Search

INTRODUCTION: In pregnancy, vitamin D deficiency is associated with increased risk of fetal growth restriction and preeclampsia. The underlying mechanisms are not known, but placental dysfunction is believed to play a role. In a Danish population, where health authorities recommend a 10 µg/day vitamin D supplement during pregnancy, we explored current use of vitamin D supplements and vitamin D status. In term placentas, alterations in vitamin D metabolism and placental growth, evaluated by the key placental growth factor pregnancy-associated plasma protein-A (PAPP-A), and their relation to vitamin D insufficiency were investigated.MATERIAL AND METHODS: We included 225 randomly selected pregnant women attending a nuchal translucency scan at gestational weeks 11-14. Information on use of vitamin D supplements and body mass index (BMI) at inclusion was obtained using self-reported questionnaires. Plasma 25-hydroxyvitamin D was measured at inclusion and correlated with pregnancy outcomes and placental biology, as judged by expression of PAPP-A and enzymes involved in vitamin D metabolism (CYP24A1, CYP27B1) in term placentas.RESULTS: Vitamin D supplements were used by 92% of the women, but 42% were vitamin D insufficient (plasma 25-hydroxyvitamin D CONCLUSIONS: Despite high compliance with official guidelines regarding vitamin D supplements, vitamin D insufficiency was frequent and the findings indicate that vitamin D insufficiency may affect placental growth. High BMI was associated with vitamin D insufficiency and increased placental vitamin D turnover, but further investigations are needed.

Objective: The spread of new psychoactive substances (NPS) has expanded rapidly in the last decade. The complexity of the pharmacological effects of NPS challenges the traditional treatment guidelines, and information of the emergence of new arrivals is valuable. Our knowledge on the actual range of recreational drugs used and NPS available in Denmark is limited as identification is possible only when consumers become patients in the healthcare system or through drug seizures. We aimed to detect classical recreational drugs and NPS in the urine of music festival attendees and evaluate if the use of NPS could have been predicted by comparing study data with drug seizure data from the previous year published by European and Danish health authorities.Methods: In a cross-sectional study, 44 urine samples were collected from three urinals at Roskilde Festival 2016—the largest Danish music festival. Two urinals were placed at music stages with late-night concerts, and one urinal was placed at a camp site. Samples were prepared using enzymatic hydrolysis followed by cationic and anionic solid phase extraction, and analysed using ultra performance liquid chromatography-high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS). Data were processed using an in-house library of 467 target substances, including legal and illegal drugs and metabolites. Urine drug-screening immunoassays were also evaluated and results were compared to UPLC-HR-TOF-MS results.Results: In total, 77 drugs, including metabolites, were qualitatively identified in the 44 urine samples. The recreational drugs identified were amphetamine (n = 30), cocaine (n = 44), MDA (n = 40), MDMA (n = 44), THC-COOH (n = 19) and ketamine (n = 17). No NPS were identified. Sample testing using the urine drug-screening immunoassays showed presence of cocaine (n = 27), methamphetamine/MDMA (n = 4), THC (n = 7), “Spice” (n = 7) and methylphenidate (n = 1). These discrepancies might be caused by differences in cut-off values between the analytical methods, limited specificity or cross-reactivity of the urine drug-screening immunoassays compared to UPLC-HR-TOFMS results.Conclusion: Widespread uses of classical recreational drugs were identified in pooled urine samples. The prevalence of NPS was not as comprehensive as expected based on the European and Danish health authorities reports on illegal drugs. Urine drug-screening immunoassays results are advised to be confirmed by chromatographic bioanalysis.

Using self-reports and blood samples from 225 unselected Danish first trimester pregnant women, the purpose of this study was to assess their use of stimulants, for example caffeine and nicotine as well as over-the-counter (OTC) and prescription drugs. According to self-reported information, 24% had used prescription drugs and 48% had used OTC drugs mainly acetaminophen (42%), 9.3% were habitual smokers, 44% stated a daily use of caffeinated beverages, and 1.3% used illegal drugs. Ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOFMS) analysis was performed on corresponding blood samples applying golden standards for use of UPLC-HR-TOFMS in forensic medicine. Traces of prescription drugs were detected in 5.3% of the samples and 8.9% contained OTC drugs (acetaminophen 7.1%). Traces of smoking were identified in 8.0%, caffeine in 83% and illegal drugs in 0.9%. These results indicate a substantial use of OTC drugs and caffeine among Danish pregnant women. Blood analysis indicated that many women could be unaware of their caffeine intake. As common substances may be associated with adverse pregnancy outcomes, healthcare professionals should inquire about such habits during pregnancy. The results also underline the need for more research into the molecular effects of such drugs on placental function and foetal development.

The number of new psychoactive substances (NPS) is constantly increasing. However, although the number might be large, most NPS have a low prevalence of use, so keeping screening libraries updated with the relevant analytical targets becomes a challenge. One way to ensure sufficient screening coverage is to use shared high resolution-mass spectrometry (HR-MS) databases, such as HighResNPS.com: a free, online, spreadsheet-format, crowd-sourced HR-MS database for NPS screening. The aims of this study were (i) to present the database to the scientific community and (ii) to verify that the HighResNPS database can be utilized in suspect screening workflows for LC–HR-MS instruments and software from four different instrument vendors. A sample was spiked with 10 NPS, and participating laboratories then analyzed the sample with their respective HR-MS vendor platforms and the HighResNPS database. The HighResNPS data were obtained via a spreadsheet converted to fit the import specifications of the different vendor platforms. Suspect screening was performed using LC–HR-MS vendor platforms from Thermo Fisher, Waters, Bruker and Agilent. All 10 NPS were identified in at least three workflows used for the four different vendor platforms. Multiple users have submitted data to HighResNPS for the same NPS, which resulted in multiple true-positive identifications for these NPS. Suspect screening with LC–HR-MS can be based on diagnostic fragment ions reported by users of different vendor platforms and can support NPS identification in biological samples and/or seizure analyses when no reference standard is available in-house. The present work clearly demonstrates that HighResNPS data is compatible with instruments and screening software from at least four different vendor platforms. The database can thus serve as a useful add-on in LC–HR-MS screening workflows.

A broad targeted screening method based on broadband collision-induced dissociation (bbCID) ultra-performance liquid chromatography high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS) was developed and evaluated for toxicological screening of whole blood samples. The acidic, neutral and basic substances covered by the method were identified in postmortem and antemortem whole blood samples from forensic autopsy cases, clinical forensic cases and driving under the influence of drugs (DUID) cases by a reverse target database search. The screening method covered 467 substances. Validation was performed on spiked whole blood samples and authentic postmortem and antemortem whole blood samples. For most of the basic drugs, the established cut-off limits were very low, ranging from 0.25 ng/g to 50 ng/g. The established cut-off limits for most neutral and acidic drugs, were in the range from 50 ng/g to 500 ng/g. Sample preparation was performed using simple protein precipitation of 300 μL of whole blood with acetonitrile and methanol. Ten microliters of the reconstituted extract were injected and separated within a 13.5 min UPLC gradient reverse-phase run. Positive electrospray ionization (ESI) was used to generate the ions in the m / z range of 50⿿1000. Fragment ions were generated by bbCID. Identification was based on retention time, accurate mass, fragment ion(s) and isotopic pattern. A very sensitive broad toxicological screening method using positive electrospray ionization UPLC-HR-TOF-MS was achieved in one injection. This method covered basic substances, substances traditionally analyzed in negative ESI (e.g., salicylic acid), small highly polar substances such as beta- and gamma-hydroxybutyric acid (BHB and GHB, respectively) and highly non-polar substances such as amiodarone. The new method was shown to combine high sensitivity with a very broad scope that has not previously been reported in toxicological whole blood screening when using only one injection.

Methoxyacetylfentanyl belongs to the group of fentanyl analogues and has been associated with several deaths in recent years. We present three case reports of deceased individuals that tested positive for methoxyacetylfentanyl consumption, as well as in vitro and in vivo metabolite profiles. Methoxyacetylfentanyl was quantified by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) in femoral blood, as well as in urine and brain tissue when these were available. Metabolite profiling was performed by incubating methoxyacetylfentanyl with pooled human hepatocytes (pHH) in Leibovitz's L-15 medium supplemented with fetal bovine serum. Metabolites were identified in vivo and in vitro using UHPLC–high resolution (HR)–MS/MS. The measured methoxyacetylfentanyl concentration was 0.022–0.056 mg/kg (N = 3) in femoral blood, 0.12 mg/kg (N = 1) in urine, and 0.074 mg/kg (N = 1) in brain tissue homogenate. A total of 10 metabolites were identified. The observed metabolic pathways were: hydroxylation(s), N-dealkylation, Odemethylation, deamination, glucuronidation, and combinations thereof. Major analytical targets in vitro and across measured biological samples in vivo were methoxyacetylfentanyl, the O-demethyl-metabolite, and the deamide-metabolite. Intoxication with methoxyacetylfentanyl was judged as the cause of death or a major contributing factor in all three presented cases.

Methoxyacetylfentanyl belongs to the group of fentanyl analogues and has been associated with several deaths in recent years. We present three case reports of deceased individuals that tested positive for methoxyacetylfentanyl consumption, as well as in vitro and in vivo metabolite profiles. Methoxyacetylfentanyl was quantified by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in femoral blood, as well as in urine and brain tissue when these were available. Metabolite profiling was performed by incubating methoxyacetylfentanyl with pooled human hepatocytes (pHH) in Leibovitz's L-15 medium supplemented with fetal bovine serum. Metabolites were identified in vivo and in vitro using UHPLC-high resolution (HR)-MS/MS. The measured methoxyacetylfentanyl concentration was 0.022-0.056mg/kg (N=3) in femoral blood, 0.12mg/kg (N=1) in urine, and 0.074mg/kg (N=1) in brain tissue homogenate. A total of 10 metabolites were identified. The observed metabolic pathways were: hydroxylation(s), N-dealkylation, O-demethylation, deamination, glucuronidation, and combinations thereof. Major analytical targets in vitro and across measured biological samples in vivo were methoxyacetylfentanyl, the O-demethyl- metabolite, and the deamide-metabolite. Intoxication with methoxyacetylfentanyl was judged as the cause of death or a major contributing factor in all three presented cases.

Among the new psychoactive substances (NPS) that have recently emerged on the market, many of the new synthetic opioids have shown to be particularly harmful. A new synthetic analogue of fentanyl, N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide (acrylfentanyl), was identified in powder from a seized capsule found at a forensic psychiatric ward in Denmark. Gas chromatography with mass spectrometry (GC-MS) identified a precursor to synthetic fentanyls, N-phenyl-1-(2-phenylethyl)piperidin-4-amine; however, the precursor 1-(2-phenethyl)piperidin-4-one, was not detected. Analysis of the electron impact mass spectrum of the main, unknown chromatographic peak (GC) tentatively identified an acryloyl analogue of fentanyl. Further analyses by quadrupole time-of-flight high resolution mass spectrometry (QTOF-MS), matrix-assisted laser ionization Orbitrap mass spectrometry (MALDI-Orbitrap-MS), nuclear magnetic resonance spectroscopy (NMR), and infra-red spectroscopy (IR) confirmed the presence of acrylfentanyl (also known as acryloylfentanyl). Quantitative analysis with liquid chromatography and triple quadrupole mass spectrometry (LC-MS/MS) determined the content of acrylfentanyl in the powder, equal to 88.3 mass-% acrylfentanyl hydrochloride. An impurity observed by NMR was identified as triethylamine hydrochloride. Acrylfentanyl is sold on the Internet as a ‘research chemical’. Like other synthetic fentanyls, such as acetylfentanyl, it poses a serious risk of fatal intoxication. Copyright © 2016 The Authors. Drug Testing and Analysis Published by John Wiley & Sons Ltd.

Udgivelsesdato: 2010-Dec-1 Research within the field of metabolite profiling has already illuminated our understanding of a variety of physiological and pathological processes. Microdialysis has added further refinement to previous models and has allowed the testing of new hypotheses. In the present study, a new ultra-performance liquid chromatography/electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS) method for the simultaneous detection and quantification of intermediary energy metabolites in microdialysates was developed. The targeted metabolites were mainly from the citric acid cycle in combination with pyruvic acid, lactic acid, and the ATP (adenosine triphosphate) hydrolysis product adenosine along with metabolites of adenosine. This method was successfully applied to analyze the microdialysates obtained from an experimental animal study giving insight into the hitherto unknown concentration of many interstitial energy metabolites, such as succinic acid and malic acid. With a total cycle time of 3 min, injection to injection, this method permits analysis of a much larger number of samples in comparison with conventional high performance liquid chromatography/tandem mass spectrometry HPLC-MS/MS strategies. With this novel combination where microdialysis and high sensitivity UPLC-MS/MS technique is combined within cardiologic research, new insights into the intermediary energy metabolism during ischemia-reperfusion is now feasible.

ObjectiveThe aim of this study was to investigate the prevalence of xenobiotic substances, such as caffeine, nicotine and illicit drugs (eg, cannabis and cocaine), in blood samples from first-trimester Danish pregnant women unaware of the screening.DesignA cross-sectional study examined 436 anonymised residual blood samples obtained during 2014 as part of the nationwide prenatal first-trimester screening programme. The samples were analysed by ultra performance liquid chromatography with high-resolution time-of-flight mass spectrometry.SettingAn antenatal clinic in a Danish city with 62 000 inhabitants, where >95% of pregnant women joined the screening programme.Primary and secondary outcome measuresThe prevalence and patterns of caffeine, nicotine, medication and illicit drug intake during the first trimester of pregnancy.ResultsThe prevalence of prescription and over-the-counter drug detection was 17.9%, including acetaminophen (8.9%) and antidepressants (3.0%), of which citalopram (0.9%) was the most frequent. The prevalence of illegal drugs, indicators of smoking (nicotine/cotinine) and caffeine was 0.9%, 9.9%, and 76.4%, respectively. Only 17.4% of women had no substance identified in their sample.ConclusionsThis study emphasises the need for further translational studies investigating lifestyle habits during pregnancy, as well as the underlying molecular mechanisms through which xenobiotic substances may affect placental function and fetal development.

The illicit drug 3,4-methylenedioxymethamphetamine (MDMA) has profound physiological cerebral, cardiac, and hepatic effects that are reflected in the blood. Screening of blood for MDMA and other narcotics are routinely performed in forensics analysis using ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOFMS). The aim of this study was to investigate whether such UPLC-HR-TOFMS data collected over a two-year period could be used for untargeted metabolomics to determine MDMA metabolites as well as endogenous changes related to drug response and toxicology. Whole blood samples from living Danish drivers' positive for MDMA in different concentrations were compared to negative control samples using various statistical methods. The untargeted identification of known MDMA metabolites was used to validate the methods. The results further revealed changes of several acylcarnitines, adenosine monophosphate, adenosine, inosine, thiomorpholine 3-carboxylate, tryptophan, S-adenosyl-l-homocysteine (SAH), and lysophospatidylcholine (lysoPC) species in response to MDMA. These endogenous metabolites could be implicated in an increased energy demand and mechanisms related to the serotonergic syndrome as well as drug induced neurotoxicity. The findings showed that it was possible to extract meaningful results from retrospective UPLC-HR-TOFMS screening data for metabolic profiling in relation to drug metabolism, endogenous physiological effects, and toxicology.

This paper reports on a fatal overdose case involving the potent hallucinogenic drug 25C-NBOMe (2-(4-chloro-2,5-dimethoxyphenyl)- N -[(2-methoxyphenyl)methyl]ethanamine). In the present case, a young male was hospitalized after the recreational use of this potent drug. He died at the hospital at approximately 12 h after ingestion, with preceding signs of serotonin toxicity. Medico-legal autopsy was performed on the deceased, during which time peripheral whole blood, urine, vitreous humor, liver and gastric content samples were submitted for toxicological examination. Further, whole blood collected at the hospital at 2–4 h following ingestion of the drug was analyzed. 25C-NBOMe and a demethylated and glucuronidated metabolite of 25C-NBOMe were identified in the urine and blood samples using ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC–HRTOF–MS). Subsequently, 25C-NBOMe was quantified in the peripheral whole blood (0.60 μg/kg), urine (2.93 μg/kg), vitreous humor (0.33 μg/kg), liver (0.82 μg/kg) and gastric content (0.32 μg total) samples collected during autopsy and in the ante-mortem whole blood (0.81 μg/kg) by ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). The autopsy findings were consistent with acute poisoning. Based on the toxicological findings, the cause of death was determined to be a fatal overdose of 25C-NBOMe in combination with amphetamine intake. To our knowledge, the present paper reports the first quantification of 25C-NBOMe in biological specimens from a fatal intoxication case.

Hydroxycinnamic acids are effective antioxidants and are abundant components of plant cell walls, especially in cereal bran. For example, wheat and rye brans are rich sources of the hydroxycinnamates ferulic acid, sinapic acid, and p-coumaric acid. These phenolics are part of human and animal diets and may contribute to the beneficial effects derived from consumption of cereal bran. However, these compounds are ester linked to the main polymers in the plant cell wall and cannot be absorbed in this complex form. The present work shows that esterases with activity toward esters of the major dietary hydroxycinnamates are distributed throughout the intestinal tract of mammals. In rats, the cinnamoyl esterase activity in the small intestine is derived mainly from the mucosa, whereas in the large intestine the esterase activity was found predominantly in the luminal microflora. Mucosa cell-free extracts obtained from human duodenum, jejunum, and ileum efficiently hydrolyzed various hydroxycinnamoyl esters, providing the first evidence of human cinnamoyl esterase(s). This study first demonstrates the release by human colonic esterase(s) (mostly of microbial origin) of sinapic acid and p-coumaric acid from rye and wheat brans. Hydrolysis by intestinal esterase(s) is very likely the major route for release of antioxidant hydroxycinnamic acids in vivo.

Dietary antioxidants that protect low-density lipoprotein (LDL) from oxidation may help to prevent atherosclerosis and coronary heart disease. The antioxidant activities of purified monomeric and dimeric hydroxycinnamates and of phenolic extracts from rye (whole grain, bran, and flour) were investigated using an in vitro copper-catalyzed human LDL oxidation assay. The most abundant ferulic acid dehydrodimer (diFA) found in rye, 8-O-4-diFA, was a slightly better antioxidant than ferulic acid and p-coumaric acid. The antioxidant activity of the 8-5-diFA was comparable to that of ferulic acid, but neither 5-5-diFA nor 8-5-benzofuran-diFA inhibited LDL oxidation when added at 10-40 microM. The antioxidant activity of the monomeric hydroxycinnamates decreased in the following order: caffeic acid > sinapic acid > ferulic acid > p-coumaric acid. The antioxidant activity of rye extracts was significantly correlated with their total content of monomeric and dimeric hydroxycinnamates, and the rye bran extract was the most potent. The data suggest that especially rye bran provides a source of dietary phenolic antioxidants that may have potential health effects.

Four ferulic acid dehydrodimers were isolated from rye bran and purified by preparative HPLC after alkaline hydrolysis. The identity of the compounds were confirmed by UV,1H and13C-NMR and mass spectroscopy. The content of the four identified dimers corresponded to a total dimer concentration of 307 μg/g (dry matter) of the whole grain. The concentrations in the bran fraction were 10–20 times higher than in the starchy endosperm. The four dimers were in decreasing amounts: ((Z)- β -{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-3-methoxy-cinnamic acid (8-O-4′-DiFA);trans -5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (8-5′-DiFA benzofuran form), (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid (5-5′-DiFA) and (E,E)-4,4′-dihydroxy-3,5′-dimethoxy-β,3′-bicinnamic acid (8-5′-DiFA). The ferulic acid dehydrodimers were also synthesised by a simple procedure from ethyl ferulate using peroxidase and hydrogen peroxide.

The efficiency of cobalt(III)-ligated peptides as acceptor nucleophiles in acyl transfer reactions catalyzed by α-chymotrypsin was examined. A series of metallopeptides with the general formula [H-(Gly)n-OCo(NH3)5]2+ (1 ≤ n ≤ 4) was tested. The aminolysis rate of acyl-chymotrypsin was measured spectrophotometrically by monitoring the concentration of unreacted nucleophile. The rate of the competing hydrolysis of acyl-chymotrypsin was obtained by automatic titration with base, using a pH-stat. The main result was that the positively charged metallopeptides, in general, were more efficient nucleophiles than the corresponding amides and free peptides that were examined for comparison. A binding model that rationalizes the findings is given.

An ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) method for simultaneous screening of 46 medicinal drugs and drugs of abuse in whole blood was developed and validated. The method includes most of the commonly used and abused drugs such as amphetamines, cocaine, benzodiazepines, and opioids. Chromatographic separation of the targeted drugs was achieved using a Waters ACQUITY UPLC coupled to a Waters Micromass LCT Premier XE time-of-flight mass spectrometer. The total chromatographic run time was 13.5 min injection to injection. The estimated method LOQ is in the range of 0.06-27 ng/g, which is below the therapeutic levels for each of the drugs analyzed but LSD. The extraction recovery ranged from 6% to 197% with median value 95% and mean value 82%. Matrix effect ranged from 81% suppression to 29% enhancement of the signals compared to signals obtained in the absence of biological matrix. The method was tested on 55 authentic forensic toxicology samples confirming the same positive results as found using the routine analytical procedures as well as some additional compounds. Recently there has been considerable attention paid to drug-facilitated sexual assault and the toxicological findings in these cases. As part of a pilot study to investigate the prevalence of medicinal drugs, drugs of abuse, and alcohol in victims of alleged sexual assault, biological specimens were obtained from 167 victims being examined at the Sexual Assault Center in Aarhus, Denmark. The obtained blood samples were analyzed using the novel screening method supported by additional analyses for e.g. THC and alcohol. 124 victims reported they have been drinking alcohol prior to the assault (74%). Alcohol analyses revealed 59 positive findings (48%). 35 of the cases were found positive for one or more drugs excluding alcohol (21%). 20 of the victims reported they have been subject to a drug-facilitated sexual assault (12%). For the victims suspecting drug-facilitated sexual assault, the toxicological analyses revealed four positive for alcohol and nine victims were positive for one or more drugs, with six of the victims found positive for benzodiazepines or other drugs with sedative effects. It was notable that victims tested positive for medicinal drugs and drugs of abuse as well as victims of alleged drug-facilitated sexual assault in average underwent medical examination later than the whole study population.

This poster reports a fatal overdose case involving Bromo-Dragonfly. Bromo-Dragonfly is a common name for 1-(8-bromobenzo[1,2-b; 4,5-b´]difuran-4-yl)-2-aminopropane. Structurally, it is closely related to phenylethylamines like 2C-B and DOB. Bromo-Dragonfly is considered an extremely potent hallucinogen, only slightly less potent than LSD. In the present case from Denmark an 18-year-old woman was found dead one morning in October 2007. The previous evening she and her boyfriend had both ingested a hallucinogen LSD-like liquid. A medico-legal autopsy was performed on the deceased. Liver, blood, urine and vitreous humour were submitted for toxicological examination. Furthermore a bottle found next to the deceased containing a colorless liquid was subjected to analysis.

A description of the illicit drug market in Denmark's second largest city is provided based upon the prevalence of narcotics and illicitly sold medicals during the years 2002 and 2003. The changes on the illicit drug market are described by comparing the results to a similar study conducted ten years earlier.The study is comprised of 469 cases of seized material by Aarhus Police during the period January 1st 2002-December 31st 2003. Additional information relating to the 341 persons charged is also included in the study.Heroine, cocaine and amphetamine were seized in 31%, 30% and 28% of the cases, respectively, and comprise the most frequently encountered hard drugs on the market. The prevalence of cocaine in Aarhus Police District has increased more than tenfold during the past ten years. The purity of the three drugs decreased significantly during the same period, although large variations in the quality of drugs were observed. Medicals were found in 16% of the seizures (containing 32 different active substances). The most frequent group of medicals was benzodiazepines, which made up a total of 74% of the medicals in the study. Anabolic steroids, ecstasy and methamphetamine were each found in 4% of the seizures. Men with an average age of 29.1 years comprised 92% of the persons charged in the study. Persons with a foreign nationality comprised 15% of the charged, while 25% had a birthplace outside Denmark.The prevalence of stimulants especially cocaine have increased significantly during the past ten years. Meanwhile the purity of the drugs has decreased. The benzodiazepines are still the most frequent group of medicals on the illicit market.

Udgivelsesdato: 2007-Jul The psychostimulant 3,4-methylendioxymethamphetamine (MDMA, "ecstasy") evokes degeneration of telencephalic serotonin innervations in rodents, nonhuman primates, and human recreational drug users. However, there has been no alternative to nonhuman primates for studies of the cognitive and neurochemical consequences of serotonin depletion in a large-bodied animal. Therefore, we used positron emission tomography (PET) with [(11)C]DASB to map the distribution of plasma membrane serotonin transporters in brain of Göttingen minipigs, first in a baseline condition, and again at 2 weeks after treatment with MDMA (i.m.), administered at a range of doses. In parallel PET studies, [(11)C]WAY-100635 was used to map the distribution of serotonin 5HT(1A) receptors. The acute MDMA treatment in awake pigs evoked 1 degrees C of hyperthermia. MDMA at total doses greater than 20 mg/kg administered over 2-4 days reduced the binding potential (pB) of [(11)C]DASB for serotonin transporters in porcine brain. A mean total dose of 42 mg/kg MDMA in four animals evoked a mean 32% decrease in [(11)C]DASB pB in mesencephalon and diencephalon, and a mean 53% decrease in telencephalic structures. However, this depletion of serotonin innervations was not associated with consistent alterations in the binding of [(11)C]WAY-100635 to serotonin 5HT(1A) receptors. Stereological cell counting of serotonin-positive neurons, which numbered 95,000 in the dorsal raphé nucleus of normal animals, was unaffected in MDMA-treated group. group.

The content and composition of dietary fibre (DF), phenolic acids and ferulic acid dehydrodimers as well as the activity of endogenous enzymes were measured in freeze-dried samples of rye wholemeal, dough and bread during the rye bread-making process. Activities of α-amylase, β-xylosidase, α-arabinofuranosidase, and cinnamoyl esterase(s) were found in extracts of wholemeal and in dough after mixing and proofing, whereas activity of endo-xylanase was only found in the wholemeal. As a result of dough mixing, the content of total DF decreased by 21%, which was caused by a decrease in the amount of water-unextractable (WUE) DF. The content of water-extractable (WE) DF was unchanged during the bread-making process, but the relative content of WE DF increased. The content of total ester-bound phenolic acids and ferulic acid dehydrodimers decreased from 1575 µg/g in the wholemeal to 1472 µg/g in rye bread. The most pronounced effect occurred in an imitated sour dough (acidified with lactic and acetic acid instead of by lactic acid bacteria). The observed changes in content and solubility of DF in the dough could be explained by the activity of endogenous enzymes and mechanical disaggregation, whereas the changes observed in the imitated sour dough indicated that some acid hydrolysis might also have taken place.

Dietary antioxidants that protect low-density lipoprotein (LDL) from oxidation may help to prevent atherosclerosis and coronary heart disease. The antioxidant activities of purified monomeric and dimeric hydroxycinnamates and of phenolic extracts from rye (whole grain, bran, and flour) were investigated using an in vitro copper-catalyzed human LDL oxidation assay. The most abundant ferulic acid dehydrodimer (diFA) found in rye, 8-O-4-diFA, was a slightly better antioxidant than ferulic acid and p-coumaric acid. The antioxidant activity of the 8-5-diFA was comparable to that of ferulic acid, but neither 5-5-diFA nor 8-5-benzofuran-diFA inhibited LDL oxidation when added at 10-40 microM. The antioxidant activity of the monomeric hydroxycinnamates decreased in the following order: caffeic acid > sinapic acid > ferulic acid > p-coumaric acid. The antioxidant activity of rye extracts was significantly correlated with their total content of monomeric and dimeric hydroxycinnamates, and the rye bran extract was the most potent. The data suggest that especially rye bran provides a source of dietary phenolic antioxidants that may have potential health effects. Udgivelsesdato: 2001-Aug

Dehydrodimers of hydroxycinnamates play an important role in the cross-linking of plant cell walls. An aqueous solution of quaternary ammonium salts with a long aliphatic chain is known to spontaneously organize itself into micelles with the ionic part at the outer sphere. It is shown that regioisomeric ferulic acid dehydrodimers can be obtained in one step from trans-ferulic acid after attachment to these micelles and using the biomimetic peroxidase-H2O2 system. The surfactant hexadecyltrimethylammonium hydroxide yielded trans-4-(4-hydroxy-3-methoxybenzylidene)-2-(4-hydroxy-3-methoxyphenyl)-5-oxotetrahydrofuran-3-carboxylic acid (25%), (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid (21%), and trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (14%), whereas the surfactant tetradecyltrimethylammonium bromide gave 4-cis, 8-cis-bis(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione (18%) as the main product. The use of micelles appears to be not only a new way to synthesize regioisomeric ferulic acid dehydrodimers but may also help to understand the regiospecificity of dimeric hydroxycinnamate formation in vivo. Udgivelsesdato: 2001-Jul

The contents of pnenolic acids and ferulic acid dehydrodimers were quantified by HPLC analysis after alkaline hydrolysis in kernels of 17 rye (Secale cereale L.) varieties grown in one location in Denmark during 1997 and 1998. Significant variations (P < 0.05) with regard to the concentration of the analyzed components were observed among the different rye varieties and also between different harvest years. However, the content of phenolic acids in the analyzed rye varieties was narrow compared to cereals such as wheat and barley. The concentration of ferulic acid, the most abundant phenolic acid ranged from 900 to 1170 microgram g(-1) dry matter. The content in sinapic acid ranged from 70 to 140 microgram g(-1) dry matter, p-coumaric acid ranged from 40 to 70 microgram g(-1) dry matter, and caffeic, p-hydroxybenzoic, protocatechuic, and vanillic acids were all detected in concentrations less than 20 microgram g(-1) dry matter. The most abundant ferulic acid dehydrodimer 8-O-4 -DiFA was quantified in concentrations from 130 to 200 microgram g(-1) dry matter followed by 8,5 -DiFA benzofuran form (50-100 microgram g(-1) dry matter), 5,5 -DiFA (40-70 microgram g(-1) dry matter), and 8,5 -DiFA (20-40 microgram g(-1) dry matter).

Whole blood samples collected by the Danish police from drivers suspected driving under the influence of drugs are routinely screened by UPLC-HR-TOFMS in forensic analysis to determine the nature of potential drugs. Data collected within a two-year period was used for untargeted metabolomics to evaluate the drug metabolism of 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”). Despite of the untraditional experimental setup, and a very heterogeneous population with different concentrations of MDMA/kg blood weight, as well as unknown information about amount and time of administration in relation to blood sampling, it was possible to extract meaningful information. Various statistical methods were tested and their predictability was validated by the positive identification of MDMA blood metabolites. In addition, endogenous metabolites that may be related to energy metabolism, the serotonergic syndrome, and drug induced neurotoxicity could be identified. The use of retrospective UPLC-HR-TOFMS forensic data is a potential new approach for identifying changes in human drug metabolism and endogenous biomarkers related to licit and illicit drug consumption and toxicology, especially in relation to new designer drugs.

Background Powder in a capsule seized during a smuggling attempt in a forensic psychiatric department in Denmark was analysed and unambiguously identified as the new synthetic opioid acryloylfentanyl. Shortly after, in the same geographical region, a 28-year-old male was found dead. The police and a forensic physician performed a crime scene investigation. The decedent was a known drug user, and he and a friend had been snorting “fentanyl and benzo”. Medico-legal autopsy was performed on the deceased and relevant samples were submitted for toxicological examination. Methods Identification of substances in the seized powder was based on (i) gas chromatography/mass spectrometry (GC-MS); (ii) liquid chromatography / triple quadrupole mass spectrometry (LC-MS/MS); (iii) ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS); (iv) orbitrap mass spectrometry; (v) nuclear magnetic resonance spectroscopy (NMR); and (vi) infra-red spectroscopy (IR). Post-mortem samples from the deceased were screened by UPLC-HR-TOF-MS and quantified using LC-MS/MS. Results A new synthetic analogue, N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide, with the synonyms acryloylfentanyl and acrylfentanyl, was identified. From the finding of a precursor for synthesis, N-phenyl-1-(2-phenetyl)piperdin-4-amine, and the identification of an impurity (triethylamine hydrochloride) of approximately 12%, a method of synthesis was proposed. Most important toxicological post mortem results were: acryloylfentanyl and 3-hydroxyfenazepam, detected in a nasal swap sample; acryloylfentanyl (0.001 mg/kg), buprenorphine (0.002 mg/kg), diazepam (0.15 mg/kg) and 7-aminoclonazepam (0.19 mg/kg), quantified in femoral blood. Based on these findings, the cause of death was suspected to be an overdose of acryloylfentanyl in combination with 3-hydroxyfenazepam, buprenorphine, clonazepam and diazepam use and aspiration of gastric content.Conclusions A new synthetic analogue of fentanyl known as acryloylfentanyl was identified in a seized powder. Soon after, it was also identified in a forensic pathology case, where it was suspected to have caused a fatal overdose in combination with aspiration. This is an example of a rapidly transforming drug market, where new synthetic opioids emerge at a growing rate and cause harm. New synthetic opioids of the fentanyl class – such as acryloylfentanyl and acetylfentanyl - are potent drugs and pose serious challenges for public health due to a high risk of fatal intoxication.

Studies have reported a widespread use of herbal medicine in the general population (6-48%) and among pregnant women (4-69%) with great geographic and socio-economic variations in the extent of utilization and compounds used. The use of herbal medicine in Denmark remains relatively undescribed. Equivalent to conventional drugs, herbal medicine has side effects, interactions and contraindications. Thus, especially pregnant women should be careful as the safety profile remains unclear. Many patients do not report their use of herbal medicine to healthcare practitioners if they are not asked directly.

Hydroxycinnamic acids are effective antioxidants and are abundant components of plant cell walls, especially in cereal bran. For example, wheat and rye brans are rich sources of the hydroxycinnamates ferulic acid, sinapic acid, and p-coumaric acid. These phenolics are part of human and animal diets and may contribute to the beneficial effects derived from consumption of cereal bran. However, these compounds are ester linked to the main polymers in the plant cell wall and cannot be absorbed in this complex form. The present work shows that esterases with activity toward esters of the major dietary hydroxycinnamates are distributed throughout the intestinal tract of mammals. In rats, the cinnamoyl esterase activity in the small intestine is derived mainly from the mucosa, whereas in the large intestine the esterase activity was found predominantly in the luminal microflora. Mucosa cell-free extracts obtained from human duodenum, jejunum, and ileum efficiently hydrolyzed various hydroxycinnamoyl esters, providing the first evidence of human cinnamoyl esterase(s). This study first demonstrates the release by human colonic esterase(s) (mostly of microbial origin) of sinapic acid and p-coumaric acid from rye and wheat brans. Hydrolysis by intestinal esterase(s) is very likely the major route for release of antioxidant hydroxycinnamic acids in vivo. Udgivelsesdato: 2001-Nov

English title: The prevalence of prescription type medicine, over-the-counter medicine and psychoactive substances in Danish pregnant women

Objective: The spread of new psychoactive substances (NPS) has expanded rapidly the last decade with 570 new substances registered the last 10 years [1]. New substances are developed to meet the demand for new variations ineffect but also to bypass legislation. Due to the complexity of the effects of NPS, traditional treatment guidelines are challenged. Thus, information of the emergence of new arrivals is of great value. Our knowledge on the actualrange of drugs used and NPS available in Denmark is limited as identification is possible only when consumers become patients in the healthcare system or through police seizures [2]. We carried out a cross-sectional study ofcollected pooled anonymous urine, sampled from urinals at the biggest music festival during the year, Roskilde Festival, with the aim to detect classic recreational drugs and NPS. The aim of this study was to identifyrecreational drugs currently used and predict the emergence of NPS by comparing study data with seizure data from the previous year published by EMCDDA [1].Methods: In total 44 urine samples were collected from three urinals at Roskilde Festival 2016. Two urinals were placed at music stages with late-night concerts and techno/electronic music, and one urinal was placed at a camp site. Samples were prepared using enzymatic hydrolysis and cationic and anionic solid phase extraction, and analysed using UPLC-HR-TOFMS. Data was processed against an in-house library of 467 target substances, including legal and illegal drugs and metabolites. Results: In total 77 drugs, including metabolites, were qualitatively identified in the 44 urine samples. The recreational drugs identified (n=number of samples) were amphetamine (n=30), cocaine (n=43), MDMA (n=44),THC-COOH (n=19), and ketamine (n=17).Conclusion: Several classical recreational drugs were identified in pooled urine samples. While the widespread use of these drugs at the festival was confirmed, the prevalence of NPS was not as comprehensive as expected based onthe EMCDDA report [1] and the Danish report on illegal drugs [2]. The limited use of NPS and the substantial dilution due to the pooled anonymous nature of the sampling scheme make it difficult to detect single intakes. Thus, the methods used did not allow for predicting the emergence of NPS in Denmark.References: [1] European Monitoring Centre for Drugs and Drug Addiction (2016), European Drug Report 2016: Trends and Developments, Publications Office of the European Union, Luxembourg. [2] The Danish Health Authority. Illegal drugs in Denmark, 2015 [Danish].