Substances detected in used syringes of injecting drug users across 7 cities in Europe in 2017 and 2018: The European Syringe Collection and Analysis Project Enterprise (ESCAPE).

BACKGROUND AND AIMS: Injecting drug use is a matter of public health concern, associated with risks of overdoses, addiction and increased risk of bloodborne viral transmissions. Self-reported data on substances injected can be inaccurate or subject to bias or drug users might be oblivious to their injected substances or adulterations. Gathering of robust analytical information on the actual composition of substances injected might provide better information about the drugs that are being used. Therefore, this study aimed to analyse the residual content of discarded syringes collected across 7 European cities, collectively called the European Syringe Collection and Analysis Project Enterprise (ESCAPE). METHODS: Used syringes were collected at street automatic injection kit dispensers or at harm-reduction services in Amsterdam, Budapest, Cologne, Glasgow, Helsinki, Lausanne and Paris. Two sampling periods were executed thus far, in 2017 and 2018. Qualitative chemical analysis of the content of used syringes was performed combining gas chromatographic (GC) and ultra(high)performance liquid chromatographic ((U)HPLC) analytical techniques with detection by mass spectrometry (MS). RESULTS: Substances detected most frequently across both campaigns were cocaine, heroin, buprenorphine, amphetamines and synthetic cathinones. In Amsterdam, Cologne, Lausanne and Glasgow heroin and cocaine were the psychoactive substances most often detected, often in conjunction with each other. Helsinki showed a high presence of buprenorphine and amphetamines. In Budapest and Paris, synthetic cathinones were frequently detected. Less synthetic cathinones and cocaine was detected in 2018, whereas buprenorphine was detected almost twice as much. Inner-city variations were found, probably reflecting the types of people who inject drugs (PWID) in different areas of the city. CONCLUSION: Overall, laboratory-confirmed local data on injected substances showed resemblance to national surveys done among PWID. However, the ESCAPE data also showed some interesting differences, showing it can be used for local interventions and complementing existing monitoring data.

Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864).

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 'designer benzodiazepines' monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864)] offered as 'research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC MS/MS), liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4'-Chlorodiazepam biotransformation consisted of N-dealkylation and hydroxylation. It has to be noted that 4'-chlorodiazepam and its metabolites show almost identical LC-MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings.

Determination of medicinal and illicit drugs in post mortem dental hard tissues and comparison with analytical results for body fluids and hair samples.

In burnt or skeletonized bodies dental hard tissue sometimes is the only remaining specimen available. Therefore, it could be used as an alternative matrix in post mortem toxicology. Additionally, analysis of dental tissues could provide a unique retrospective window of detection. For forensic interpretation, routes and rates of incorporation of different drugs as well as physicochemical differences between tooth root, tooth crown and carious material have to be taken into account. In a pilot study, one post mortem tooth each from three drug users was analyzed for medicinal and illicit drugs. The pulp was removed in two cases; in one case the tooth was root canal treated. The teeth were separated into root, crown and carious material and drugs were extracted from the powdered material with methanol under ultrasonication. The extracts were screened for drugs by LC-MS(n) (ToxTyper™) and quantitatively analyzed with LC-ESI-MS/MS in MRM mode. The findings were compared to the analytical results for cardiac blood, femoral blood, urine, stomach content and hair. In dental hard tissues, 11 drugs (amphetamine, MDMA, morphine, codeine, norcodeine, methadone, EDDP, fentanyl, tramadol, diazepam, nordazepam, and promethazine) could be detected and concentrations ranged from approximately 0.13pg/mg to 2,400pg/mg. The concentrations declined in the following order: carious material>root>crown. Only the root canal treated tooth showed higher concentrations in the crown than in the root. In post mortem toxicology, dental hard tissue could be a useful alternative matrix facilitating a more differentiated consideration of drug consumption patterns, as the window of detection seems to overlap those for body fluids and hair.

Argon Mediates Anti-Apoptotic Signaling and Neuroprotection via Inhibition of Toll-Like Receptor 2 and 4.

PURPOSE: Recently, the noble gas argon attracted significant attention due to its neuroprotective properties. However, the underlying molecular mechanism is still poorly understood. There is growing evidence that the extracellular regulated kinase 1/2 (ERK1/2) is involved in Argon´s protective effect. We hypothesized that argon mediates its protective effects via the upstream located toll-like receptors (TLRs) 2 and 4. METHODS: Apoptosis in a human neuroblastoma cell line (SH-SY5Y) was induced using rotenone. Argon treatment was performed after induction of apoptosis with different concentrations (25, 50 and 75 Vol% in oxygen 21 Vol%, carbon dioxide and nitrogen) for 2 or 4 hours respectively. Apoptosis was analyzed using flow cytometry (annexin-V (AV)/propidiumiodide (PI)) staining, caspase-3 activity and caspase cleavage. TLR density on the cells' surface was analyzed using FACS and immunohistochemistry. Inhibition of TLR signaling and extracellular regulated kinase 1/2 (ERK1/2) were assessed by western blot, activity assays and FACS analysis. RESULTS: Argon 75 Vol% treatment abolished rotenone-induced apoptosis. This effect was attenuated dose- and time-dependently. Argon treatment was accompanied with a significant reduction of TLR2 and TLR4 receptor density and protein expression. Moreover, argon mediated increase in ERK1/2 phosphorylation was attenuated after inhibition of TLR signaling. ERK1/2 and TLR signaling inhibitors abolished the anti-apoptotic and cytoprotective effects of argon. Immunohistochemistry results strengthened these findings. CONCLUSION: These findings suggest that argon-mediated anti-apoptotic and neuroprotective effects are mediated via inhibition of TLR2 and TLR4.

Correction: Neuroprotection by Argon Ventilation after Perinatal Asphyxia: A Safety Study in Newborn Piglets.

[This corrects the article DOI: 10.1371/journal.pone.0113575.].

Neuroprotection by argon ventilation after perinatal asphyxia: a safety study in newborn piglets.

UNLABELLED: Hypothermia is ineffective in 45% of neonates with hypoxic-ischemic encephalopathy. Xenon has additive neuroprotective properties, but is expensive, and its application complicated. Argon gas is cheaper, easier to apply, and also has neuroprotective properties in experimental settings. The aim was to explore the safety of argon ventilation in newborn piglets. METHODS: Eight newborn piglets (weight 1.4-3.0 kg) were used. Heart rate, blood pressure, regional cerebral saturation, and electrocortical brain activity were measured continuously. All experiments had a 30 min. baseline period, followed by three 60 min. periods of argon ventilation alternated with 30 min argon washout periods. Two animals were ventilated with increasing concentrations of argon (1h 30%, 1 h 50%, and 1 h 80%), two were subjected to 60 min. hypoxia (FiO2 0.08) before commencing 50% argon ventilation, and two animals received hypothermia following hypoxia as well as 50% argon ventilation. Two animals served as home cage controls and were terminated immediately. RESULTS: Argon ventilation did not result in a significant change of heart rate (mean ± s.d. -3.5 ± 3.6 bpm), blood pressure (-0.60 ± 1.11 mmHg), cerebral oxygen saturation (0.3 ± 0.9%), electrocortical brain activity (-0.4 ± 0.7 µV), or blood gas values. Argon ventilation resulted in elevated argon concentrations compared to the home cage controls (34.5, 25.4, and 22.4 vs. 7.3 µl/ml). CONCLUSION: Ventilation with up to 80% argon during normoxia, and 50% argon after hypoxia did not affect heart rate, blood pressure, cerebral saturation and electrocortical brain activity. Clinical safety studies of argon ventilation in humans seem justified.

'Psychotropics caught in a trap' - adopting a screening approach to specific needs.

In the field of forensic toxicology, numerous strategies using different types of LC-MS platforms have been developed to set up an ultimate comprehensive screening method. Despite all this research, the question for the detection of a dedicated set of substances arises quite often in daily routine work. In this project, a screening method for the detection of psychotropic drugs based on the open library concept of a recently developed LC-MS(n) screening approach was developed and the effectiveness of a heated ESI-source was evaluated. To set up an individual spectral library all available data of psychotropics from the Toxtyper™ library was transferred to a new library format and complemented by MS, MS(2) and MS(3) data of additional psychotropic compounds. Precursor masses and retention time information of the library were used to trigger data dependent acquisition of MS(n)-spectra. Serum samples were analysed after alkaline liquid-liquid extraction on a Dionex RSLC (Acclaim™ C18 100×2.1C) coupled to a Bruker amaZon speed ion trap. A conventional ESI-source and an ionBooster™ source (IB) were used for ionization. All other LC and MS parameters were adopted from the original screening approach. Identification and result reporting was carried out by a fully automated software script. This screening method finally contains the individual precursor mass and retention time of 105 psychotropic substances and metabolites. Method evaluation was performed using pooled serum samples fortified with 12 different mixtures containing a total of 99 compounds at low therapeutic concentrations (cLOW and 2×cLOW). The customized method (ESI/IB) led to a higher rate of identifications (92%) - especially at low concentration levels (cLOW) - as the comprehensive screening approach (87%). Results from routine analysis with known intake of psychotropic drugs were confirmed with positive findings, if the concentration range was above or around the assumed limit of detection from this evaluation study. The Toxtyper open library concept enables fast and easy generation of new screening methods. The generated screening method is a fast and robust tool for the detection and identification of 105 psychotropics in human serum. Use of the ionBooster source led to a significant increase of the ionization efficiency within this sort of substance class. Evaluation in spiked human serum samples showed detection of low therapeutic levels for the majority of compounds, making the screening applicable for clinical and forensic samples (intoxication and post mortem cases).

A comprehensive library-based, automated screening procedure for 46 synthetic cannabinoids in serum employing liquid chromatography-quadrupole ion trap mass spectrometry with high-temperature electrospray ionization.

Considering the vast variety of synthetic cannabinoids and herbal mixtures - commonly known as 'Spice' or 'K2' - on the market and the resulting increase of severe intoxications related to their consumption, there is a need in clinical and forensic toxicology for comprehensive up-to-date screening methods. The focus of this project aimed at developing and implementing an automated screening procedure for the detection of synthetic cannabinoids in serum using a liquid chromatography-ion trap-MS (LC-MS(n)) system and a spectra library-based approach, currently including 46 synthetic cannabinoids and 8 isotope labelled analogues. In the process of method development, a high-temperature ESI source (IonBooster(TM), Bruker Daltonik) and its effects on the ionization efficiency of the investigated synthetic cannabinoids were evaluated and compared to a conventional ESI source. Despite their structural diversity, all investigated synthetic cannabinoids benefitted from high-temperature ionization by showing remarkably higher MS intensities compared to conventional ESI. The employed search algorithm matches retention time, MS and MS(2)/MS(3) spectra. With the utilization of the ionBooster source, limits for the automated detection comparable to cut-off values of routine MRM methods were achieved for the majority of analytes. Even compounds not identified when using a conventional ESI source were detected using the ionBooster-source. LODs in serum range from 0.1 ng/ml to 0.5 ng/ml. The use of parent compounds as analytical targets offers the possibility of instantly adding new emerging compounds to the library and immediately applying the updated method to serum samples, allowing the rapid adaptation of the screening method to ongoing forensic or clinical requirements. The presented approach can also be applied to other specimens, such as oral fluid or hair, and herbal mixtures and was successfully applied to authentic serum samples. Quantitative MRM results of samples with analyte concentrations above the determined LOD were confirmed as positive findings by the presented method.

Concentrations of amoxicillin and clindamycin in teeth following a single dose of oral medication.

OBJECTIVES: The main purpose of this study is the detection of amoxicillin and clindamycin concentrations in teeth. MATERIALS AND METHODS: Eleven patients received 2 g of amoxicillin, and 11 patients received 600 mg of clindamycin in a single dose of oral medication at least 60 min prior to tooth extraction due to systemic diseases. The concentrations were determined in crowns and roots separately using liquid chromatography-tandem mass spectrometry (LC-MS-MS). RESULTS: Amoxicillin (13 samples) and clindamycin (12 samples) were detected in the samples of the root and crown preparations of the extracted teeth. The mean concentration of amoxicillin was 0.502 µg/g in the roots and 0.171 µg/g in the crowns. The mean concentration of clindamycin was 0.270 µg/g in the roots and 0.064 µg/g in the crowns. CONCLUSIONS: A single dose of oral amoxicillin and clindamycin leads to concentrations of both antibiotics in teeth which exceed the minimal inhibition concentration of some oral bacteria. CLINICAL RELEVANCE: The proof of antibacterial activity in dental hard tissue after oral single-dose application is new. The antimicrobial effect of amoxicillin and clindamycin concentrations in roots of teeth may be of clinical relevance to bacterial reinfection from dentinal tubules.

Ethanol concentration in breastmilk after the consumption of non-alcoholic beer.

BACKGROUND: During lactation, the consumption of ethanol is discussed controversially. After women drink alcoholic beverages, ethanol can be found in breastmilk with a time lag. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular, non-alcoholic beer has become popular in recent years. According to regulations in the United States and most European countries, these "alcohol-free" beverages may still contain ethanol up to 1.2% by volume. To determine how much of this ethanol may reach the breastfed child, a drinking experiment with non-alcoholic beer was performed. SUBJECTS AND METHODS: Fifteen healthy breastfeeding women participated in the study. After at least 5 days of abstinence from ethanol and the donation of a void breastmilk sample, they were asked to drink 1.5 L of non-alcoholic beer within 1 hour. Breastmilk samples were collected using electronic breast pumps immediately after the end of drinking as well as 1 and 3 hours later. The milk was analyzed for ethanol by headspace-gas chromatography-flame ionization detection using a fully validated method. RESULTS: In two women, trace amounts of ethanol (up to 0.0021 g/L) were found in the samples gained immediately after the drinking period. In the other samples ethanol could not be detected (limit of detection=0.0006 g/L). CONCLUSIONS: The mother's consumption of non-alcoholic beer is likely innocuous for the breastfed infant.

A case of a distinct difference between the measured blood ethanol concentration and the concentration estimated by Widmark's equation.

In the last century, several mathematical models have been developed to calculate blood ethanol concentrations (BAC) from the amount of ingested ethanol and vice versa. The most common one in the field of forensic sciences is Widmark's equation. A drinking experiment with 10 voluntary test persons was performed with a target BAC of 1.2 g/kg estimated using Widmark's equation as well as Watson's factor. The ethanol concentrations in the blood were measured using headspace gas chromatography/flame ionization and additionally with an alcohol dehydrogenase (ADH)-based method. In a healthy 75-year-old man a distinct discrepancy between the intended and the determined blood ethanol concentration was observed. A blood ethanol concentration of 1.83 g/kg was measured and the man showed signs of intoxication. A possible explanation for the discrepancy is a reduction of the total body water content in older people. The incident showed that caution is advised when using the different mathematical models in aged people. When estimating ethanol concentrations, caution is recommended with calculated results due to potential discrepancies between mathematical models and biological systems.

Analysis of 30 synthetic cannabinoids in oral fluid using liquid chromatography-electrospray ionization tandem mass spectrometry.

In recent years, the analysis of synthetic cannabinoids in human specimens has gained enormous importance in the broad field of drug testing. Nevertheless, the considerable structural diversity among synthetic cannabinoids already identified in 'herbal mixtures' hampers the development of comprehensive analytical methods. As the identification of the main metabolites of newly appearing substances is very laborious and time-consuming, the detection of the parent compounds in blood samples is the current approach of choice for drug abstinence testing. Whenever blood sampling is not possible however, the need for alternative matrices arises. In this article, we present a fully validated liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the analysis of 30 synthetic cannabinoids in oral fluid samples collected with the Dräger DCD 5000 collection device. The method proved to be suitable for the quantification of 28 substances. The limits of detection were in the range from 0.015 to 0.9 ng/ml, while the lower limits of quantification ranged from 0.15 to 3.0 ng/ml. The method was successfully applied to 264 authentic samples during routine analysis. A total of 31 samples (12%) was tested positive for at least one of the following synthetic cannabinoids: AM-694, AM-2201, JWH-018, JWH-019, JWH-081, JWH-122, JWH-203, JWH-210, JWH-250, JWH-307, MAM-2201, and RCS-4. Given that stabilization of the collection pads after sampling is warranted, the collection device provides satisfactory sensitivity. Hence, whenever blood sampling is not possible, the Dräger DCD 5000 collection device offers a good tool for the analysis of synthetic cannabinoids in oral fluid in the broad field of drug testing.

Ethyl sulphate and ethyl glucuronide in vitreous humor as postmortem evidence marker for ethanol consumption prior to death.

To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices.

Pre-analytical stability of selected benzodiazepines on a polymeric oral fluid sampling device.

Oral fluid field tests are designed to provide preliminary results with a high grade of reliability in order to meet analytical and forensic standards. Some test systems additionally offer the possibility of an independent confirmatory analysis of a test sample. The pre-analytical stability of 11 frequently abused benzodiazepines on an oral fluid collecting device (Dräger DCD 5000) has been investigated. The collection device was designed to complement a special mobile testing system (Dräger DrugTest 5000) to be sent to a laboratory for further confirmatory analysis. Blank oral fluid pool was spiked with a mixture of eleven frequently abused benzodiazepines and given onto a collection device. To simulate possible sample shipping, the collection devices were stored in the dark up to 14 days at ambient temperature in a plastic tube. The collection device was simultaneously stored without further treatment after oral fluid collection ('native') and with addition of 950muL of methanol, respectively. At different storage intervals repeat determination was carried out for every sample using a modified version of our standard LC-MS/MS method for the detection of benzodiazepines in serum. Different recoveries of benzodiazepines due to degradation and/or adsorption to the collection device during the 14 days of 'native' storage were found. Major loss of analytes was found for benzodiazepines containing a nitro-group such as flunitrazepam and clonazepam. This could be prevented almost completely by methanolic storage of the collection device after sampling. Therefore, we recommend the centrifugation of the collection device and separation from the polymer unit prior to sample shipping. If this should not be possible, addition of methanol immediately after sample collection can be used to avoid degradation of benzodiazepines during shipment.

Identification of sinicuichi alkaloids in human serum after intoxication caused by oral intake of a Heimia salicifolia extract.

A 26-year-old male came to hospital around midnight complaining about muscle pain of the extremities as well as the tongue and slightly raised temperature. He reported the intake of an unknown amount of sinicuichi tea he had fermented over 24 h by adding yeast and sugar. The patient was treated with Vomex A (dimenhydrinate) and released from hospital the following afternoon. A blood sample taken shortly after submission and a small amount of the used plant material were available for analysis. Herbal drugs are widely used as stimulants as a legal alternative to illegal psychoactive drugs or in traditional context. Among many others like Sassafras officinalis, Salvia divinorum or Ephedra, Heimia salicifolia ("sinicuichi"), a species of the lythraceae family, is available via several online shops. Brewed up or fermented and consumed, the so-called sinicuichi tea may cause exhilarating feelings and an alteration of awareness accompanied by bradycardia, relaxation of the muscles and a pleasant faintness. Therefore Sinicuichi brew and heimia leaves are widely used for medication by the natives of Central and South America. After liquid extraction with acetone five different alkaloids were detected in the plant material by LC-MS/MS operated in the Q1 scan mode applying a TurboIonSpray source. Subsequently, Product Ion Spectra were recorded and after confirming the molecular formula by determining the accurate masses, possible structures of H. salicifolia alkaloids were assigned. The information of the Product Ion Spectra was then used to set up a sensitive multiple reaction monitoring (MRM) method. Applying the MRM method to the patient's serum sample after alkaline liquid-liquid extraction all of the five heimia alkaloids detected in the plant material were also detected qualitatively in the serum extract, confirming the ingestion.

Electrospray-ionization MS/MS library of drugs as database for method development and drug identification.

An ESI MS/MS library of 800 compounds has been developed and a collection of data is now available for Analyst 1.4 and higher. Compounds include forensically important drugs, such as illegal drugs, some deuterated analogues, hypnotics, amphetamines, benzodiazepines, neuroleptics, antidepressants and many others. For setting up the library of product ion spectra, 20-200 ng of the compounds have been injected either by flow injection or via a short LC-column, the precursor ions were chosen from the Q1 scan spectra, and product ion spectra were generated by CID in the collision cell using three different collision energies (20, 35 and 50 eV). Three spectra of each compound have been collected and compound names, CAS numbers, formulas and molecular weights have been added in the database, which has been generated by the Analyst software. The library can be used for compound identification during general unknown screening analysis by combination of Q1 scan techniques and subsequent MS/MS analysis in a second analytical run. Quantitative procedures for multi drug analysis using Multiple Reaction Monitoring can be established by selection of product ions and suitable collision energies from the library. For publication of the spectra, PDF-files have been generated and can be viewed on-line as supplementary data or from the website in alphabetical order: (supplementary data, should be made available via ELSEVIER-WEBSITE or via ).