In September 2019, a 22-year-old man with a history of drug abuse presented to the hospital with altered mental status. Due to a suspected drug overdose, a blood sample taken on admission and a urine sample collected 30 h thereafter were submitted to our laboratory to test for illegal drugs, pharmaceutical substances, and designer drugs. During the routine toxicological analysis of the serum sample, morphine and phenobarbital were identified by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Additionally, two compounds showing identical accurate masses and isotope ratios as the designer benzodiazepine diclazepam and the benzodiazepine lormetazepam were found. However, retention times differed significantly from the expected values, and the acquired MS/MS spectra did not match the library entries of the two compounds, indicating the presence of two previously unknown substances. After further investigation, SL-164 (5-chloro-3-(4-chloro-2-methylphenyl)-2-methyl-4(3H)-quinazolinone), a methaqualone analog, which has recently emerged on the research chemical market, and its hydroxy metabolite were tentatively identified by accurate mass, isotope matching, and plausible fragmentation. However, for unequivocal confirmation and quantification, a reference standard is required. As no reference material was available by the end of 2019, SL-164 was obtained from an online shop, and its identity and purity (97.8%) were confirmed by nuclear magnetic resonance spectroscopy. The subsequent quantitative analysis revealed a concentration of 390 ng/mL SL-164 in serum. In the urine sample, the parent compound was not detected, but three suspected monohydroxylated metabolites were found. This example shows that LC-QTOF-MS is a powerful approach for the (tentative) identification of unknown compounds in biological matrices.

In forensics, entomological specimens can be used as additional/alternative matrices to detect xenobiotics when human specimens are limited in their application. Despite some advantages over implementing putrefied human remains, most medico-legal laboratories do not include entomotoxicological procedures as routine analytical methods. We thus applied two authentic cases to evaluate necrophagous larvae’s potential as complementary matrices for toxicological analysis after extensive postmortem decomposition. Larvae and postmortem human samples, including hair, stomach contents, pericardial fluid, liver, lung, and skeletal muscle, were collected at autopsy. Samples were analyzed by liquid chromatography–tandem mass spectrometry and liquid chromatography–quadrupole time-of-flight mass spectrometry for pharmaceutical substances, illicit drugs, and new psychoactive substances, including synthetic cannabinoids, benzodiazepines, new synthetic opioids, and stimulants. Nearly all substances detected in human specimens, including several benzodiazepines and synthetic cannabinoids, were also detected in larvae. Surprisingly, some drugs, including the new psychoactive substances EAM-2201 and U-47700, were found exclusively in larvae and hair. The benzodiazepine etizolam was detected only in liver, lungs, and stomach contents, possibly resulting from characteristic tissue distribution in humans and/or larvae. Antemortem external hair contamination with synthetic cannabinoids from side-stream smoke and postmortem hair contamination with substances in putrefaction fluids can be supposed in these cases. Our findings suggest that supplementary information can indeed be gained from analyzing larvae additional to those human specimens that are typically used for toxicological analysis after extensive postmortem decomposition. Nevertheless, these results represent merely two cases, requiring in-depth studies to determine whether such findings can identify acute intoxications as possible causes of death.

One of the major challenges of testing drugs of abuse is the detection of highly diluted urine samples. The ingestion of a large amount of fluid can considerably reduce the concentration of substances, possibly resulting in inaccurate drug testing. For detection, determination of urinary creatinine is a widely established procedure. In this study, results from the most popular methods, including photospectrometry (Jaffe) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been compared regarding 327 urine abstinence control samples. Since samples with creatinine concentrations close to the cut-off of 20 mg/dL are of particular interest, only samples below 50 mg/dL were considered. Results revealed a close correlation of creatinine concentrations by both analytical methods with an R2 value of 0.9005. A mean concentration difference of 3.30 ± 3.45 mg/dL was observed, indicating a moderate underestimation by the Jaffe reaction. Graphical analyses showed high accordance between both methods with only a few outliers. Due to easy handling and for economic reasons, the spectrometric method is often preferred over LC-MS/MS. For urine samples with creatinine concentrations close to the cut-off, confirmation through a second method should be performed to avoid a possible disadvantage or even severe consequences for the respective individual. It is recommended that each laboratory establishes a reliable verification method.

Background: A 22-year-old male with a known history of drug abuse presented to our department with prolonged agitated delirium, myocloni, tachycardia and subfebrile temperature after the deliberate ingestion of opium poppy tea (Papaver somniferum L.) together with the methaqualone analog SL-164 (5-chloro-3-(4-chloro-2-methylphenyl)-2-methyl-4(3H)-quinazolinone) which is sold online as a designer drug. Methods: SL-164 and its hydroxy metabolites were detected in serum and urine via liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Results: The pronounced delirium was treated with benzodiazepines and neuroleptics; temporary medical restraint had to be applied. Symptoms completely resolved over the next 72 h and the patient was discharged on day three able to give consent. Conclusions: Although methaqualone was a popular and widespread sedative in the 1950s and 60 s before its discontinuation in the USA in 1985, derivatives of the methaqualone class have not previously played a large role as drugs of abuse in the rapidly growing market of new psychoactive substances. To our knowledge, this is the first case of agitated delirium with detection of SL-164 and hydroxylated metabolites in a patient's serum and urine.

5F-ADB is an indazole-based synthetic cannabinoid. In recent years, it has been detected in legal high products as well as in biological samples and is associated with serious adverse health, behavioral effects and even death. Due to the fast pace of the market of synthetic cannabinoids, data on such newly appearing substances are scarce. As pharmacological properties are often investigated in vitro or by using animal experiments, reports on synthetic cannabinoid findings in human samples along with corresponding case history descriptions are valuable for the interpretation of upcoming routine cases. Herein we report five cases with verified 5F-ADB consumption, including three fatalities, a case of driving under the influence of drugs as well as a case of grievous bodily harm. In four cases, 5F-ADB could be detected in blood or plasma. Concentrations were in the range of 0.11-0.57 μg/L. In one instance 5F-ADB consumption was verified by the detection of 5F-ADB metabolites in postmortem body fluids. The described cases illustrate various adverse effects including confusion (possibly even psychosis), collapse, loss of consciousness, unsafe driving style or changing moods that might be attributed to 5F-ADB.

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 (>= 0.1 ng 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. (c) 2018 Elsevier B.V. All rights reserved.

The risk of earth burials for the environment and public health is a matter of controversial debate. The aim of the present study is to characterise the drainage of cemeteries with regard to the concentration of a number of pharmaceuticals and to the soil’s hydrochemical properties, and to discuss these data in comparison with data obtained for surface waters located upstream of the cemeteries. Of the 12 drainage samples analysed using LC-ESI-MS/MS, seven contained carbamazepine (< 225 ng l−1), five contained hydrochlorothiazide, one contained metoprolol (23 ng l−1) and one contained traces of ibuprofen. The surface water samples contained a larger number of different drugs (8 of the 12 drugs under investigation) and higher concentrations (e.g. metropolol 2230 ng l−1). The NO3, NH4, PO4 and DOC concentrations and the electrical conductivity of the cemetery drainages were in several samples higher than those of the surface water samples. The NO3 and NH4 concentrations exceeded the legal contaminant limits of drinking water in only one case. The present study found that the release of drugs and nutrients from cemeteries, measured in surface water drug loads, presents a low environmental risk. However, the study is only a snapshot and long-term monitoring of cemetery drainages, including a broad range of pharmaceuticals and detailed hydrological investigations, will have to be carried out before more substantiated statements can be made.

Aims: Hair testing is a common technique for the determination of drug abuse. As drugs which are commonly smoked or sniffed (e.g. cocaine), can contaminate the hair through smoke or dust, testing for metabolites, especially hydroxy metabolites, is highly recommended. To check if the detection of hydroxy metabolites gives a definite proof of ingestion, the presence of these metabolites in street cocaine samples has to be checked. Methods: For this study 451 cocaine (COC) (COC >0.1 ng/mg) positive hair samples were analysed by LCMS/MS for the metabolites benzoylecgonine (BE), norcocaine (NC), cocaethylene (CE), ortho-, metaand para-hydroxy-cocaine (o-, m-, p-OH-COC), metaand para-hydroxybenzoylecgonine (m-, p-OH-BE), and metaand para-hydroxy-norcocaine (m-, p-OH-NC). The results were compared with the cocaine metabolite concentrations in 146 street cocaine samples, confiscated by the Bavarian police, by comparison of the concentration ratios for BE/COC, NC/COC and CE/COC and the area ratios for hydroxy-metabolites/COC. Results and Discussion: The following concentration/area ratios were found in the street cocaine samples: BE/COC 0.03 1.2%, NC/COC 0 4.1%, p-OH-COC up to 0.04 %, m-OH-COC up to 0.09% and o-OH-COC 0.18%. CE, OH-BE or OH-NC were not detected in any of the seized samples. Similar area ratios for the hydroxy-metabolites were found for p-OH-COC in 5.1 % of the hair samples, in 6.8 % of the samples for m-OH-COC and in 88.7 % for o-OHCOC. Conclusion: mand p-OH-COC area ratios which exceed the ratios of street cocaine more than twice plus additional identification of OH-BE and OH-NC will be implemented as new in-house criteria to distinguish between contamination and ingestion. It is expected that the more hydrophilic hydroxy-metabolites of cocaine are incorporated into the hair-matrix in a lesser extent than the cocaine itself. Detection of the metaand para-hydroxy-metabolites using the above mentioned criteria is a reliable tool to distinguish between ingestion and external contamination.

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.

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.

Aim: This study shows 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 post-mortem urine samples. Methods: After adding a mixture of various internal standards, 100 μL of the urine sample was treated with β-glucuronidase and diluted 1:10 with a buffer solution. Ten μL of the prepared sample was directly injected into the chromatographic system. 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 identification of the compounds was based on accurate mass (< 5 ppm), retention time (± 2%) if available, isotopic pattern fit (± 10%) and library match (> 70%). These four parameters served as identification criteria. Results and Discussion: In routine casework, a target library, consisting of 1253 exact monoisotopic masses (“Weinmann” ESI-MS/MS library) as well as additional in-house compounds, was used. A retention time for 320 compounds was available. The limits of detection (LOD), determined for 34 substances, were < 10 ng/mL for 91% of the compounds. The limits of quantitation (LOQ) were < 20 ng/mL for 91% of the analytes. 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. The procedure is accredited according to ISO 17025. Conclusion: LCQTOF-MS is an attractive technique for the fast and specific identification of drugs and toxic compounds as well as their metabolites in urine samples with the additional advantage of possible retrospective data analysis. Basic validation and identification criteria are discussed.

The effects of tetrahydrocannabinol (THC) and endogenous cannabinoids (endocannabinoids, ECs) are both mediated by activation of the cannabinoid receptors CB1 and CB2. Exogenous activation of these receptors by THC could therefore alter EC levels. We tested this hypothesis in healthy volunteers (n = 25) who received a large intravenous dose of THC (0.10 mg/kg). Effects on the EC system were quantified by serial measurements of plasma ECs after THC administration. Eleven blood samples were drawn during the first 5 h after THC administration and two more samples after 24 and 48 h. THC, its metabolites THC-OH (biologically active) and THC-COOH (non-active), and the ECs anandamide and 2-arachidonoylglycerol (2-AG) were quantified by liquid chromatography-mass spectrometry. EC-plasma levels showed a biphasic response after THC injection reaching maximal values at 30 min. Anandamide increased slightly from 0.58 ± 0.21 ng/ml at baseline to 0.64 ± 0.24 ng/ml (p < 0.05) and 2-AG from 7.60 ± 4.30 ng/ml to 9.50 ± 5.90 ng/ml (p < 0.05). After reaching maximal concentrations, EC plasma levels decreased markedly to a nadir of 300 min after THC administration (to 0.32 ± 0.15 ng/ml for anandamide and to 5.50 ± 3.01 ng/ml for 2-AG, p < 0.05). EC plasma concentrations returned to near baseline levels until 48 h after the experiment. THC (0.76 ± 0.16 ng/ml) and THC-OH (0.36 ± 0.17 ng/ml) were still measurable at 24 h and remained detectible until 48 h after THC administration. Although the underlying mechanism is not clear, high doses of intravenous THC appear to influence endogenous cannabinoid concentrations and presumably EC-signalling.