Organ distribution of diclazepam, pyrazolam and 3-fluorophenmetrazine.

The organ distribution of 3-fluorophenmetrazine (3-FPM), pyrazolam, diclazepam as well as its main metabolites delorazepam, lormetazepam and lorazepam, was investigated. A solid phase extraction (SPE) and a QuEChERS (acronym for quick, easy, cheap, effective, rugged and safe) - approach were used for the extraction of the analytes from human tissues, body fluids and stomach contents. The detection was performed on a liquid chromatography-tandem mass spectrometry system (LCMS/MS). The analytes of interest were detected in all body fluids and tissues. Results showed femoral blood concentrations of 10 µg/L for 3-FPM, 28 µg/L for pyrazolam, 1 µg/L for diclazepam, 100 µg/L for delorazepam, 6 µg/L for lormetazepam, and 22 µg/L for lorazepam. Tissues (muscle, kidney and liver) and bile exhibited higher concentrations of the mentioned analytes than in blood. Additional positive findings in femoral blood were for 2-fluoroamphetamine (2-FA, approx. 89 µg/L), 2-flourometamphetamine (2-FMA, hint), methiopropamine (approx. 2.2 µg/L), amphetamine (approx. 21 µg/L) and caffeine (positive). Delorazepam showed the highest ratio of heart (C) and femoral blood (P) concentration (C/P ratio = 2.5), supported by the concentrations detected in psoas muscle (430 µg/kg) and stomach content (approx. 210 µg/L, absolute 84 µg). The C/P ratio indicates that delorazepam displays susceptibility for post-mortem redistribution (PMR), supported by the findings in muscle tissue. 3-FPM, pyrazolam, diclazepam, lorazepam and lormetazepam did apparently not exhibit any PMR. The cause of death, in conjunction with autopsy findings was concluded as a positional asphyxia promoted by poly-drug intoxication by arising from designer benzodiazepines and the presence of synthetic stimulants.

Validation of an LC-MS/MS method for the quantitative analysis of 1P-LSD and its tentative metabolite LSD in fortified urine and serum samples including stability tests for 1P-LSD under different storage conditions.

A variety of hallucinogens of the lysergamide type has emerged on the drug market in recent years and one such uncontrolled derivative of lysergic acid diethylamide (LSD) is 1-propionyl-LSD (1P-LSD). Due to the high potency of LSD and some of its derivatives (common doses: 50-200 µg), sensitive methods are required for the analysis of biological samples such as serum and urine. The occurrence of an intoxication case required the development of a fully validated, highly sensitive method for the quantification of 1P-LSD and LSD in urine and serum using LC-MS/MS. Given that LSD is unstable in biological samples when exposed to light or elevated temperatures, we also conducted stability tests for 1P-LSD in urine and serum under different storage conditions. The validation results revealed that the analysis method was accurate and precise with good linearity over a wide calibration range (0.015-0.4 ng mL-1). The limit of detection (LOD) and the lower limit of quantification (LLOQ) of 1P-LSD and LSD in serum and urine were 0.005 ng mL-1 and 0.015 ng mL-1, respectively. The stability tests showed no major degradation of 1P-LSD in urine and serum stored at -20 °C, 5 °C or at room temperature for up to five days, regardless of protection from light. However, LSD was detected in all samples stored at room temperature showing a temperature-dependent hydrolysis of 1P-LSD to LSD to some extent (up to 21% in serum). Serum samples were particularly prone to hydrolysis possibly due to enzymatically catalyzed reactions. The addition of sodium fluoride prevented the enzymatic formation of LSD. The method was applied to samples obtained from the intoxication case involving 1P-LSD. The analysis uncovered 0.51 ng mL-1 LSD in urine and 3.4 ng mL-1 LSD in serum, whereas 1P-LSD remained undetected. So far pharmacokinetic data of 1P-LSD is missing, but with respect to the results of our stability tests and the investigated case rapid hydrolysis to LSD in-vivo seems more likely than instabilities of 1P-LSD in urine and serum samples.

Method validation and preliminary pharmacokinetic studies on the new designer stimulant 3-fluorophenmetrazine (3-FPM).

Pharmaceutical research not only provides the basis for the development of new medicinal products but also for the synthesis of new drugs of abuse. 3-Fluorophenmetrazine (3-FPM), a fluorinated derivative of the anorectic phenmetrazine, was first patented in 2011 and appeared on the drug market in 2014. Though invented for potential medical purposes, pharmacokinetic data on this compound, crucial for interpreting forensic as well as clinical cases, are not available. Therefore, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the detection of 3-FPM in serum, urine, and oral fluid was developed, validated for urine and serum, and used to quantify 3-FPM in samples obtained during a controlled self-experiment. The method proved to be linear, selective and sufficiently sensitive. The limits of detection (LODs) were 0.1 ng/mL, 0.2 ng/mL, and 0.05 ng/mL in serum, urine, and oral fluid. Inter-day precision and intra-day precision (RSD) in serum samples were below 6.3% and below 8.5%, respectively. The highest serum concentration (cmax ) of 210 ng/mL was reached 2.5 hours (tmax ) after ingestion. The elimination half-life and the volume of distribution were calculated to be approx. 8.8 hours and 400 L (5.3 L/kg). 3-FPM could be detected in serum and urine up to 82 hours and 116 hours, respectively. It was still detected in the last oral fluid sample taken 55 hours after ingestion. 3-FPM was mainly excreted unchanged. Main metabolic reactions were aryl-hydroxylation and N-hydroxylation. Interestingly, the product of oxidative ring opening (2-amino-1-(3-fluorophenyl)propan-1-ol) showed the largest window of detection in the self-experiment.

Novel psychoactive substances: overdose of 3-fluorophenmetrazine (3-FPM) and etizolam in a 33-year-old man.

Though illegal in the UK, in many countries novel psychoactive substances are quasi-legal synthetic compounds that are widely available online under the guise of research chemicals. These substances are relatively cheap and are often undetectable in standard drug screens. Nearly 200 such compounds are introduced yearly, and little is usually known about their metabolism or physiological effects. Consequently, managing patients in overdose situations on largely unknown substances usually involves supportive care, however anticipating and managing atypical side effects are challenging in the absence of knowledge of these compounds. In this report, we discuss our encounter with a 33-year-old unconscious man presenting with coingestion of a novel stimulant 3-fluorophenmetrazine with a rarely used benzodiazepine etizolam. This patient developed seizure-like activity and delayed widespread T-wave inversions, both of which ultimately resolved without sequelae.

Synthesis, analytical characterization, and monoamine transporter activity of the new psychoactive substance 4-methylphenmetrazine (4-MPM), with differentiation from its ortho- and meta- positional isomers.

The availability of new psychoactive substances (NPS) on the recreational drug market continues to create challenges for scientists in the forensic, clinical and toxicology fields. Phenmetrazine (3-methyl-2-phenylmorpholine) and an array of its analogs form a class of psychostimulants that are well documented in the patent and scientific literature. The present study reports on two phenmetrazine analogs that have been encountered on the NPS market following the introduction of 3-fluorophenmetrazine (3-FPM), namely 4-methylphenmetrazine (4-MPM), and 3-methylphenmetrazine (3-MPM). This study describes the syntheses, analytical characterization, and pharmacological evaluation of the positional isomers of MPM. Analytical characterizations employed various chromatographic, spectroscopic, and mass spectrometric platforms. Pharmacological studies were conducted to assess whether MPM isomers might display stimulant-like effects similar to the parent compound phenmetrazine. The isomers were tested for their ability to inhibit uptake or stimulate release of tritiated substrates at dopamine, norepinephrine and serotonin transporters using in vitro transporter assays in rat brain synaptosomes. The analytical characterization of three vendor samples revealed the presence of 4-MPM in two of the samples and 3-MPM in the third sample, which agreed with the product label. The pharmacological findings suggest that 2-MPM and 3-MPM will exhibit stimulant properties similar to the parent compound phenmetrazine, whereas 4-MPM may display entactogen properties more similar to 3,4-methylenedioxymethamphetamine (MDMA). The combination of test purchases, analytical characterization, targeted organic synthesis, and pharmacological evaluation of NPS and their isomers is an effective approach for the provision of data on these substances as they emerge in the marketplace.

Multiple Drug-Toxicity Involving Novel Psychoactive Substances, 3-Fluorophenmetrazine and U-47700.

3-Fluorophenmetrazine (3-FPM) is a stimulant-like novel psychoactive substance (NPS) and fluorinated analog of phenmetrazine that has recently appeared on the recreational drug market, with limited published information. Likewise, the synthetic opioid U-47700 has gained popularity among recreational drug users and is frequently detected in postmortem casework. We present the case history, autopsy and toxicological findings of a fatality involving the designer drugs 3-FPM and U-47700 for the first time in the literature. A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of 3-FPM in whole blood, with a 0.001-0.100 mg/L analytical range. The method met the requirements for acceptable linearity, bias and precision. 3-FPM was detected along with U-47700 and other drugs including amitriptyline, nortriptyline, methamphetamine, amphetamine, diazepam, nordiazepam, temazepam, and the designer benzodiazepines flubromazolam and delorazepam. 3-FPM was quantified in the decedent's peripheral (femoral) and central (aortic) blood at 2.4 and 2.6 mg/L, respectively. These concentrations are similar to reported concentrations in non-fatal intoxications. U-47700 was present in peripheral blood at a semi-quantitative concentration of 0.36 mg/L, consistent with reported U-47700 postmortem concentrations. The cause of death was considered multiple drug-toxicity (3-FPM, U-47700, amitriptyline, methamphetamine, diazepam, temazepam, flubromazolam and delorazepam) and the manner of death ruled an accident. This case illustrates the dangers of polysubstance use and discusses the potential overlap between recreational and fatal concentrations for some NPS.

Test purchase, synthesis and characterization of 3-fluorophenmetrazine (3-FPM) and differentiation from its ortho- and para-substituted isomers.

The knowledge captured in patent and scientific research literature stimulates new ideas and fosters new drug development efforts. Manufacturers and entrepreneurs dedicated to the sale of 'research chemicals' and/or new psychoactive substances (NPS) also make use of access to information to identify, prepare, and launch a range of new substances. One of the most recent compounds to appear on the NPS market is the phenmetrazine analog 3-fluorophenmetrazine (3-FPM) which represents one of many phenylmorpholines designed to explore treatment options in areas such as obesity and drug dependence. The anorectic drug analogs phenmetrazine and phendimetrazine, used as prescription medicines before they were withdrawn, feature amphetamine-like properties associated with monoamine release. Available data on 3-FPM suggest that the effects might show mechanistic overlaps. This study describes the synthesis and extensive analytical characterization of 3-FPM and its differentiation from synthesized ortho- and para- substituted isomers, 2-FPM and 4-FPM, respectively. This study was triggered by the purchase of five powdered samples advertised as 3-FPM by five different Internet vendors based in the United Kingdom. The analytical data obtained for the vendor samples were consistent with the synthesized 3-FPM standard and differentiation between all three isomers was possible. The presence of positional isomers and the absence of suitable reference material can cause difficulties in the day-to-day operation of forensic work and given the rate at which many of the newly emerging NPS appear on the market, a comprehensive approach is needed when attempting to decipher the identity of NPS arriving onto the drug market. Copyright © 2016 John Wiley & Sons, Ltd.

Adverse events related to the new psychoactive substance 3-fluorophenmetrazine - results from the Swedish STRIDA project.

BACKGROUND: New psychoactive substances (NPS) are often poorly pharmacologically documented and the production is unregulated, implying high risks for toxic side effects. This report from the STRIDA project describes analytically confirmed non-fatal intoxications involving the phenmetrazine analogue 3-fluorophenmetrazine (3-FPM). STUDY DESIGN AND METHODS: Observational case series of patients with suspected acute NPS exposure requiring hospital care. Blood and urine samples were collected from patients presenting in emergency departments (ED) or intensive care units (ICU), after consultation with the Swedish Poisons Information Centre (PIC). Laboratory analysis was performed by multi-component liquid chromatography-mass spectrometry. Clinical data were collected during PIC consultations and retrieved from medical records. RESULTS: From November 2014 to October 2015, eight cases were registered as 3-FPM or "phenmetrazine" intoxications at the PIC after consultation. During the same period, analysis of STRIDA project samples confirmed 3-FPM use in a total of 19 patients (84% men) aged 22-54 (median 30) years. 3-FPM was detected in 15 out of 19 serum (2.7-1416 ng/mL) and in 14 out of 14 urine (1.0-6857 µg/mmol creatinine) samples. All patients were also tested positive for other psychoactive substances, with benzodiazepines being most common (57% of the cases). Ten patients were monitored in the ED for <4 h, while six needed ICU monitoring of which five were graded as severe intoxications (Poisoning Severity Score 3). Prominent clinical signs were tachycardia (47%), depressed consciousness (42%), agitation/anxiety (37%), delirium (37%), dilated pupils (26%), and seizures (16%). All patients survived. CONCLUSION: In 19 patients testing positive for 3-FPM, a high incidence of severe clinical features was demonstrated. However, as all patients had also used other psychoactive substances, it was difficult to identify a unique toxidrome for 3-FPM. The results further showed that many 3-FPM intoxications would have been missed, if relying solely on information from PIC consultations. These results emphasize the importance of performing bioanalytical investigation in cases of suspected NPS intoxication.

3-Fluorophenmetrazine, a fluorinated analogue of phenmetrazine: Studies on in vivo metabolism in rat and human, in vitro metabolism in human CYP isoenzymes and microbial biotransformation in Pseudomonas Putida and wastewater using GC and LC coupled to (HR)-MS techniques.

Wastewater-based epidemiology (WBE) as means to estimate illicit drug and new psychoactive substance (NPS) consumption with spatial and temporal resolution is gaining increasing attention. In order to evaluate a given NPS using WBE, in vivo metabolism and microbial biotransformation of excretion products and unchanged compounds need evaluation. The aims of this study were to identify in vivo phase I and II metabolites of the NPS 3-fluorophenmetrazine (3-FPM) in human and rat urine and study the in vitro contribution of Cytochrome P450 (CYP) isoenzymes in phase I metabolism. Additionally, to study microbial biotransformation products (MBPs) of 3-FPM from incubations in wastewater and in a wastewater isolated Pseudomonas Putida strain. To these aims gas chromatography and liquid chromatography coupled to mass spectrometry were applied. Metabolites and MBPs were isolated from urine and microbial incubations after solid phase extraction and precipitation with or without enzymatic conjungate cleaving. The main transformation pathways were N-oxidation, aryl hydroxylation and subsequent O-methylation, alkyl hydroxylation, oxidation, and degradation of the ethyl-bridge yielding the O/N-bis-dealkylated metabolite, combinations thereof and further glucuronidation or sulfations. The main excretion products in the human urine sample were the unchanged compound and the N-oxide, and the main MBPs were the N-oxide and hydroxylation with subsequent oxidations on the alpha-methyl position. Based on these findings, the proposed strategy for WBE analysis of 3-FPM is quantitative determination of unchanged 3-FPM together with qualitative verification of a number of selected metabolites to verify consumption and rule out discharge.

Synthesis and resolution of the novel appetite suppressant 2-benzylmorpholine, a nonstimulant isomer of phenmetrazine.

The synthesis of 2-benzylmorpholine from allylbenzene together with its resolution into its (+)- and (-)-enantiomers is reported. Oral dosing of the racemate to dogs caused appetite suppression with an ED50 of 3 and 5.5 mg kg-1 at 1 and 2 h, respectively, after access to a meat meal. No stimulant activity was observed in dogs given oral doses of 200 mg kg-1 but the appetite suppressant effect in dogs declined during 20 days of chronic oral dosing at 10 mg kg-1. Appetite suppression activity was shown to reside in the (+)-enantiomer.

The effect of acetyldexphenmetrazine and dexphenmetrazine on the susceptibility to audiogenic seizures in mice.

The antiepileptic effect of dexphenmetrazine (DP) and acetyldexphenmetrazine (ADP) was tested on audiogenic seizures in a 100% susceptible strain of mice. DP had no antiepileptic effect, however, it markedly suppressed the postparoxysmal motor inhibition. ADP had a distinct anticonvulsive effect--it suppressed the convulsive component of the seizure, leaving its running component unaffected. The results are compared with the effect of both drugs on electrographic epileptic phenomena in the turtle brain (Servít and Strejcková 1976).

The effect of dexphenmetrazine and acetyldexphenmetrazine upon epileptic electrographic activities in the brain of the turtle.

The effect of acetyldexphenmetrazine (ADP) and dexphenmetrazine (DP) on normal and epileptic electrographic activities in the cortical and thalamic structures of the turtle brain were studied in curarized and artificially ventilated animals. Both drugs almost exclusively influenced cortical activities. The effect of low doses of ADP and DP was similar--they desynchronized cortical activity and suppressed the activity of a cortical penicillin focus. They also elevated the cortical response to optic stimuli. Higher doses of ADP continued to suppress both normal and epileptic cortical activities. Higher doses of DP had a two phase effect with enhancement of epileptic activity in the first phase. Continuous trains of theta activity appeared after low doses of ADP and very often after both low and high doses of DP. The findings are discussed in terms of comparative physiology of the brain.