the well-known matrices--femoral blood and urine--the specimen vitreous humour and the injection site promise the best possibilities for a proof of insulin at autopsy. In addition to insulin analyses, the parameters C-peptide, proinsulin, glucose, lactate, and sulfonylureas should be measured in case of suspected fatal hypoglycaemia.

Nurse induced respiratory depression by succinylcholine--the 'hero syndrome'.

A nurse administered the neuromuscular blocking agent succinylcholine (SUX) to at least one patient and gave first aid in the therapy of unexpected respiratory depression. SUX is regarded as an undetectable and thus perfect poison due to its short half-life and degradation to the endogenous compounds choline and succinic acid. However, SUX and especially its metabolite succinylmonocholine (SMC) were found in plasma and urine a few hours after administration by means of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Compared to clinical studies, the window of detection was sufficient to gain definite proof; in other cases no samples were collected. The nurse enjoyed high reputation with the doctors. According to the court she wanted to present herself spectacularly as the first and decisive rescuer to demonstrate her special abilities and capacities, perhaps to receive a better job in the hospital. Considering the actual case, the hero syndrome is not limited to fire-fighters.

Detection of diabetic metabolism disorders post-mortem--forensic case reports on cause of death hyperglycaemia.

Diabetic coma is the most severe form of hyperglycaemic metabolic disorders. The post-mortem diagnosis of this disorder of glucose metabolism can be difficult and vague due to a lack of characteristic morphological findings. Six death cases caused by diabetic coma are described with special focus on biochemical (and histological) findings. The possible glycaemia markers glucose, lactate, HbA1c, fructosamine, anhydroglucitol, and ketone bodies were measured and the usefulness of these parameters is evaluated and discussed. Estimations of glucose concentrations in vitreous humour or cerebrospinal fluid and of ketone bodies in blood or other matrices are obligatory while measurements of HbA1c, fructosamine, or anhydroglucitol can only provide additional information on the long-term adjustment of diabetes in the deceased. Lactate concentrations (addition of glucose and lactate levels to form the sum formula of Traub) do not give more information than the glucose concentration itself and can be therefore omitted.

Optimization and validation of CEDIA drugs of abuse immunoassay tests in serum and urine on an Olympus AU 400.

A preliminary initial cloned enzyme donor immunoassay (CEDIA) was optimized for serum and urine drug testing with respect to the German per se limits for driving under the influence of drugs (serum) and lowered cut-offs in cases of driving licence re-granting (urine). The tests were performed on an Olympus AU 400 auto analyzer. Validation revealed sensitivities between 93% and 100% based on comparison with data from gas or liquid chromatography coupled with mass spectrometry. Even if specificity ranged between 83% and 98 %, the tests can be considered useful for forensic purposes. Receiver operating characteristic (ROC) curves, Youden indices, as well as positive and negative predictive values are presented.

Estimation of gamma-hydroxybutyrate (GHB) co-consumption in serum samples of drivers positive for amphetamine or ecstasy.

There is no toxicological analysis of gamma-hydroxybutyrate (GHB) applied routinely in cases of driving under influence (DUI); therefore the extent of consumption of this drug might be underestimated. Its consumption is described as occurring often concurrently with amphetamine or ecstasy. This study examines 196 serum samples which were collected by police during road side testing for GHB. The samples subject to this study have already been found to be positive for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and/or 3,4-methylenedioxyethamphetamine (MDEA). Analysis has been performed by LC/MS/MS in the multiple reaction monitoring (MRM) mode. Due to its polarity, chromatographic separation of GHB was achieved by a HILIC column. To differentiate endogenous and exogenous levels of GHB, a cut-off concentration of 4µg/ml was applied. Of the 196 samples, two have been found to be positive for GHB. Of these samples, one sample was also positive for amphetamine and one for MDMA. Whilst other amphetamine derivates were not detected in these samples, both samples were found to be positive for cannabinoids. These results suggest that co-consumption of GHB with amphetamine or ecstasy is relatively low (1%) for the collective of this study.

Investigations on the influence of different grinding procedures on measured ethyl glucuronide concentrations in hair determined with an optimized and validated LC-MS/MS method.

Ethyl glucuronide (EtG) analysis in hair is a suitable method for the retrospective determination of previous alcohol consumption. According to the German guidelines, EtG abstinence is improbable at c(EtG) > 7 pg/mg in the proximal 3 cm of scalp hair. The chromatography of the routinely used liquid chromatography-tandem mass spectrometry procedure was optimized by replacing the stationary phase. To simplify sample preparation, two different mills were tested, and an optimized grinding process was developed. The new method was successfully validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry. Despite a simple extraction procedure without any cleaning steps, a very high sensitivity (limit of detection, 1.7 pg/mg; limit of quantitation, 2.3 pg/mg) could be achieved. Competitive analysis showed significantly higher EtG concentrations in pulverized versus cut hair samples. The strong impact of sample preparation on the determined EtG concentrations suggests the introduction of a standardized sample preparation method to produce comparable results.

A high-performance liquid chromatographic-tandem mass spectrometric method for the determination of ethyl glucuronide and ethyl sulfate in urine validated according to forensic guidelines.

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are powerful markers for alcohol intake and abuse. Several analytical procedures for the quantification of EtG and EtG in serum and urine have been developed so far. Many of the published methods show limits of detections (LODs) or limits of quantifications (LOQs) for EtG in urine within the range of 0.1 mg/L or higher. Since this is the actual cutoff value for proving abstinence in Germany, problems may occur if urine samples are highly diluted. In this paper, the validation of a highly sensitive, fast and simple LC-MS-MS for the determination of EtG and EtS in urine is described. The calibration curves for EtG and EtS is linear over the whole range (0.025-2.0 mg/L). Very low detection limits can be achieved (LOD: EtG 0.005 mg/L, EtS 0.005 mg/L; and LOQ: EtG 0.019 mg/L, EtS 0.015 mg/L). All data for selectivity, precision and accuracy, recovery, as well as for the processed sample and the freeze/thaw stability, comply with the guidelines of the German Society of Toxicological and Forensic Chemistry. Strong matrix-related effects can be compensated for by using an internal standard. Finally, the applicability of the procedure is proven by analysis of 87 human urine samples and by successful participation in interlaboratory comparison tests.

Evaluation of two immunoassay procedures for drug testing in hair samples.

A preliminary initial enzyme-linked immunosorbent assay (LUCIO-Direct ELISA kit) and a preliminary DRI enzyme immunoassay were evaluated for drug detection in head hair with respect to lowered cutoff values recommended in Germany for the control of abstinence in cases of re-granting of drivers' licences. Following drug classes were included: cannabinoids, opiates, cocaine like substances, amphetamine, methamphetamine (and methylenedioxyamphetamines), methadone, and benzodiazepines. 759 analyses were performed using LUCIO-Direct ELISA kits and 936 analyses using DRI enzyme immunoassay tests. Sample size for each drug group and immunoassay test reached from 74 to 178. The LUCIO-Direct ELISA kit revealed a sensitivity of 91% for amphetamine up to 98% for methadone (methamphetamine 92%, cocaine 94%, opiates 94%, benzodiazepines 96%) and values of specificity of 72% for methadone up to 89% for amphetamine and benzodiazepines. The test was not useful for a preliminary screening for tetrahydrocannabinol (sensitivity of 65%) in consideration of a suggested cutoff of 0.02 ng/mg. The DRI enzyme immunoassay test was only useful for morphine and cocaine testing at low recommended new cutoff values (0.1 ng/mg) revealing sensitivities of 94% and 99%, respectively.

Variations in vitreous humor chemical values as a result of pre-analytical treatment.

Vitreous humor (VH) is used for postmortem diagnosis of metabolic diseases and to clarify the postmortem interval. Because of its viscous nature, this fluid has to be liquefied prior to analysis; however variations in measured concentrations of the analytes are ascribed to different pre-analytical treatment methods with regard to. The aim of this study was to compare different pre-analytical methods. Centrifugation, heat treatment, enzymatic digestion and liquefying by ultrasound were compared using a collection of 120 samples obtained from 2003 to 2007. The determined parameters were: sodium, potassium, chloride, calcium, glucose, creatinine, urea and lactate. Analyses were performed either photometrically or by using ion-selective electrodes. Heat and hyaluronidase treatment generate slightly higher and lower values in the measurement of electrolytes and glucose. However, in the determination of calcium concentration, both methods (heat especially) are associated with extreme low or high values. Only differences between ultrasound and centrifugation treatment show comparatively small variations and are close to instrument accuracy. Therefore, we recommend centrifugation, combined with mixing, as the best and easiest method in which to prepare frozen samples for analysis. Additionally, the measurement of lactate shows that analytical methods, calibrated for serum and urine, cannot be easily applied for VH.

Comparison of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) concentrations in hair for testing abstinence.

Hair analysis is a powerful tool for retrospective drug analysis. By determining the minor ethanol metabolites ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) in hair, even a previous consumption of alcohol is detectable. However, previous studies showed a lack of correlation if both parameters are determined simultaneously. A further study was conducted to confirm or refute these results. One hundred and sixty hair samples were analyzed for EtG and FAEE in the context of driving ability. In 109 cases, alcohol abstinence was clearly proven and was excluded in 15 cases. In 36 cases, ambiguous results were found. Possible reasons for the deviating results are discussed. It is recommended, that in context of driving ability diagnostics the EtG result is determinant. In critical cases FAEE concentrations can be determined for checking purposes, but a negative FAEE result cannot refute a determined EtG concentration >7 pg/mg.

An uncommon case of a suicide with inhalation of hydrogen cyanide.

An uncommon suicide by oral ingestion of potassium cyanide salts and contemporaneous inhalation of hydrogen cyanide is presented. A 48-year-old tradesman was found dead sitting in his car. A penetrating odor of bitter almonds was noticed when opening the doors. A camping stove and a cooking pot containing large amounts of dark blue crystals were found in the footwell of the car. White powder adhered to his fingers and to the area around the mouth. Furthermore bottles containing potassium ferrocyanide and different kinds of acid and leach were found in the car together with internet information about, e.g. potassium ferrocyanide and potassium cyanide. At autopsy hemorrhages and erosions of the mucosa of the respiratory tract, esophagus and stomach were found. Concentrations of cyanide were 0.2mg/l in stomach contents, 0.96mg/kg in brain tissue, 2.79mg/kg in lungs, and 5.3mg/l in blood. The white and toxic powder potassium cyanide was formed by heating of the yellow crystals of potassium ferrocyanide on the camping stove. This powder was probably ingested orally. Addition of acid converted the salt into the highly toxic gas hydrogen cyanide. Oxidation with atmospheric oxygen built the dark blue ferrous compound Prussian blue. This case report of a person who was not familiar with chemicals demonstrates the acquisition of professional information via the internet, enabling a suicide with a complex procedure.

Enantioselective determination of ondansetron and 8-hydroxyondansetron in human plasma from recovered surgery patients by liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-mass spectrometric assay with atmospheric pressure chemical ionization for quantification of ondansetron and its main metabolite 8-hydroxyondansetron in human plasma was presented. The enantiomeric separation was achieved on a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate). The validation data were within the required limits. The assay was successfully applied to authentic plasma samples. Quantitative results from postoperative patients receiving ondansetron demonstrated a great interindividual variability in postoperative plasma drug concentrations, the metabolites were not detected in their unconjugated form. A wide variation in the S-(+)-/R-(-)-ondansetron concentration ratio between 0.14 and 7.18 is indicative for a stereoselective disposition or metabolism. In further studies CYP2D6 and CYP3A4 genotype dependent metabolism of ondansetron enantiomers as well as of co-administered drugs and clinical efficacy of the medication should be tested.

Cocaine and benzoylecgonine concentrations in fluorinated plasma samples of drivers under suspicion of driving under influence.

Due to an in vitro decomposition of cocaine (COC), usually an analytical detection in unstabilized blood taking systems is impossible and for this reason the main metabolite benzoylecgonine (BZE) is determined. In a few regions in Germany the local authorities use systems containing sodium fluoride for taking a blood sample. Caused by inhibition of esterases in the taken sample COC is still detectable in blood samples. Cases of driving under the influence (DUI) with positive findings for COC and/or BZE were evaluated and substance concentrations in plasma were correlated with documented deficiencies in the psycho-physical performance. In 51.5% of all cases positive for cocaine-like substances besides BZE a positive result was also given for COC. If both substances were found (mean COC concentration 0.836 mg/L) the concentration of BZE was significantly higher (mean 0.669 mg/L) compared to cases with a single detection of BE (mean 0.209 mg/L) (p=0.001). In 72 cases without any detection of further drugs cocaine users seemed rather excited and stimulated towards intervening police officers, in particular when COC was present in the blood (17.8%). Also in the medical investigation reports a rather stimulative effect (25% vs. 3.6% sedated) was to be registered. Whereas with a sole determination of BZE, a stimulated (19%) as well as a sedated impression (14.9%) was described. Definite concentration-effect relations could not be recognized. Indeed, more peculiarities were to be registered with the simultaneous detection of COC than with a sole BZE determination. The determination of COC and the differences in the BZE concentration can be explained by the fact that the simultaneous detection of both substances is indicative for of a consumption shortly before the blood sampling. A sole detection of BZE is more likely indicative for a consumption already some time ago. Therefore, in the first case one would rather suggest an acute intoxication phase. A determination of BZE without COC is more likely indicative for a transition to the drug-induced exhaustion phase which is also to be expected after the consumption of COC. The absence of COC can be seen within the scope of a validity check as an indication of a possible exhaustion reaction. A use of fluoride stabilized blood sampling systems is advised. This makes it easier to investigate the state of intoxication or to appraise the temporal connection between COC consumption and incident or blood sampling.

A fully validated high-performance liquid chromatography-tandem mass spectrometry method for the determination of ethyl glucuronide in hair for the proof of strict alcohol abstinence.

Hair analysis has become a powerful tool for the detection of chronic and past drug consumption. For several years, it has been possible to determine even the intake of ethanol in hair samples by detecting the ethanol metabolites ethyl glucuronide or fatty acid ethyl esters. Recently, new requirements were published for the use of EtG as an abstinence test (c(EtG) < 7 pg/mg) as well as for heavy-drinking detection (c(EtG) > 30 pg/mg). In order to perform abstinence tests, a sensitive LC-MS/MS procedure has been developed and fully validated according to the guidelines of forensic toxicology. The nine-point calibration curve showed linearity over the range of concentrations from 2-1,000 pg/mg. Detection and quantification limits were 1 and 4 pg/mg respectively. The intra- and inter-day precision and accuracy were always better than 20%. The validated procedure has successfully been applied to perform abstinence tests and to analyze hair samples from persons in withdrawal treatment. Concentrations between

Human amygdala reactivity is diminished by the ß-noradrenergic antagonist propranolol.

BACKGROUND: Animal models of anxiety disorders emphasize the crucial role of locus ceruleus-noradrenergic (norepinephrine, NE) signaling, the basolateral amygdala (BLA) and their interactions in the expression of anxiety-like behavioral responses to stress. Despite clinical evidence for the efficacy of a ß-noradrenergic receptor blockade with propranolol in the alleviation of anxiety symptoms and the secondary prevention of post traumatic stress disorder, preclinical evidence for a ß-noradrenergic modulation of BLA activity in humans is missing. METHOD: We combined functional magnetic resonance imaging in healthy volunteers with probabilistic mapping of intra-amygdalar responses to fearful, neutral and happy facial expressions to test the hypothesis that a ß-noradrenergic receptor blockade with propranolol would inactivate the BLA. RESULTS: Consistent with our a priori hypothesis, propranolol diminished BLA responses to facial expressions, independent of their emotional valence. The absence of activity changes in probabilistically defined visual control regions underscores the specific action of propranolol in the BLA. CONCLUSIONS: Our findings provide the missing link between the anxiolytic potential of propranolol and the biological basis of ß-noradrenergic activation in the human BLA as a key target for the pharmacological inhibition of anxiety neurocircuitry. Moreover, our findings add to emerging evidence that NE modulates both the reactivity (sensitivity) and the operating characteristics (specificity) of the BLA via ß-noradrenergic receptors.

Determination of glycyrrhetic acid after consumption of liquorice and application to a fatality.

Besides alcohol and drugs of abuse, several popular foods contain potentially toxic substances and cases of intoxication after consumption of these foods attract notice of forensic toxicology. This is also true for the case of a 34-year-old woman who was suspected to have suffered lethal acute intoxication from eating nothing but liquorice over a period of several months. The liquorice ingredient glycyrrhizin and its metabolite glycyrrhetic acid, which elicits a mineralocorticoid effect, were determined in the sort of liquorice the woman had consumed by using LC-MS/MS. In addition, a fast and sensitive procedure for the quantification of glycyrrhetic acid including a simple sample preparation was developed. The method was proven to be accurate and precise. In a liquorice ingestion experiment, 200 g of liquorice had to be eaten. Afterwards, concentrations of glycyrrhetic acid in the blood of up to 434 ng/ml were measured. Since only traces of glycyrrhetic acid had been found in the blood and stomach content of the deceased woman, the possibility of acute lethal glycyrrhetic acid intoxication could be eliminated. Excluding other causes of death, the woman is believed to have died from a lethal hyperglycemic coma. Nonetheless, the influence of harmful and toxic substances in food should be taken into consideration in special cases.