A proposed approach to confirm heroin administration - Regional differences in heroin purity is a major factor.

In forensic toxicology, a marker of street heroin use is urgent especially in the absence of urinary 6-monoacetylmorphine. ATM4G, the Glucuronide of Acetylated product of Thebaine compound 4 Metabolite (ATM4), arising from byproducts of street heroin synthesis has been considered as a useful marker in some European studies. However, whether ATM4G is a universal marker particularly in Southeast Asia due to 'street' heroin with high purity, it's still unclear. To investigate putative markers for different regions, ATM4G and other metabolites including the Acetylated product of Thebaine compound 3 Metabolite (ATM3) and thebaol, also originated from thebaine were detected in 552 urine samples from heroin users in Taiwan. Results were compared with that from samples collected in the UK and Germany. Only a sulfo-conjugate of ATM4, ATM4S, was detected in 28 Taiwanese users using a sensitive MS3 method whilst out of 351 samples from the UK and Germany, ATM4G was present in 91. Thebaol-glucuronide was first time detected in 118. No markers were detected in urine following herbal medicine use or poppy seed ingestion. The presence of ATM4S/ATM4G might be affected by ethnicities and heroin supplied in regions. Thebaol-glucuronide is another putative marker with ATM4G and ATM4S for street heroin use.

Hyphenated high-resolution mass spectrometry-the "all-in-one" device in analytical toxicology?

This trend article reviews papers with hyphenated high-resolution mass spectrometry (HRMS) approaches applied in analytical toxicology, particularly in clinical and forensic toxicology published since 2016 and referenced in PubMed. The article focuses on the question of whether HRMS has or will become the all-in-one device in these fields as supposed by the increasing number of HRMS presentations at scientific meetings, corresponding original papers, and review articles. Typical examples for the different application fields are discussed such as targeted or untargeted drug screening, quantification, drug metabolism studies, and metabolomics approaches. Considering the reviewed papers, HRMS is currently the only technique that fulfills the criteria of an all-in-one device for the various applications needed in analytical toxicology.Graphical abstract.

Is adipose tissue suitable for detection of (synthetic) cannabinoids? A comparative study analyzing antemortem and postmortem specimens following pulmonary administration of JWH-210, RCS-4, as well as ∆9-tetrahydrocannabinol to pigs.

Examining fatal poisonings, chronic exposure may be reflected by the concentration in tissues known for long-term storage of drugs. Δ9-tetrahydrocannabinol (THC) persists in adipose tissue (AT), but sparse data on synthetic cannabinoids (SC) are available. Thus, a controlled pig study evaluating antemortem (AM) disposition and postmortem (PM) concentration changes of the SC 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210) and 2-(4-methoxyphenyl)-1-(1-pentyl-indole-3-yl)methanone (RCS-4) as well as THC in AT was performed. The drugs were administered pulmonarily (200 µg/kg body weight) to twelve pigs. Subcutaneous (s.c.) AT specimens were collected after 15 and 30 min and then hourly up to 8 h. At the end, pigs were sacrificed and s.c., perirenal, and dorsal AT specimens were collected. The carcasses were stored at room temperature (RT; n = 6) or 4 °C (n = 6) and specimens were collected after 24, 48, and 72 h. After homogenization in acetonitrile and standard addition, LC-MS/MS was performed. Maximum concentrations were reached 0.5-2 h after administration amounting to 21 ± 13 ng/g (JWH-210), 24 ± 13 ng/g (RCS-4), and 22 ± 20 ng/g (THC) and stayed at a plateau level. Regarding the metabolites, very low concentrations of N-hydroxypentyl-RCS-4 (HO-RCS-4) were detected from 0.5 to 8 h. PM concentrations of parent compounds did not change significantly (p > 0.05) over time under both storage conditions. Concentrations of HO-RCS-4 significantly (p < 0.05) increased in perirenal AT during storage at RT. These results suggest a rapid distribution and persistence in s.c. AT. Furthermore, AT might be resistant to PM redistribution of parent compounds. However, significant PM increases of metabolite concentrations might be considered in perirenal AT.

Time- and temperature-dependent postmortem concentration changes of the (synthetic) cannabinoids JWH-210, RCS-4, as well as ∆9-tetrahydrocannabinol following pulmonary administration to pigs.

In forensic toxicology, interpretation of postmortem (PM) drug concentrations might be complicated due to the lack of data concerning drug stability or PM redistribution (PMR). Regarding synthetic cannabinoids (SC), only sparse data are available, which derived from single case reports without any knowledge of dose and time of consumption. Thus, a controlled pig toxicokinetic study allowing for examination of PMR of SC was performed. Twelve pigs received a pulmonary dose of 200 µg/kg BW each of 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210), 2-(4-methoxyphenyl)-1-(1-pentyl-indole-3-yl)methanone (RCS-4), and Δ9-tetrahydrocannabinol via an ultrasonic nebulizer. Eight hours after, the pigs were put to death with T61 and specimens of relevant tissues and body fluids were collected. Subsequently, the animals were stored at room temperature (n = 6) or 4 °C (n = 6) and further samples were collected after 24, 48, and 72 h each. Concentrations were determined following enzymatic cleavage and solid-phase extraction by liquid-chromatography tandem mass spectrometry applying the standard addition approach. High concentrations of the parent compounds were observed in lung, liver, kidney and bile fluid/duodenum content as well as brain. HO-RCS-4 was the most prevalent metabolite detected in PM specimens. In general, changes of PM concentrations were found in every tissue and body fluid depending on the PM interval as well as storage temperature.

Development and application of a strategy for analyzing eight biomarkers in human urine to verify toxic mushroom or ricinus communis ingestions by means of hydrophilic interaction LC coupled to HRMS/MS.

The analytical proof of a toxic mushroom and/or plant ingestion at an early stage of a suspected intoxication can be crucial for fast therapeutic decision making. Therefore, comprehensive analytical procedures need to be available. This study aimed to develop a strategy for the qualitative analysis of α- and ß-amanitin, psilocin, bufotenine, muscarine, muscimol, ibotenic acid, and ricinine in human urine by means of hydrophilic interaction liquid chromatography-high resolution MS/MS (HILIC-HRMS/MS). Urine samples were prepared by hydrophilic-phase liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction and precipitation, performed in parallel. Separation and identification of the biomarkers were achieved by HILIC using acetonitrile and methanol as main eluents and Orbitrap-based mass spectrometry, respectively. The method was validated as recommended for qualitative procedures and tests for selectivity, carryover, and extraction recoveries were included to also estimate the robustness and reproducibility of the sample preparation. Limits of identification were 1 ng/mL for α- and ß-amanitin, 5 ng/mL for psilocin, bufotenine, muscarine, and ricinine, and 1500 ng/mL and 2000 ng/mL for ibotenic acid and muscimol, respectively. Using γ-amanitin, l-tryptophan-d5, and psilocin-d10 as internal standards, compensation for variations of matrix effects was shown to be acceptable for most of the toxins. In eight urine samples obtained from intoxicated individuals, α- and ß-amanitin, psilocin, psilocin-O-glucuronide, muscimol, ibotenic acid, and muscarine could be identified. Moreover, psilocin-O-glucuronide and bufotenine-O-glucuronide were found to be suitable additional targets. The analytical strategy developed was thus well suited for analyzing several biomarkers of toxic mushrooms and plants in human urine to support therapeutic decision making in a clinical toxicology setting. To our knowledge, the presented method is by far the most comprehensive approach for identification of the included biomarkers in a human matrix.

Pitfalls in drug testing by hyphenated low- and high-resolution mass spectrometry.

This paper reviews various pitfalls observed during developing, validation, application, and interpretation of drug testing approaches using GC-MS and low- and high-resolution LC-MS. They include sampling and storage of body samples, sample adulteration and contamination, analyte stability, sample preparation without or with cleavage of conjugates, extraction, derivatization, internal standardization, false negative and positive results by GC-MS or LC-MS screening and/or confirmation procedures including artifact formation, ion suppression or enhancement by electrospray ionization, and finally pitfalls in data interpretation. Conclusions and prospects close the Tutorial.

Evaluation of novel organosilane modifications of paper spray mass spectrometry substrates for analyzing polar compounds.

Paper spray mass spectrometry (PSMS) is currently used in different analytical fields, but less effort has been made so far to use PSMS for highly polar compounds. Such analytes usually show poor performance in PSMS due to their high affinity for common paper substrates in addition to high matrix effects. In this study, strategies for hydrophobic modifications of commercially available paper substrates using ten different organosilanes were developed. The modified substrates were generated, characterized, and applied for PSMS analysis of polar toxins. By using the modified paper, PSMS performance of some of the toxins could be considerably increased, especially for orellanine, showing a more than 80-fold signal enhancement when substrates modified with chlorotrimethylsilane were used. For other toxins like ricinine, only small beneficial effects could be shown on PSMS performance when using modified substrates. Statistical equivalence tests showed sufficient ruggedness of the developed procedures also compared to conventional substrates. Thus, further systematic development of paper substrates modified with organosilane derivatives based on the presented study for application in PSMS should be encouraged.

Distribution of the (synthetic) cannabinoids JWH-210, RCS-4, as well as ∆9-tetrahydrocannabinol following pulmonary administration to pigs.

New psychoactive substances, especially synthetic cannabinoids (SC), are gaining increasing relevance in postmortem forensic toxicology. Particularly, the interpretation of analytical results is challenging, as usually, no toxicokinetic (TK) data concerning distribution in organs and tissues are available. Thus, a controlled pig TK study allowing for examination of organ and tissue distribution of SC was performed. For this purpose, 12 pigs received a single pulmonary dose of 200 µg/kg body weight each of 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210), 2-(4-methoxyphenyl)-1-(1-pentylindole-3-yl)methanone (RCS-4), and Δ9-tetrahydrocannabinol (THC) via an ultrasonic nebulizer. Eight hours after administration, the animals were put to death by the administration of T61. Thereupon, relevant organs, important body fluids such as bile and colon content, and tissues such as muscle tissue were collected. After enzymatic hydrolysis and solid-phase extraction, analysis was performed by liquid chromatography-tandem mass spectrometry. For quantification, a standard addition method was applied. The parent compounds could be detected in every analyzed specimen with the exception of colon content. Regarding JWH-210, the kidneys and lungs are viable matrices for postmortem analysis. In terms of RCS-4, the lungs were found to be an appropriate matrix. Concerning THC, the liver, bile fluid as well as duodenum content were suitable matrices for detection. Metabolites were only detected in tissues/body fluids involved in metabolism and/or elimination. Bile fluid and duodenum content were shown, as the most appropriate specimens for quantification of metabolites.

In vitro metabolic fate of nine LSD-based new psychoactive substances and their analytical detectability in different urinary screening procedures.

The market of new psychoactive substances (NPS) is characterized by a high turnover and thus provides several challenges for analytical toxicology. The analysis of urine samples often requires detailed knowledge about metabolism given that parent compounds either may be present only in small amounts or may not even be excreted. Hence, knowledge of the metabolism of NPS is a prerequisite for the development of reliable analytical methods. The main aim of this work was to elucidate for the first time the pooled human liver S9 fraction metabolism of the nine d-lysergic acid diethylamide (LSD) derivatives 1-acetyl-LSD (ALD-52), 1-propionyl-LSD (1P-LSD), 1-butyryl-LSD (1B-LSD), N6-ethyl-nor-LSD (ETH-LAD), 1-propionyl-N6-ethyl-nor-LSD (1P-ETH-LAD), N6-allyl-nor-LSD (AL-LAD), N-ethyl-N-cyclopropyl lysergamide (ECPLA), (2'S,4'S)-lysergic acid 2,4-dimethylazetidide (LSZ), and lysergic acid morpholide (LSM-775) by means of liquid chromatography coupled to high-resolution tandem mass spectrometry. Identification of the monooxygenase enzymes involved in the initial metabolic steps was performed using recombinant human enzymes and their contribution confirmed by inhibition experiments. Overall, N-dealkylation and hydroxylation, as well as combinations of these steps predominantly catalyzed by CYP1A2 and CYP3A4, were found. For ALD-52, 1P-LSD, and 1B-LSD, deacylation to LSD was observed. The obtained mass spectral data of all metabolites are essential for reliable analytical detection particularly in urinalysis and for differentiation of the LSD-like compounds as biotransformations also led to structurally identical metabolites. However, in urine of rats after the administration of expected recreational doses and using standard urine screening approaches, parent drugs or metabolites could not be detected.

Blood plasma level determination using an automated LC-MSn screening system and electronically stored calibrations exemplified for 22 drugs and two active metabolites often requested in emergency toxicology.

Fast and comprehensive qualitative and quantitative methods preferably by gas chromatography-mass spectrometry (GC-MS) and/or liquid chromatography-mass spectrometry (LC-MS) are needed to support the (differential) diagnosis of acute poisonings in emergency toxicology. One option is a commercially available qualitative screening solution based on LC-MSn (Bruker Daltonik Toxtyper™, TT). Identified and toxicologically relevant compounds should be quantified to assess severity of poisonings. The aim of the present study was to test the TT system for quantification simultaneous with the screening process in blood plasma exemplified for 22 relevant drugs and two active metabolites. A standard liquid-liquid extraction was used for sample work-up followed by 1:5 dilution of the final extracts. They were analyzed using the TT system consisting of a Bruker amaZon speed ion trap and a Thermo Fisher Dionex Ultimate 3000 LC system. Plasma levels were assessed using full-scan data and an electronically stored five-point calibration. The calibration model was linear for the studied ranges and could be used for at least two months. The method was validated according to international guidelines. The acceptance criteria recommended for emergency toxicology for accuracy and precision were fulfilled for all tested compounds, but bromazepam, lorazepam, oxycodone, and prothipendyl could reliably be determined only above the therapeutic range. In conclusion, the presented procedure allowed the combination of a comprehensive LC-MSn screening with fast automated assessment of plasma levels for emergency toxicology of tested compounds.

Cytotoxicity of new psychoactive substances and other drugs of abuse studied in human HepG2 cells using an adopted high content screening assay.

New psychoactive substances (NPS) are still an emerging issue in clinical and forensic toxicology. Information about their cytotoxic potential is limited or even unavailable before distribution and thus their intake can be of high risk for consumers. The aim of the presented study was to develop a strategy to identify cytotoxic potential of NPS based on a high content screening assay (HCSA) using HepG2 cell line and four fluorescent dyes, namely Hoechst33342, TMRM, CAL-520, and TOTO-3. The HCSA was optimized to work without an automated analyzer by using the model compounds fluvastatin, paracetamol, propranolol, and simvastatin. The following parameters were monitored: stained nuclei as a measure for cell count as well as nuclear size and nuclear intensity (all Hoechst33342), mitochondrial membrane potential (TMRM), cytosolic calcium level (CAL-520), and plasma membrane integrity (TOTO-3). The present study showed strong cytotoxic potential for the NPS 5F-PB-22 and MDAI, moderate effects for MDMA, MDPV, methylone, cathinone, 4-MEC, and mephedrone, and no toxic effects for methamphetamine. To assess the metabolic suitability of HepG2 cells under the chosen conditions, cell culture supernatants were analyzed by liquid chromatography-high resolution-tandem mass spectrometry. Metabolites were merely detected for lipophilic drugs such as 5F-PB-22 and MDPV and in addition with a much lower abundance in comparison to the parent compound but the study only allowed a qualitative look for metabolites and the used liver cell line might not ideal when considering metabolism.

Interactions of phenethylamine-derived psychoactive substances of the 2C-series with human monoamine oxidases.

Psychoactive substances of the 2C-series (2Cs) are phenethylamine-derived designer drugs that can induce psychostimulant and hallucinogenic effects. Chemically, the classic 2Cs contain two methoxy groups in positions 2 and 5 of the phenyl ring, whereas substances of the so-called FLY series contain rigidified methoxy groups integrated in a 2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran core. One of the pharmacological features that has not been investigated in detail is the inhibition of monoamine oxidase (MAO). Inhibition of this enzyme can cause elevated monoamine levels that have been associated with adverse events such as agitation, nausea, vomiting, tachycardia, hypertension, or seizures. The aim of this study was to extend the knowledge surrounding the potential of MAO inhibition for 17 test drugs, which consisted of 12 2Cs (2C-B, 2C-D, 2C-E, 2C-H, 2C-I, 2C-N, 2C-P, 2C-T-2, 2C-T-7, 2C-T-21, bk-2C-B, and bk-2C-I) and five FLY analogs (2C-B-FLY, 2C-E-FLY, 2C-EF-FLY, 2C-I-FLY, and 2C-T-7-FLY). The extent of MAO inhibition was assessed using an established in vitro procedure based on heterologously expressed enzymes and analysis by hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry. Thirteen test drugs showed inhibition potential for MAO-A and 11 showed inhibition of MAO-B. In cases where MAO-A IC50 values were determined, values ranged from 10 to 125 µM (7 drugs) and from 1.7 to 180 µM for MAO-B (9 drugs). In the absence of detailed clinical information on most test drugs, it is concluded that a pharmacological contribution of MAO inhibition cannot be excluded and that further studies are warranted.

In vitro glucuronidation of designer benzodiazepines by human UDP-glucuronyltransferases.

Multiple new psychoactive substances (NPS) are released into the recreational drug market each year. One NPS drug class that has become more common in recent years is that of the benzodiazepines (designer benzodiazepines, DBZ). Several metabolism studies have been performed to improve their bioanalytical detection via the best target. These studies have shown the presence of parent glucuronides and, as polymorphisms have been noted for the catalyzing enzymes (UDP-glucuronyltransferases) responsible for glucuronide conjugation reactions, it is important to keep this in mind when interpreting DBZ cases in clinical and/or forensic toxicology. Therefore, the aim of this study was to determine the UDP-glucuronyltransferases (UGTs) responsible for parent compound conjugation of nine DBZ to facilitate interpretation of related cases. Clonazolam, deschloroetizolam, etizolam, flubromazolam, flunitrazolam, metizolam, nifoxipam, nitrazolam, and pyrazolam were incubated with pooled human liver microsomes (pHLM) or 13 different human UGTs. The samples were analyzed using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Glucuronide conjugates of flunitrazolam and nifoxipam were only detected in pHLM, suggesting that these reactions are performed by dimer complexes of several UGTs or complexes between UGTs and other metabolizing enzymes contained in pHLM. Nitrazolam or pyrazolam glucuronides were not detected. Glucuronidation of clonazolam, deschloroetizolam, etizolam, flubromazolam, and metizolam was catalyzed exclusively by UGT1A4. The conjugation of the majority of the DBZ was performed by the UGT isoform 1A4 for which polymorphisms have been described. This underlines the importance of taking glucuronidation polymorphism into consideration when interpreting intoxication cases.

Metabolic fate of the new synthetic cannabinoid 7'N-5F-ADB in rat, human, and pooled human S9 studied by means of hyphenated high-resolution mass spectrometry.

New psychoactive substances (NPS) are an important issue in clinical/forensic toxicology. 7'N-5F-ADB, a synthetic cannabinoid derived from 5F-ADB, appeared recently on the market. Up to now, no data about its mass spectral fragmentation pattern, metabolism, and thus suitable targets for toxicological urine screenings have been available. Therefore, the aim of this study was to elucidate the metabolic fate of 7'N-5F-ADB in rat, human, and pooled human S9 (pS9). The main human urinary excretion products, which can be used as targets for toxicological screening procedures, were identified by Orbitrap (OT)-based liquid chromatography-high resolution-tandem mass spectrometry (LC-HRMS/MS). In addition, possible differentiation of 7'N-5F-ADB and 5F-ADB via LC-HRMS/MS was studied. Using the in vivo and in vitro models for metabolism studies, 36 metabolites were tentatively identified. 7'N-5F-ABD was extensively metabolized in rat and human with minor species differences observed. The unchanged parent compound could be found in human urine but metabolites were far more abundant. The most abundant ones were the hydrolyzed ester (M5), the hydrolyzed ester in combination with hydroxylation of the tertiary butyl part (M11), and the hydrolyzed ester in addition to glucuronidation (M30). Besides the parent compound, these metabolites should be used as targets for urine-based toxicological screening procedures. Two urine-paired human plasma samples contained mainly the parent compound (c = 205 µg/L, 157 µg/L) and, at a higher abundance, the compound after ester hydrolysis (M5). In pS9 incubations, the parent compound, M5, and M30 were detectable among others. Furthermore, a differentiation of both compounds was possible due to different retention times and fragmentation patterns.

Inhibition and stimulation of the human breast cancer resistance protein as in vitro predictor of drug-drug interactions of drugs of abuse.

Transporter-mediated drug-drug interactions (DDI) may induce adverse clinical events. As drugs of abuse (DOA) are marketed without preclinical safety studies, only very limited information about interplay with membrane transporters are available. Therefore, 13 DOA of various classes were tested for their in vitro affinity to the human breast cancer resistance protein (hBCRP), an important efflux transporter. As adenosine 5'-triphosphate (ATP) hydrolysis is crucial for hBCRP activity, adenosine 5'-diphosphate (ADP) formation was measured and used as in vitro marker for hBCRP ATPase activity. ADP quantification was performed by hydrophilic interaction liquid chromatography coupled to high-resolution tandem mass spectrometry and its amount in test compound incubations was compared to that in reference incubations using the hBCRP substrate sulfasalazine or the hBCRP inhibitor orthovanadate. If DOA caused stimulation or inhibition, further investigations such as Michaelis-Menten kinetic modeling or IC50 value determination were conducted. Among the tested DOA, seven compounds showed statistically significant hBCRP ATPase stimulation. The entactogen 3,4-BDB and the plant alkaloid mitragynine were identified as strongest stimulators. Their affinity to the hBCRP ATPase was lower than that of sulfasalazine but comparable to that of rosuvastatin, another hBCRP model substrate. Five DOA showed statistically significant hBCRP ATPase inhibition. Determination of IC50 values identified the synthetic cannabinoid receptor agonists JWH-200 and WIN 55,212-2 as the strongest inhibitors comparable to orthovanadate. The present study clearly demonstrated that tested DOA show in part high affinities to the hBCRP within the range of model substrates or inhibitors. Thus, there is a risk of hBCRP-mediated DDI, which needs to be considered in clinical settings.

Nano liquid chromatography-high-resolution mass spectrometry for the identification of metabolites of the two new psychoactive substances N-(ortho-methoxybenzyl)-3,4-dimethoxyamphetamine and N-(ortho-methoxybenzyl)-4-methylmethamphetamine.

Among the emerging new psychoactive substances (NPS), compounds carrying an N-ortho-methoxybenzyl substituent, the so-called NBOMes, represented a highly potent group of new hallucinogens. Recently, 3,4-dimethoxyamphetamine (3,4-DMA)-NBOMe and 4-methylmethamphetamine (4-MMA)-NBOMe occurred, but no data on their pharmacokinetics were available. According to other NBOMes, they are expected to be extensively metabolized. For detection and identification of their phase I and II metabolites, nano liquid chromatography coupled to high resolution tandem mass spectrometry (nanoLC-HRMS/MS) was used. Rat urine was prepared by simple dilution and incubation mixtures with pooled human liver S9 fraction by precipitation. Furthermore, the results concerning detectability using the new nanoLC approach were compared to those obtained by conventional ultra-high performance LC (UHPLC). In addition, the detectability of the compounds by standard urine screening approaches (SUSAs) routinely used by the authors with UHPLC-HRMS/MS, LC-MSn, and GC-MS was tested. Both NBOMes were extensively metabolized mainly by O-demethylation and conjugation with glucuronic acid (3,4-DMA-NBOMe) or oxidation of the tolyl group to the corresponding carboxylic acid (4-MMA-NBOMe). The developed nanoLC-HRMS/MS approach was successfully applied for identification of 38 3,4-DMA-NBOMe metabolites and 33 4-MMA-NBOMe metabolites confirming its detection power. Furthermore, the solvent saving nanoLC system showed comparable results to the UHPLC-HRMS/MS approach. In addition, an intake of an estimated low common user's dose of the compounds was detectable by all SUSAs only via their metabolites. Suggested targets for urine screening procedures were O-demethyl- and O,O-bis-demethyl-3,4-DMA-NBOMe and their glucuronides and carboxy-4-MMA-NBOMe and its glucuronide and N-demethyl-carboxy-4-MMA-NBOMe.

Can toxicokinetics of (synthetic) cannabinoids in pigs after pulmonary administration be upscaled to humans by allometric techniques?

Being advertised and distributed as attractive substitutes of cannabis, synthetic cannabinoids (SC) are gaining increasing relevance in forensic and clinical toxicology. As no data from controlled human studies are available, SC are sold and consumed without the knowledge of their toxicokinetic (TK) and toxicodynamic properties. Hence, animal models coupled with mathematical approaches should be used to ascertain those properties. Therefore, a controlled pig TK study allowing for extrapolation to human data was performed. For this purpose, eleven pigs received a single pulmonary dose of 200 µg/kg body weight each of Δ9-tetrahydrocannabinol (THC), 4-ethylnaphthalene-1-yl-(1-pentylindole-3-yl)methanone (JWH-210) as well as 2-(4-methoxyphenyl)-1-(1-pentyl-indole-3-yl)methanone (RCS-4) via an ultrasonic nebulizer. Blood and urine samples were repeatedly drawn over 8 h. Serum-concentration-time profiles of the parent compounds were determined using LC-MS/MS. Urine specimens were analyzed by LC-HR-MS/MS in order to elucidate the main metabolites. Maximum serum concentrations were reached 10-15 min after beginning of nebulization and amounted to 66 ±â€¯36 ng/mL for THC, 41 ±â€¯11 ng/mL for JWH-210, and 34 ±â€¯8.9 ng/mL for RCS-4. The serum-concentration-time profiles of THC, JWH-210, and RCS-4 were best described by three-compartment models with first order absorption and elimination processes. Absorption from the lungs to serum was modeled by first-order processes. The determination of the bioavailability yielded 23.0%, 24.2%, and 45.7% for THC, JWH-210, and RCS-4, respectively. Furthermore, the developed THC model was upscaled to humans using allometric scaling techniques. A successful prediction of human concentration-time profiles could be done. Also the metabolic patterns were in good agreement with human data. In conclusion, these findings are the first reported regarding the TK properties of SC after pulmonary administration to pigs. The presented method of TK serves as an appropriate predictor of human TK of cannabinoids.

Mass Spectrometry for Research and Application in Therapeutic Drug Monitoring or Clinical and Forensic Toxicology.

This article reviews current applications of various hyphenated low- and high-resolution mass spectrometry techniques in the field of therapeutic drug monitoring and clinical/forensic toxicology in both research and practice. They cover gas chromatography, liquid chromatography, matrix-assisted laser desorption ionization, or paper spray ionization coupled to quadrupole, ion trap, time-of-flight, or Orbitrap mass analyzers.

Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia.

Inducible DNA recombination of floxed alleles in vivo by liver metabolites of tamoxifen (TAM) is an important tool to study gene functions. Here, we describe protocols for optimal DNA recombination in astrocytes, based on the GLAST-CreERT2/loxP system. In addition, we demonstrate that quantification of genomic recombination allows to determine the proportion of cell types in various brain regions. We analyzed the presence and clearance of TAM and its metabolites (N-desmethyl-tamoxifen, 4-hydroxytamoxifen and endoxifen) in brain and serum of mice by liquid chromatographic-high resolution-tandem mass spectrometry (LC-HR-MS/MS) and assessed optimal injection protocols by quantitative RT-PCR of several floxed target genes (p2ry1, gria1, gabbr1 and Rosa26-tdTomato locus). Maximal recombination could be achieved in cortex and cerebellum by single daily injections for five and three consecutive days, respectively. Furthermore, quantifying the loss of floxed alleles predicted the percentage of GLAST-positive cells (astroglia) per brain region. We found that astrocytes contributed 20 to 30% of the total cell number in cortex, hippocampus, brainstem and optic nerve, while in the cerebellum Bergmann glia, velate astrocytes and white matter astrocytes accounted only for 8% of all cells.

Development of an In-vitro Drug Delivery Efficiency Test for a Pulmonary Toxicokinetic Pig Study.

BACKGROUND: Since their appearance on the drugs of abuse market, synthetic cannabinoids (SCs) are gaining increasing toxicological relevance. They are consumed without knowledge of their toxicokinetic (TK) and toxicodynamic properties and human studies are not allowed due to ethical reasons. A controlled animal TK study following nebulization of 4-ethylnaphthalene-1-yl-(1-pentylindole- 3-yl)methanone (JWH-210), 2-(4-methoxyphenyl)-1-(1-pentyl-indole-3-yl)methanone (RCS-4) as well as Δ9-tetrahydrocannabinol (THC) to pigs should be helpful for better interpretation of analytical results in cases of misuse or poisoning. As a prerequisite, an in-vitro test system mimicking a ventilated pig had to be developed to determine the quantity and reproducibility of which drug dose is delivered to the pig lung. METHODS: JWH-210, RCS-4, and THC (1 mg in 2 mL ethanol each) were nebulized during ventilation using an ultrasonic nebulizer. The drug aerosol was delivered via the inspiratory limb and the endotracheal tube passing through a glass fiber filter (n = 6). The drugs were extracted from the filters using ethanol and ultrasonication. After several dilution steps and adding an internal standard solution, the extracts were analyzed by LC-MS/MS. RESULTS: Extraction of the nebulized drugs revealed delivery efficiencies of 78.8 ± 5.0% for JWH-210, 70.5 ± 6.9% for RCS-4, and 70.8 ± 7.9% for THC. The loss of about 20-30% of the administered dose might be attributable to retention in the nebulizer device or adhesion of the aerosol particles to the tube wall. CONCLUSION: Nevertheless, regarding delivery efficiencies, the minor standard deviations indicate an acceptable reproducibility, suggesting that this administration system is suitable for application in TK studies.