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Hair analysis can provide chronological insights into past drug use for months to years after drug administration. In comparison to analyses from other biological matrices, such as blood and urine, sample pretreatment is often tedious and not environmental friendly. In this study, we present a more environmental friendly approach to hair analysis using micropulverized hair and electromembrane extraction for the efficient extraction of 15 drugs of abuse, prescription drugs, and metabolites from hair. The optimized extraction method, involving micropulverization, demonstrated comparable yields to the standard approach of cutting and overnight incubation. A 15-min extraction method using a commercial electromembrane extraction prototype was developed and validated according to forensic guidelines, using only 10 µL of organic solvent per sample. The final method, employing HPLC-MS-MS with a biphenyl column, exhibited good linearity, precision, and sensitivity. An AgreePrep assessment comparing the environmental impact of our method with the standard routine method, involving overnight incubation and conventional liquid-liquid extraction, was conducted. This is the first time micropulverized hair has been subjected to electromembrane extraction., (© The Author(s) 2024. Published by Oxford University Press.)

Objective: People regularly contact emergency medicine services concerned that they have been exposed to drink spiking, i.e., exposure to drugs without their knowledge or permission. We identified drugs in blood and urine samples from patients suspecting exposure to drink spiking, with special consideration for drugs not reported taken by the patient (unreported drugs)., Methods: From September 2018 to May 2019, we collected blood and urine samples from patients 16 years or older presenting at an emergency clinic in Oslo, Norway, within 48 hours of suspected exposure to drink spiking. We also collected information on ethanol ingestion and drugs taken. Blood samples were analyzed for 20 classical recreational drugs using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and an automated enzymatic method for ethanol. Urine samples were analyzed using immunoassay methods and a specific gas chromatography mass spectrometry (GCMS) method for gammahydroxybutyrate (GHB)., Results: From 100 included patients (median age 24 years, 62 females), we collected 100 blood samples and 72 urine samples. Median time since exposure was 5 hours. Unreported drugs were found in 15 patients. Unreported drugs in the blood samples were clonazepam in 3, methylenedioxymethamphetamine (MDMA) in 3, amphetamine in 2, tetrahydrocannabinol (THC) in 2, tramadol in 1, cocaine in 1, and methamphetamine in 1. Unreported drugs in the urine samples were cocaine in 5, amphetamine in 4, ecstasy in 3, and cannabis in 2. Ethanol was found in 69 patients, all reporting ethanol ingestion. Median blood ethanol concentration was higher in patients with no unreported drugs detected, 1.00‰ (interquartile range (IQR) 0-1.52) vs. 0‰ (IQR 0-0.46) (p<0.001). GHB was not detected., Conclusion: Unreported drugs, possibly used for drink spiking, were found in 15% of patients. Blood ethanol concentration was higher when no unreported drugs were found. GHB was not detected in any patient., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dalaker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Illegal amphetamine is usually composed of a racemic mixture of the two enantiomers (S)- and (R)-amphetamine. However, when amphetamine is used in medical treatment, the more potent (S)-amphetamine enantiomer is used. Enantiomer-specific analysis of (S)- and (R)-amphetamine is therefore used to separate legal medical use from illegal recreational use. The aim of the present study was to describe our experience with enantiomer-specific analysis of amphetamine in urine and oral fluid, as well as blood, and examine whether the distribution of the two enantiomers seems to be the same in different matrices. We investigated 1,722 urine samples and 1,977 oral fluid samples from prison inmates, and 652 blood samples from suspected drugged drivers, where prescription of amphetamine was reported. Analyses were performed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). The enantiomer separation was achieved by using a chiral column, and results from the method validation are reported. Samples containing <60% (S)-amphetamine were interpreted as representing illegal use of amphetamine. The distribution of the two enantiomers was compared between different matrices. In urine and oral fluid, the mean amount of (S)-amphetamine was 45.2 and 43.7%, respectively, while in blood, the mean amount of (S)-amphetamine was 45.8%. There was no statistically significant difference in the amount of (S)-amphetamine between urine and oral fluid samples and between urine and blood samples, but the difference was significant in blood compared to oral fluid samples (P < 0.001). Comparison of urine and oral fluid between similar populations indicated that enantiomers of amphetamine can be interpreted in the same way, although marginally higher amounts of (R)-amphetamine may occur in oral fluid. Oral fluid, having several advantages, especially during collection, could be a preferred matrix in testing for illegal amphetamine intake in users of medical amphetamine., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Parallel artificial liquid membrane extraction (PALME) is a 96-well plate setup variant of liquid-phase microextraction. Basic or acidic analytes are extracted in neutral form from the sample, through a supported liquid membrane (SLM), and into aqueous acceptor. PALME is already considered a green extraction technique, but in the current conceptual work, we sought to make it even greener by replacing the use of organic solvents with essential oils (EO). PALME was combined with LC-MS/MS for analysis of plasma samples and multiple drugs of abuse with toxicological relevance (amphetamines, phenethylamines, synthetic cathinones, designer benzodiazepines, ayahuasca alkaloids, lysergic acid diethylamide, and ketamine)., Results: Fourteen EO were compared to organic solvents frequently used in PALME. The EO termed smart & sassy yielded the best analyte recovery for all drugs studied and was thus selected as SLM. Then, factorial screening and Box-Behnken were employed to optimize the technique. The extraction time, concentration of base, sample volume, and percentage of trioctylamine significantly impacted analyte recovery. The optimum values were defined as 120 min, 10 mmol/L of NaOH, 150 μL, and 0%, respectively. Once optimized, validation parameters were 1-100 ng mL-1 as linear range, accuracy ±16.4%, precision >83%, 1 ng mL-1 as limit of quantitation, 0.1-0.75 ng mL-1 as limit of detection, matrix effect <20%, and recovery 20-106%. Additionally, EO purchased from different production batches were tested and achieved acceptable reproducibility. Data were in compliance with requirements set by internationally accepted validation guidelines and the applicability of the technique was proven using authentic samples., Significance: In this study, the use of an EO provided a solvent-free sample preparation technique suited to extract different classes of drugs of abuse from plasma samples, dismissing the use of hazardous organic solvents. The method also provided excellent sample clean-up, thus being a simple and efficient tool for toxicological applications that is in agreement with the principles of sustainable chemistry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Benzimidazole opioids, often referred to as nitazenes, represent a subgroup of new psychoactive substances with a recent increase in fatal overdoses in the USA and Europe. With a variety of analogs emerging on the illicit drug market, forensic laboratories are challenged to identify these potent drugs. We here present a simple quantitative approach for the determination of nine nitazene analogs, namely, clonitazene, etodesnitazene, etonitazene, etonitazepyne, flunitazene, isotonitazene, metodesnitazene, metonitazene and protonitazene in whole blood using liquid-phase microextraction and electromembrane extraction in a 96-well format and liquid chromatography-tandem mass spectrometry. Green and efficient sample preparation was accomplished by liquid-phase microextraction in a 96-well format and resulted in high extraction yields for all analytes (>81%). Here, blood diluted with buffer (1:1, %v) was extracted from a donor compartment across a thin organic liquid membrane and into an aqueous acceptor solution. The acceptor solution was collected and directly injected into the analysis platform. Chromatographic separation was accomplished with a biphenyl column, allowing for a baseline separation of the structural isomers isotonitazene and protonitazene before detection by multiple reaction monitoring. Validation was performed according to Scientific Working Group of Forensic Toxicology guidelines. The calibration range was from 0.5 to 50 nM (except for protonitazene and clonitazene from 0.1 nM) with good linearity and limits of detection down to 0.01 nM. An AGREEprep assessment was performed to evaluate sample preparation greenness, with a final score of 0.71. Nitazenes represent a current threat to public health, and analytical methods that cover a wide range of these analogs are limited. Here, the described method may assist in the detection of nitazenes in whole blood and prevent these substances from being missed in postmortem investigations., (© The Author(s) 2023. Published by Oxford University Press.)

The use of oral fluid as sample matrix has gained significance in the analysis of drugs of abuse due to its non-invasive nature. In this study, the 13 opioids morphine, oxycodone, codeine, O-desmethyl tramadol, ethylmorphine, tramadol, pethidine, ketobemidone, buprenorphine, fentanyl, cyclopropylfentanyl, etonitazepyne, and methadone were extracted from oral fluid using electromembrane extraction based on conductive vials prior to analysis with ultra-high performance liquid chromatography-tandem mass spectrometry. Oral fluid was collected using Quantisal collection kits. By applying voltage, target analytes were extracted from oral fluid samples diluted with 0.1% formic acid, across a liquid membrane and into a 300 μL 0.1% (v/v) formic acid solution. The liquid membrane comprised 8 μL membrane solvent immobilized in the pores of a flat porous polypropylene membrane. The membrane solvent was a mixture of 6-methylcoumarin, thymol, and 2-nitrophenyloctyl ether. The composition of the membrane solvent was found to be the most important parameter to achieve simultaneous extraction of all target opioids, which had predicted log P values in the range from 0.7 to 5.0. The method was validated in accordance to the guidelines by the European Medical Agency with satisfactory results. Intra- and inter-day precision and bias were within guideline limits of ± 15% for 12 of 13 compounds. Extraction recoveries ranged from 39 to 104% (CV ≤ 23%). Internal standard normalized matrix effects were in the range from 88 to 103% (CV ≤ 5%). Quantitative results of authentic oral fluid samples were in accordance with a routine screening method, and external quality control samples for both hydrophilic and lipophilic compounds were within acceptable limits., (© 2023. The Author(s).)

Separation and quantification of amphetamine enantiomers are commonly used to distinguish between consumption of prescription amphetamine (mostly S-amphetamine) and illicit forms of the drug (racemate). In this study, electromembrane extraction with prototype conductive vials was combined with ultra-high performance supercritical fluid chromatography (UHPSFC-MS/MS) to quantify R- and S-amphetamine in urine. Amphetamine was extracted from 100 μL urine, diluted with 25 μL internal standard solution and 175 μL 130 mM formic acid, across a supported liquid membrane (SLM) consisting of 9 μL of a 1:1(w/w) mixture of 2-nitrophenyloctyl ether (NPOE) and bis(2-ethylhexyl)phosphite (DEHPi) into an acceptor phase containing 300 μL 130 mM formic acid. The extraction was facilitated by the application of 30 V for 15 min. Enantiomeric separation was achieved using UHPSFC-MS/MS with a chiral stationary phase. The calibration range was 50-10,000 ng/mL for each enantiomer. The between-assay CV was ≤5%, within-assay CV ≤ 1.5%, and bias within ±2%. Recoveries were 83%-90% (CV ≤ 6%), and internal standard corrected matrix effects were 99-105 (CV ≤ 2%). The matrix effects ranged from 96% to 98% (CV ≤ 8%) when not corrected by the internal standard. The EME method was compared with a chiral routine method that employed liquid-liquid extraction (LLE) for sample preparation. Assay results were in agreement with the routine method, and the mean deviation between methods was 3%, ranging from -21% to 31%. Finally, sample preparation greenness was assessed using the AGREEprep tool, which resulted in a greenness score of 0.54 for conductive vial EME, opposed to 0.47 for semi-automated 96-well LLE., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Benzodiazepines and z-hypnotics are detected in the majority of fatal overdose cases in Norway, often in combination with other drugs of abuse, and their concentrations in peripheral blood (PB) might be important to elucidate the cause of death. In some forensic autopsies, PB is however not available. The aim of the present study was to compare concentrations of benzodiazepines and z-hypnotics in five alternative matrices to assess whether these concentrations are comparable to concentrations in PB. A total of 109 forensic autopsy cases were included. PB, cardiac blood (CB), pericardial fluid (PF), psoas muscle (PM), lateral vastus muscle (LVM) and vitreous humor (VH) from each case were analyzed using ultra high performance liquid chromatography--tandem mass spectrometry. We were able to detect clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, nitrazepam, 7-aminonitrazepam, diazepam, nordiazepam, oxazepam, alprazolam, midazolam, zopiclone and zolpidem in all the analyzed matrices. Concentrations measured in VH were generally much lower than those of PB for all compounds except zopiclone. 7-Amino metabolite concentrations were high compared to the parent compounds, although less so for the muscle samples. Concentrations of the parent nitrobenzodiazepines in muscles were higher than those in PB, but for the other compounds, concentrations in muscle showed good correspondence with PB. Both CB and PF were viable alternative matrices for PB, although a larger variation and a tendency for higher concentrations in PF were observed. This study shows that CB, PM, LVM and PF can give comparable concentrations to PB for benzodiazepines and z-hypnotics, while VH was less suitable. The concentrations in alternative matrices must, however, be interpreted carefully., (© The Author(s) 2023. Published by Oxford University Press.)

The present paper defines the optimal extraction window (OEW) for three-phase membrane-based liquid-phase microextraction (MP-LPME) in terms of analyte polarity (log P), and anchors this to existing theories for equilibrium partitioning and kinetics. Using deep eutectic solvents (DES) as supported liquid membranes (SLM), we investigated how the OEW was affected by ionic-, hydrogen bond and π-π interactions between the SLM and analyte. Eleven basic model analytes in the range -0.4 < log P < 5.0 were extracted by MB-LPME in a 96-well format. Extraction was performed from 250 µL standard solution in 25 mM phosphate buffer (pH 7.0) into 50 µL of 10 mM HCl acceptor solution (pH 2.0) with mixtures of coumarin, camphor, DL-menthol, and thymol, with and without the ionic carrier di(2-ethylhexyl) phosphate (DEHP), as the SLM. The OEW with pure DES was in the range 2 < log P < 5, and low SLM aromaticity was favorable for the extraction of non-polar analytes. Here, extraction recoveries up to 98% were obtained. Upon addition of DEHP to the SLMs, the OEW shifted to the range -0.5 < log P < 2, and a combination of 5% DEHP and moderate aromaticity resulted in extraction recoveries up to 80% for the polar analytes. Extraction with ionic carrier was inefficient for the non-polar analytes, due to excessive trapping in the SLM. The results from our study show that LPME performs optimally in a relatively narrow log P-window of ≈ 2-3 units and that the OEW is primarily affected by ionic carrier and aromaticity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cannabis is the most widely used illicit substance worldwide. A limited number of studies have investigated whether tetrahydrocannabinol (THC) and cannabidiol (CBD) can be detected in other postmortem matrices than blood and urine. The aim of this study was to investigate the distribution of THC and CBD in several different postmortem matrices. Concentrations in peripheral blood were compared to those in cardiac blood, pericardial fluid, psoas muscle, vastus lateralis muscle, and vitreous humor. A total of 39 postmortem forensic autopsy cases were included. THC and CBD were analyzed using gas chromatography-mass spectrometry. We were able to detect both THC and CBD in most of the analyzed matrices. For vitreous humor, however, only approximately 50% of the cases were available for analysis, and only two were found to be positive. Median concentrations in peripheral blood were 0.0040 (0.00042-0.056) mg/L for THC and 0.0013 (0-0.023) mg/L for CBD. The concentration ratios between pericardial fluid and cardiac blood compared to peripheral blood were< 1 for both THC and CBD for the majority of the cases. For THC, a median ratio of 0.60 (0.063-7.2) and 0.65 (0.068-4.8) were found for pericardial fluid and cardiac blood, respectively, compared to peripheral blood, whereas for CBD the corresponding median ratios were 0.40 (0.010-1.9) and 0.80 (0.017-2.4). The THC concentrations in psoas muscle and vastus lateralis muscle were high compared to those in peripheral blood in several cases, and large variations in the muscles to peripheral blood concentration ratios were seen. This was also the case for CBD. Our study shows that THC and CBD can be detected in postmortem matrices other than peripheral blood, and results from other matrices might provide important information in forensic cases where peripheral blood is not available. However, vitreous humor was not suitable for detecting neither THC nor CBD., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Conductive vial electromembrane extraction (EME) with prototype equipment was applied for the first time to extract lipophilic basic drugs from serum. With this equipment, traditional platinum electrodes were replaced with sample and acceptor vials made from a conductive polymer, making the electrodes fully integrated and disposable. EME was combined with UHPLC-MS/MS, and a method to determine selected psychoactive drugs (alimemazine, amitriptyline, atomoxetine, clomipramine, doxepin, duloxetine, fluvoxamine, levomepromazine, nortriptyline and trimipramine) and metabolites (desmethyl clomipramine and desmethyl doxepin) in serum was developed, optimized, and validated. Extractions were carried out with 50 V for 15 min from serum samples (100 µL) diluted 1:3 with formic acid (0.1% v/v), using 2-nitrophenyl octyl ether as the supported liquid membrane (SLM), and formic acid (0.1% v/v, 300 µL) as acceptor phase. Using conductive vial EME, the extraction of lipophilic drugs reached exhaustive or near-exhaustive conditions, with recoveries in the range 75-117%. The method demonstrated excellent accuracy and precision, with bias within ± 6%, and intra- and inter-day CVs ranging 0.9 - 6% and 2 - 6%, respectively. In addition, acceptor phases were completely free of glycerophosphocholines. EME-UHPLC-MS/MS was successfully applied in determination of psychoactive drugs in 30 patient samples, and the results were in agreement with the current hospital routine method at St. Olav University Hospital (Trondheim, Norway). Obtaining comparable results to well-established routine methods is highly important for future implementation of EME into routine laboratories. These results thus serve as motivation for further advancing the EME technology. Until now, EME has been carried out with laboratory-build equipment, and the introduction of commercially available standardized equipment is expected to have a positive impact on future research activity., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Electromembrane extraction (EME) of the polar zwitterionic drugs, anthracyclines (ANT, doxorubicin, daunorubicin and its metabolite daunorubicinol), from rabbit plasma was investigated. The optimized EME was compared to conventional sample pretreatment techniques such as protein precipitation (PP) and liquid-liquid extraction (LLE), mainly in terms of extraction reliability, recovery and matrix effect. In addition, phospholipids profile in the individual extracts was evaluated. The extracted samples were analyzed using UHPLC-MS/MS with electrospray ionization in positive ion mode. The method was validated within the concentration range of 0.25-1000 ng/mL for all tested ANT. Compared with PP and LLE, the EME provided high extraction recovery (more than 80% for all ANT) and excellent sample clean-up (matrix effect were 100 ± 10% with RSD values lower than 4% for all ANT). Furthermore, only negligible amounts of phospholipids were detected in the EME samples. Finally, practical applicability of EME was proved by analysis of plasma samples taken from a pilot in vivo study in rabbits. Consistent results were obtained when using both EME and LLE to extract the plasma prior to the analysis, which further confirmed high reliability of EME. This study clearly showed that EME is a simple, rapid, repeatable technique for extraction of ANT from plasma and it is an up to date alternative to routine conventional extraction techniques., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

In this paper, we review recent research articles on liquid-phase microextraction of drug substances from biological fluids, such as plasma, serum, urine, and saliva. We focus on papers where liquid-phase microextraction is combined with liquid chromatography coupled with mass spectrometry (LC-MS), published in the period 2019-2020. First, we discuss different configurations of liquid-phase microextraction, including dispersive liquid-liquid microextraction (DLLME), dispersive liquid-liquid microextraction based on solidified floating organic droplet (DLLME-SFO), single-drop microextraction (SDME), hollow-fibre liquid-phase microextraction (HF-LPME), solvent bar microextraction (SBME), and electromembrane extraction (EME). Second, we discuss new types of solvents used in liquid-phase microextraction, including ionic liquids, deep eutectic solvents, and nanostructured supramolecular solvents. Especially, we focus on the potential for implementation in routine laboratories, which we consider as the next step for liquid-phase microextraction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Exposure of wildlife and domestic animals to anticoagulant rodenticides (ARs) is a worldwide concern, but few methods exist to determine residue levels in live animals. Traditional liver detection methods preclude determining exposure in live wildlife. To determine the value of assessing AR exposure by fecal analysis, we compared fecal and liver residues of ARs in the same animals. We collected liver and fecal samples from 40 apparently healthy red foxes ( Vulpes vulpes ) potentially exposed to ARs, and quantified brodifacoum, bromadiolone, coumatetralyl, difenacoum, difethialone, and flocoumafen residues by liquid chromatography-tandem mass spectrometry. Residues of ARs were detected in 53% of the fecal samples and 83% of the liver samples. We found good concordance between AR residues in feces and liver for coumatetralyl, difenacoum, and difethialone. Bromadiolone occurred in significantly greater frequency in livers compared to feces, but no significant difference in concentration between feces and liver in individual foxes could be detected. Brodifacoum displayed a significant difference in concentration and occurrence of positive samples between liver and feces. Our findings demonstrate that fecal analysis of ARs provides a feasible and valuable non-lethal means of determine AR exposure in live wildlife.

Background: Exposure to anticoagulant rodenticides (ARs) in dogs is among the most common causes of poisoning in small animal practice, but information about toxicokinetic of these rodenticides in dogs is lacking. We analysed blood and faeces from five accidentally exposed dogs and 110 healthy dogs by reversed phase ultra-high performance liquid chromatography-tandem mass spectrometry. The aim of the study was to estimate elimination of brodifacoum, bromadiolone and difenacoum after acute exposure, calculate the half-lives of these rodenticides in dogs, estimate faecal elimination in a litter of puppies born, and further to identify the extent of AR exposure in a healthy dog population., Results: Three dogs were included after single ingestions of brodifacoum; two dogs ingested bromadiolone and one dog ingested difenacoum. Maximum concentrations in faeces were found after day 2-3 for all ARs. The distribution half-lives were 1-10 days for brodifacoum, 1-2 days for bromadiolone and 10 days for difenacoum. Brodifacoum and difenacoum had estimated terminal half-lives of 200-330 days and 190 days, respectively. In contrast, bromadiolone had an estimated terminal half-life of 30 days. No clinical signs of poisoning or coagulopathy were observed in terminal elimination period. In blood, the terminal half-life of brodifacoum was estimated to 8 days. Faeces from a litter of puppies born from one of the poisoned dogs were examined, and measurable concentrations of brodifacoum were detected in all samples for at least 28 days after parturition. A cross-sectional study of 110 healthy domestic dogs was performed to estimate ARs exposure in a dog population. Difenacoum was detected in faeces of one dog. Blood and faecal samples from the remaining dogs were negative for all ARs., Conclusions: Based on the limited pharmacokinetic data from these dogs, our results suggest that ARs have a biphasic elimination in faeces using a two-compartment elimination kinetics model. We have shown that faecal analysis is suitable and reliable for the assessment of ARs exposure in dogs and a tool for estimating the AR half-lives. Half-lives of ARs could be a valuable indicator in the exposed dogs and provides important information for veterinarians monitoring AR exposure and assessment of treatment length in dogs.

Similarly to many other sample extraction techniques, efficient extraction of very polar compounds with electromembrane extraction (EME) is difficult. To date, the best known strategy to improve the mass transfer of these compounds is the addition of an ionic carrier, often bis(2-ethylhexyl) phosphate (DEHP) to the supported liquid membrane (SLM). DEHP is known to work by providing ionic interactions with basic compounds, to improve the partitioning into the SLM. In this work, the behavior of DEHP during extractions was studied for the first time. Interestingly, substantial amounts of DEHP was found to leak from the SLM into the aqueous sample at pH > 4. Due to this leakage, the ion-pair formation between analytes and DEHP was moved from the sample/SLM interface (interfacial complexation) to the bulk of the sample solution (bulk-sample complexation), which improved the mass transfer of polar bases considerably. Based on this, an extraction procedure for eight polar bases with log P values from +0.7 to -5.9 was developed and optimized. The optimization demonstrated that extraction of more polar analytes was favored by bulk-sample complexation. With optimized conditions, extraction from biological samples such as urine, protein-precipitated plasma, and raw plasma were performed with recoveries >40%, except for a few analytes. In addition, the extraction system could be operated under robust conditions with relatively low current (<70 μA for plasma), and provided low variability (<16% RSD), as well as good clean-up efficiency. These findings are an important step in further scientific anchoring of EME, and development of the technique towards selective extraction of very polar substances from complex biological matrices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

High occurrence of anticoagulant rodenticides (ARs) in wildlife is a rising concern, with numerous reports of secondary exposure through predation. Because of widespread distribution of the red fox ( Vulpes vulpes ), they may act as sentinels for small mammal-hunting predators in rural, suburban, and urban areas. No AR surveillance in wild mammals with analyses of residues in feces has been conducted throughout a single country. We collected 163 fecal samples from presumed healthy red foxes from 18 out of 19 counties in Norway. The foxes were shot during regular hunting between January and December 2016 and samples collected directly after death. Fecal samples were analyzed for six ARs: brodifacoum, bromadiolone, coumatetralyl, difenacoum, difethialone, and flocoumafen. We detected ARs in 54% (75/139) of the animals. Brodifacoum was most frequently detected (46%; 64/139), followed by coumatetralyl (17%; 23/139), bromadiolone (16%; 22/139), difenacoum (5%; 7/139), difethialone (1%; 2/139), and flocoumafen (1%; 2/139). More than one substance was detected in 40% (30/75) of the positive foxes, and 7% (5/75) of these animals were exposed to four different ARs. There were no statistically significant seasonal, age, or sex differences in foxes after exposure to one AR compound. We found a significant difference in occurrence of brodifacoum and coumatetralyl in foxes from different geographical areas. These findings demonstrate fecal analyses as a valuable method of detecting AR exposure in red foxes. We suggest using direct fecal sampling with analyses as a method to evaluate the occurrence of ARs in live endangered wildlife in connection with radio tagging or collaring operations.

The use of miniaturized systems with the possibility for automation has become increasingly popular in the field of bioanalysis. As a new approach to liquid phase microextraction in the 96-well format, parallel artificial liquid membrane extraction (PALME) was introduced in 2013. In the present work, the reliability of the quantitative data obtained with PALME was thoroughly evaluated. Amitriptyline, nortriptyline, quetiapine, venlafaxine, o-desmethylvenlafaxine, and fluoxetine were selected as model analytes. The analytes were extracted under non-exhaustive conditions from human plasma samples and the extracts were analyzed directly by LC-MS/MS. Accuracy was within ±15% and precision was <15% when the QC samples were prepared in both pooled plasma and in plasma from multiple sources. Accuracy and precision was superior when stable isotopically labelled (SIL) internal standards were used, as compared to structural analogue internal standards in the plasma samples from multiple sources. SIL internal standards are therefore recommended as the first choice. Assessment of accuracy and precision was also carried out with four different operators performing the extraction procedure, providing accuracy within ±15% and precision <15%. The extraction recoveries were in the range from 48 to 85 %, and non-exhaustive extraction of the analytes did not affect the accuracy and precision of the method. With the method described, up to 96 samples can be extracted with a total extraction time of 60 min and with a total consumption of organic solvent less than 0.4 mL for the whole wellplate. PALME is therefore a new approach to high-throughput sample preparation, providing accurate quantification, along with simple workflow, low consumption of organic solvent, and extensive sample clean-up., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aim: Electromembrane extraction (EME) of 37 drugs of abuse with significant differences in terms of polarity (0.68 < log P < 4.3) and basicity (1.17 < pK a  < 10.38) was investigated from human plasma. Materials & methods: EME was performed with 250 mM trifluoroacetic acid and DMSO (1:1 v/v) in the acceptor solution. Results & conclusion: The analytes were extracted efficiently with pure 2-nitrophenyloctyl ether as supported liquid membrane when the acceptor solution was modified with DMSO. Thus, using DMSO mixed with 250 mM trifluoroacetic acid (1:1, v/v) as acceptor solution, recoveries from 40 to 105% (relative standard deviation <20%) were obtained for 33 of the analytes under optimized conditions. EME followed by ultra-HPLC-MS/MS analysis was evaluated from human plasma, and the results were satisfactory.

Escalating opioid use among fertile women has increased the number of children being exposed to opioids during fetal life. Furthermore, accumulating evidence links prenatal opioid exposure, including opioid maintenance treatment, to long-term negative effects on cognition and behavior, and presses the need to explore novel treatment strategies for pregnant opioid users. The present study examined the potential of a monoclonal antibody (mAb) targeting heroin's first metabolite, 6-acetylmorphine (6-AM), in providing fetal protection against harmful effects of prenatal heroin exposure in mice. First, we examined anti-6-AM mAb's ability to block materno-fetal transfer of active metabolites after maternal heroin administration. Next, we studied whether maternal mAb pretreatment could prevent adverse effects in neonatal and adolescent offspring exposed to intrauterine heroin (3 × 1.05 mg/kg). Anti-6-AM mAb pretreatment of pregnant dams profoundly reduced the distribution of active heroin metabolites to the fetal brain. Furthermore, maternal mAb administration prevented hyperactivity and drug sensitization in adolescent female offspring prenatally exposed to heroin. Our findings demonstrate that passive immunization with a 6-AM-specific antibody during pregnancy provides fetal neuroprotection against heroin metabolites, and thereby prevents persistent adverse behavioral effects in the offspring. An immunotherapeutic approach to protect the fetus against long-term effects of prenatal drug exposure has not been reported previously, and should be further explored as prophylactic treatment of pregnant heroin users susceptible to relapse., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Background and Objectives: Even though nightlife studies with potentially intoxicated participants provide the much needed information on drug use, they face additional methodological challenges. This study aimed to explore the utility of such studies by (i) classifying nightlife attendees based on their self-reported drug use and by (ii) examining whether these classifications were meaningful when assessed against other sources of data, including oral fluid drug tests., Methods: Self-reported questionnaires, oral fluid samples and blood alcohol concentration readings were collected in a sample of 1,085 nightlife patrons recruited outside 12 popular nightclubs in Oslo, Norway, in 2014. Patrons were classified using multiple approaches, including latent class analysis. Group differences were examined by logistic regression models., Results: Participants were classified into 5 mutually exclusive groups: 2 among current non-users ("Never-users"; "Previous users"), 2 among current users ("Multiple drugs"; "Cannabis mainly") and one "Incomplete information" group. Meaningful differences across these groups were observed. For instance, positive tests for any illicit drug were more common in "Multiple drugs" group than in "Cannabis mainly" (62.7 vs. 29.1%, adjusted OR [aOR] 3.77 [2.42-5.84]) or "Incomplete information" groups (62.7 vs. 34.4%, aOR 2.46 [1.26-4.79]). Despite their self-declared non-use, illicit substances were detected in oral fluids of "Never-users" (13.1%; 95% CI 9.9-17.2) and "Previous users" (7.9%; 95% CI 5.1-12.1)., Conclusions: Despite some discrepancies between self-reports and biological tests, self-reports proved both suitable and useful in identification of substantively different drug-user typologies, potentially informing targeted policy responses. Still, methodological challenges associated with onsite studies of illicit drug use should be further explored., (© 2019 S. Karger AG, Basel.)

Peripheral blood (PB) is considered to be the golden standard for measuring postmortem drug concentrations. In several cases, PB is however not available, but information regarding drug findings might still be crucial in order to determine the cause of death. Antidepressants are frequently detected in postmortem samples from forensic toxicology cases, but the literature investigating concentrations in other matrices than peripheral and heart blood is limited.We here describe a study for comparison of concentrations for a large number of different drugs in six different matrices. A total of 173 postmortem cases were included in the study material. The results from 44 cases with findings of antidepressants (amitriptyline/nortriptyline, citalopram, mianserin, mirtazapine, paroxetine, sertraline, trimipramine and venlafaxine) are presented in this article. Concentrations in peripheral and cardiac blood (CB), pericardial fluid (PF), two muscle samples and vitreous humour (VH) are compared. Ratios between concentrations in different matrices have also been compiled from available literature.All the investigated antidepressants were detected in all different matrices, and comparable concentration levels were found in the different matrices with a few exceptions. Concentrations in VH were generally lower than in the other matrices, and in a few cases with low concentrations in blood the antidepressants were not detected in VH. For most of the cases, ratios of 0.5-2 were found between concentration in PB and that in the other matrices. Some deviant concentrations where however found.This study shows that CB, PF, muscle and VH can provide important indications of the corresponding concentrations in PB when PB is not available.

Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R 2  > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL -1 and 3.2 to 160 ng mL -1 , respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and - 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis. Graphical abstract.

In this paper, parallel artificial liquid membrane extraction (PALME) was used for the first time to clean-up dried blood spots (DBS) prior to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Fundamental studies exploring amongst others desorption from the DBS in alkaline or acidic aqueous conditions, total extraction time and absolute recoveries were executed. Desorption and PALME were performed using a set of two 96-well plates, one of them housing the sample and the other comprising the supported liquid membrane (SLM) and the acceptor solution. In one procedure, amitriptyline and quetiapine (basic model analytes) were desorbed from the DBS using 250 μL of 10 mM sodium hydroxide solution (aqueous), and subsequently extracted through the SLM consisting of 4 μL of 1% trioctylamine in dodecyl acetate, and further into an acceptor solution consisting of 50 μL of 20 mM formic acid. In a second procedure, ketoprofen, fenoprofen, flurbiprofen, and ibuprofen (acidic model analytes) were desorbed from the DBS into 20 mM formic acid, extracted through an SLM with dihexyl ether, and further into an acceptor solution of 25 mM ammonia. Within 60 min of PALME, both basic and acidic model analytes were effectively desorbed from the DBS and extracted into the acceptor solution, which was injected directly into the analytical instrument. Recoveries between 63 and 85% for the six model analytes were obtained. PALME provided excellent clean-up from the DBS samples, and acceptor solutions were free from phospholipids. Linearity was obtained with r 2  > 0.99 for five of the six analytes. Accuracy, precision and UHPLC-MS/MS matrix effects were in accordance with the European Medicines Agency (EMA) guideline. Based on these experiments, PALME shows great potential for future processing of DBS in a short and simple way, and with the presented setup, up to 96 DBS can be processed within a total extraction time of 60 min., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In some forensic autopsies blood is not available, and other matrices are sampled for toxicological analysis. The aims of the present study were to examine whether heroin metabolites can be detected in different post-mortem matrices, and investigate whether analyses in other matrices can give useful information about concentrations in peripheral blood. Effects of ethanol on the metabolism and distribution of heroin metabolites were also investigated. We included 45 forensic autopsies where morphine was detected in peripheral blood, concomitantly with 6-acetylmorphine (6-AM) detected in any matrix. Samples were collected from peripheral blood, cardiac blood, pericardial fluid, psoas muscle, lateral vastus muscle, vitreous humor and urine. Opioid analysis included 6-AM, morphine, codeine, and morphine glucuronides. The 6-AM was most often detected in urine (n = 39) and vitreous humor (n = 38). The median morphine concentration ratio relative to peripheral blood was 1.3 (range 0-3.6) for cardiac blood, 1.4 (range 0.07-5.3) for pericardial fluid, 1.2 (range 0-19.2) for psoas muscle, 1.1 (range 0-1.7) for lateral vastus muscle and 0.4 (range 0.2-3.2) for vitreous humor. The number of 6-AM positive cases was significantly higher (P = 0.03) in the ethanol positive group (n = 6; 86%) compared to the ethanol negative group (n = 14; 37%) in peripheral blood. The distribution of heroin metabolites to the different matrices was not significantly different between the ethanol positive and the ethanol negative group. This study shows that toxicological analyses of several matrices could be useful in heroin-related deaths. Urine and vitreous humor are superior for detection of 6-AM, while concentrations of morphine could be assessed from peripheral or cardiac blood, pericardial fluid, psoas muscle and lateral vastus muscle.

Aim: Electromembrane extraction (EME) of weakly basic benzodiazepines was investigated (-1.47 < pKa < 5.01)., Materials & Methods: 96-well EME was performed with strongly acidic conditions in the acceptor solution using 250-mM trifluoroacetic acid to maximize ionization., Results & Conclusion: Recoveries more than 80% were obtained for analytes with pKa > 2, whereas EME was less efficient for substances with pKa < 2. The latter was trapped in the supported liquid membrane due to less acidic pH conditions in the acceptor solution close to the supported liquid membrane. EME followed by UHPLC-MS/MS analysis was evaluated from human plasma, and the results were in compliance with EMA guidelines. Both electrokinetic migration and passive diffusion contributed to mass transfer when performing EME of weakly basic analytes.

Para-methoxymethamphetamine (PMMA) has caused numerous fatal poisonings worldwide and appears to be more toxic than other ring-substituted amphetamines. Systemic metabolism is suggested to be important for PMMA neurotoxicity, possibly through activation of minor catechol metabolites to neurotoxic conjugates. The aim of this study was to examine the metabolism of PMMA in humans; for this purpose, we used human liver microsomes (HLMs) and blood samples from three cases of fatal PMMA intoxication. We also examined the impact of CYP2D6 genetics on PMMA metabolism by using genotyped HLMs isolated from CYP2D6 poor, population-average, and ultrarapid metabolizers. In HLMs, PMMA was metabolized mainly to 4-hydroxymethamphetamine (OH-MA), whereas low concentrations of para-methoxyamphetamine (PMA), 4-hydroxyamphetamine (OH-A), dihydroxymethamphetamine (di-OH-MA), and oxilofrine were formed. The metabolite profile in the fatal PMMA intoxications were in accordance with the HLM study, with OH-MA and PMA being the major metabolites, whereas OH-A, oxilofrine, HM-MA and HM-A were detected in low concentrations. A significant influence of CYP2D6 genetics on PMMA metabolism in HLMs was found. The catechol metabolite di-OH-MA has previously been suggested to be involved in PMMA toxicity. Our studies show that the formation of di-OH-MA from PMMA was two to seven times lower than from an equimolar dose of the less toxic drug MDMA, and do not support the hypothesis of catechol metabolites as major determinants of fatal PMMA toxicity. The present study revealed the metabolite pattern of PMMA in humans and demonstrated a great impact of CYP2D6 genetics on human PMMA metabolism., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Purpose: Little is known about the relationship between concentrations in oral fluid (OF) and blood for the widely prescribed hypnotic drug zopiclone. The purpose of this study was to investigate the usefulness of OF zopiclone concentrations to predict blood zopiclone concentrations in order to introduce OF testing as an alternative to more cumbersome blood testing., Methods: 16 healthy young male volunteers received capsules of either 5 or 10mg zopiclone on two different study days separated by at least one week. Blood and OF were collected simultaneously at baseline and 9 times after intake of zopiclone on each study day. In addition an OF sample was collected 24-81h after intake. Lunch was served between samples taken 2.5 and 3.5h after intake. All samples were analysed for zopiclone, and the cut-off was 10ng/ml in blood and 0.2ng/ml in OF-buffer mixture., Results: Zopiclone was detected in all OF samples during the study day. After 24-81h, all subjects were also positive for zopiclone in OF, except from three subjects ingesting the 5mg dose. In a single case zopiclone was detected in a baseline OF sample 14days after intake on an earlier study day. Zopiclone was detected in both OF and blood in 231 OF/blood pairs, and a significant but weak correlation between OF and blood concentration was seen (R 2 of 0.30). The median (range) zopiclone OF/blood concentration ratio (ZOBCR) for all samples were 3.3 (0.8-18). The ZOBCR decreased when the OF volume increased. After 30 of 31 given doses of zopiclone, the ZOBCR was higher in samples collected before lunch than samples collected after lunch., Discussion: Vast intra- and interindividual differences in ZOBCR were found, and the correlation between OF and blood concentration was less pronounced than reported in former studies. In accordance with earlier studies we found a negative correlation between ZOBCR and OF volume. The ZOBCR decreases in relation to recent intake of a meal, probably because stimulated saliva production causes "dilution" of saliva. OF zopiclone concentration appeared unsuitable for estimation of blood zopiclone concentration. Due to long detection time, analysis of zopiclone in OF might be useful to detect non-recent, previous intake., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Context: Recreational drug toxicity is frequent. Availability of new psychoactive substances is steadily increasing. However, data with verified analyses from clinical settings are limited. To evaluate the impact of novel psychoactive substances (NPS) on recreational drug toxicity in Oslo, Norway, we analysed samples from a selection of patients., Methods: All the patients presenting with recreational drug toxicity at the Oslo Accident and Emergency Outpatient Clinic (OAEOC) and at the Oslo University Hospital (OUH) were registered from April through September 2014. Oral fluid samples were collected at the OAEOC. Blood samples were collected at the OUH. The samples were screened using ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS)., Results: Nine hundred and sixty-four cases were included, 841 (87.2%) at the OAEOC and 123 (12.8%) at the OUH. A total of 55 oral fluid samples (OAEOC) and 103 blood samples (OUH) could be analysed. NPS were not clinically suspected in any of the screened cases. At the outpatient clinic, the most commonly found substances were clonazepam in 42/55 (76.4%) cases, amfetamines in 40/55 (72.7%) and heroin in 39/55 (70.9%). In seven (12.7%) cases NPS were detected: 4-methylamfetamine in three cases, dimethyltryptamine in two, methylone in one, and N,N-dimethyl-3,4-methylenedioxyamfetamine in one. Among the hospital patients, the most commonly found substances were clonazepam in 51/103 (49.5%) cases, amfetamines in 48/103 (46.6%), heroin in 31/103 (30.1%), and diazepam in 30/103 (29.1%). In five (4.9%) cases NPS were detected: JWH-210 in two cases, AM-2201 in two, and 5-EAPB in one., Conclusion: NPS were clinically not suspected, though found in eight percent of cases. Still, the vast majority of patients treated for recreational drug toxicity in Oslo have taken classical drugs. Management of these patients should be based on their clinical condition. However, it is highly important to be alert to atypical presentations possibly resulting from unsuspected drugs.

Aim: For the first time, extracts obtained from human plasma samples by electromembrane extraction (EME) were investigated comprehensively with particular respect to phospholipids using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Thhe purpose was to investigate the potential of EME for phospholipid cleanup in different EME systems., Results & Discussion: No traces of phospholipids were detected in any of the acceptor solutions, whereas the model analytes were extracted with recoveries up to 50%. Thus, the EME systems tested in this work were found to be highly efficient for providing phospholipid-free extracts., Conclusion: Ultra-HPLC-MS/MS analysis of the donor solutions revealed that the phospholipids principally remained in the plasma samples. This proved that the phospholipids did not migrate in the electrical field and they were prevented from penetrating the supported liquid membrane.

Parallel artificial liquid membrane extraction (PALME) was combined with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the potential for screening of new psychoactive substances (NPS) was investigated for the first time. PALME was performed in 96-well format comprising a donor plate, a supported liquid membrane (SLM), and an acceptor plate. Uncharged NPS were extracted from plasma or whole blood, across an organic SLM, and into an aqueous acceptor solution, facilitated by a pH gradient. MDAI (5,6-methylenedioxy-2-aminoindane), methylone, PFA (para-fluoroamphetamine), mCPP (meta-chlorophenylpiperazine), pentedrone, methoxetamine, MDPV (methylenedioxypyrovalerone), ethylphenidate, 2C-E (2,5-dimethoxy-4-ethylphenethylamine), bromo-dragonfly, and AH-7921 (3,4-dichloro-N-{[1-(dimethylamino)cyclohexyl]methyl}benzamide) were selected as representative NPS. Optimization of operational parameters was necessary as the NPS were novel to PALME, and because PALME was performed from whole blood for the very first time. In the PALME method developed for plasma, NPS were extracted from a 250μL alkalized donor solution consisting of 125μL plasma sample, 115μL 40mM NaOH, and 10μL internal standard. In the PALME method from whole blood, the 250μL alkalized donor solution consisted of 100μL whole blood, 50μL deionized water, 75μL 80mM NaOH, and 25μL internal standard. In both methods, extraction was accomplished across an SLM of 5μL dodecyl acetate with 1% trioctylamine (w/w), and further into an acidic acceptor solution of 50μL 20mM formic acid. The extraction was promoted by agitation at 900rpm and was carried out for 120min. Method validation was performed and the following parameters were considered: linearity, limits of quantification (LOQ), intra- and inter-day precision, accuracy, extraction recoveries, carry-over, and matrix effects. The validation results were in accordance with FDA guidelines., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Immunotherapy can provide a supplemental treatment strategy against heroin use on the principle of sequestering the active drug in the bloodstream, thereby reducing its distribution to the brain. Previous studies have shown that heroin's first metabolite, 6-monoacetylmorphine (6-MAM), is the main mediator of acute heroin effects. The objective of the present study was to characterize the pharmacological potential of a monoclonal antibody against 6-MAM (anti-6-MAM mAb) to counteract the heroin response. The individual contributions from heroin and 6-MAM to heroin effects were also examined by pretreating mice with anti-6-MAM mAb (10-100 mg/kg) prior to either heroin or 6-MAM injection (1.25-2.5 μmol/kg). The opioid-induced behavioral response was assessed in a locomotor activity test, followed by opioid and antibody quantification in blood and brain tissue. Pretreatment with mAb caused a profound reduction of heroin- and 6-MAM-induced behavior, accompanied by correspondingly decreased levels of 6-MAM in brain tissue. mAb pretreatment was more efficient against 6-MAM injection than against heroin, leading to an almost complete blockade of 6-MAM-induced effects. mAb pretreatment was unable to block the immediate (5-minute) transport of active metabolites across the blood-brain barrier after heroin injection, indicating that heroin itself appears to enhance the immediate delivery of 6-MAM to the brain. The current study provides additional evidence that 6-MAM sequestration is crucial for counteracting the acute heroin response, and demonstrates the pharmacological potential of immunotherapy against heroin use., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

The aim of this study was to investigate psychoactive drug use among nightclub patrons by analysing samples of oral fluid and compare with findings in blood samples from criminal suspects. We hypothesized that the profile of illicit drug use among nightclub patrons is different from what we observe in those forensic cases. Research stations were established outside nine popular nightclubs with different profiles and patron-characteristics in downtown Oslo. Data and sample collection was conducted on Fridays and Saturdays in March and May 2014. Individuals and groups who entered defined recruitment zones from 23:00 to 03:30 were invited to participate in this voluntary and anonymous study. Oral fluid was collected using the Intercept Oral Fluid Sampling Device. Methanol was added to increase the recovery of cannabinoids from the device. Sample preparation was performed using liquid-liquid extraction with ethyl acetate/heptane (4:1) after adding internal standards, ammonium carbonate buffer pH 9.3 and Triton X100. The first 80 samples were analysed for 122 substances, which included psychoactive medicinal drugs, classical illicit drugs and new psychoactive substances (NPS). Based on the findings and discussions with police and customs authorities, the remaining oral fluid samples were analysed for 46 substances. Among the 500 samples collected during the study period, we found illicit drugs in 25.4% and medicinal drugs in 4.2% of the samples. The most prevalent substances were: cocaine 14.6%, THC 12.4%, amphetamine/methamphetamine 2.8%, diazepam 1.2% and clonazepam 1.0%. Various NPS were found in 1.4% of the samples. The prevalence of drugs in blood samples from criminal suspects were for cocaine 3.4%, THC 34.7%, amphetamine/methamphetamine 37.0%, diazepam 12.0%, and clonazepam 29.3%. Multi-drug use was more common among criminal suspects (41.3%) than among club patrons (6.8%). The results showed that the drug use pattern among nightclub patrons was substantially different from the drug use pattern manifested by individuals apprehended by the police suspected for criminal conduct., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

There has been a significant increase in the number of new intoxicants on the illegal drugs market globally, also in Norway. The substances are given the name NPS: Novel Psychoactive Substances, and are mainly sold over the Internet. Uncertain dosage of potent substances entails a risk of accidental overdose, and therefore serious intoxication and death. In this article we provide an overview of current knowledge with regard to these substances.

Oral fluid has become an important matrix for drugs of abuse analysis. These days the applicability is challenged by the fact that an increasing number of new psychoactive drugs are coming on the market. Synthetic cannabinoids and synthetic cathinones have been the main drug classes, but the diversity is increasing and other drugs like piperazines, phenethylamines, tryptamines, designer opioids and designer benzodiazepines are becoming more prevalent. Many of the substances are very potent, and low doses ingested will lead to low concentrations in biological media, including oral fluid. This review will highlight the phenomenon of new psychoactive substances and review methods for oral fluid drug testing analysis using on-site tests, immunoassays and chromatographic methods.

In 2010-2013, 29 fatal intoxications related to the designer drug paramethoxymethamphetamine (PMMA, 4-methoxymethamphetamine) occurred in Norway. The current knowledge about metabolism and toxicity of PMMA in humans is limited. Metabolism by the polymorphic cytochrome P450 (CYP) 2D6 enzyme to the psychoactive metabolite 4-hydroxymethamphetamine (OH-MA), and possibly by additional enzymes, is suggested to be involved in its toxicity. The aim of this work was to study the association between CYP genetics, PMMA metabolism and risk of fatal PMMA toxicity in humans. The frequency distribution of clinically relevant gene variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A5, and the phenotypic blood CYP2D6 metabolic ratio (OH-MA/PMMA) in particular, were compared in fatal PMMA intoxications (n=17) and nonfatal PMMA abuse controls (n=30), using non-abusers (n=305) as references for the expected genotype frequencies in the Norwegian population. Our study demonstrated that the CYP2D6 enzyme and genotype are important in the metabolism of PMMA to OH-MA in humans, but that other enzymes are also involved in this biotransformation. In the fatal PMMA intoxications, the blood concentrations of PMMA were higher and the CYP2D6 metabolic ratios were lower, than in the nonfatal PMMA abuse controls (median (range) 2.1 (0.03-5.0) vs 0.3 (0.1-0.9) mg/L, and ratio 0.6 (0.0-4.6) vs 2.1 (0.2-7.4) p=0.021, respectively). Overall, our findings indicated that, in most cases, PMMA death occurred rapidly and at an early stage of PMMA metabolism, following the ingestion of large and toxic PMMA doses. We could not identify any genetic CYP2D6, CYP2C9, CYP2C19 or CYP3A5 predictive marker on fatal toxicity of PMMA in humans. The overrepresentation of the CYP2D6 poor metabolizer (PM) genotype found in the nonfatal PMMA abuse controls warrants further investigations., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Amphetamine and methamphetamine are popular drugs of abuse worldwide and are important components of drug monitoring programs. Windows of detection for amphetamine and methamphetamine in oral fluid after high doses have not been investigated. Repeated high-dose ingestions are likely to cause positive samples for extended periods. Common routes of administration of amphetamine/methamphetamine in Norway are oral intake or injection. The aim of this study was to investigate windows of detection for amphetamine and methamphetamine in oral fluid from drug addicts under sustained abstinence during detoxification., Methods: Twenty-five patients admitted to a closed detoxification unit were included in this study. Oral fluid samples were collected daily in the morning and evening, and urine every morning for 10 days. A blood sample was drawn during the first 5 days after admission if the patient consented. Oral fluid results were compared with urine results to determine whether a new ingestion occurred. Oral fluid was collected with the Intercept oral fluid collection device. In-house cutoff concentrations for amphetamine and methamphetamine were 6.8 and 7.5 mcg/L, respectively, in oral fluid, and 135 and 149 mcg/L, respectively, in urine., Results: Amphetamines were detected in 11 oral fluid, 5 urine, and 2 blood specimens from 25 patients. Patients self-reported amphetamines intake of up to 0.5-2 g daily. Windows of detection for amphetamine and methamphetamine in oral fluid were up to 8 days, longer than in urine at the applied cutoff values., Conclusions: These data confirm that oral fluid is a viable alternative to urine for monitoring amphetamine abuse, and that these substances might be detected in oral fluid for at least 1 week after ingestion of high doses. Such long detection times were, as far as we are aware, never reported previously for oral fluid amphetamines.

Background: Clonazepam, diazepam, and alprazolam are benzodiazepines with sedative, anticonvulsant, and anxiolytic effects, but their prevalence in drug abuse and drug overdoses has long been recognized. When detection times for psychoactive drugs in oral fluid are reported, they are most often based on therapeutic doses administered in clinical studies. Repeated ingestions of high doses, as seen after drug abuse, are however likely to cause positive samples for extended time periods. Findings of drugs of abuse in oral fluid collected from imprisoned persons might lead to negative sanctions, and the knowledge of detection times of these drugs is thus important to ensure correct interpretation. The aim of this study was to investigate the time window of detection for diazepam, clonazepam, and alprazolam in oral fluid from drug addicts admitted to detoxification., Methods: Twenty-five patients with a history of heavy drug abuse admitted to a detoxification ward were included. Oral fluid was collected daily in the morning and the evening and urine samples every morning for 10 days, using the Intercept device. Whole blood samples were collected if the patient accepted. The cutoff levels in oral fluid were 1.3 ng/mL for diazepam, N-desmethyldiazepam, and 7-aminoclonazepam and 1 ng/mL for clonazepam and alprazolam. In urine, the cutoff levels for quantifications were 30 ng/mL for alprazolam, alpha-OH-alprazolam, and 7-aminoclonazepam, 135 ng/mL for N-desmethyldizepam, and 150 ng/mL for 3-OH-diazepam and for all the compounds, the cutoff for the screening analyses were 200 ng/mL., Results: The maximum detection times for diazepam and N-desmethyldiazepam in oral fluid were 7 and 9 days, respectively. For clonazepam and 7-aminoclonazepam, the maximum detection times in oral fluid were 5 and 6 days, respectively. The maximum detection time for alprazolam in oral fluid was 2.5 days. New ingestions were not suspected in any of the cases, because the corresponding concentrations in urine were decreasing. Results from blood samples revealed that high doses of benzodiazepines had been ingested before admission, and explains the longer detection times in oral fluids than reported previously after intake of therapeutic doses of these drugs., Conclusions: This study has shown that oral fluid might be a viable alternative medium to urine when the abuse of benzodiazepines is suspected.

Introduction: An increase in striatal dopamine is considered essential for the rewarding and reinforcing effects of drugs of abuse. We have developed and validated an ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the analysis of dopamine in rat brain extracellular fluid (ECF) sampled with microdialysis. The method was applied to monitor changes in dopamine concentrations over time after an intravenous bolus injection of heroin., Methods: Dopamine and dopamine-d3 were analyzed using a 2.1×100mm Aquity T3 column, 1.7μm particle size, with a formic acid and methanol gradient. The run time of the method was 2.5min including equilibration time., Results: The method had an LOQ of 0.15ng/mL, which equals 0.55pg on column. The calibration curves were linear in the tested area of 0.15 to 16ng/mL. Inter-assay coefficients of variation varied between 5-17%, with an accuracy expressed as bias of -10 to 5%. The intra-assay coefficients of variation varied between 9-15%, with an accuracy of -3-7%., Discussion: Heroin metabolism is very rapid. Sampling intervals of only 2min were thus required to obtain an adequate number of samples of dopamine analysis accompanying the concentration-time profile of opioids in the brain. Applying a flow of 2μL/min, 4μL of dialysate were sampled at 2min intervals, in 7μL internal standard. The injection volume onto the UPLC column was 10μL. Analyses of microdialysate samples from a rat given heroin i.v. showed that it was possible to measure baseline levels and rapid changes in dopamine concentrations with very short sampling periods., (Copyright © 2015. Published by Elsevier Inc.)

A large number of new psychoactive substances are available at the illicit drug market and the synthetic cannabinoids (SCs) are among the substances that have led to serious side effects and death. Knowledge about common concentrations of these drugs are however sparse. Concentrations of APINACA and 5F-APINACA in biological matrixes have previously not been reported, and concentrations of UR-144 and its degradant product in blood samples from driving under the influence of drug (DUID) cases have not been published. The aims of this study were to report concentrations of APINACA, 5F-APINACA, UR-144 and UR-144 degradant from DUID cases analyzed at the Norwegian Institute of Public Health (NIPH), and also previously unpublished concentrations of AM-2201 in cases from our Institute. We have further summarized all the former published studies where concentrations of SCs have been reported, to compare with the results from these newer SCs. In whole blood from one driver we have found 5F-APINACA and from three drivers both APINACA and 5F-APINACA in concentrations from 0.24 to 24.5 and 0.9 to 6.5 μg/L, respectively, and UR-144 in two cases in concentrations of 0.22 and 0.47 μg/L. UR-144 degradant in a concentration of 0.15 μg/L was found in one of the cases. A summary of the literature reveals major deficiencies regarding concentrations of most of the SCs. The SCs most frequently detected in DUID cases were (n≥8) AM-2201, JWH-122, JWH-018 and JWH-210. In intoxication cases AM-2201 (n=517) was the most often detected SC, followed by JWH-122, JWH-210, UR-144, JWH-018, and MAM-2201 (n>100). Four studies regarding concentrations in autopsy cases have been published, and concentrations of four different SCs have been reported (JWH-018, JWH-073, JWH-210, AM-2201 and the metabolites of AM-2201; 4-OH-pentyl, JWH-018 5-OH-pentyl and JWH-018 pentanoic acid). Pharmacokinetic data are only available for JWH-018 (n=3), JWH-073 (n=1) and the metabolites of AM-2201; 4-OH-pentyl, 6-OH-indole, JWH-018 5-OH-pentyl and JWH-018 pentanoic acid (n=1)., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: The use of oral fluid for detecting drugs of abuse has become increasingly more frequent. Few studies have, however, investigated the detection times for drugs of abuse in oral fluid, compared with that of in urine or in blood. Cannabis is the world's most widely used drug of abuse, and the detection times for cannabis, in different types of matrixes, are therefore important information to the laboratories or institutions performing and evaluating drugs of abuse analyses. It is well known that frequent use of high dosages of cannabis, for longer periods of time, might lead to prolonged detection times for THC-COOH in urine. Cannabis intake is detected in oral fluid as THC, and a positive finding is considered to be a result of recent smoking, although some studies have already reported longer detection times. The aim of this study was to investigate the detection time for THC in oral fluid, collected from drug addicts admitted for detoxification. Findings in oral fluid were compared with findings in urine, among 26 patients admitted to a closed detoxification unit., Methods: The study, being the first in doing so, describes the concentration-time profiles for THC in oral fluid among chronic cannabis users, during monitored abstinence, using the Intercept collection kit. The study also includes the concentration-time profiles for creatinine-corrected THC-COOH ratios in urine samples, included to monitor for the possibility of new intakes., Results: THC was detected in oral fluid collected from 11 of the 26 patients in the study. The elimination curves for THC in oral fluid revealed that negative samples could be interspersed among positive samples several days after cessation, whereas the THC-COOH concentrations in urine were decreasing. THC was, in this study, detected in oral fluid for up to 8 days after admission., Conclusions: The study shows that frequent use of high dosages of cannabis may lead to prolonged detection times, and that positive samples can be interspersed among negative samples. These results are of great importance when THC results from oral fluid analyses are to be interpreted.

After injection, heroin is rapidly metabolized to 6-monoacetylmorphine (6-MAM) and further to morphine. As morphine has been shown to increase striatal dopamine, whereas 6-MAM has not been studied in this respect, we gave i.v. injections of 3 μmol 6-MAM, morphine or heroin to rats. Opioids were measured in blood, and dopamine and opioids in microdialysate from brain striatal extracellular fluid (ECF), by UPLC-MS/MS. After 6-MAM injection, 6-MAM ECF concentrations increased rapidly, and reached Cmax of 4.4 μM after 8 min. After heroin injection, 6-MAM increased rapidly in blood and reached Cmax of 6.4 μM in ECF after 8 min, while ECF Cmax for heroin was 1.2 μM after 2 min. T max for morphine in ECF was 29 and 24 min following 6-MAM and heroin administration, respectively, with corresponding Cmax levels of 1 and 2 μM. Dopamine levels peaked after 8 and 14 min following 6-MAM and heroin administration, respectively. The dopamine responses were equal, indicating no dopamine release by heroin per se. Furthermore, 6-MAM, and not morphine, appeared to mediate the early dopamine response, whereas morphine administration, giving rise to morphine ECF concentrations similar to those observed shortly after 6-MAM injection, did not increase ECF dopamine. 6-MAM appeared accordingly to be the substance responsible for the early increase in dopamine observed after heroin injection. As 6-MAM was formed rapidly from heroin in blood, and was the major substance reaching the brain after heroin administration, this also indicates that factors influencing blood 6-MAM concentrations might change the behavioural effects of heroin.

The relationship of drug concentrations between oral fluid and whole blood was evaluated by studying the linear correlation of concentrations and calculating the oral fluid to blood concentration ratios (OF/B) for different substances. Paired oral fluid and whole blood samples were collected from volunteers and persons suspected of drug use in four European countries. Oral fluid samples were collected with the Saliva∙Sampler™ device. All samples were analyzed for drugs of abuse and psychoactive medicines with validated gas and liquid chromatography-mass spectrometric methods. The median OF/B ratios were, for amphetamines 19-22, for opioids 1.8-11, for cocaine and metabolites 1.7-17, for tetrahydrocannabinol (THC) 14, for benzodiazepines 0.035-0.33, and for other psychoactive medicines 0.24-3.7. Most of the these results were close to theoretical values based on the physicochemical properties of the drugs and to values presented earlier, but there was a lot of inter-individual variation in the OF/B ratios. For all substances, except for lorazepam (R(2)  = 0.031) and THC (R(2)  = 0.030), a correlation between the oral fluid and whole blood concentrations was observed. Due to large variation seen here, drug findings in oral fluid should not be used to estimate the corresponding concentrations in whole blood (or vice versa). However, detection of drugs in oral fluid is a sign of recent drug use and oral fluid can be used for qualitative detection of several drugs, e.g. in epidemiological prevalence studies. By optimizing the sampling and the analytical cut-offs, the potential of oral fluid as a confirmation matrix could be enhanced., (Copyright © 2013 John Wiley & Sons, Ltd.)

In drug-facilitated sexual assault (DFSA) cases, drug residues may be detected in beverage remains found in cups or glasses known to have been used by the victims. In this small naturalistic study, the possibility of beverages being contaminated, either by drug concentrated oral fluid or by oral cavity contained tablets, was investigated. Analysis of residues from cups containing soft drinks was performed by immunoassay and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Beverage with both added tablets and spiked oral fluid was investigated, as well as simulation of swallowing tablets. Only the residues from added tablets were positive with immunoassay, while drugs were detectable in all cups using more sensitive UPLC-MS/MS. In conclusion, the possibility of detecting drug residues in beverages due to a contamination, from either drug concentrated oral fluid or oral cavity contained tablets at a time of consumption, should be kept in mind when performing sensitive analysis., (© 2013 American Academy of Forensic Sciences.)

From early year 2000 different herbal products containing synthetic cannabinoids (SC) have appeared on the drug market all over the world, and new substances are frequently introduced. The prevalence of SC use in different populations is however still mainly unknown, also in Norway. This information is difficult to obtain, but studies of drivers suspected of driving under the influence of drugs (DUID), might provide important information. The aim of this study was to assess the prevalence of SC in drivers suspected of being under the influence of drugs in Norway, and investigate if SCs impair driving performance. For two periods of three and four weeks all blood samples from drivers suspected of DUID in Norway were analyzed for the presence of 12 and 18 different SCs, respectively. A new ultra performance liquid chromatography tandem mass spectrometry method was developed. A total of 726 cases were analyzed during our study period, and SCs were detected in 16 cases (2.2%) in total. The mean age of these drivers was 29.6 years. High concentrations of other psychoactive drugs were detected in all the blood samples where a SC was found. AM-2201 and JWH-018 were the most frequently detected SCs, each found in five cases. In addition RSC-4, JWH-122, JWH-081 and JWH-250 were detected. None of the drivers had reported using SCs prior to driving. Despite the limited number of SCs investigated in this 7 week study period, a considerable percent of the cases were positive. Other psychoactive drugs of abuse were always found concomitant with the SCs, and the age of these drivers indicates that experienced drug users also ingest SCs. Since other drugs were found in all the samples, the psychomotor impairment caused by the SCs is difficult to estimate. Our study shows the importance of screening analyses of biological samples from different populations to assess the prevalence of drug use, since self-reporting might be encumbered with significant under-reporting., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background and Purpose: Heroin, with low affinity for μ-opioid receptors, has been considered to act as a prodrug. In order to study the pharmacokinetics of heroin and its active metabolites after i.v. administration, we gave a bolus injection of heroin to rats and measured the concentration of heroin and its metabolites in blood and brain extracellular fluid (ECF)., Experimental Approach: After an i.v. bolus injection of heroin to freely moving Sprague-Dawley rats, the concentrations of heroin and metabolites in blood samples from the vena jugularis and in microdialysis samples from striatal brain ECF were measured by ultraperformance LC-MS/MS., Key Results: Heroin levels decreased very fast, both in blood and brain ECF, and could not be detected after 18 and 10 min respectively. 6-Monoacetylmorphine (6-MAM) increased very rapidly, reaching its maximal concentrations after 2.0 and 4.3 min, respectively, and falling thereafter. Morphine increased very slowly, reaching its maximal levels, which were six times lower than the highest 6-MAM concentrations, after 12.6 and 21.3 min, with a very slow decline during the rest of the experiment and only surpassing 6-MAM levels at least 30 min after injection., Conclusions and Implications: After an i.v. heroin injection, 6-MAM was the predominant opioid present shortly after injection and during the first 30 min, not only in the blood but also in rat brain ECF. 6-MAM might therefore mediate most of the effects observed shortly after heroin intake, and this finding questions the general assumption that morphine is the main and most important metabolite of heroin., (© 2013 The British Pharmacological Society.)

Background: ''Spice'' is the term used for various products that contain synthetic cannabinoids. In recent years a growing number of products have been reported on the illegal market, also in Norway. The substances are sold over the internet as 'legal' cannabis. A number of the substances have gradually been classified as narcotics, also in Norway, but new variants continue to be developed. An overview is provided here of current knowledge of the efficacy and occurrence of synthetic cannabinoids., Method: The article is based on a discretionary selection of relevant articles found by means of a literature search in PubMed and on reports from Norwegian and European authorities and research institutions., Results: Synthetic cannabinoids are a large group of drugs of abuse that have an effect similar to cannabis, but may be considerably more potent. The contents of the various Spice products vary with respect to potency, purity and the number and types of additives, and this implies a risk of unintentional overdose. There are reports from abroad of cardiac infarction in teenagers, severe psychoses, anxiety, unconsciousness and deaths following use., Interpretation: Synthetic cannabinoids are marketed over the internet as legal and harmless cannabis, but can cause severe intoxication and death. There is a considerable need for more knowledge about the action and harmful effects of these substances.