Drug Recognition Evaluation and Chemical Confirmation of a 25C-NBOMe-Impaired Driver.

This case report details an individual arrested for drug-impaired driving after leaving the scene of multiple motor vehicle collisions and evading police. The driver was examined by a drug recognition expert and failed the drug recognition evaluation. The driver admitted to using cocaine, marijuana, an antidepressant medication and "N-bomb," a novel psychoactive substance that possesses hallucinogenic properties. Toxicological analyses at the Centre of Forensic Sciences' Toronto laboratory revealed only the substance 2-[4-chloro-2,5-dimethoxyphenyl]-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in the accused's urine. This is the first report in which 25C-NBOMe was identified through DRE and toxicological analyses in a drug-impaired driver.

25C-NBOMe and 25I-NBOMe metabolite studies in human hepatocytes, in vivo mouse and human urine with high-resolution mass spectrometry.

25C-NBOMe and 25I-NBOMe are potent hallucinogenic drugs that recently emerged as new psychoactive substances. To date, a few metabolism studies were conducted for 25I-NBOMe, whereas 25C-NBOMe metabolism data are scarce. Therefore, we investigated the metabolic profile of these compounds in human hepatocytes, an in vivo mouse model and authentic human urine samples from forensic cases. Cryopreserved human hepatocytes were incubated for 3 h with 10 µM 25C-NBOMe and 25I-NBOMe; samples were analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) on an Accucore C18 column with a Thermo QExactive; data analysis was performed with Compound Discoverer software (Thermo Scientific). Mice were administered 1.0 mg drug/kg body weight intraperitoneally, urine was collected for 24 h and analyzed (with or without hydrolysis) by LC-HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF; data were analyzed manually and with WebMetabase software (Molecular Discovery). Human urine samples were analyzed similarly. In vitro and in vivo results matched well. 25C-NBOMe and 25I-NBOMe were predominantly metabolized by O-demethylation, followed by O-di-demethylation and hydroxylation. All methoxy groups could be demethylated; hydroxylation preferably occurred at the NBOMe ring. Phase I metabolites were extensively conjugated in human urine with glucuronic acid and sulfate. Based on these data and a comparison with synthesized reference standards for potential metabolites, specific and abundant 25C-NBOMe urine targets are 5'-desmethyl 25C-NBOMe, 25C-NBOMe and 5-hydroxy 25C-NBOMe, and for 25I-NBOMe 2' and 5'-desmethyl 25I-NBOMe and hydroxy 25I-NBOMe. These data will help clinical and forensic laboratories to develop analytical methods and to interpret results. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and 2-(4-chloro-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in human and rat urine by GC-MS, LC-MSn, and LC-HR-MS/MS approaches.

25B-NBOMe and 25C-NBOMe are potent 5-HT2A receptor agonists that have been associated with inducing hallucinogenic effects in drug users and severe intoxications. This paper describes the identification of their metabolites in rat and human urine by liquid chromatography (LC)-high resolution (HR)-MS/MS, the comparison of metabolite formation in vitro and in vivo and in different species, the general involvement of human cytochrome-P450 (CYP) isoenzymes on their metabolism steps, and their detectability by standard urine screening approaches (SUSAs) using GC-MS, LC-MSn, or LC-HR-MS/MS. Both NBOMe derivatives were mainly metabolized by O-demethylation, O,O-bis-demethylation, hydroxylation, and combinations as well as by glucuronidation and sulfation of the main phase I metabolites. For 25B-NBOMe, 66 metabolites could be identified and 69 for 25C-NBOMe. After application of low doses of both substances to rats, they were detectable mainly via their metabolites by both LC-based SUSAs. In case of acute intoxication, it was possible to detect 25B-NBOMe and its metabolites in an authentic human urine sample when using the GC-MS SUSA in addition to the LC-based SUSAs. Initial CYP activity screening revealed the involvement of CYP1A2 and CYP3A4 in hydroxylation and CYP2C9 and CYP2C19 in O-demethylation. The presented study demonstrated that 25B-NBOMe and 25C-NBOMe were extensively metabolized and detectable by both LC-based SUSAs.

A data-independent acquisition workflow for qualitative screening of new psychoactive substances in biological samples.

Identification of new psychoactive substances (NPS) is challenging. Developing targeted methods for their analysis can be difficult and costly due to their impermanence on the drug scene. Accurate-mass mass spectrometry (AMMS) using a quadrupole time-of-flight (QTOF) analyzer can be useful for wide-scope screening since it provides sensitive, full-spectrum MS data. Our article presents a qualitative screening workflow based on data-independent acquisition mode (all-ions MS/MS) on liquid chromatography (LC) coupled to QTOFMS for the detection and identification of NPS in biological matrices. The workflow combines and structures fundamentals of target and suspect screening data processing techniques in a structured algorithm. This allows the detection and tentative identification of NPS and their metabolites. We have applied the workflow to two actual case studies involving drug intoxications where we detected and confirmed the parent compounds ketamine, 25B-NBOMe, 25C-NBOMe, and several predicted phase I and II metabolites not previously reported in urine and serum samples. The screening workflow demonstrates the added value for the detection and identification of NPS in biological matrices.

Pathological findings in 2 cases of fatal 25I-NBOMe toxicity.

The research compound 25I-NBOMe, also known as CIMBI-5 or INBMeO, was created in academic laboratories as a potent serotonin 2A receptor agonist. Because of its high affinity and ambiguous legal status, recreational drug enthusiasts have used this compound as a powerful alternative to other hallucinogenic drugs such as lysergic acid diethylamide. We report 2 deaths after 25I-NBOMe ingestion by decedents who attended separate "rave" parties. The first case involved a 21-year-old male who admitted taking "acid" to his friend. A sudden violent rage caused him to flail about, and he subsequently became unresponsive. The postmortem examination revealed numerous external injuries that were consistent with physical aggression. The second case involved a 15-year-old female who was socializing outside a rave party, became ill, and rapidly deteriorated as her friend transported her to the hospital. The postmortem assessment was similar to the first case in that external contusions featured prominently. Comprehensive toxicology screens in both cases revealed only evidence of marijuana use. A deeper analysis using time-of-flight mass spectrometry revealed the presence of 25I-NBOMe, which was further confirmed by tandem-mass spectrometry. The behavior and injuries in these cases reveal a consistent pattern preceding fatal 25I-NBOMe toxicity.

Disposition and metabolism of safinamide, a novel drug for Parkinson's disease, in healthy male volunteers.

BACKGROUND/AIMS: Absorption, biotransformation and elimination of safinamide, an enantiomeric α-aminoamide derivative developed as an add-on therapy for Parkinson's disease patients, were studied in healthy volunteers administered a single oral dose of 400 mg (14)C safinamide methanesulphonate, labelled in metabolically stable positions. METHODS: Pharmacokinetics of the parent compound were investigated up to 96 h, of (14)C radioactivity up to 192/200 h post-dose. RESULTS/CONCLUSIONS: Maximum concentration was achieved at 1 h (plasma, median Tmax) for parent drug and at 7 and 1.5 h for plasma and whole blood (14)C radioactivity, respectively. Terminal half-lives were about 22 h for unchanged safinamide and 80 h for radioactivity. Safinamide deaminated acid and the N-dealkylated acid were identified as major metabolites in urine and plasma. In urine, the ß-glucuronide of the N-dealkylated acid and the monohydroxy safinamide were also characterized. In addition, the glycine conjugate of the N-dealkylated acid and 2-[4-hydroxybenzylamino]propanamide were tentatively identified as minor urinary metabolites.

Pharmacokinetics of dapoxetine hydrochloride in healthy Chinese, Japanese, and Caucasian men.

Dapoxetine is a short-acting selective serotonin reuptake inhibitor developed for the on-demand treatment of premature ejaculation and is approved in some European Union countries, as well as Mexico and Korea, for this indication. The pharmacokinetics of dapoxetine 30 mg and 60 mg in healthy Chinese (single dose), Japanese, and Caucasian men (single and multiple dose) were assessed in 2 studies. In the 3 ethnic groups, dapoxetine was rapidly absorbed following oral administration, with peak plasma concentrations evident approximately 1 hour after dosing, independent of dose, dosing frequency (single or multiple dosing), or ethnicity. Dapoxetine was eliminated in a biphasic manner with an apparent mean terminal half-life of 14 to 17 hours. There was a dose-proportional increase in dapoxetine maximum plasma concentration (C(max)) and area under concentration-time curves (AUCs). The single-dose pharmacokinetic parameters of dapoxetine metabolites were also similar for the 3 ethnic groups, as were the pharmacokinetics of dapoxetine and its metabolites following single and multiple dosing in Caucasian and Japanese men. Dapoxetine was well tolerated by all 3 ethnic groups.

Metabolism, pharmacokinetics, and excretion of a nonpeptidic substance P receptor antagonist, ezlopitant, in normal healthy male volunteers: characterization of polar metabolites by chemical derivatization with dansyl chloride.

The excretion, biotransformation, and pharmacokinetics of ezlopitant [(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropyl-2-methoxy-benzyl)-amine], a substance P receptor antagonist, were investigated in healthy male volunteers after oral administration of a single 200-mg (approximately 93 microCi/subject) dose of [(14)C]ezlopitant. The total recovery of administered radioactive dose was 82.8 +/- 5.1, with 32.0 +/- 4.2% in the urine and 50.8 +/- 1.4% in the feces. Mean observed maximal serum concentrations for ezlopitant and total radioactivity were achieved at approximately 2 h after oral administration; thus, ezlopitant was rapidly absorbed. Ezlopitant was extensively metabolized in humans, since no unchanged drug was detected in urine and feces. The major pathway of ezlopitant in humans was the result of the oxidation of the isopropyl side chain to form the omega-hydroxy and omega-1-hydroxy (M16) metabolites. M16 and omega,omega-1-dihydroxy (1,2-dihydroxy, M12) were identified as the major circulating metabolites accounting for 64.6 and 15.4% of total circulating radioactivity, respectively. In feces, the major metabolite M14 was characterized as the propionic acid metabolite and formed by further oxidation of the omega-hydroxy metabolite. The urinary metabolites were the result of cleaved metabolites caused by oxidative dealkylation of the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl moiety. The metabolites (M1A, M1B, and M4), approximately 34% of the total radioactivity in urine, were identified as benzyl amine derivatives. These were polar metabolites that were further characterized using the reaction with dansyl chloride to derivatize the primary amines and phenol moieties to less polar analytes. The other metabolites were the result of O-demethylation, dehydrogenation of the isopropyl group, and oxidation on the quinuclidine moiety.

Mass spectrometric structure determination of metabolites of 3-[2-(N,N-dimethylaminomethyl)phenylthio]phenol.

Metabolites of 3-[2-(N,N-dimethylaminoethyl)phenylthio]phenol (I) were isolated from the urine of rats, mice, rabbit and dog and from the faeces of rats by extraction and thin-layer chromatography. From the mass spectrum of I and using characteristics mass shifts in the spectra of metabolites, the structures of four metabolites were determined, whereas for two other isomeric metabolites the structures were resolved by means of IR spectra. Conclusions regarding the structures derived from the spectra were confirmed by comparing them with synthetic standards. Generally, metabolic changes of I can be characterized by demethylation, hydroxylation combined with methylation, S-oxidation and by a combination of these metabolic reactions.

Effects of liver disease on the disposition of ciramadol in humans.

To determine the effects of liver disease on the disposition of ciramadol, an analgesic that undergoes ether glucuronidation, we studied its plasma pharmacokinetics in 10 patients with stable cirrhosis, 8 with acute viral hepatitis, and 16 age-matched healthy controls. Renal excretion of the glucuronides was also determined. In healthy controls given a single intravenous dose of the drug the t 1/2 was 3.4 +/- 0.3 hrs and the systemic clearance was 668 +/- 109 ml/min of which renal clearance was 320 +/- 73 ml/min and non-renal clearance 349 +/- 74 ml/min. The corresponding values after an oral dose were similar. Renal clearance was related directly to the estimated creatinine clearance. Moreover, the renal clearance of ciramadol exceeded creatinine clearance, suggesting that the drug was excreted not only by glomerular filtration but also by tubular secretion. The systemic clearance of intravenous ciramadol was diminished by 40% in cirrhosis, P less than 0.05, due to a reduction in renal clearance, while non-renal clearance remained normal. Renal clearance of the inactive glucuronides, on the other hand, was not affected. In patients with acute viral hepatitis, systemic clearance was unchanged, but renal clearance of ciramadol tended to increase during the acute phase of the disease and to return toward normal after recovery. Renal excretion of the inactive glucuronides was decreased by 48% (P less than .05). These findings suggest that the non-renal ether glucuronidation of ciramadol remains intact in patients with stable cirrhosis or acute viral hepatitis. However, the renal clearance of the drug may be impaired in cirrhosis, but tends to be enhanced in acute hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

The trans-cis isomerization of trans-4'-(2-hydroxy-3,5-dibromo-benzylamino)cyclohexanol in vivo and in vitro in different species.

Isomerization of trans-4'-(2-hydroxy-3,5-dibromo-benzylamino)cyclohexanol (HDBC) in vivo has been investigated in horse, cow, dog, rat and man. Following oral administration of 4'-trans-HDBC to the horse, a very efficient first-pass trans-cis isomerization was observed. In the urine of the horse and cow, 40% and 29% respectively of the conjugated alcohols consisted of the 4'-cis isomer. Isomerization in rat and dog took place only to a small extent, and in man no 4'-cis isomer was detected. Oxidation of HDBC to the corresponding ketone, at pH 9.0, was highest with horse- and rat-liver 10 000 g supernatants and lowest with dog-liver supernatant. Reduction of the ketone with 10 000 g liver supernatants and with cryst. horse-liver alcohol dehydrogenase led to the formation of the alcohol containing 42-69% as the 4'-cis isomer, whereas after reduction with NaBH4 the alcohol contained only 20% of the 4'-cis isomer. This indicates that the conformer with the lower energy (1' and 4' position equatorially substituted) preferentially formed only during chemical reduction. A correlation between the formation of the ketone in vitro and the formation of 4'-cis-HDBC in vivo was observed in the horse, cow and dog. No similar correlation was found in the rat, where a high in vivo trans-cis isomerization might have been expected from the in vitro data.

The metabolic disposition of 14C-ciramadol in humans.

Twelve subjects received single 15 mg oral doses of 14C-ciramadol. Excretion of the dose occurred almost entirely by the renal route (93.5 +/- 11.7 (S.D.)% of the dose), and only 0.7 +/- 0.6% of the dose was recovered in faeces indicating that absorption was essentially complete. More than 90% of the amount recovered in urine was excreted within 24 h after dosing. Unchanged drug accounted for 43.9 +/- 6.5% of the dose, while a phenolic glucuronide conjugate was the only major urinary metabolite accounting for a further 37.9 +/- 7.8%. A second glucuronide that was conjugated with the alicyclic ring was also identified but constituted only 2.3 +/- 0.6% of the dose. Concentrations of radioactivity in plasma reached a peak at 2 h after dosing and declined with a terminal disposition half life of 4.9 h. Only ciramadol and the aryl-O-glucuronide were detected in substantial amounts in plasma. Renal clearance of ciramadol amounted to 298 +/- 54 ml/min suggesting tubular secretion in addition to glomerular filtration.

N-Methyl-benzylamine, a metabolite of pargyline in man.

1. N-methyl-benzylamine was identified in human urine and plasma after the administration of pargyline in man. This metabolite was identified by thin layer chromatography mobility, gas chromatographic retention time and mass spectrum relative to authentic N-methyl-benzylamine. 2. [14C]-N-methyl-benzylamine, together with a small amount of [14C]-benzylamine, was formed in vitro from [14C]-pargyline when this was incubated with human and rat liver microsomes. 3. The urinary excretion rates of pargyline and its metabolite have also been described.

Metabolism of beclamide, N-benzyl-3-chloropropionamide, in man.

1. Following oral administration of [14C]beclamide to human subjects there was almost complete elimination of radioactivity in the urine within 48 h. 2. Less than 1% of the administered dose was excreted unchanged. 3. The major metabolites were (a) N-(4-hydroxybenzyl)-3-chloropropionamide, (4-hydroxybeclamide; c. 56%), which was eliminated to a small extent in the free form and as a sulphate but mainly as a glucuronide, and (b) hippuric acid (c. 20%). 4. Six minor metabolites were also detected, of which the following were identified: benzoic acid (up to c. 7%), N-(4-hydroxybenzyl-3-chloro-2-hydroxypropionamide, (dihydroxy-beclamide; c. 2%, mostly as a glucuronide) and 4-hydroxybenzoic acid (c. 0.6%, partly as a glucuronide).

Mass spectral identification of the metabolites of alpha,alpha-dimethyl-4-(alpha,alpha,beta,beta-tetrafluorophenethyl)-benzylamine (MK-251), a novel antiarrhythmic agent, in various species.

The identification of a number of metabolites of the novel antiarrhythmic agent, alpha,alpha-dimethyl-4-(alpha,alpha,beta,beta-tetrafluorophenethyl)benzylamine (MK-251), is presented. The compound is extensively metabolized by dog, monkey, baboon and man. Similar metabolic profiles were obtained for all species. Isolation and purification were accomplished by solvent extraction and chromatographic (column, gas and thin-layer) procedures. Gas chromatography, derivatization, infrared, nuclear magnetic resonance and particularly combined gas chromatography low and high resolution mass spectrometry techniques were employed to characterize the metabolites. The major urinary and plasma metabolites were identified as 2-[4-(alpha,alpha,beta,beta-tetrafluorophenethyl)phenyl]-2-propanol and its glucuronide conjugate. Other metabolites characterized were: the N-glucuronide of MK-251; 2-[4-(alpha,alpha,beta,beta-tetrafluorophenethyl)phenyl]propene; 2-nitro-2-[4-)alpha,alpha,beta,beta-tetrafluorophenethyl)phenyl]propane; alpha,alpha-dimethyl-4(alpha,alpha,beta,beta-tetrafluorophenethyl)benzyl methyl ether; and 4-(alpha,alpha,beta,beta-tetrafluorophenethyl)acetophenone. The 0-methyl ether metabolite represents the first instance of in vivo alkylation of a tertiary alcohol. Tentative identification was made for the N-hydroxy analog of MK-251 and for the glycol analog of 2-[4-(alpha,alpha,beta,beta-tetrafluorophenethyl)phenyl]-2-propanol. The observed pharmacological response appears to result mainly from MK-251 and not from the four metabolites.

Physiological disposition and metabolic fate of a new antiarrhythmic agent, alpha, alpha-dimethyl-4-(alpha, alpha, beta, beta-tetrafluorophenethyl) benzylamine in the rat, dog, monkey, baboon, and man.

The physiological disposition of a new orally active antiarrhythmic drug, alpha, alpha-dimethyl-4-(alpha, alpha, beta, beta-tetrafluorophenethyl)benzylamine (MK-251) was investigated in the rat, dog, rhesus monkey, baboon, and man. MK-251 was extensively absorbed after oral administration in all species. Fecal excretion was the major route of tracer elimination in the rat (70%) and dog (80%), whereas the monkey, baboon, and man excreted the majority of the dose via the urine (40-80%). MK-251 and/or its metabolites were widely distributed in rat tissues and exhibited tissue/plasma ratios greater than one in most instances. The lung, liver, and kidney possessed a high tissue affinity for drug and metabolites. The plasma and urinary profile of radioactivity indicated extensive metabolism of MK-251 in all species. Less than 5% of the plasma and urinary radioactivity was identified as unchanged drug. In spite of extensive metabolic transformations, a remarkable feature of this drug is its persistence in the plasma for long periods of time. This is thought to be due to tissue affinity. The metabolic pattern for MK-251 was essentially the same in all species. The major metabolites present in the plasma and the urine were identified as the carbinol analog of MK-251, 2-[4-(alpha, alpha, beta, beta-tetrafluorophenethyl)phenyl]-2-propanol (I), and its glucuronide conjugate. Other metabolites characterized in the urine and plasma were: the N-glucuronide of MK-251, 2-[4-(alpha, alpha, beta, beta-tetrafluorophenethyl)phenyl]propene (II), 2-nitro-2-[4-(alpha, alpha, beta, beta-tetrafluorophenethyl)phenyl]propane (III), alpha, alpha-dimethyl-4-(alpha, alpha, beta, beta-tetrafluorophenethyl)benzyl methyl ether (IV-1) and 4-(alpha, alpha, beta, beta-tetrafluorophenethyl), acetophenone (IV-2). Two minor urinary metabolites were tentatively identified as the N-hydroxy analog of MK-251 and the glycol analog of carbinol I. The in vivo formation of the methyl ether represents the first report of alkylation of a tertiary alcohol.