Ketamine-derived designer drug methoxetamine: metabolism including isoenzyme kinetics and toxicological detectability using GC-MS and LC-(HR-)MSn.

Methoxetamine (MXE; 2-(3-methoxyphenyl)-2-(N-ethylamino)-cyclohexanone), a ketamine analog, is a new designer drug and synthesized for its longer lasting and favorable pharmacological effects over ketamine. The aims of the presented study were to identify the phases I and II metabolites of MXE in rat and human urine by GC-MS and LC-high-resolution (HR)-MS(n) and to evaluate their detectability by GC-MS and LC-MS(n) using authors' standard urine screening approaches (SUSAs). Furthermore, human cytochrome P450 (CYP) enzymes were identified to be involved in the initial metabolic steps of MXE in vitro, and respective enzyme kinetic studies using the metabolite formation and substrate depletion approach were conducted. Finally, human urine samples from forensic cases, where the ingestion of MXE was suspected, were analyzed. Eight metabolites were identified in rat and different human urines allowing postulation of the following metabolic pathways: N-deethylation, O-demethylation, hydroxylation, and combinations as well as glucuronidation or sulfation. The enzyme kinetic studies showed that the initial metabolic step in humans, the N-deethylation, was catalyzed by CYP2B6 and CYP3A4. Both SUSAs using GC-MS or LC-MS(n) allowed monitoring an MXE intake in urine.

[Acute methoxetamine intoxication--a case report with serum and urine concentrations]. / Ostre zatrucie metoksetamina--opis przypadku z wyznaczonymi stezeniami w surowicy i moczu.

Methoxetamine (MXE) is a novel synthetic drug, structurally related to phencyclidine, with ketamine-like properties. Available in Poland since 2010, with no legal control, is adverti. sed as the "ideal dissociation drug". The aim of this study was to present a case of nasal methoxetamine acute poisoning in a 28-year-old man, the course of treatment, and the method of identification of this substance in serum and urine. In the course of this intoxication extreme agitation and aggression with slight response to benzodiazepines were observed. The patient was confused, hallucinated. In addition, the physical examination re. vealed tachycardia 120/min and normal blood pressure (130/80 mm Hg). The period of acute poisoning was covered by amnesia. The MXE concentrations in serum and urine were determined using liquid chromatography-mass spectrometry (LC-MS-MS) method, and were respectively 270 ng/ml and 660 ng/ml. Confirmed MXE poisoning increases our knowledge about this new substance, providing relevant clinical and analytical data.

Metabolism and toxicological detection of a new designer drug, N-(1-phenylcyclohexyl)propanamine, in rat urine using gas chromatography-mass spectrometry.

Studies are described on the metabolism and the toxicological detection of the phencyclidine-derived designer drug N-(1-phenylcyclohexyl)-propanamine (PCPR) in rat urine using gas chromatographic-mass spectrometric techniques. The identified metabolites indicated that PCPR was metabolized by hydroxylation of the cyclohexyl ring at different positions, hydroxylation of the phenyl ring, N-dealkylation, and combinations of these steps. Parts of the metabolites were excreted in conjugated form. The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of an intake of a common drug users' dose of PCPR in rat urine. Assuming similar metabolism in humans, the STA should be suitable for proof of an intake of PCPR in human urine.

Metabolism of cyclamate and its conversion to cyclohexylamine.

Since cyclamates were first introduced in the early 1950s, arguments have raged over the potential carcinogenicity of this artificial sweetener. Concern over the safety of cyclamates arises from observations that some individuals and experimental animals can metabolize cyclamate to cyclohexylamine and that cyclohexylamine has been shown to produce testicular atrophy in experimental animals. This study examines the absorption, excretion, and metabolism of cyclamate, particularly its conversion to cyclohexylamine. In addition, the potential toxicity and pharmacology of cyclohexylamine are discussed.

Fatal intoxication with methoxetamine.

Methoxetamine (MXE) is a new synthetic drug of abuse structurally related to ketamine and phencyclidine. A case of a 29-year-old male with acute toxicity related to the analytically confirmed use of MXE is reported. The man was found dead at his residence. Biological material was analyzed using liquid chromatography-tandem mass spectrometry. The concentration of MXE in urine of the deceased was 85 µg/mL. Despite the vial containing the blood sample being destroyed during transportation and the blood leaking out into the cardboard packaging, the blood level of MXE was estimated. After determination of the cardboard grammage (approx. 400 g/m(3) ) and the mean mass of the blood obtained after drying (0.1785 ± 0.0173 g per 1 mL), the estimated blood concentration of MXE was found to be 5.8 µg/mL. The high concentration of MXE in blood and urine and the circumstances of the case indicate an unintentional, fatal intoxication with this substance.

Biodistribution and renal excretion of isomers of the cationic tracer, (99m)Tc diaminocyclohexane (DACH): biodistribution of cationic renal tracers.

The buildup of organic anions in the plasma in the uremic state can competitively inhibit the tubular extraction of para-aminohippurate or (131)I ortho-iodohippurate (OIH) and lead to spuriously low measurements of effective renal plasma flow (ERPF). This problem can be circumvented by the use of cationic tracers. The cationic renal tracer, (99m)Tc labeled diaminocyclohexane ((99m)Tc DACH), has a clearance of 80% of OIH in mice but its clearance in humans is relatively low, only 30% of OIH. The (99m)Tc DACH isomer(s) used in prior studies, however, was not clearly defined and may have consisted of a single isomer or a combination of isomers. Since the anionic isomers of some (99m)Tc renal tracers have been shown to have widely different clearances, the biodistribution and urine excretion of the (99m)Tc cis-, trans-S,S, trans-R,R and +/-trans-DACH isomers were compared in Sprague-Dawley rats at 10 minutes and 60 minutes postinjection to determine if one of the (99m)Tc DACH isomers may be a significantly better renal tracer than the others. The red cell binding of (99m)Tc +/- trans-DACH was also determined. All of the isomers showed a high degree of specificity for the kidney with minimal secretion into the gastrointestinal tract. Urine excretion of the 4 tracers, however, was only 38-48% that of OIH at 10 minutes and 66-84% that of OIH at 60 minutes. Red cell binding was 6.9%. Cationic renal tracers have the potential to provide a more accurate measurement of ERPF than anionic tracers. Based on the animal data, however, it is unlikely that any of the (99m)Tc DACH isomers will have a substantially higher clearance in humans than the form of (99m)Tc DACH originally tested. Development of alternative cationic renal tracers is warranted.

Quantification of cyclamate and cyclohexylamine in urine samples using high-performance liquid chromatography with trinitrobenzenesulfonic acid pre-column derivatization.

An HPLC isocratic method with pre-column derivatization and UV detection for the quantification of cyclamate and cyclohexylamine in urine samples is described. The method requires very little sample preparation. Free cyclohexylamine is analysed in a first run and subsequently cyclamate is analysed as cyclohexylamine, after the simple process of oxidation of the sample by means of hydrogen peroxide. Cycloheptylamine is used as internal standard. Trinitrobenzenesulfonic acid (TNBS) appears to be a good reagent for the pre-column derivatization. The time per run is 15 min; the coefficients of variation of the assays range from 1.1 to 5.5%; the limits of detection are 0.09 and 0.11 ppm for cyclohexylamine and cyclamate anion, respectively. The system described has always performed efficiently, with a high degree of stability, in daily routine work.

Quantitative liquid chromatographic determination of bromadoline and its N-demethylated metabolites in blood, plasma, serum, and urine samples.

Bromadoline and its two N-demethylated metabolites were extracted into ether:butyl chloride after the addition of internal standard and basification of the various biological fluids (blood, plasma, serum, and urine). These compounds were then extracted into dilute phosphoric acid from the organic phase and separated on a reversed-phase chromatographic system using a mobile phase containing acetonitrile and a buffer of 1,4-dimethylpiperazine and perchloric acid. The overall absolute extraction recoveries of these compounds were approximately 50-80%. The background interferences from the biological fluids were negligible and allowed quantitative determination of bromadoline and the metabolites at levels as low as 2-5 ng/mL. At mobile phase flow rate of 1 mL/min, the sample components and the internal standard were eluted at the retention times within approximately 7-12 min. The drug- and metabolite-to-internal standard peak height ratios showed excellent linear relationships with their corresponding concentrations. The analytical method showed satisfactory within- and between-run assay precision and accuracy, and has been utilized in the simultaneous determination of bromadoline and its two N-demethylated metabolites in biological fluids collected from humans and from dogs after administration of bromadoline maleate.

Effects of cyclamate calcium on the immune response in rabbits.

To determine the effect of cyclamate calcium on the humoral immune response in rabbits, one group was fed a 3% and another a 5% water solution of cyclamate calcium. A third group served as controls. After 150 days, the animals were tested for their ability to respond immunologically to bovine serum albumin (BSA). Other aspects of immunity investigated were the presence or absence of neoplasia of the urinary bladder, spleen, thymus, and lymph nodes, the number of white and red blood cells, and cyclamate conversion and excretion in the urine. The results indicated that the 5% group had a delayed and suppressed response to BSA. No neoplasia was detected. This group had fewer red cells and excreted increased concentrations of cyclohexylamine in the urine.

The dispositional kinetics of phencyclidine and its N-ethylamine analogue in rats.

The uptake kinetics of [3H]-labelled phencyclidine (PCP) and N-ethyl-l-phenycyclohexylamine (PCE) in rats, measured in terms of decreases in the blood concentrations of the drugs after i.v. administration of a single 1.09 mumol dose, were not significantly different. Within a week of administration, the rats excreted about 93% of the [3H]-PCP and about 65% of [3H]-PCE via their urine and faeces; their urine contained nore [3H], mainly as metabolites of [3H]-PCP and of [3H]-PCE, than their faeces. Similarly, more [3H] remained in the tissues of rats treated with [3H]-PCE than in the tissues of [3H4-PCP-treated rats. The fact that PCE is metabolized and excreted more slowly than PCP may account for the higher psychotropic effects of PCE.

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

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.

Analytical findings of an acute intoxication after inhalation of methoxetamine.

Methoxetamine (MXE) is increasingly used and abused, as it is frequently presented as being safer than ketamine, and legal. Cases of only MXE consumption being associated with the occurrence of seizures are rarely reported. A single MXE intoxication case by inhalation is described concerning a 21-year-old man, not known to be epileptic, who was found collapsed in his bedroom, supposedly after an epileptic seizure. He was transferred to the Emergency Department of the Henri Mondor Hospital, Aurillac, France. He was conscious, but with a sinus bradycardia (48/min) and an ST-segment elevation on the electrocardiogram, and a slightly increased creatine kinase level (270 U/L) and hyponatremia (127 mmol/L). New seizure activity occurred during hospitalization, but the clinical course in the intensive care unit was favorable. Quantitation of MXE in serum and urine using gas chromatography coupled to mass spectrometry (GC-MS) was developed, as well as a liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) method for the determination of MXE in hair. Limits of detection and quantification were, respectively, 2 and 10 µg/L for the GC-MS method and both 0.5 pg/mg for the LC-MS-MS method. Concentrations of 30 and 408 µg/L were, respectively, measured in serum and urine. Concentrations of 135 and 145 pg/mg were detected in two 2.5 cm hair strands, consistent with one or several consumptions during the 2 ½ months prior to sampling. A sample of the powder consumed was available and also analyzed. This case illustrates the dangers of this drug, which justify its classification as a narcotic in France since August 2013.

Metabolism and toxicological detection of the designer drug N-(1-phenylcyclohexyl)-3-methoxypropanamine (PCMPA) in rat urine using gas chromatography-mass spectrometry.

Studies on the metabolism and the toxicological detection of the phencyclidine-derived designer drug N-(1-phenylcyclohexyl)-3-methoxypropanamine (PCMPA) in rat urine are described using gas chromatographic-mass spectrometric (GC-MS) techniques. Based on the identified metabolites, the following metabolic pathways could be postulated: N-dealkylation, O-demethylation partially followed by oxidation of the resulting alcohol to the corresponding carboxylic acid, hydroxylation of the cyclohexyl ring at different positions, and aromatic hydroxylation. The formed metabolites were identical to those of the homologue N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA) with exception of the mono hydroxyl metabolites of PCEPA. All PCMPA metabolites were partially excreted in conjugated form. An intake of a common drug users' dose of PCMPA could be detected in rat urine by the authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation. The STA should be suitable for proof of an intake of PCMPA also in human urine assuming similar metabolism.

The metabolism of cyclamate to cyclohexylamine in humans during long-term administration.

A group of 14 subjects, who had been identified from 261 volunteers in a 1-week screen as being able to metabolize the sweetener cyclamate to cyclohexylamine (>0.2% of a daily dose), and 31 nonconverters (<0.2% metabolism) were given calcium cyclamate tablets (equivalent to 250 mg cyclamic acid, 3 times daily) for a period of 13 weeks. The metabolism of cyclamate to cyclohexylamine was determined using twice-weekly timed (3 h) urine collections during week 1-3 and 7-13. Urine specimens were collected on all other study days to investigate day-to-day fluctuations in cyclohexylamine excretion. Analyses of the twice weekly timed urine collections showed that subjects recruited as nonconverters essentially remained nonconverters. Of the converters, three showed consistently low metabolism, five showed erratic metabolism, five showed low metabolism initially, which increased during the latter part of the study, and one subject showed consistently high metabolism throughout the study. Analysis of the day-to-day urine specimens showed marked intrasubject variability. The plasma concentrations of cyclohexylamine measured on weeks 1-3 and 7-13 reflected the urine profiles. The highest individual long-term average steady-state excretion values based on the 3-h urine collections and daily samples were 21%, 23%, 25%, 29%, 34%, and 38%. The maximum % metabolism detected in the high converters occasionally reached the value of 60% reported in previous short-term studies, but this high activity was not maintained, and was followed by periods of lower metabolism. The results of this metabolism study support an acceptable daily intake (ADI) of 0-11 mg/kg body weight per day.

Biological monitoring of hexamethylene- and isophorone-diisocyanate by the determination of hexamethylene- and isophorone-diamine in hydrolysed urine using liquid chromatography and mass spectrometry.

Hexamethylene-1,6-diamine (HDA) and isophoronediamine (IPDA) in hydrolysed human urine were studied as their perfluorofatty anhydride derivatives. Liquid chromatography and mass spectrometry with thermospray ionization were used. For quantitative analysis, the negative ions monitored were the m/z = 407 and 461, corresponding to the (M-1)- ions of the HDA-pentafluoropropionic anhydride (HDA-PFPA) and the IPDA-PFPA derivatives, respectively, and the m/z = 411 ions of the tetradeuterium-labelled HDA-PFPA (internal standard). Human urine was spiked with HDA and IPDA to six different concentrations in the range 2.5-20 micrograms l-1. Tetradeuterated HDA was used as the internal standard for the determination of both HDA and IPDA. The linear calibration curves obtained passed virtually through the origin, and the correlation coefficients were 0.998 for HDA and 0.973 for IPDA. The over-all precision for human urine spiked to a concentration of 5 micrograms l-1 of HDA and 25 micrograms l-1 of IPDA was found to be 5 and 14% (n = 5), respectively. The m/z = (M-1)- fragments, defined as twice the signal-to-noise ratio, were at the 0.5-1 pg level for the HDA and IPDA derivatives. The method presented made it possible to perform about 400 chromatographic runs during 24 h.

Studies on the formation of cyclohexylamine and N-methylcyclohexylamine from bromhexine in animals and man, and simultaneous determination of cyclohexylamine and N-methylcyclohexylamine by gas chromatography.

In order to detect cyclohexylamine and N-methylcyclohexylamine simultaneously in urine, a gas chromatographic method was developed in which Chromosorb 103 (porous polymer) was found to be suitable as the column packing. The detection limits were 0.1 mug/ml of cyclohexylamine and 0.4 mug/ml of N-methylcyclohexylamine in urine. In tests on animals administered bromhexine, which contains N-methylcyclohexylamine as part of its side-chain, neither cyclohexylamine nor N-methylcyclohexylamine was detected in the urine.