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Testing hair for cannabis use has increasingly been scrutinised due to exposure to second-hand smoke or environmental contamination. Confirmation of drug use involving detection of metabolites such as 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) and 11-hydroxy-delta-9-tetrahydrocannabinol (THC-OH) having very rarely been considered. We developed a new, simplified procedure with regard to expenditure of time and material to determine delta-9-tetrahydrocannabinol (THC, qualitatively), as well as THC-OH and THC-COOH (quantitatively) from 587 hair samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) which was compared to hitherto established methods (n = 3). Compared to conventional methanolic extraction alkaline dissolution resulted in higher concentrations for THC-OH. Concentrations determined from specimens ranged from 0.01 to 18.7 ng THC/mg hair, 0.05-37.6 pg THC-OH/mg hair, and from 0.1 to 54.3 pg THC-COOH/mg hair. THC was detectable in 70.4% samples along with both metabolites from more than half of these samples. In 12.9% of THC-positive cases, neither THC-OH nor THC-COOH were present. In 8.9% of THC-negative cases, it was possible to detect metabolites either alone or in combination. THC-OH could more frequently be detected than THC-COOH and appeared to be less susceptible to cosmetic treatment. In summary, THC-OH turned out to be a further suitable marker to prove cannabis use. Determination of both metabolites is recommended to unequivocally differentiate consumption from external exposure or contamination., (© 2018 John Wiley & Sons, Ltd.)

Since there are no characteristic morphological findings post mortem diagnosis of diabetes mellitus and identification of diabetic coma need to be confirmed by suitable biomarkers. The postmortem identification of preexisting hyperglycemia or diabetic coma can be difficult if the matrices for the determination of the established biomarkers are not available or the obtained results are close to the established cut-off values. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for renal reabsorption. Therefore low serum concentrations of 1,5-AG, reflect hyperglycemic excursions over the prior 1-2 weeks in diabetic patients. To evaluate postmortem 1,5-AG concentrations in vitreous humor (VH) and cerebrospinal fluid (CSF), a liquid chromatographic mass spectrometric method for the quantification of 1,5-AG in VH and CSF was developed and validated according to international guidelines. In order to establish a cut-off for the identification of an ante mortem existing diabetes and the diagnosis of a diabetic coma in deceased the relationships between 1,5-AG concentrations in VH and CSF to other diabetes associated biochemical parameters of 47 non-diabetic, 86 diabetic and 9 cases of diabetic coma were examined. In 83 of these cases, both matrices could be obtained and analyzed. Comparisons of the respective HbA1c, Glucose in VH or Sum-formula of Traub to 1,5-AG concentrations in VH and CSF resulted in correlation coefficients R 2 ≤0.2. For the application of 1,5-AG concentrations in VH against CSF, a linear regression gave a correlation coefficient of R 2 =0.955. Comparable linear correlations of 1,5-AG concentrations could be observed between VH and femoral venous blood (FVB) (R 2 =0.839) as well as between CSF and FVB (R 2 =0.756). Due to overlapping concentration ranges, the determination of a reliable cut-off for the differentiation of diabetic disease to diabetic coma cases was not possible. However, the 1,5-AG concentrations in VH and CSF in cases of deceased diabetics were significantly lower (p<0.05) than in non-diabetic deceased and therefore indicate a pre-existing diabetes or even a diabetic coma as the cause of death., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Drugs which are commonly smoked or sniffed (e.g. cocaine), can contaminate hair through smoke or dust; therefore testing for metabolites, especially hydroxy metabolites, is highly recommended. The presence of hydroxy metabolites in street-cocaine (COC) has been discussed. To check if detection of hydroxy metabolites definitely proves ingestion, the presence of these metabolites in street COC samples has to be checked. It is expected that the more hydrophilic hydroxy metabolites of COC are incorporated into the hair-matrix to a lesser extent. For this study 576 COC positive hair samples (≥0.1ng COC/mg hair) were analysed by LC-MS/MS for benzoylecgonine (BE), norcocaine (NC), cocaethylene (CE), ortho-, meta- and para-hydroxy COC (o-, m-, p-OH-COC), meta- and para-hydroxy BE (m-, p-OH-BE), and meta- and para-hydroxy NC (m-, p-OH-NC). The results were compared with the respective metabolite/COC concentration ratios in 146 street COC samples, confiscated by the Bavarian police. Peak areas were used to estimate BE/COC, NC/COC, CE/COC and hydroxy metabolites/COC. Similar metabolic ratios were found for o-OH-COC in 88% of the samples, but for p-OH-COC and m-OH-COC only in 5.1% and 6.8%, respectively. Notably, p- and m-OH-BE as well as p- and m-OH-NC could not be identified from seized samples. We propose that area ratios exceeding the ratios of street COC more than twice or identification of OH-BE and OH-NC enable to differentiate COC consumption from contamination. Using these criteria, consumption of the drug could be proven in 92% of COC positive samples. As detection of meta- and para-hydroxy metabolites using the above mentioned criteria is a reliable tool to distinguish between ingestion and external contamination, it is recommended to implement their measurement into daily routine work., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Because of the lack of characteristic morphological findings post mortem diagnosis of diabetes mellitus and identification of diabetic coma can be complicated. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for renal reabsorption. Therefore low serum concentrations of 1,5-AG, reflect hyperglycemic excursions over the prior 1-2 weeks in diabetic patients. Next to clinical applications determination of 1,5-AG can also be used in forensic analysis. To investigate the elimination of 1,5-AG, a liquid chromatographic-mass spectrometric method for the determination of 1,5-AG and creatinine in urine was developed and validated according to international guidelines. To evaluate ante mortem concentrations of 1,5-AG spot urine samples of 30 healthy subjects, 46 type 1 and 46 type 2 diabetic patients were analyzed. 1,5-AG urine concentrations of diabetic patients were significantly (p<0.001) lower (mean: 1.54μg/ml, n=92) compared to concentrations of healthy subjects (mean: 4.76μg/ml, n=30) which led to the idea that 1,5-AG urine concentrations post mortem might help in the interpretation of a diabetic coma post mortem. Urine of 47 deceased non-diabetics, 37 deceased diabetic and 9 cases of diabetic coma were measured. Comparison of blood and urine 1,5-AG concentrations in clinic samples (linear, R 2 =0.13) and forensic samples (linear, R 2 =0.02) showed no correlation. Urinary levels of 1,5-AG in deceased diabetic (mean 6.9μg/ml) and in non-diabetic patients (mean 6.3μg/ml) did not show a significant difference (p=0.752). However, urinary 1,5-AG concentrations in deceased due to diabetic coma (mean: 1.7μg/ml) were significantly lower than in non-diabetic (mean: 6.3μg/ml, p=0.039) and lower than in diabetic cases (mean: 4.7μg/ml, p=0.058). The determination of a reliable cut-off for the differentiation of diabetic to diabetic coma cases was not possible. Normalization of urinary 1,5-AG concentrations with the respective creatinine concentrations did not show any gain of information. In clinical (serum) and forensic blood samples a significant difference between all groups could be detected (p<0.05). Comparison of blood and urine 1,5-AG concentrations in clinical samples (linear, R 2 =0.13) and forensic samples (linear, R 2 =0.02) showed no correlation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination. This especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens, thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC-positive samples for these compounds was performed by liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS). Once identified, mass transitions for o-, p- and m-isomers of hydroxy COC as well as p- and m-isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by liquid chromatography-tandem mass spectrometry (LC-MS/MS) which was applied to 576 hair samples. Hydroxy metabolites were present in 92.2% of COC-positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p-OH-BE, m-OH-BE as well as p-OH-NCOC and m-OH-NCOC have been identified for the first time in COC-positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC-positive hair samples., (Copyright © 2017 John Wiley & Sons, Ltd.)

In this study, two fatalities associated with the synthetic opioids AH-7921 and MT-45 are reported. Within the last few years, both compounds have emerged on the recreational drug market and are sold as "research chemicals" on the internet. In the first case, a 22-year-old woman was found dead in the bedroom of her apartment by two of her friends. A plastic bag labeled "AH-7921" was found in the apartment and the two friends stated that the deceased had consumed AH-7921 prior to her death. The woman was a known drug addict. In the second case, a 24-year-old man was found dead in his room by his mother. The deceased was sitting on a chair in front of his desk slumped over. Several bags of white powder labeled "MT-45", "Methoxmetamine" and "Methoxphenidine" were found in his room. Toxicological analyses of femoral blood, heart blood, liver, pericardial fluid, urine, vitreous humor and stomach content of the deceased were performed using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Time-of-flight mass spectrometry was carried out on an LC-Triple TOF 5600 system (AB Sciex) with electrospray ionization operated in positive mode. In the first case, additional hair analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-QTOF-MS. In both cases, the relevant synthetic opioid could be detected in all analyzed samples. The concentration of AH-7921 was determined to be 450μg/L in femoral blood. MT-45 was present at a concentration of 2900μg/L in femoral blood. Besides methoxmetamine which could qualitatively be detected in femoral blood, urine and stomach content no methoxphenidine was found. In summary, deaths of the young individuals could be, by exclusion of other causes of death, attributed to the consumption of an overdose of AH-7921 and MT-45, respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This work represents the development, validation, and application of a liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) screening method for the detection of pharmaceutical substances and illicit drugs (acidic, basic, and neutral organic drugs) in urine samples. Time-of-flight mass spectrometry was performed using an LC-Triple TOF 5600 system with electrospray ionization operated in both positive and negative mode, respectively. The limits of detection (LODs), determined for 34 substances, were < 10 ng/mL for 91% of the compounds. The limits of quantitation (LOQs) were < 20 ng/mL for 91% of the substances. The identification of the compounds was based on exact mass (< ± 5 ppm), retention time (<2%) if available, isotopic pattern fit (<10%) and library hit (>70%). These four parameters served as identification criteria and are discussed according to their role in identifying compounds even without reference substances. In routine casework, two in-house XIC (extracted ion chromatogram) lists, consisting of 456 protonated and 26 deprotonated compounds were used and retention times for 365 compounds were available. Compared to the results found with the established gas chromatography-mass spectrometry (GC-MS) procedure, the findings with the LC-QTOF-MS screening method showed a good comparability. Results that were not detected by LC-QTOF-MS because of a missing entry in the targeted XIC list could retrospectively be confirmed by simply entering the elemental formula of the relevant substance into the software and reprocessing the sample. LC-QTOF-MS offers an attractive technique for the fast and specific identification of illicit drugs and toxic compounds in urine samples. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Discrimination between street heroin consumption and poppy seed ingestion represents a major toxicological challenge in daily routine work. Several difficulties associated with conventional street heroin markers originate from their versatile occurrence in various poppy seed products and medications, respectively, as well as to small windows of detection. A novel opportunity to overcome these hindrances is represented by the new potential street heroin marker acetylated-thebaine-4-metabolite glucuronide (ATM4G), originating from thebaine during street heroin synthesis followed by metabolic reactions after administration. In this study, urine samples after consumption of different German poppy seed products and urine samples from subjects with suspicion of preceding heroin consumption were tested for ATM4G, 6-AC (6-acetylcodeine), papaverine, noscapine, 6-MAM (6-monoacetylmorphine), morphine, and codeine. Neither 6-AC and 6-MAM nor ATM4G but morphine and codeine could be detected in urine samples following poppy seed ingestion. As well, neither papaverine nor noscapine could be observed even after consumption of poppy seeds containing up to 37 µg noscapine and up to 9.8 µg papaverine, respectively. Concerning the urine samples with suspicion of preceding heroin consumption, ATM4G could be detected in 9 of 43 cases. By contrast, evidence of 6-AC and 6-MAM, respectively, could only be seen in 7 urine samples. In conclusion, ATM4G should be measured additionally in cases requiring discrimination of street heroin consumption from poppy seed intake. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Two cases of fatalities are reported of which the recreational use of Mitragyna speciosa ("kratom") could be confirmed. One of these cases presents with one of the highest postmortem mitragynine concentrations published to date. Our results show that even extremely high mitragynine blood concentrations following the consumption of kratom do not necessarily have to be the direct cause of death in such fatalities as a result of an acute overdose. The two cases are compared with regard to the differences in mitragynine concentrations detected and the role of mitragynine in the death of the subjects. Irrespective of the big differences in mitragynine concentrations in the postmortem blood samples, mitragynine was not the primary cause of death in either of the two cases reported here. Additionally, by rough estimation, a significant difference in ratio of mitragynine to its diastereomers in the blood and urine samples between the two cases could be seen., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The Mombello Psychiatric Hospital in Limbiate, near Milan, replaced the old Senavra Hospital as the Psychiatric Hospital for the Province of Milan in the 19th century. During the early 20th century, bodies of several Mombello patients were dissected and preserved by Giuseppe Paravicini, an anatomist who operated within the asylum. The aim of the present study was to examine and memorialize this important assemblage. To this end, we were allowed to sample the head hair of six such preparations for toxicological analysis. By means of high performance liquid chromatography, cocaine and its main metabolite, benzoylecgonine, were detected in two out of six hair samples. The concentrations for cocaine were 0.151 and 0.09ng/mg and for benzoylecgonine 0.103 and 0.147ng/mg, respectively. Given that cocaine was a commonly used medicine, beginning in the mid-19th century and persisting into the 20th century, it is not surprising that some patients may have ingested this drug. In addition to the detection of cocaine, these analyses also provided evidence of nicotine and caffeine intake., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The organic cation transporter OCT1 (SLC22A1) mediates uptake and metabolism of the active tramadol metabolite (+)O-desmethyltramadol in the liver. In this study, the influence of OCT1 genetic polymorphisms on pharmacokinetics and analgesic efficacy of tramadol in patients recovering from surgery was analyzed in addition to the CYP2D6 genotype. Postoperative patients who received tramadol through patient-controlled analgesia were enrolled. Genotypes resulting in 0, 1, or 2 active OCT1 alleles were determined as well as CYP2D6 genotypes. The primary endpoint was the 24-hour postoperative tramadol consumption in patients with 0 vs at least 1 active OCT1 allele. Secondary endpoint was the OCT1-dependent plasma concentration (areas under the concentration-time curves) of the active tramadol metabolite (+)O-desmethyltramadol. Of 205 patients, 19, 82, and 104 carried 0, 1, and 2 active OCT1 alleles, respectively. Cumulative tramadol consumption through patient-controlled analgesia was lowest in patients with 0 active OCT1 allele compared with the group of patients with 1 or 2 active alleles (343 ± 235 vs 484 ± 276 mg; P = 0.03). Multiple regression revealed that the number of active OCT1 alleles (P = 0.014), CYP2D6 (P = 0.001), pain scores (P < 0.001), and the extent of surgery (0.034) had a significant influence on tramadol consumption. Plasma areas under the concentration-time curves of (+)O-desmethyltramadol were 111.8 (95% confidence interval: 63.4-160.1), 80.2 (65.1-95.3), and 64.5 (51.9-77.2) h·ng·mL in carriers of 0, 1, or 2 active OCT1 alleles (P = 0.03). Loss of OCT1 function resulted in reduced tramadol consumption and increased plasma concentrations of (+)O-desmethyltramadol in patients recovering from surgery. Therefore, analyzing OCT1 next to CYP2D6 genotype might further improve future genotype-dependent dose recommendations for tramadol.

Synthetic amphetamines such as 3,4-methylenedioxy-N-methylamphetamine (MDMA, Ecstasy) have become recreational drugs in German discotheques because of their euphoric and mood-brightening effects. However, their consumption involves considerable risks, which may even be lethal under certain circumstances. A 19-year-old man was taken to a university hospital for suspected intoxication with a narcotic drug, where he died the next day. As cause of death "fulminant liver failure" was diagnosed. In blood from the femoral vein, MDMA was found in a concentration of 4.27 mg/l. Histological examination showed acute necrosis of the liver and parenchymatous bleeding. The second case is that of a 39-year-old man who collapsed at his workplace and died in hospital shortly afterwards. In his rucksack, a small bag with 1.6 g of amphetamine was found. Analysis of blood from the femoral vein showed an amphetamine concentration of 1.08 mg/l.

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., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Levamisole is an anti-helminthic drug and gained forensic interest after it was found that it was used as a cocaine adulterant. A liquid chromatography-mass spectrometry (LC-MS) method for the determination of levamisole and its metabolite aminorex in human plasma is described. Selectivity is given; calibration curves were linear within a calibration range of 1 ng/mL-500 ng/mL. Limits of detection and quantification (LODs, LOQs) were 0.85 ng/mL for levamisole and 0.09 ng/mL, and 0.34 ng/mL for aminorex, respectively. Precision data was in accordance with the GTFCh guidelines. The validated method was successfully applied to study the pharmacokinetics of levamisole after administration of 100 mg of levamisole orally. Levamisole could be detected up to 36 h after ingestion in serum, while aminorex never exceeded the LOQ. A one-compartment model best described levamisole pharmacokinetics. The following parameters were calculated: ka = 1.2 [1/h], CL/F = 52 l/h, V/F = 347 l, f (renal) = 0.0005, t ½ = 2.0 h, AUC = 1923 ng/mL*h, cmax = 214 ng/mL, tmax = 1.98 h. Levamisole could be quantified in 42.5% of cocaine--positive plasma samples (2.2 to 224 ng/mL). Aminorex was positive in only 11.3% of the cases; however, it was never found higher than the LOQ. Pemoline, another stimulant detected in horse urine samples after administration of levamisole, was not found either in serum or in urine of this pharmacokinetic study. In post-mortem cases, levamisole and aminorex could be detected in femoral blood and the urine of cocaine users. Pemoline was not detected., (Copyright © 2014 John Wiley & Sons, Ltd.)

A massive lethal overdose with beta-metildigoxin in a 36-week-old infant is presented. Determination of beta-metildigoxin and its metabolites digoxin, digoxigenin and digoxigenin-monodigitoxosid is achieved by a liquid chromatographic mass spectrometric (LC-MS/MS) method. Measured concentrations for beta-metildigoxin and digoxin in peripheral blood were 40.2 ng/ml and 25.6 ng/ml, respectively. Tissue distribution showed highest concentrations in kidney tissue and gastric content. The metabolite digoxigenin-monodigitoxosid could be detected in heart blood, duodenal content, gastric content and fat tissue while the metabolite digoxigenin could only be detected in gastric content since the drug was given by a stomach tube., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

There is a need for quick and reliable methods for rapid screening of drug-influenced drivers on the roadside by police. Because the window of detection in oral fluid is more similar to blood than to urine, this matrix should therefore be appropriate for screening procedures. The performance of the Rapid STAT(®) (Mavand Solution GmbH, Mössingen, Germany), DrugWipe5/5+(®) (Securetec Detektions-Systeme AG, Brunnthal, Germany) and Dräger DrugTest(®) 5000 (Draeger Safety AG & Co. KGaA, Luebeck, Germany) on-site oral fluid devices was evaluated with random oral fluid specimens from car drivers in North Rhine-Westphalia (Germany). Additionally, some drivers were checked using an on-site urine device (DrugScreen(®), NAL von Minden, Regensburg, Germany). During a 11-month period, 1.212 drivers were tested. Both OF and urine on-site tests were compared to serum results. The following sensitivities were obtained by the oral fluid devices: THC 71% (DrugWipe(®)), 87% (Dräger), 91% (RapidSTAT); opiates 95% (Dräger), 100% (DrugWipe(®), RapidSTAT(®)); amphetamine 84% (DrugTest(®) 5000), 90% (RapidSTAT(®)), 100% (DrugTest(®) 5000); methamphetamine 50% (DrugTest(®) 5000), 100% (RapidSTAT(®)); cocaine 76% (DrugTest(®) 5000), 100% (DrugWipe(®), RapidSTAT(®)); methadone 33-63%, and benzodiazepines 0-33% (both with a low number of positives). THC specificity was especially low (29% [DrugWipe(®)] and 47% [DrugTest(®) 5000]) due to low cut-off concentrations. These data were similar to those obtained from the literature (e.g., DRUID project). The urine screening device showed a good sensitivity (THC 93%, opiate 94%, amphetamine 94%, methamphetamine 75% (low number of positives), cocaine 100%) and also an acceptable specificity (39%, 86%, 63%, 77%, 47%, respectively). Although oral fluid may be a useful matrix for on-site testing of drugged drivers, it is evident that oral fluid devices still show a lack of sensitivity (methamphetamine, benzodiazepines) and specificity (THC). Poor results for benzodiazepines may be explained by the small positive test number. Although the sensitivity for THC came out higher than compared to the literature, specificity is not yet satisfactory (only <90%). Furthermore, specificity was poor due to lowered cut-offs resulting in multiple false positive tests., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The imidazoline derivative xylometazoline, an alpha-2-adrenergic agonist, is used as non-prescription nasal preparation due to its vasoconstrictive and decongestive properties. Especially in children, an overdose can quickly cause severe central nervous system depression and cardiovascular adverse effects. In three 3-year-old boys (triplets) a xylometazoline intoxication was diagnosed by toxicological analysis. On admission to an emergency unit all three children were still unresponsive. One triplet showed respiration of 15-20 breaths/min and required oxygen support (3L/min) via face mask; his electrocardiogram revealed sinus bradycardia of 64 beats/min with supraventricular extrasystoles. However, no interventions were necessary except fluid management via intravenous lines. Eleven hours after the event, two of the triplets were awake but still not fully oriented. The third triplet woke up 20h after instillation of nose drops. Intoxication was caused by a compounding error in a pharmacy resulting in a concentration 40 times above the adequate dosage for children. In general, physicians, pharmacists and the public should be educated about the toxicity of over-the-counter preparations., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Recreational use of synthetic cannabinoid receptor agonists-so-called "Spice" products-became very popular during the last few years. Several reports on clinical symptoms and poisonings were published. Unfortunately, most of these reports do not contain any analytical data on synthetic cannabinoids in body fluids, and no or only a limited number of cases were reported concerning driving under the influence (DUI) of this kind of drugs. In this article, several cases of DUI of synthetic cannabinoids (AM-2201, JWH-018, JWH-019, JWH-122, JWH-210, JWH-307, MAM-2201 (JWH-122 5-fluoropentyl derivative), and UR-144) are presented, focusing on analytical results and signs of impairment documented by the police or the physicians who had taken the blood sample from the suspects. Consumption of synthetic cannabinoids can lead to impairment similar to typical performance deficits caused by cannabis use which are not compatible with safe driving. These deficits include centrally sedating effects and impairment of fine motor skills necessary for keeping the vehicle on track. Police as well as forensic toxicologists and other groups should become familiar with the effects of synthetic cannabinoid use, and be aware of the fact that drug users may shift to these "legal" alternatives due to their nondetectability by commonly used drug screening tests based on antibodies. Sophisticated screening procedures covering the complete range of available compounds or their metabolites have to be developed for both blood/serum and urine testing.

The Capuchin Catacombs of Palermo contain over 1800 preserved bodies: friars, priests and laypeople including men, women, and children. The bodies were accessible to family members who could visit the deceased and commemorate them through prayers. The "Sicily Mummy Project" analyzed hair samples from 38 mummies to determine the presence of ethyl glucuronide (EtG) using a routine procedure in our accredited laboratory of liquid chromatography coupled with mass spectrometry. The limit of quantification was 2.3 pg/mg. The hair samples were from 1.5 to 12 cm in length. All samples were analyzed in 2 segments (seg. A 0-3 cm and seg. B the remainder). Samples <4 cm in length were cut in half. In 31 out of 76 segments positive results were obtained for EtG, with concentrations between 2.5 and 531.3 pg/mg (mean 73.8, median 13.3 pg/mg). In 14 cases positive results were obtained for both segments. In one sample a positive result was obtained for segment A but not for segment B and in a further two samples only for segment B. The results indicate that EtG analyses can be performed on mummy hair samples even several hundred years after death to identify evidence for significant alcohol consumption during life., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2013 John Wiley & Sons, Ltd.)

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., (Copyright © 2013 John Wiley & Sons, Ltd.)

The appearance of dangerous and insufficiently studied designer drugs has increased substantially within the last few years. Mixtures containing centrally active compounds are often declared as "bath salt", "incense", "plant food", "bong cleaners" and are marketed in head shops and on the Internet. As the majority of the ingredients of such products are not subject to regulations of the German Narcotics Law (Betäubungsmittelgesetz, BtMG), the vendors and consumers mistake the sale of such products for legal. An alternative possibility to prosecute the distribution of so-called "legal highs" arises from the regulations of the German Medicinal Products Act (Arzneimittelgesetz, AMG). Indicating a private address, several products were purchased via the Internet. The products were analyzed by gas chromatography- mass spectrometry using computer-assisted database search and potential hits were checked for plausibility. The analysis of 100 samples revealed centrally acting compounds (including caffeine) in 98 % (75 % of all samples positive for caffeine). In 16 % of the samples, drugs subject to the BtMG at the time of purchase (end of 2011) were found including 2,5-dimethoxy-4-methylamphetamine, amphetamine, etilamphetamine, N-benzylpiperazine, mephedrone, methcathinone, and phenobarbital. In 55 % of the samples, drugs subject to the current BtMG were found (after its amendment on 20 July 2012). In 37 % of the samples, substances subject to the AMG were found (e.g. ephedrine). In 35 % of the samples, drugs with a potential psychotropic effect were found. In 57.3 % of the positive samples, more than one active ingredient was determined and in some cases up to five active components were found. Other interesting pharmacologically active ingredients found were 4-methylcathinone (n=13), flephedrone (n=8), trifluoromethylphenyl-piperazine (n=7), methylone (n=5), butylone (n=2), hordenine (n=2), and harmane (n=2). Most of the substances not covered by the BtMG can be classified as "unsafe" drugs. The distribution of unsafe drugs is illegal in Germany. However, the easy availability of real or potential drugs has to be seen critically. Little is known about the toxicological and pharmacological effects of those single substances, let alone of interactions in mixtures of such substances. In chat rooms, users advertise such drugs and blaze abroad their own experiences.

This article presents a case of suicide by intoxication with various pharmaceuticals, particularly anticonvulsants, combined with the fungicide captan. A cause of death could not be ascertained at autopsy. However, systematic toxicological analysis (STA) including a screening via solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) for (semi) volatile organic compounds revealed results suggesting a possible cause of death. The effects of captan on the human organism, its metabolism, and distribution will be discussed. Macroscopically, the cause of death was unascertained. STA revealed clonazepam, citalopram, and its metabolites, lamotrigine, levetiracetam, lacosamide, clonazepam, captan, and its metabolite tetrahydrophthalimide (THPI). For the first time, it was detected in human viscera. A quantification of THPI was performed to obtain distribution in the organs. The significance of a complete STA must be emphasized. The presence of THPI would have been missed without previous detection of captan. Consequently, this fatality would not have been investigated satisfactorily., (© 2013 American Academy of Forensic Sciences.)

A 42-year old-man was found lying in his bed having seizures. Later he became unconscious and hypotonic developing mydriasis as well as rigidity. The body core temperature (rectal temperature) was above 42 °C. Blood pH was decreased during treatment, and his general condition deteriorated. The patient developed gasping respiration, ventricular fibrillation, and died. During autopsy and histological investigation cerebral and pulmonary edema were noted together with general congestion of the internal organs. Further observations included contraction bands of myocytes, a contracted spleen, fibrosis of the liver, and gall stones. Toxicological analyses of peripheral blood revealed the following results: amisulpride 4.65 mg/l, biperiden 0.12 mg/l, imipramine 0.33 mg/l, and desipramine 0.68 mg/l. An amisulpride-induced neuroleptic malignant syndrome was therefore diagnosed as the patho-physiological mechanism leading to death.

In a preliminary test, four single hairs of a drug abuser were analyzed for the presence of drugs by MALDI Fourier transform mass spectrometry (MALDI-FTMS). Washed hair strains were directly fixed on a sample plate using pressure stable, double-sided adhesive tape; α-cyano-4-hydroxycinnamic acid matrix was manually spotted onto the hair strains. FTMS full scans were obtained moving from the hair root region towards the hair tip. Cocaine (accurate m/z ratio 304.15433) was identified mostly from the root of the hair and then later again towards the hair tip. This was confirmed by analysis of a second hair. Additionally cocaine metabolites with m/z ratio 290.13868 (benzoylecgonine), and m/z 318.16998 (cocaethylene) were detected for plausibility control. Using the MALDI technique, time-related information was obtained concerning the behavioural pattern of the consumer with high resolution compared to conventional procedures. However, in two hairs of the same individual which were analyzed under the same conditions, negative results were achieved. These preliminary results confirm the applicability of MALDI-MS for the determination of drugs and pharmaceuticals in hair samples being useful in forensic toxicology. The high chronological resolution allows an enhanced interpretation concerning the periods of drug administration. However, the negative results with two negative hairs have also demonstrated that hair analysis of single hairs can lead to misinterpretation. Different growth rates have to be considered, and particularly the phenomenon of different growth phases (anagen, catagen, telogen) require attention., (Copyright © 2013 John Wiley & Sons, Ltd.)

The antihelminthic drug Levamisole can enhance cocaine effects by conversion into the amphetamine-like drug aminorex. We describe an LC-MS method for the determination of levamisole and its metabolite aminorex in human urine. Selectivity is given, calibration curves were linear within the calibration range 2.5-250 ng/mL; limits of the method were LoD 0.51 ng/mL, LoQ 1.02 ng/mL for levamisole and LoD 0.65 ng/mL, LoQ 0.76 ng/mL for aminorex. Precision data was in accordance with the guidelines (intraday precision for aminorex ranged between 5.75 and 11.0 % for levamisole between 8.36 and 10.9 %; interday precision for levamisole 10.9-16.9 % and for aminorex 7.64-12.7 %; accuracy data for levamisole -1.96 to -14.3 % and for aminorex-11.9 to-18.5 %). The validated method was successfully applied to study the urinary excretion of levamisole after the administration of 100 mg of levamisole orally. Levamisole and aminorex could be detected in post-administration urine samples. Levamisole could be detected up to 39 h after ingestion, while aminorex was detectable up to 54 h. Maximum aminorex concentrations were 45 ng/mL urine. Further metabolites of levamisole after oral ingestion by means of liquid chromatography hybrid quadrupole time-of-flight high-resolution mass spectrometry (LC-QTOF-HRMS) were identified. Only 0.5 % of the ingested drug was quantified as unchanged levamisole in urine. Besides aminorex, five isomers of aminorex and 4 hydroxy-metabolites of aminorex or its isomers were found. Furthermore, levamisole is also hydroxylated and eliminated free or conjugated with sulfate or glucuronide into urine.

An interrelation between consumption of illegal drugs and committing an indictable offence has been repeatedly discussed in literature. In a retrospective study serum concentrations of illegal and legal drugs as well as data originating from police reports and examinations by physicians taking blood from individuals being suspected to be under the influence of drugs were evaluated. Results from 4816 cases were available. Property offences were the most frequent type (36%) as well as consumption of cannabinoids (55%). Psychophysiological conditions of consumers were compared with according serum concentrations. Close correlations between stimulating drugs and violence associated crime could not be found. Stimulated as well as sedated behaviour occurring following the consumption of various drugs might be the reason for no clear correlation between types of offence and consumed illegal or legal drugs in this study., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The postmortem determination of hyperglycaemic coma is quite difficult because of the lack of morphological findings and the difficult interpretation of biochemical parameters. Methylglyoxal (MG) is a reactive oxoaldehyde, which is mainly derived from glycolysis. An electrospray ionisation liquid chromatography-tandem mass spectrometric procedure for the determination of methylglyoxal in human serum and postmortem blood was developed. It involves protein precipitation with perchloric acid and a derivatisation step with 2,3-diaminonaphthalene. The assay was validated according to international guidelines. Serum samples from diabetics obtained at a diabetes clinic and from non-diabetics were used to assess data about reference concentrations in human serum. The assay showed linearity within the physiological concentrations in serum (5-500 ng/ml). Intraday imprecision at three concentrations was 10.3, 9.2 and 8.3 %, and interday imprecision was 15.3, 14.2 and 9.4 %; the limit of detection was 1.3 ng/ml, and limit of quantification, 3.2 ng/ml. One hundred and eighteen clinical (100 diabetics, 18 non-diabetics) and 98 forensic samples (84 non-diabetics, 14 in a status of hyperglycaemic coma) were measured. During life, diabetics showed significantly (p < 0.001) higher serum concentrations of MG than non-diabetics. After death, concentrations of MG increased significantly (p < 0.001). However, there was no correlation between the sum formula of Traub in vitreous humour and MG femoral blood concentrations (R = 0.237). This indicates that MG concentrations in the deceased cannot distinguish deaths due to a hyperglycaemic coma from other causes of death.

Background: The impact of polymorphic cytochrome P450 CYP2D6 enzyme on oxycodone's metabolism and clinical efficacy is currently being discussed. However, there are only spare data from postoperative settings. The hypothesis of this study is that genotype dependent CYP2D6 activity influences plasma concentrations of oxycodone and its metabolites and impacts analgesic consumption., Methods: Patients received oxycodone 0.05 mg/kg before emerging from anesthesia and patient-controlled analgesia (PCA) for the subsequent 48 postoperative hours. Blood samples were drawn at 30, 90 and 180 minutes after the initial oxycodone dose. Plasma concentrations of oxycodone and its metabolites oxymorphone, noroxycodone and noroxymorphone were analyzed by liquid chromatography-mass spectrometry with electrospray ionization. CYP2D6 genotyping was performed and 121 patients were allocated to the following genotype groups: PM (poor metabolizer: no functionally active CYP2D6 allele), HZ/IM (heterozygous subjects, intermediate metabolizers with decreased CYP2D6 activity), EM (extensive metabolizers, normal CYP2D6 activity) and UM (ultrarapid metabolizers, increased CYP2D6 activity). Primary endpoint was the genotype dependent metabolite ratio of plasma concentrations oxymorphone/oxycodone. Secondary endpoint was the genotype dependent analgesic consumption with calculation of equianalgesic doses compared to the standard non-CYP dependent opioid piritramide., Results: Metabolism differed between CYP2D6 genotypes. Mean (95%-CI) oxymophone/oxycodone ratios were 0.10 (0.02/0.19), 0.13 (0.11/0.16), 0.18 (0.16/0.20) and 0.28 (0.07/0.49) in PM, HZ/IM, EM and UM, respectively (p = 0.005). Oxycodone consumption up to the 12(th) hour was highest in PM (p = 0.005), resulting in lowest equianalgesic doses of piritramide versus oxycodone for PM (1.6 (1.4/1.8); EM and UM 2.2 (2.1/2.3); p<0.001). Pain scores did not differ between genotypes., Conclusions: In this postoperative setting, the number of functionally active CYP2D6 alleles had an impact on oxycodone metabolism. The genotype also impacted analgesic consumption, thereby causing variation of equianalgesic doses piritramide : oxycodone. Different analgesic needs by genotypes were met by PCA technology in this postoperative cohort.

Numerous death cases due to suffocation in a toxic or oxygen deficient gas atmosphere have been described in the literature, but unfortunately especially cases involving inert gases like helium are often presented without detailed toxicological findings. Observations on two suicides are reported, one by helium and the other by argon inhalation. During autopsies gas samples from the lungs were collected directly into headspace vials by a procedure ensuring minimal loss and dilution. Qualitative gas analyses were performed using headspace gas chromatography-mass spectrometry (HS-GC/MS). For carrier gas the commonly used helium was replaced by hydrogen. Qualitative positive results were obtained in the argon case, but the case involving helium revealed negative findings. The use of HS-GC/MS enables in principle to detect inert gases like argon or helium. However, a number of factors may later influence the results as, e.g. a longer period of time between death and sampling or pre-analytical artefacts during sampling of such highly volatile substances. In absence of analytical data supporting helium exposure, the causes of death in the actual cases were found to be asphyxia and in both cases the manner was suicide., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Postmortem diagnosis of diabetes and a diabetic coma can be difficult because of the lack of characteristic morphological findings. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for reabsorption in the kidneys. Therefore, diabetics with a permanent hyperglycemia show significantly lower serum concentrations of 1,5-AG than non-diabetics. A liquid chromatography-mass spectrometric method for the determination of 1,5-AG in serum and postmortem blood was developed and validated according to international guidelines. Linearity was given between 1 μg/ml and 50 μg/ml. Recovery rates ranged between 70.8% and 89.8%, the limit of quantification of the procedure was 0.20 μg/ml, limit of quantification was 0.55 μg/ml. Serum of 199 diabetics and 116 non-diabetics and femoral blood of 31 diabetic and 27 non-diabetic deceased was measured. Average concentrations were significantly (p<0.001) higher in non-diabetics compared to diabetics ante and postmortem. Seven of the diabetics may have died because of a hyperglycemic coma indicated by a sum formula of Traub>450 mg/dl. 1,5-AG average concentrations in these deceased were not significantly different to diabetics which did not die because of a diabetic coma. Concentrations of 1,5-AG give a hint for not well controlled diabetes antemortem and postmortem and can be assumed as an additional and alternative information postmortem to the measurement of HbA1c or fructosamine., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are commonly used alcohol markers for previous alcohol consumption. Nevertheless, the optimum EtG cutoff for urinary abstinence tests is still being discussed, and no cutoff has been recommended for EtS yet. The aim of this study was to verify cutoffs by investigating EtG and EtS concentrations (c(EtG) and c(EtS)) in the urine of healthy persons after drinking small, but realistic amounts of alcohol (one or two glasses of beer or white wine), and to look for the window of detection in strongly alcohol-intoxicated patients who were beginning withdrawal treatment. Very high EtG and EtS concentrations were measured in the first urine samples of patients under withdrawal treatment. However, 24 h later, concentrations decreased considerably, and c (EtG) < 0.5 mg/l and c (EtS) < 0.1 mg/l were determined in 26.7 % (4/13) and 13.3 % (2/13) of the samples, respectively. Concentrations above 0.1 mg/l (EtG) and 0.05 mg/l (EtS) were measured for 23.5 and 20.5 h after consuming 0.1 l of white wine or 0.33 l of beer, and 24 h after the experiment, 75 % (9/12) of the urine samples were tested negative for EtG and EtS using the following cutoffs: EtG 0.5 mg/l and EtS 0.1 mg/l. In half of the samples, concentrations below 0.1 mg/l (EtG) and 0.05 mg/l (EtS) were detected. Urinary cutoffs for EtG of 0.5 mg/l or higher are not suitable for testing abstinence. Even 0.1 mg/l is not effective to detect the intake of small amounts of alcohol in the context of abstinence tests. For EtS, 0.05 mg/l were found to be a potential cutoff to exclude the repeated intake of alcohol. Yet, further research is required to verify this cutoff. For a limited time period, EtG and EtS concentrations within the range of these cutoffs are also detectable after unintentional consumption of alcohol. Participants of abstinence programs have to be informed about the alcohol content of certain foods and beverages whose consumption is in conflict with strict abstinence.

Endurance exercise is known to promote sustained antinociceptive effects, and there is evidence that the reduction of pain perception mediated by exercise is driven by central opioidergic neurotransmission. To directly investigate the involved brain areas and the underlying neural mechanisms in humans, thermal heat-pain challenges were applied to 20 athletes during 4 separate functional magnetic resonance imaging (fMRI) scans, i.e., before and after 2 hours of running (exercise condition) and walking (control condition), respectively. Imaging revealed a reproducible pattern of distributed pain-related activation in all 4 conditions, including the mesial and lateral pain systems, and the periaqueductal gray (PAG) as a key region of the descending antinociceptive pathway. At the behavioral level, running as compared with walking decreased affective pain ratings. The influence of exercise on pain-related activation was reflected in a significant time × treatment interaction in the PAG, along with similar trends in the pregenual anterior cingulate cortex and the middle insular cortex, where pain-induced activation levels were elevated after walking, but decreased or unchanged after running. Our findings indicate that enhanced reactive recruitment of endogenous antinociceptive mechanisms after aversive repeated pain exposure is attenuated by exercise. The fact that running, but not walking, reproducibly elevated β-endorphin levels in plasma indicates involvement of the opioidergic system in exercise. This may argue for an elevated opioidergic tone in the brain of athletes, mediating antinociceptive mechanisms. Our findings provide the first evidence using functional imaging to support the role of endurance exercise in pain modulation., (Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

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.

Scientific opinions differ whether the use of stimulants causes deterioration in driving skills. In 1857 of 8709 cases of driving under the influence of drugs, amphetamine-like drugs (amphetamine, methamphetamine, and methylendioxyamphetamine) were present either alone or together with other licit or illicit drugs. In 338 cases, amphetamines were the only psychoactive substance group in plasma at mean, median, and highest concentrations of 0.18, 0.12, and 1.05 mg/L, respectively. A widespread opinion is that after the consumption of amphetamines, centrally stimulating effects with corresponding consequences on safe driving are expected. In contrast, many cases were observed that rather suggested an influence of centrally sedating substances when considering the psycho-physical conditions. Relations between concentration and effect could not be established. The apparent sedation is probably the consequence of sleep deprivation during an amphetamine binge and the after-effects of the drug., (© 2012 American Academy of Forensic Sciences.)

The muscle relaxant succinylcholine (SUX) evokes respiratory paralysis, and numerous cases of fatal SUX intoxication have been reported. Detection of SUX and its metabolite succinylmonocholine (SMC) is difficult, both due to their (bis-) quaternary structure and the extreme hydrolytic susceptibility of SUX, and data on degradation kinetics of SUX and SMC is scarce. The present study investigates the in vivo and in vitro degradation as well as elimination of both target analytes using authentic blood and urine samples from anesthetized patients. With a special focus on the urinary data and stabilization issues, this work intends to considerably enhance the forensic knowledge concerning SUX intoxications and to present the reader with practical analytical strategies to cope with such difficult cases. Eighteen subjects undergoing surgery and requiring arterial as well as bladder catheters were included in this study. Muscle relaxation was initialized with a bolus injection of 80-100 mg SUX. Blood and urine samples were either collected using paraoxonized (n = 15) or non-modified (n = 3) tubes. Sampling was performed within 6 h after SUX application following a pre-assigned schedule. Samples were processed according to a validated isotope dilution HPLC-MS/MS method using ion-pair solid-phase extraction. In blood, SUX was usually detectable for up to 10 min post-injection, while detection of SMC was possible over the whole observation period of 6 h. Effectiveness of organophosphate stabilization was proven for both analytes and is therefore recommended. In freshly secreted urine, detection windows of a minimum of 2 h as opposed to 6 h have been determined for SUX versus SMC, respectively. Considering SMC plasma kinetics, detection of the metabolite in blood and freshly secreted urine appears to be possible over a period of at least 8-24 h. Paraoxon did not enhance the stability of either target substance in urine, stabilization of urine samples is nonetheless recommended. In summary, SMC was proven to be the most promising target analyte in SUX analysis, with urine being the proposed matrix of choice for forensic applications. Furthermore, our work defines meaningful detection windows for SUX and SMC in blood and urine as routine matrices and presents sampling recommendations as well as guideline values for forensic toxicological analysis.

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Due to sensitive limits of detection of chromatographic methods and low limit values regarding the screening of drugs under the terms of impairment in safe driving (§ 24a StVG, Street Traffic Law in Germany), preliminary immunoassay (IA) tests should be able to detect also low concentrations of legal and illegal drugs in serum in forensic cases. False-negatives should be avoided, the rate of false-positive samples should be low due to cost and time. An optimization of IA cutoff values and a validation of the assay is required for each laboratory. In a retrospective study results for serum samples containing amphetamine, methylenedioxy derivatives, cannabinoids, benzodiazepines, cocaine (metabolites), methadone and opiates obtained with CEDIA drugs of abuse reagents on a Hitachi 912 autoanalyzer were compared with quantitative results of chromatographic methods (gas or liquid chromatography coupled with mass spectrometry (GC/MS or LC/MS)). Firstly sensitivity, specificity, positive and negative predictive values and overall misclassification rates were evaluated by contingency tables and compared to ROC-analyses and Youden-Indices. Secondly ideal cutoffs were statistically calculated on the basis of sensitivity and specificity as decisive statistical criteria with focus on a high sensitivity (low rates of false-negatives), i.e. using the Youden-Index. Immunoassay (IA) and confirmatory results were available for 3014 blood samples. Sensitivity was 90% or more for nearly all analytes: amphetamines (IA cutoff 9.5 ng/ml), methylenedioxy derivatives (IA cutoff 5.5 ng/ml), cannabinoids (IA cutoff 14.5 ng/ml), benzodiazepines (IA cutoff >0 ng/ml). Test of opiates showed a sensitivity of 86% for a IA cutoff value of >0 ng/ml. Values for specificity ranged between 33% (methadone, IA cutoff 10 ng/ml) and 90% (cocaine, IA cutoff 20 ng/ml). Lower cutoff values as recommended by ROC analyses were chosen for most tests to decrease the rate of false-negatives. Analyses enabled the definition of cutoff values with good values for sensitivity. Small rates of false-positives can be accepted in forensic cases., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

By means of in situ proton magnetic resonance spectroscopy ((1)H-MRS) time-dependent metabolic changes in brain are measured for the estimation of post-mortem intervals (PMIs). An isolated whole head and a whole young sheep from an abattoir with known time of death were stored at approximately 21°C, and localized (1)H-MRS (TR/TE 2,500/25 ms) of the brain was performed at various PMIs with a clinical 1.5 T whole-body scanner using a quadratur head coil. In the isolated head, additional metabolites including free trimethylammonium (fTMA), propionate, and butyrate, and especially acetate, could be detected as described in previous studies. In the head of the whole animal, especially fTMAs and lactate were found in the late PMI but no further metabolites of relevance. These preliminary findings support the hypothesis that in a whole-body corpse, the distribution of bacteria from the gastrointestinal tract up to the brain in the late post-mortem interval can influence the metabolic decomposition of brain tissues. In further studies, it should be kept in mind that an isolated head does not represent authentic circumstances and is, therefore, not an ideal model for brain decomposition in intact corpses.

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: An influence of polymorphic cytochromes P450 (CYP) 2D6 genetic variants on antiemetic efficacy of ondansetron has been suggested. However, the role of CYP3A in ondansetron metabolism and efficacy has been unclear. In this study, we evaluated the hypothesis that genotype-dependent CYP2D6 and CYP3A activity selectively influences plasma concentrations of ondansetron enantiomers. Additionally, the effects of doubling the ondansetron dose on genotype-dependent plasma concentrations were investigated., Methods: Patients received IV ondansetron 4 or 8 mg for emesis prophylaxis before emergence from anesthesia. The CYP2D6-dependent activity score representing no, decreased, normal, or increased CYP2D6 enzyme activity as well as CYP3A low (CYP3A5*3/*3) and high expressor status (CYP3A5 wt/wt or wt/*3) were determined. Plasma concentrations of R- and S-ondansetron enantiomers were measured by liquid chromatography-tandem mass spectrometry. Area under the plasma concentration-time curves (AUCs) of R- and S-ondansetron were associated with CYP2D6 and CYP3A5 genotype-dependent enzyme activity., Results: Complete data of 141 subjects were analyzed. Concentrations of S-ondansetron differed between CYP2D6 activity groups (P = 0.01) with highest values in patients with no CYP2D6 activity (mean [95% confidence interval]: 362.5 [238.3/486.7] h · ng/mL) and lowest values in those with increased activity (149.6 [114.5/184.8] h · ng/mL) compared with subjects displaying genotypes resulting in reduced or normal CYP2D6 activity (263.6 [228.8/298.8], 255.4 [228.2/282.7] h · ng/mL). AUC of R-ondansetron was 2 times higher in CYP3A5 low expressors compared with high expressors (281.5 [248.6/314.3] vs 142.5 [92.4/192.7] h · ng/mL; P = 0.003). Doubling the ondansetron dose increased plasma concentrations only in individuals with low CYP3A activity, but not in individuals with high enzyme activity (P < 0.001)., Conclusions: The metabolism of ondansetron seems to be enantioselective. In this postoperative setting, CYP2D6 activity scores correlated with concentrations of S-ondansetron, whereas CYP3A5 expressor status mainly influenced concentrations of R-ondansetron. Genetically and environmentally determined CYP2D6 and CYP3A enzyme activity might have implications for antiemetic efficacy.

Intoxications with succinylcholine (SUX) lead to a potentially lethal respiratory paralysis, and forensic cases involving accidental or deliberate SUX-application have been reported. Detection of SUX as well as its metabolite succinylmonocholine (SMC) is difficult: both substances are analytically challenging, and the extremely short plasma half-life of SUX additionally hampers detection of the parent compound. Pharmacokinetic data are scarce on SUX and non-existent on SMC. To enhance forensic knowledge concerning SUX intoxications, plasma pharmacokinetics of SMC were investigated in anesthetized patients. Fifteen subjects scheduled for a surgical procedure were included in this study. Muscle-relaxation was initialized with a bolus injection of 80-100 mg SUX. Blood sampling was performed within 6 h after SUX application using paraoxonized tubes. Collected plasma was processed according to a validated isotope dilution high-performance liquid chromatography-tandem mass spectrometry method using ion-pair solid-phase extraction. Pharmacokinetic parameters were derived from a user-defined as well as a three-compartment model. For SMC, peak plasma concentrations were reached after 0.03-2.0 min. In contrast to SUX, SMC was more slowly and more extensively distributed, featuring triphasic plasma concentration time profiles. Pharmacokinetic key parameters were subject to interindividual variation of potential forensic importance, with terminal half-lives of 1-3 h indicating a detection interval of 8-24 h for SMC in plasma. SMC was proven to be the only realistic SUX marker in a forensic context. The present work defines meaningful detection windows for plasmatic SMC after SUX application and offers guideline values for forensic toxicological casework.

Doubts concerning the applicability of succinylmonocholine (SMC) as a succinylcholine (SUX) marker have been issued. A comparative analysis of previously discussed tissues, i.e. brain, liver and kidney, was conducted to further elucidate this question by searching for diagnostically useful differences in analyte content in samples of SUX- versus non-SUX-associated fatalities. Furthermore, possible advantages of vitreous humor as a novel and promising target matrix for SUX analytics were assessed. Sample material of SUX-negative controls as well as the fatal SUX-intoxication was derived from frozen archive material and current autopsies. Samples were analyzed according to a modified protocol of a previously published and validated method employing ion-pairing solid-phase extraction and subsequent HPLC-MS/MS analysis. Standard addition was employed for quantification as well as an estimation of the analytical limits of the method. In all tested matrices, the method was proven to be sufficiently sensitive for the intended application. No indication of native SMC was found in controls of fresh tissues, nor in fresh or frozen vitreous humor. However, most of the samples were found to be positive for a previously reported interference with SMC's main ion transition, thereby falsely suggesting an SMC content of up to 139 ng/g, 126 ng/g, 165 ng/g and 93 ng/ml in brain, liver, kidney and vitreous humor, respectively. Contrasting the results for fresh sample material, SMC was detectable in some of the initially non-putrefied liver samples after long-term storage, as well as in massively decomposed SUX-negative control bodies. In this context, a microbial origin of the analyte may be assumed. All tissues as well as the vitreous humor of the fatal SUX-intoxication were negative for SUX and SMC. Just like serum, tissue and vitreous humor samples therefore do not allow a reliable diagnosis of a SUX-intoxication: in tissues this is due to the pronounced instability of both target analytes in these esterase-containing matrices, for vitreous humor an additional reason could be their insufficient incorporation into this medium., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

In 2009 cutoff values of assessment criteria to testify abstinence control in order to estimate driving ability were standardized in Germany. The cutoff values are lower than required in existing guidelines like SAMHSA and there is critical discussion about detection of low concentrations by using immunoassay, especially concerning amphetamines in urine (50 ng/ml). In this study Direct ELISA kits were tested for their applicability to identify the absence of amphetamines, cannabinoids, opiates, cocaine, methadone and benzodiazepines in urine. Results were confirmed by LC/MS or GC/MS analyses. Sensitivity, specificity, predictive values (positive as well as negative) and overall misclassification rates were evaluated by contingency tables and were compared to ROC-analyses. Sensitivity results as well as specificity results were satisfying showing sensitivity values higher than 96% for each analyte. The amphetamine test we used showed sensitivity and specificity of 100% and 88%, respectively, even if amphetamine tests usually react with high cross-reactivity. Our study results include high discrimination at required cutoff values between positives and negatives for each drug group and demonstrate that immunological tests complying with requirements of current decreased urine cutoff values for assessment of driving ability do exist., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

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.

The detection of diabetic metabolism disorders raises problems in forensic practice and sudden death with a subsequent negative autopsy finding is a common problem. In the case of an unclear hypoglycaemia, the detection of oral antidiabetics allows the differentiation of hypoglycaemia due to oral antidiabetics from that due to other reasons (insulin-induced, insulinoma). The development of an electrospray ionisation (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure for the simultaneous identification and quantification of oral antidiabetics of the sulfonylurea, the glinide, the thiazolidinedione and the gliptin types in human plasma is desired. The following analytes were included: glimepiride, glibenclamide, gliquidone, glibornuride, glisoxepide, glipizide and gliclazide (sulfonylurea type), nateglinide and repaglinide (glinide type), rosiglitazone and pioglitazone (thiazolidinedione type) and the dipeptidyl peptidase inhibitors vildagliptin, sitagliptin and saxagliptin. After a liquid-liquid extraction with tert-butyl methyl ether at two pHs, the oral antidiabetics were separated with fast gradient elution over a C(8) column. Identification of the oral antidiabetics was achieved by two specific ion transitions of each analyte in multiple reaction monitoring mode. Quantification was performed by referring the most intense ion transition peak areas to peak areas of the ion transitions of deuterated oral antidiabetics (hydroxytolbutamide-d(9) for the sulfonylureas, repaglinide-ethyl-d(5) for the glinides, pioglitazone-d(4) for the thiazolidinediones and vildagliptin-d(3) for the gliptins). The assay was validated according to international guidelines. The LC-MS/MS assay allows the simultaneous identification of 14 oral antidiabetics and quantification of 11 oral antidiabetics in plasma in the ESI mode in a single run. Linearity is shown up to overdose concentrations. The limits of detection with a signal-noise-ratio greater than 3 were below 1 ng/ml for all analytes. Recoveries ranged from 78 to 105%; for vildagliptin and saxagliptin recoveries were worse (45%) owing to their hydrophilic character. Intraday and interday precision and accuracy were below 20% for 11 drugs at three concentrations. For the gliptins, several validation parameters were out of range and, therefore, quantitatively this method is inappropriate.