Assessment of total uncertainty in cocaine and benzoylecgonine wastewater load measurements

To check the effectiveness of campaigns preventing drug abuse or indicating local effects of efforts against drug trafficking, it is beneficial to know consumed amounts of substances in a high spatial and temporal resolution. The analysis of drugs of abuse in wastewater (WW) has the potential to provide this information. In this study, the reliability of WW drug consumption estimates is assessed and a novel method presented to calculate the total uncertainty in observed WW cocaine (COC) and benzoylecgonine (BE) loads. Specifically, uncertainties resulting from discharge measurements, chemical analysis and the applied sampling scheme were addressed and three approaches presented. These consist of (i) a generic model-based procedure to investigate the influence of the sampling scheme on the uncertainty of observed or expected drug loads, (ii) a comparative analysis of two analytical methods (high performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry), including an extended cross-validation by influent profiling over several days, and (iii) monitoring COC and BE concentrations in WW of the largest Swiss sewage treatment plants. In addition, the COC and BE loads observed in the sewage treatment plant of the city of Berne were used to back-calculate the COC consumption. The estimated mean daily consumed amount was 107 ± 21 g of pure COC, corresponding to 321 g of street-grade COC.

Population Normalization with Ammonium in Wastewater-Based Epidemiology: Application to Illicit Drug Monitoring

Fluctuations in ammonium (NH4+), measured as NH4-N loads using an ion-selective electrode installed at the inlet of a sewage treatment plant, showed a distinctive pattern which was associated to weekly (i.e., commuters) and seasonal (i.e., holidays) fluctuations of the population. Moreover, population size estimates based on NH4-N loads were lower compared to census data. Diurnal profiles of benzoylecgonine (BE) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) were shown to be strongly correlated to NH4-N. Characteristic patterns, which reflect the prolonged nocturnal activity of people during the weekend, could be observed for BE, cocaine, and a major metabolite of MDMA (i.e., 4-hydroxy-3-methoxymethamphetamine). Additional 24 h composite samples were collected between February and September 2013. Per-capita loads (i.e., grams per day per 1000 inhabitants) were computed using census data and NH4-N measurements. Normalization with NH4-N did not modify the overall pattern, suggesting that the magnitude of fluctuations in the size of the population is negligible compared to those of illicit drug loads. Results show that fluctuations in the size of the population over longer periods of time or during major events can be monitored using NH4-N loads: either using raw NH4-N loads or population size estimates based on NH4-N loads, if information about site-specific NH4-N population equivalents is available.

A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography-mass spectrometry(HILIC-MS).

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.

Driving under the influence of cocaine or therapeutic administration of cocaine?

Introduction: In forensic toxicology, cocaine is better known for its powerful stimulating effects of nervous system and its high potential for recreational abuse, than for his therapeutic use. However, cocaine is still use as a topical anesthetic and peripheral vasoconstrictor in surgeries of eye, ear, nose and throat. Last decade, an increase of the presence of cocaine and metabolites in blood samples of drivers suspected to drive under the influence of drugs (DUID) was observed in Switzerland (Augsburger et al., Forensic Sci Int 153 (2005) 11-15; Senna et al., Forensic Sci Int 198 (2010) 11-16). Observed blood concentration ranges of cocaine and benzoylecgonine were 10-925 μg/L and 20-5200 μg/L, respectively. Since 2005, zero-tolerance approach was introduced in the Swiss legislation for different substances, especially cocaine (analytical cutoff: 15 μg/L). Thus, the interpretation often amounts to determine if the concentration is situated above or under the limit. However, it is important for the interpretation to take into account the context and to be critical with the obtained results, at the risk of ending in erroneous conclusions. Methods: Systematical toxicological analyses were performed on blood and urine, if available, for 5 DUID cases, as already published (Augsburger et al., Forensic Sci Int 153 (2005)). Positive results were confirmed and drugs were quantified in biological samples by GCMS, GC-MS/MS or LC-MS/MS. Results: Administration of cocaine after traffic accident was identified in five cases. All people were admitted to the emergency room because of severe trauma. Maxillofacial surgery was done shortly after admission to the emergency room, involving use of nasal application of cocaine (swab). For all cases, use of cocaine swab was not mentioned in the document filled by the police and by medical staff requested for blood and urine sampling. The information was obtained retrospectively after consultation of the medical records, without precise indication of the application time or dose. Case 1. A 83-year old man (pedestrian) was hit by a car. Blood (+11h after the accident): cocaine (16 μg/L), benzoylecgonine (370 μg/L). Urine: cocaine (1700 μg/L), benzoylecgonine (560 μg/L). Case 2. A 84-year old woman (pedestrian) was hit by a car. Blood (+1.5h after the accident): cocaine (230 μg/L), benzoylecgonine (370 μg/L). Urine was not available. Hair (+4 months after the accident): segment 1 (0-2 cm), cocaine not detected; segment 2 (2-4 cm), cocaine: <0.5 ng/mg. Case 3. A 66-year old man was involved in a car/car accident. He died 2 hours and 5 minutes after the crash. Blood (+1.5h after the accident): cocaine and metabolites not detected. Blood (+2h after the accident): cocaine (1750 μg/L), benzoylecgonine (460 μg/L). Blood (post-mortem): cocaine (370 μg/L), benzoylecgonine (200 μg/L). Urine (+1.5h after the accident): cocaine not detected. Case 4. A 57-year old woman on a motor scooter was hit by a car. She died 2 hours and 10 minutes after the crash. Blood (+0.5h after the accident): cocaine and metabolites not detected. Urine (post-mortem): cocaine (<20 μg/L), benzoylecgonine (120 μg/L). Case 5. A 30-year old man was involved in a car accident. Blood (+4h after the accident): cocaine (29 μg/L), benzoylecgonine (< 20 μg/L). Urine (+4h after the accident): cocaine and metabolites not detected. Ethanol (1,32 g/kg) and cannabinoids (THC (2,0 μg/L), THCCOOH (38 μg/L)) were also detected in blood. Conclusion: To our knowledge, this is the first description of DUID cases involving therapeutic use of cocaine after an accident. These results indicate that even if a per se law is effective for prosecution case of DUID, a critical interpretation of the results is always needed, especially if a medical intervention occurs after an accident.

Etude de la toxicité hépatique de la cocaïne associée à de l'alcool: Effets de l'éthanol sur le métabolisme de la cocaïne dan les hépatocytes de rat en suspension et dans un modèle enzymatique humain

Le but essentiel de notre travail a été d?étudier la capacité du foie, premier organe de métabolisation des xénobiotiques, à dégrader la cocaïne en présence d?éthanol, à l?aide de deux modèles expérimentaux, à savoir un modèle cellulaire (les hépatocytes de rat en suspension) et un modèle acellulaire (modèle reconstitué in vitro à partir d?enzymes purifiées de foie humain). La première partie a pour objectifs de rechercher les voies de métabolisation de la cocaïne qui sont inhibées et / ou stimulées en présence d?éthanol, sur hépatocytes isolés de rat. Dans ce but, une méthode originale permettant de séparer et de quantifier simultanément la cocaïne, le cocaéthylène et huit de leurs métabolites respectifs a été développée par Chromatographie Phase Gazeuse couplée à la Spectrométrie de Masse (CPG / SM). Nos résultats préliminaires indiquent que l?éthanol aux trois concentrations testées (20, 40 et 80 mM) n?a aucun effet sur la cinétique de métabolisation de la cocaïne. Notre étude confirme que l?addition d?éthanol à des cellules hépatiques de rat en suspension supplémentées en cocaïne résulte en la formation précoce de benzoylecgonine et de cocaéthylène. L?apparition retardée d?ecgonine méthyl ester démontre l?activation d?une deuxième voie de détoxification. La production tardive d?ecgonine indique une dégradation de la benzoylecgonine et de l?ecgonine méthyl ester. De plus, la voie d?oxydation intervenant dans l?induction du stress oxydant en produisant de la norcocaïne est tardivement stimulée. Enfin, notre étude montre une métabolisation complète de la concentration initiale en éthanol par les hépatocytes de rat en suspension. La deuxième partie a pour but de déterminer s?il existe d?autres enzymes que les carboxylesterases formes 1 et 2 humaines ayant une capacité à métaboliser la cocaïne seule ou associée à de l?éthanol. Pour ce faire, une méthode de micropurification par chromatographie liquide (Smart System®) a été mise au point. Dans le cadre de nos dosages in situ de la cocaïne, du cocaéthylène, de la benzoylecgonine, de l?acide benzoïque et de la lidocaïne, une technique par Chromatographie Liquide Haute Performance couplée à une Détection par Barrette de Diode (CLHP / DBD) et une méthode de dosage de l?éthanol par Chromatographie Phase Gazeuse couplée à une Détection par Ionisation de Flamme équipée d?un injecteur à espace de tête (espace de tête CPG / DIF) ont été développées. La procédure de purification nous a permis de suspecter la présence d?autres enzymes que les carboxylesterases formes 1 et 2 de foie humain impliquées dans le métabolisme de la cocaïne et déjà isolées. A partir d?un modèle enzymatique reconstitué in vitro, nos résultats préliminaires indiquent que d?autres esterases que les formes 1 et 2 de foie humain sont impliquées dans l?élimination de la cocaïne, produisant benzoylecgonine et ecgonine méthyl ester. De plus, nous avons montré que les sensibilités de ces enzymes à l?éthanol sont variables.<br/><br/>The main purpose of our work was to study the ability of the liver, as the first organ to metabolise xenobiotic substances, to degrade cocaine in the presence of ethanol. In order to do this, we used two experimental models, namely a cellular model (rat liver cells in suspension) and an a-cellular model (model reconstructed in vitro from purified human liver enzymes). The purpose of the first part of our study was to look for cocaine metabolising processes which were inhibited and / or stimulated by the presence of ethanol, in isolated rat liver cells. With this aim in mind, an original method for simultaneously separating and quantifying cocaine, cocaethylene and eight of their respective metabolites was developed by Vapour Phase Chromatography coupled with Mass Spectrometry (VPC / MS). Our preliminary results point out that ethanol at three tested concentrations (20, 40 et 80 mM) have no effect on the kinetic of metabolisation of cocaine. Our study confirms that the addition of alcohol to rat liver cells in suspension, supplemented with cocaine, results in the premature formation of ecgonine benzoyl ester and cocaethylene. The delayed appearance of ecgonine methyl ester shows that a second detoxification process is activated. The delayed production of ecgonine indicates a degradation of the ecgonine benzoyl ester and the ecgonine methyl ester. Moreover, the oxidising process which occurs during the induction of the oxidising stress, producing norcocaine, is stimulated at a late stage. Finally, our study shows the complete metabolisation of the initial alcohol concentration by the rat liver cells in suspension. The second part consisted in determining if enzymes other than human carboxylesterases 1 and 2, able to metabolise cocaine on its own or with alcohol, existed. To do this, a micropurification method us ing liquid phase chromatography (Smart System®) was developed. A technique based on High Performance Liquid Chromatography coupled with a Diode Array Detection (HPLC / DAD) in the in situ proportioning of cocaine, cocaethylene, ecgonine benzoyl ester, benzoic acid and lidocaine, and a method for proportioning alcohol by quantifying the head space using Vapour Phase Chromatography coupled with a Flame Ionisation Detection (head space VPC / FID) were used. The purification procedure pointed to the presence of enzymes other than the human liver carboxylesterases, forms 1 and 2, involved in the metabolism of cocaine and already isolated. The preliminary results drawn from an enzymatic model reconstructed in vitro indicate that human liver carboxylesterases, other than forms 1 and 2, are involved in the elimination of cocaine, producing ecgonine benzoyl ester and ecgonine methyl ester. Moreover, we have shown that the sensitivity of these enzymes to alcohol is variable.

Thoracic aortic dissection associated with cocaine abuse.

Cardiovascular complications of cocaine abuse include myocardial ischemia and infarction, dysrhythmias, cardiomyopathies and aortic dissection. The case in point pertains to a 26-year-old, Caucasian male, substance abuser who suffered a thoracic aortic dissection following the use of crack cocaine. The autopsy and histological findings showed a connective tissue abnormality including a focal microcystic medial necrosis and a fragmentation of the elastic fibers in the arterial walls. Blood concentrations of cocaine and benzoylecgonine, taken individually, were considered to be within a potentially toxic range. Blood concentrations of methadone also indicated use of this drug at the same time. The small amounts of morphine found in the blood and urine were compatible with heroine or morphine use more than 24 h before death.

Simple method for determination of cocaine and main metabolites in urine by CE coupled to MS

in this work, a simple method for the simultaneous determination of cocaine (COC) and five COC metabolites (benzoylecgonine, cocaethylene (CET), anhydroecgonine, anhydroecgonine methyl ester and ecgonine methyl ester) in human urine using CE coupled to MS via electrospray ionization (CE-ESI-MS) was developed and validated. Formic acid at 1 mol/L concentration was used as electrolyte whereas formic acid at 0.05 mol/L concentration in 1:1 methanol:water composed the coaxial sheath liquid at the ESI nozzle. The developed method presented good linearity in the dynamic range from 250 ng/mL to 5000 ng/mL (coefficient of determination greater than 0.98 for all compounds). LODs (signal-to-noise ratio of 3) were 100 ng/mL for COC and CET and 250 ng/mL for the other studied metabolites whereas LOQ`s (signal-to-noise ratio of 10) were 250 ng/mL for COC and CET and 500 ng/mL for all other compounds. Intra-day precision and recovery tests estimated at three different concentration levels (500, 1500 and 5000 ng/mL) provided RSD lower than 10% (except anhydroecgonine, 18% RSD) and recoveries from 83-109% for all analytes. The method was successfully applied to real cases. For the positive urine samples, the presence of COC and its` metabolites was further confirmed by MS/MS experiments.

Determination of the highest no-effect dose (HNED) and of the elimination pattern for cocaine in horses

Cocaine is one of the most widespread illegal stimulants utilized by the human population throughout the world. The aim of this study was to establish the highest no-effect dose (HNED) of cocaine on the spontaneous locomotor activity (SLA) of horses in a behavior chamber, and thereby to determine the maximal acceptable threshold of the urinary drug concentration in horses. Twelve English thoroughbred mares received 0.02, 0.03, 0.04, 0.08 or 0.12 mg kg(-1) cocaine i.v. or saline solution (control). It was noted that doses above 0.04 mg kg(-1) induced a significant increase in SLA (P < 0.05, Tukey's test). No significant increase in SLA was seen in the mares that received 0.03 mg kg(-1), but the animals showed important behavioral changes that did not occur after the 0.02 mg kg(-1) dose. It was concluded that the HNED of cocaine for horses in a behavior chamber is 0.02 mg kg(-1). After injection of this dose in five horses, urine samples were collected at predetermined intervals through vesical catheterization. The concentrations of cocaine, norcocaine, benzoylecgonine and ecgonine methyl ester were quantified by liquid chromatography/electrospray ionization tandem mass spectrometry. Cocaine and norcocaine concentrations remained consistently below the level of detection. Benzoylecgonine reached a mean (+/- SEM) maximum concentration of 531.9 +/- 168.7 ng ml(-1) after 4 h, whereas ecgonine methyl ester peaked 2 h after injection at a concentration of 97.2 +/- 26.5 ng ml(-1). The maximum admissible concentration for cocaine and/or metabolites in the urine of horses is difficult to establish unequivocally because of the substantial individual variation in the drug elimination pattern observed in horses, which can be inferred by the large standard error of the means obtained. Copyright (C) 2002 John Wiley Sons, Ltd.

The effect of sodium fluoride preservative and storage temperature on the stability of cocaine in horse blood, sheep vitreous and deer muscle

The in vitro stability of cocaine in horse blood, sheep vitreous humour (VH) and homogenised deer muscle is described. The stability of cocaine in horse blood was of interest because many toxicology laboratories utilise horse blood for the preparation of calibration and check standards and the latter are typically stored during routine use. The storage stability of cocaine in human VH and muscle has not been previously reported. In the absence of blank human VH and muscle, cocaine stability under varying conditions was demonstrated in animal tissues. Blood and VH were stored with and without addition of NaF at room temperature (RT), 4 degrees C and -18 degrees C for 84 days. Muscle homogenates were prepared in water, water/2% NaF, and phosphate buffer (pH 6.0)/2% NaF, and stored for 31 days at RT, 4 degrees C and -18 degrees C. Cocaine stability in human muscle obtained from cocaine positive forensic cases was assessed following storage at -18 degrees C for 13 months. Cocaine and benzoylecgonine (BZE) were extracted using SPE and quantified by GC-MS/MS. Cocaine was stable for 7 days in refrigerated (4 degrees C) horse blood fortified with 1 and 2% NaF. In the absence of NaF, cocaine was not detectable by day 7 in blood stored at RT and 4 degrees C and had declined by 81% following storage at -18 degrees C. At 4 degrees C the rate of cocaine degradation in blood preserved with 2% NaF was significantly slower than with 1% NaF. The stability of cocaine in horse blood appeared to be less than that reported for human blood, probably attributable to the presence of carboxylesterase in horse plasma. Cocaine stored in VH at -18 degrees C was essentially stable for the study period whereas at 4 degrees C concentrations decreased by >50% in preserved and unpreserved VH stored for longer than 14 days. Fluoride did not significantly affect cocaine stability in VH. The stability of cocaine in muscle tissue homogenates significantly exceeded that in blood and VH at every temperature. In preserved and unpreserved samples stored at 4 degrees C and below, cocaine loss did not exceed 2%. The increased stability of cocaine in muscle was attributed to the low initial pH of post-mortem muscle. In tissue from one human case stored for 13 months at -18 degrees C the muscle cocaine concentration declined by only 15% (range: 5-22%). These findings promote the use of human muscle as a toxicological specimen in which cocaine may be detected for longer compared with blood or VH. (C) 2011 Elsevier Ireland Ltd. All rights reserved.