Simon Hudson

Trend analysis of anonymised pooled urine from portable street urinals in central London identifies variation in the use of novel psychoactive substances

Abstract Context. There is increasing interest in the analysis of waste water at sewage treatment plants to monitor recreational drug use. This technique is limited for novel psychoactive substances (NPS) due to limited knowledge on their human and bacterial metabolism and stability in waste water. Small studies have reported the detection of NPS using pooled anonymous urine samples, which eliminates some of these potential confounders. Objective. To determine patterns of recreational drug, including NPS, use by confirming their presence in analysis of pooled urine from portable street urinals across a wide geographical area in central London over a 6-month period. Materials and methods. Pooled anonymous urine samples were collected from 12 four-bay stand-alone portable urinals distributed once a month across central London for six consecutive months. Samples were analysed using high-performance liquid chromatography coupled to high-resolution accurate mass spectrometry (LC-HRAM-MS); acquired data were processed against target compound databases. Results. With regards to Classical Recreational Drugs, there was consistency of detection of cathine, cocaine, morphine, MDMA over the 6 months, with variability of detection of amphetamine, ketamine and cannabis. With regards to NPS, a total of 13 NPS were detected during the six months. Mephedrone and methylhexaneamine were detected consistently each month. Other commonly detected NPS included methiopropamine (5 months), pipradrol (4 months), cathinone (4 months), 5-(2-aminopropyl)benzofuran (3 months) and 4-methyethcathinone (3 months). Of note, methoxetamine and the synthetic cannabinoid receptor agonists were not detected in any samples. Discussion. Previous studies using the same method detected three and five NPS in a nightclub and pissoir setting, respectively, on a single night. The longer sampling time of 6 months has allowed detection of 13 NPS. Of note, mephedrone showed the least month-to-month variation in detection over the 6-month sampling period. With regards to classical recreational drugs, those detected were consistent with use-data from UK population surveys. The only exception is amphetamine which these surveys have shown a steady decline in use since 1996 but our study showed some variation in the frequency of its detection. However, the sampling period was too short and a longer study is needed to detect the trend in decreasing use. Conclusion. This study demonstrates that analysis of anonymous pooled urine samples from stand-alone urinals can be used to detect and monitor trends in the use of classical recreational drugs and NPS in a large city centre over time. This technique has the potential to be a novel key indicator alongside other existing indicators to provide a more robust picture of the use of recreational drugs including NPS.

The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance

The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap.Using high resolution accurate mass full-scan analysis on the Orbitrap, the in vitro systems were found to generate at least the two most abundant phase I metabolites observed in vitro for all eight drugs studied. In the majority of cases, in vitro experiments were also able to generate the minor in vivo metabolites and sometimes metabolites that were only observed in vitro. More detailed analyses of fentanyl incubates using LC-MS/MS showed that it was possible to generate good quality spectra from the metabolites generated in vitro. These data support the suggestion of using in vitro incubates as metabolite reference material in place of in vivo post-administration samples in accordance with new qualitative identification guidelines in the 2009 International Laboratory Accreditation Cooperation-G7 (ILAC-G7) document.In summary, the in vitro and in vivo phase I metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment, refine and reduce the existing equine in vivo paradigm.

The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine.

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine. In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.

Characterizing metabolites and potential metabolic pathways for the novel psychoactive substance methoxetamine

The classic approach of controlled volunteer studies to study drug metabolism is difficult or impossible to undertake for novel psychoactive substances (NPS), as there is generally very limited information available to allow appropriate dose finding and safety. A viable and powerful alternative is the identification and characterization of phase I and II metabolites of such drugs by examining the concordance of data gathered from analysis of microsomal incubates with that from analysis of specimens collected from individuals with analytically confirmed use of NPS. Liquid chromatography-high resolution mass spectrometry provides the ability to reliably identify such metabolites. We used this technique here to study the metabolism of the ketamine analogue methoxetamine. A large number of metabolites were identified in the in vitro studies including normethoxetamine, O-desmethylmethoxetamine, dihydromethoxetamine, dehydromethoxetamine and several structural isomers of hydroxymethoxetamine and hydroxynormethoxetamine. pH dependent liquid-liquid extraction was used to discriminate phenolic from alcoholic metabolites. Phase II glucuronide conjugates included those of O-desmethylmethoxetamine, O-desmethylnormethoxetamine and O-desmethylhydroxymethoxetamine. The majority of these phase I and II metabolites were confirmed to be present in urine collected from three individuals presenting with acute methoxetamine toxicity.

A competitive enzyme immunoassay for the quantitative detection of cocaine from banknotes and latent fingermarks

A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162ngmL(-1) achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrate the effectiveness and robustness of the assay. The immunoassay presents a simple and cost-effective alternative to the current mass spectrometry based techniques for the quantitation of cocaine at forensically significant concentrations.

Taking the Pissoir – a novel and reliable way of knowing what drugs are being used in nightclubs

Background: The epidemiology on recreational drug use is based on self-reported user surveys. The scope of this is limited as users are often not aware of exactly what drug(s) they are using. Waste water (sewage plant) analysis has been used to identify “regional” recreational drug use but is limited by a lack of understanding of the metabolism and stability of novel recreational drugs. Aims: The feasibility of collecting pooled urine samples from a sub-population attending a night-club using a portal urinal to confirm the classical and novel recreational drugs being used. Design and Methods: Urine samples were collected from a nightclub over one weekend for analysis by various chromatographic techniques involving mass spectrometry. Results: Classical recreational drugs and novel psychoactive substance, including mephedrone, 3-trifluoromethylphenylpiperazine and 2-aminoindane were found. Parent drug/metabolites were also detected for amphetamine, cocaine, ketamine, MDMA, mephedrone and 3-trifluoromethylphenylpiperazine. Conclusion: Anonymous pooled urine samples from within a nightclub can be used to confirm the actual drugs being used by some individuals within this sub-population. Metabolite detection indicates drugs were being used and not simply discarded into the urinal. This methodology could be used to monitor recreational drug trend in other environments, e.g. schools, geographical regions/areas and compare drug use over time.

Nopaine no gain: recreational ethylphenidate toxicity.

To the Editor: Availability of novel psychoactive substances (NPS), or ‘legal highs’, has increased markedly over recent years, with an average of 1–2 new NPSs reported to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) every week in 2013/2014. 1 Ethylphenidate (ethyl 2-phenyl-2-(piperidin-2-yl)acetate) is a psychostimulant NPS that inhibits reuptake of both dopamine and noradrenaline. 2 It is an analogue of methylphenidate that was first reported to the EMCDDA in 2011 from the UK. 3 It is often sold under the street name ‘Nopaine’. We present a series of three cases of acute ethylphenidate toxicity, including one with analytical confirmation. The first case was a previously well 21-year-old male brought to the Emergency Department (ED) by ambulance with anxiety, paranoia, agitation and visual disturbances. He also described leftsided, retrosternal chest pain lasting for ‘seconds’ associated with palpitations and ‘tingling’ in both hands. He reported using 500 mg of ethylphenidate in incremental doses over several hours on the evening of presentation, predominantly by nasal insufflation but also by smoking, and had also consumed seven cans of beer; he had purchased the ethylphenidate from a UK-based internet supplier. His symptoms developed 3 h after using ethylphenidate, for which he had self-medicated with 3 mg of etizolam, purchased from the same internet supplier. On arrival to the ED 17 h after the first ethylphenidate use, he was restless with tachycardia (heart rate: 114 bpm), hypertension (blood pressure: 184/98 mmHg) and dilated (4 mm) pupils; there was no hypertonia or clonus and he was afebrile (37.1°C). Electrocardiogram demonstrated sinus tachycardia with normal corrected QT interval. Venous blood gas, full blood count and renal profile were normal; creatine kinase was 290 I U/L (normal range: 0–229 IU/L). He was treated with 15 mg of oral diazepam in divided doses and admitted for observation; his symptoms resolved and he was discharged 10 h after presentation to the ED. Paired serum and urine samples were collected with informed consent 20 h after ethylphenidate and etizolam use. Analysis by highresolution liquid chromatography–mass spectrometry (LC–MS) identified ethylphenidate (0.24 microg/mL in blood and 0.98 microg/ mL in urine). Methylphenidate and ritalinic acid were not detected in blood; low concentrations of methylphenidate (0.059 microg/mL) and ritalinic acid (0.098 microg/mL) were detected in urine, consistent with in vivo de-ethylation of ethylphenidate to methylphenidate and its subsequent metabolism to ritalinic acid. Diazepam and etizolam were the only other substances detected, in trace amounts. The second case was a 37-year-old female brought to the ED by ambulance following intravenous injection of 1 g of ethylphenidate. She was feeling drowsy, had palpitations and felt feverish. Assessment in the ED demonstrated tachycardia (heart rate: 143 bpm), hypertension (blood pressure: 186/96 mmHg) and fever

Multiple fatalities in the North of England associated with synthetic fentanyl analogue exposure: Detection and quantitation a case series from early 2017

BACKGROUND Synthetic fentanyl analogues are highly potent opioid drugs which have no pharmaceutical use in humans. We detected the synthetic fentanyl analogues; carfentanil, butyryl fentanyl, fluorobutyrylfentanyl, furanylfentanyl, and alfentanil as well as fentanyl itself in 25 cases in early 2017. There have been no previous reports of synthetic fentanyl deaths in the United Kingdom (UK). METHODS Cases in which the history clearly stated drug use but where a post mortem blood morphine concentration was lower than would be expected to explain the sudden death, were referred for further analysis by high resolution accurate mass (HRAM) mass spectrometry. RESULTS 25 post mortem cases in which synthetic fentanyl analogues were implicated in the cause of death were reported from January to May 2017. No cases were seen in June 2017. The age range was 21-54 years and 22 were male. There was a history of heroin use, or markers of heroin use on toxicology screening in 21/25 cases. Carfentanil and fentanyl were detected in 7 cases. Multiple synthetic fentanyl analogues were present in 13 cases, with the remaining 5 cases having only carfentanil present. Synthetic fentanyl analogues were detected in combination with other drugs in all cases. Significant concentrations of ethanol were detected in only 2 cases. The concentration range of carfentanil in blood was 90-4004pg/mL. Of note, the 3 cases in which ante mortem carfentanil was quantified ranged from 21 to 98pg/mL. In all cases, death was attributed to combined central nervous system depression. CONCLUSIONS This paper highlights a new and rapid emergence of these drugs into the UK illicit drug arena. Synthetic fentanyl analogues represent a significant challenge both analytically and clinically within the groups who misuse drugs. It is worthwhile considering the possibility of the presence of these drugs in cases in which a toxicological cause of death is not apparent analytically but there is a history of drug use and circumstantial evidence exists to support a drug-related death as the most likely cause. It may be that synthetic fentanyl analogues should be screened for routinely to avoid reporting any false negative results, but the cost implications and viability of this have not been fully evaluated.