Conductive vial electromembrane extraction of opioids from oral fluid.

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.

A Monoclonal Antibody against 6-Acetylmorphine Protects Female Mice Offspring from Adverse Behavioral Effects Induced by Prenatal Heroin Exposure.

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.

Identification and Assessment of Drug-User Groups Among Nightlife Attendees: Self-Reports, Breathalyzer-Tests and Oral Fluid Drug Tests.

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.

Rapid determination of designer benzodiazepines, benzodiazepines, and Z-hypnotics in whole blood using parallel artificial liquid membrane extraction and UHPLC-MS/MS.

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 (R2 > 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.

Studies on Para-Methoxymethamphetamine (PMMA) Metabolite Pattern and Influence of CYP2D6 Genetics in Human Liver Microsomes and Authentic Samples from Fatal PMMA Intoxications.

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.

Pharmacological Effects of a Monoclonal Antibody against 6-Monoacetylmorphine upon Heroin-Induced Locomotor Activity and Pharmacokinetics in Mice.

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.

Electromembrane extraction of drugs of abuse and prescription drugs from micropulverized hair.

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.

Enantiomer-specific analysis of amphetamine in urine, oral fluid and blood.

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.

Solvent-free parallel artificial liquid membrane extraction for drugs of abuse in plasma samples using LC-MS/MS.

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.

Drugs in blood and urine samples from victims of suspected exposure to drink spiking: A prospective observational study from Oslo, Norway.

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.

Determination of amphetamine enantiomers in urine by conductive vial electromembrane extraction and ultra-high performance supercritical fluid chromatography tandem mass spectrometry.

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.

High-throughput quantification of emerging "nitazene" benzimidazole opioid analogs by microextraction and UHPLC-MS-MS.

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.

Comparative Study of Postmortem Concentrations of Benzodiazepines and Z-Hypnotics in Several Different Matrices.

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.

[Effect and occurrence of synthetic cannabinoids]. / Syntetiske cannabinoider--effekt og forekomst.

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.

Membrane-based liquid-phase microextraction of basic pharmaceuticals - A study on the optimal extraction window.

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.

Determination of psychoactive drugs in serum using conductive vial electromembrane extraction combined with UHPLC-MS/MS.

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.

Distribution of tetrahydrocannabinol and cannabidiol in several different postmortem matrices.

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.

Electromembrane extraction of anthracyclines from plasma: Comparison with conventional extraction techniques.

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.

Bioanalysis of pharmaceuticals using liquid-phase microextraction combined with liquid chromatography-mass spectrometry.

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.

Electromembrane extraction of highly polar bases from biological samples - Deeper insight into bis(2-ethylhexyl) phosphate as ionic carrier.

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.