Spatio-temporal assessment of illicit drug use at large scale: evidence from 7 years of international wastewater monitoring.

BACKGROUND AND AIMS: Wastewater-based epidemiology is an additional indicator of drug use that is gaining reliability to complement the current established panel of indicators. The aims of this study were to: (i) assess spatial and temporal trends of population-normalized mass loads of benzoylecgonine, amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) in raw wastewater over 7 years (2011-17); (ii) address overall drug use by estimating the average number of combined doses consumed per day in each city; and (iii) compare these with existing prevalence and seizure data. DESIGN: Analysis of daily raw wastewater composite samples collected over 1 week per year from 2011 to 2017. SETTING AND PARTICIPANTS: Catchment areas of 143 wastewater treatment plants in 120 cities in 37 countries. MEASUREMENTS: Parent substances (amphetamine, methamphetamine and MDMA) and the metabolites of cocaine (benzoylecgonine) and of Δ9 -tetrahydrocannabinol (11-nor-9-carboxy-Δ9 -tetrahydrocannabinol) were measured in wastewater using liquid chromatography-tandem mass spectrometry. Daily mass loads (mg/day) were normalized to catchment population (mg/1000 people/day) and converted to the number of combined doses consumed per day. Spatial differences were assessed world-wide, and temporal trends were discerned at European level by comparing 2011-13 drug loads versus 2014-17 loads. FINDINGS: Benzoylecgonine was the stimulant metabolite detected at higher loads in southern and western Europe, and amphetamine, MDMA and methamphetamine in East and North-Central Europe. In other continents, methamphetamine showed the highest levels in the United States and Australia and benzoylecgonine in South America. During the reporting period, benzoylecgonine loads increased in general across Europe, amphetamine and methamphetamine levels fluctuated and MDMA underwent an intermittent upsurge. CONCLUSIONS: The analysis of wastewater to quantify drug loads provides near real-time drug use estimates that globally correspond to prevalence and seizure data.

Severe clinical toxicity caused by 25I-NBOMe confirmed analytically using LC-MS-MS method.

Rhabdomyolysis is a relatively rare, but potentially serious complication of various diseases. Muscular injury and resultant release of electrolytes, myoglobin and other enzymatic proteins e.g. creatine kinase (CK) into circulation may result in multi-organ failure requiring an extensive treatment. Non-traumatic causes of rhabdomyolysis, like poisonings, appear to be much more frequent than traumatic causes. We present the case of a patient who developed exceptionally massive rhabdomyolysis, with CK up to 516 455 U/l, after ingestion of a relatively small dose of a novel psychoactive substance known as "Alice in Wonderland". Laboratory quantification was performed using a validated LC-MS/MS method in a whole blood sample.

Elevated hydroxylactam of hemopyrrole level in urine in perpetrators of extremely violent acts diagnosed with psychosis. / Podwyzszone stezenie hydroksylaktamu hemopyrolu u psychotycznych sprawców czynów szczególnie agresywnych.

OBJECTIVES: Hydroxylactam of hemopyrrole (HPL) is an abnormal side product of porphyrin biosynthetic pathway, which may have a devastating impact on the behavior. The link between an aggressive behavior and an increased HPL level was postulated in the 1960s. Further researches concerning HPL brought contrary results and did not clarify its function and possible role in the pathogenesis of aggression. In our research we hypothesize that a heightened level of HPL may correspond to an extreme aggressive behavior in subjects diagnosed with psychosis. METHODS: We performed an analysis of HPL level in urine samples, collected from 36 male subjects diagnosed with a mental illness who presented an extreme aggressive behavior. The control group included 22 male subjects, matched with age. RESULTS: The variable HPL/creatinine quotient differs significantly between the study group and the control group. CONCLUSIONS: We used successfully proprietary method for marking HPL level in urine, developed for the purposes of the project. The results of our study indicate that in a group of subjects with a history of an extreme aggressive behavior a corrected level of HPL may be elevated, compared to subject without history of extreme aggressive behavior. Further studies are needed to evaluate the reasons of HPL elevation and its clinical implications in this group of patients.

The effects of synthetic cannabinoid UR-144 on the human body-A review of 39 cases.

UR-144 [(1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone] is a synthetic cannabinoid, which has been detected in many 'legal highs', seized from the global drug market since the beginning of 2012. It has gained popularity as a 'legal' alternative to classic cannabis in countries where it was not controlled. Despite the widespread distribution of this substance, the data on its effects on the human body are scarce. Therefore, this paper describes the results of analysis and observed effects in 39 cases in which UR-144 was determined in blood. Symptoms were noted from the blood sampling forms filled out by the representative doctor. The determined concentrations of UR-144 were in the range of trace amounts (LOD-0.15ng/mL; LOQ-0.5ng/mL) up to 17ng/mL. The most common observed effects included slurred speech, dilated pupils, sluggish and abnormal pupillary reaction, cheerful behaviour, poor coordination, and staggering. Less frequently observed were: verbosity, narrow pupils, loss of consciousness, pale or reddened facial skin, blackout, euphoria, agitation, hallucinations, hindered communication, shaking hands, seizures, convulsions, somnolence, delayed movements, redness of the conjunctiva, and tachycardia. The discussed cases show the effects observed after UR-144 use. This study can assist in the recognition of possible effects caused by this substance.

3-Methylmethcathinone--Interpretation of Blood Concentrations Based on Analysis of 95 Cases.

3-Methylmethcathinone (3-MMC) has been one of the most popular new psychoactive substances (NPS) in Poland in recent years. 3-MMC was found in blood in 95 cases sent to the Institute of Forensic Research (IFR) during the two and a half year period, from 2013 to half of 2015. 3-MMC was determined in 13 and 48 cases in 2013 and 2014 year-round casework, respectively, while only in the first half of 2015 year it was present in 34 cases. In most cases, 3-MMC was detected together with other novel psychoactive substances and conventional drugs. Blood analyses for 3-MMC were carried out using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The concentrations of 3-MMC in all 95 cases were in the range from traces (<1 ng/mL) up to 1.6 µg/mL (mean concentration 51.3 ng/mL, median 18.5 ng/mL). Concentration ranges in particular types of cases were respectively: DUID cases: 1-171 ng/mL; traffic accidents: <1-29 ng/mL; drug possession: 2-408 ng/mL; intoxication: <1-1600 ng/mL and other: <1-61 ng/mL. The parameters of the developed method such as the LOD (0.02 ng/mL) and LOQ (1 ng/mL) demonstrate that the method is well suited for the analysis of blood samples for 3-MMC and covers the range of typical blood concentrations.

The prevalence of new psychoactive substances in biological material - a three-year review of casework in Poland.

New psychoactive substances (NPS) pose a challenge for forensic and clinical toxicologists, as well as for legislators. We present our findings from cases where NPS have been detected in biological material. During the three-year period 2012-2014 we found NPS in 112 cases (out of 1058 analyzed), with 75 cases in 2014 alone. The prevalence of all NPS (15.1-17.6%) was similar to amphetamine alone that was detected in 15.1-16.5% of cases. The new drugs found belonged to the following classes: cathinones (88%), synthetic cannabinoids (5%), phenethylamines (3%), piperazines and piperidines (3%), arylalkylamines (1%) and other (1%). The drugs detected were (in the order of decreased frequency): 3-MMC (50), α-pyrrolidinopentiophenone (α-PVP) (23), pentedrone (16), 3',4'-methylenedioxy-α-pyrrolidinobutyrophenone (MDPBP) (12), synthetic cannabinoid UR-144 (7), ethcathinone (5), mephedrone (5), methylenedioxypyrovalerone (MDPV) (4), 4-methylethcathinone (4-MEC) (3), buphedrone (3), desoxypipradrol (2-DPMP) (3), methylone (2) and 2C-B (2). In single cases, 2-methylmethcathinone (2-MMC), 2C-P, eutylone, 25I-NBOMe, meta-chlorophenylpiperazine (mCPP), ephedrone, methiopropamine (MPA), and 5-(2-aminopropyl)benzofuran (5-APB) were found. One NPS was the sole agent in 35% of all cases, and two or more NPS were present in 19% of cases. NPS (one or more) with other conventional drugs (like amphetamines, cannabinoids, cocaine, and benzodiazepines) were detected in most (65%) of the cases. NPS were very often detected in the blood of drivers which was a challenge for toxicologists due to a lack of data on their influence on psychomotor performance. A review of concentrations showed a wide range of values in different types of cases, especially driving under the influence of drugs (DUID) and intoxication.

The Influence of Synthetic Cannabinoid UR-144 on Human Psychomotor Performance--A Case Report Demonstrating Road Traffic Risks.

OBJECTIVE: UR-144 [(1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)-methanone] is a synthetic cannabinoid, which has been detected in many "legal highs" seized from the global drug market since the beginning of 2012. It gained popularity as a "legal" alternative to classic cannabis in countries where it was not controlled. The popularity of UR-144 means that this substance is also abused by individuals driving motor vehicles. This article describes a case of driving under the influence (DUI) of UR-144. The aim of the undertaken case analysis and presenting description of pharmacological similarity of THC and UR-144 is to answer the question whether UR-144 can produce effects incompatible with safe driving. METHODS: Blood from the driver was obtained by a physician approximately 2 h after the collision and 4.5 h after self-reported dosing. Police from the crash site provided behavioral observations, and the physician performed medical examination. Blood was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The developed method was described in detail. The method was linear in the range of 0.5-50 ng/mL; the precision and accuracy values obtained were less than 15%. The symptoms observed by police and physician who collected the blood sample were described. RESULTS: In the blood sample collected from the driver, UR-144 and its major pyrolysis product [1-(1-pentyl-1H-indol-3-yl)-3-methyl-2-(propan-2-yl)but-3-en-1-one] were detected. Whole-blood concentration of UR-144 was 14.6 ng/mL. The result of blood analysis and observed symptoms clearly indicated that the driver was under the influence of UR-144. CONCLUSIONS: UR-144 produces effects and impairment similar to or even more dangerous than delta-9-tetrahydrocannabinol (Δ(9)-THC), making it unsafe for driving. Therefore, UR-144 should be treated as a potentially dangerous substance in traffic safety.

Acute intoxication due to tert-amyl alcohol--a case report.

We presented a case of 28 year-old male, who was found in a deep coma complicated with acute respiratory failure because of recreational intoxication with tert-amyl alcohol (TAA). The TAA blood level at the admission was 83 µg/mL determined by gas chromatography-mass spectrometry (GC-MS). In the last few months popularity of TAA among alcohol and drug addicted people in Europe is still growing. The main reasons of these are: self-healing of addiction, low price of this xenobiotic compare to alcohol, and problem to detect this xenobiotic in generally used screening tests.

[Resurgence in paramethoxamphetamine use. Review of poisonings and two recent cases study]. / Powrót do uzywania parametoksyamfetaminy. Przeglad zatruc i studium dwóch nowych przypadków.

Two recent cases of death due to p-methoxyamphetamine (PAM) intake, a methoxylated phenethylamine derivative, were described and compared with previous PMA related deaths that occurred in many countries. Following a review of the available literature from 1974 to 2011 three periods of resurgence of an unsuspected increase of lethal PMA causation were considered. Signs of intoxications, concentrations of PMA found in biological materials were discussed. Based on the case reports can be concluded the great number of victims were unconscious of taking PMA as substitute of MDMA. Two new methods for screening, identification and quantification of amphetamine derivatives in biosamples (blood and urine) using LC-MS/MS techniques were developed. The methods have proven to be appropriate for clinical and forensic toxicology purposes.

[Toxicological analysis of biological material originating from the body of general Wladyslaw Sikorski for organic poisons]. / Badania toksykologiczne materialu biologicznego pochodzacego ze zwlok generala Wladyslawa Sikorskiego na obecnosc trucizn organicznych.

Toxicological analyses performed in individuals who died in unclear circumstances constitute a key element of research aiming at providing a complete explanation of cause of death. The entire panel of examinations of the corpse of general Sikorski also included toxicological analyses for drugs and organic poisons of synthetic and natural origin. Attention was focused on fast-acting and potent poisons known and used in the forties of the century. The internal organs (stomach, liver, lung, brain) and hair, as well as other materials collected from the body and found in the coffin were analyzed. The classic method of sample preparation, i.e. homogenization, deproteinization, headspace and liquid-liquid extraction were applied. Hyphenated methods, mainly chromatographic with mass spectrometry were used for identification of the analytes. Organic poisons were not identified in the material as a result of the research.

Stress-opioid interactions: a comparison of morphine and methadone.

The utility of methadone and morphine for analgesia and of methadone for substitution therapy for heroin addiction is a consequence of these drugs acting as opioid receptor agonists.We compared the cataleptogenic and antinociceptive effects of single subcutaneous doses of methadone hydrochloride (1-4 mg/kg) and morphine sulfate (2.5-10 mg/kg) using catalepsy and hot-plate tests, and examined the effects of the highest doses of the drugs on Fos protein expression in selected brain regions in male Sprague-Dawley rats. Methadone had greater cataleptogenic and analgesic potency than morphine. Fos immunohistochemistry revealed substantial effects on the Fos response of both the stress induced by the experimental procedures and of the drug exposure itself. There were three response patterns identified: 1) drug exposure, but not stress, significantly elevated Fos-positive cell counts in the caudate-putamen; 2) stress alone and stress combined with drug exposure similarly elevated Fos-positive cell counts in the nucleus accumbens and cingulate cortex; and 3) methadone and morphine (to a lesser extent) counteracted the stimulatory effect of nonpharmacological stressors on Fos protein expression in the somatosensory cortex barrel field, and Fos-positive cell counts in this region correlated negatively with both the duration of catalepsy and the latency time in the hot-plate test. The overlap between brain regions reacting to nonpharmacological stressors and those responding to exogenous opioids suggests that stress contributes to opioid-induced neuronal activation.

Developing of LC/MS method with microwave assisted extraction for determination of methadone and it major metabolite (EDDP) in the blood of patients of methadone maintenance treatment.

Opium derivatives as the addictive substances cause many medical and social problems. In treatment of opiates addiction except psychotherapy a long time pharmacotherapy with methadone or buprenorphine are apply. In a recent study both moderate- and high-dose methadone treatment resulted in reductions in illicit opioid use. The aim of the study was develop fast high sensitive liquid chromatography coupled with mass spectrometry for determination of methadone and EDDP after microwave assisted extraction. Developed method for methadone and EDDP determination were linear over the range 5 to 1000 ng/ml for methadone and its metabolite. The method exhibit a good intra and inter day precision, the microwave assisted extraction was also satisfactory with recovery superior to 80%. The mean concentration of methadone in plasma of patients receiving 40 mg of drug per day was 194 ng/ml but in second group (receiving 90 mg of methadone/day) 263 ng/ml. The ratio of the doses was 2.25 but for methadone concentration only 1.36. The much higher ratio (3.25) of concentrations was noticed for EDDP. This observation can support opinion other authoress about polymorphism of methadone pharmacokinetics and pharmacodynamics.

[Determination of fentanyl, atropine and scopolamine in biological material using LC-MS/APCI methods]. / Oznaczanie fentanylu, atropiny i skopolaminy w materiale biologicznym metodami LC-MS/APCI.

In this paper methods for determination of fentanyl (FL) and its three analogues: alfentanyl (AL), sufentanyl (SL) and remifentanyl (RL), atropine (AT) and scopolamine (SK) in biological material (whole blood and urine) using liquid chromatography atmospheric pressure chemical ionisation mass spectrometry technique (LC-MS/ APCI) are presented. Separation of analytes was performed in gradient conditions, using a LiChroCART LiChrospher 60 RP-select B column. The mobile phase consisted of 0.1% (v/v) formic acid in water and in acetonitrile. Target analytes were isolated from biological matrices using liquid-liquid extraction technique with n-butyl chloride or diethyl ether as extraction solvents. The validation data of the methods were: limit of detection (LOD, S/N = 3) and limit of quantification (LOQ, S/N = 10) - 0.05 and 0.25 ng/ml for FL, and 0.7 and 0.9 ng/ml for AT, both in blood, whereas 1.9 and 2.1 ng/ ml for FL, and 0.6 and 0.9 ng/ml for AT in urine. Calibration curves showed linearity in concentration ranges from LOQ to 25 ng/ml in blood and from LOQ to 50 ng/ml in urine. Determination coefficients (R2) of linear regression equation were higher then 0.98. Extraction recovery, intra-day precision (CV(w.g.)) and inter-day precision (CV(m.g.)) were determined at analytes and internal standard (I.S.) concentration of 5 ng/ml for blood, and at analytes and I.S. concentrations of 20 and 5 ng/ml, respectively for urine. Extraction recovery ranged from 76 to 100% for blood and 53--72% for urine. CV (n=5) and CV(m.g.) (n=15) equal from 4.8 to 7.5% and from 6.8 to 16.2% respectively for blood, and from 4.3 to 5.4% and from 5.8 to 9.5% for urine. The application of elaborated methods of the determination of FL, AT and SK in blood and urine for 8 expert opinions elaborated at the Institute of Forensic Research in Krakow is described. FL was detected and quantified in 3 cases, whereas AT and SK in 7.

[Cotinine in urine of mother and their newborn and in cord serum and placenta as a biomarker of foetal exposure to tobacco smoke]. / Kotynina w moczu matki i noworodka oraz w surowicy krwi pepowinowej i lozysku jako marker narazenia plodu na dym tytoniowy.

In Poland, tobacco smoking by women in a procreational age as well as the pregnant women is a common phenomenon. The aim of the conducted research was to assess the usefulness of cotinine markers in different biological materials--mother and newborn's urine, cord blood se. rum, placenta--as a biomarker of tobacco smoking by delivering women, and dependence between these biomarkers and the newborn's health state. 218 pregnant women (117 smokers and 101 non-smokers), who were checked in at the Perinatology and Gynecology Clinic of the University of Medical Sciences in Poznan, took part in the first stage of the research (period between the twelfth and sixteenth week of pregnancy) carried out between years 2004-2006. In the second stage, 201 pairs of women (89 smokers and 112 non-smokers) and their newborns were checked after the women came to hospital to deliver. The research that was conducted showed that both cotinine in the urine of delivering women and in the urine of newborns as well as in the cord blood serum may be used as a biomarker of exposure of a foetus to tobacco smoke. For practical reasons, it must be assumed that the delivering women urine should be the material from choice. The research did not indicate the usefulness of the determination of cotinine in the placenta, in order to assess the exposure of the foetus to the components of tobacco smoke. On the other hand, again it confirmed the influence of tobacco smoking on the newborn's birth parameters, a correlation between the birth weight, body length and cotinine concentration in the urine of a mother makes it possible to predict the lowering of the antropometric parameters of the newborn as a result of tobacco smoking by pregnant women.

[Determination of 4-(methylnirosamino) -4-(3-pyridyl)-1-butanol (NNA) in urine of pregnant women by means of LC/MS/MS]. / Oznaczanie 4-(metylonirozoamino) -4-(3-pirydylo)-1-butanolu (NNAL) w moczu kobiet ciezarnych metoda LC/MS/MS.

An important problem, particularly from the point of view of the medicine, is active tobacco smoking and passive exposure to tobacco smoke of pregnant women and unfavourable health aftermath for the foetus and a newborns resulting from this. The aim of this study was to evaluate the exposure of smoking mothers to tobacco-specific N-nitrosamines, through the measurement of 4-(metylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine. The evaluation of tobacco smoking was performed on the basis of questionnaire studies and determination of cotinine in urine. The questionnaire studies that have been carried out, allowed the patients to be divided into two groups: women who do not smoke and are not exposed to ETS (17 women), and the ones who smoke (14 women). The women who smoked 11-15 cigarettes a day and more than 16 cigarettes constituted the largest percentage. The conformance of the declaration with the real exposure to tobacco smoke within the group of non-smoking women was 88%. Within the group of non-smoking women, the level of cotinine in 15 cases was 0 ng/ mg of creatinine, whereas in two cases, it amounted to 19.4 and 215.4 ng/mg of creatinine respectively. Among the persons, who were smoking, there were 4 cases, where the biomarker's level was 0 ng/mg of creatinine, and in 10 cases, the mean concentration of cotinine was 169.1 +/- 121.8 ng/mg of creatinine. The measurement of the content of tobacco-specific N-nitrosamines (NNAL- metabolite of NNK) in the urine of pregnant woman was carried out by means of the liquid chromatography/tandem mass spectrometry (LC/MS/MS). The analysis of obtained results showed that the mean concentration of NNAL in urine of smoking women was 59.2 +/- 40.2 pg/ ml. In the case of two patients, who declared to be non-smokers, and in the case of whom, the presence of cotinine was detected (19.4 and 215.4 ng/mg of and 88.4 pg/ml. The presence of 4-(metylnitrosamino)-1-(3-pyridyl)-1-butanol in the mother's urine, speaks volumes for the possible risk of exposure of a foetus to the cancerogenic components of tobacco smoke. The applied LC/MS/ MS method of determination of NNAL in urine, which allows the quantity of several pg/ml of the smoker's urine to be determined, can be used to mark the biomarker of exposure to the tobacco-specific N-nitrosamines. The lack of correlation between cotinine and NNAL concentration in urine, and the number of smoked cigarettes, speaks volumes for the lapse of quite a long time from the moment of cigarette smoking to the moment of collection of the biological material for research and the possible incredible declaration as to the number of smoked cigarettes. Only a slight tendency for the increase in the concentration of NNAL as well as the increase in the cotinine level could be caused by the difference in pharmacokinetic parameters of marked metabolites at the moment of smoking cessation.

Instability of pancuronium in postmortem blood and liver taken after a fatal intramuscular Pavulon injection.

The present study was designed to determine the stability of pancuronium in postmortem blood and liver during storage. Results were obtained using the method by Kerskes et al. [C.H.M. Kerskes, K.J. Lusthof, P.G.M. Zweipfenning, J.P. Franke, The detection and identification of quaternary nitrogen muscle relaxants in biological fluids and tissues by ion-trap LC-ESI-MS, J. Anal. Toxicol. 26 (2002) 29-34.], modified and validated in our laboratory. Target analytes were isolated after enzymatic hydrolysis followed by solid phase extraction (BondElut C18 column). Internal standardisation was carried out using laudanosine and the target ions were monitored by LC-ESI-MS (monitoring ions m/z 358 for IS and 286 for pancuronium). Materials were taken from a 46-year-old woman, who had been found dead. A syringe (2 ml) and an empty ampoule of Pavulon (4 mg/2 mL) were found in her hand. The residual volume of fluid in the syringe was 0.7 ml. An autopsy was performed six days after death. It revealed a needle mark on the left thigh. Postmortem materials (muscle from the injection site, blood and liver) and the syringe with fluid were stored for four months in a freezer at -20 degrees C. The initial pancuronium concentrations were 81 ng/mL in blood and 532 ng/g in liver. The analyte was stable when stored at -20 degrees C in blood even up to seven months. In liver samples its concentrations were variable. Pancuronium in blood stored at 20 degrees C underwent degradation very rapidly. After three months of storage these blood samples had concentrations not greater about 10% of the initial value. The degradation patterns of pancuronium depended on temperature and the biological matrix.