N,N-dimethyl-2-phenylpropan-1-amine quantification in urine: application to excretion study following single oral dietary supplement dose.

N,N-dimethyl-2-phenylpropan-1-amine (NN-DMPPA) is a new designer stimulant prohibited in sport in-competition according to the List of Prohibited Substances and Methods published by the World Anti-Doping Agency (WADA). The first published data on the excretion study of NN-DMPPA to support the knowledge of NN-DMPPA in routine anti-doping control have been presented. The reliable gas chromatography-mass spectrometry quantitative method (GC-MS) has been validated and applied to the excretion study of NN-DMPPA. The validation parameters of the GC-MS method for determination of NN-DMPPA in human urine were the linear calibration range of 100 to 7500 ng/mL, the LOD of 13.9 ng/mL and the LOQ of 42.2 ng/mL. According to the obtained repeatability, intermediate precision, and trueness, the applied GC-MS method was precise and accurate. Urine samples from three volunteers in the excretion study were collected for 5 days after single oral administration of the supplement NOXPUMP containing NN-DMPPA. The obtained results showed the maximum concentration of NN-DMPPA (189-303 ng/mL) in urine samples at a time of 2-3 h post-administration. The NN-DMPPA concentration in urine samples was higher than 50 ng/mL until 22-23 h after the dietary supplement ingestion. This means that according to the WADA rules the use of a supplement containing NN-DMPPA may be related to a positive case when athletes took this supplement in-competition. Moreover, excretion results demonstrate also that NN-DMPPA may be detected in urine samples by the applied GC-MS method till 46 h after supplement administration. Additionally, the excretion study of ß-methylphenethylamine as the second prohibited substance present in the supplement NOXPUMP has been investigated. Graphical Abstract Excretion study of new designer stimulant, N,N-dimethyl-2-phenylpropan-1-amine, and ß-methylphenethylamine following single oral NOXPUMP supplement dose.

Metabolic fate, mass spectral fragmentation, detectability, and differentiation in urine of the benzofuran designer drugs 6-APB and 6-MAPB in comparison to their 5-isomers using GC-MS and LC-(HR)-MS(n) techniques.

The number of so-called new psychoactive substances (NPS) is still increasing by modification of the chemical structure of known (scheduled) drugs. As analogues of amphetamines, 2-aminopropyl-benzofurans were sold. They were consumed because of their euphoric and empathogenic effects. After the 5-(2-aminopropyl)benzofurans, the 6-(2-aminopropyl)benzofuran isomers appeared. Thus, the question arose whether the metabolic fate, the mass spectral fragmentation, and the detectability in urine are comparable or different and how an intake can be differentiated. In the present study, 6-(2-aminopropyl)benzofuran (6-APB) and its N-methyl derivative 6-MAPB (N-methyl-6-(2-aminopropyl)benzofuran) were investigated to answer these questions. The metabolites of both drugs were identified in rat urine and human liver preparations using GC-MS and/or liquid chromatography-high resolution-mass spectrometry (LC-HR-MS(n)). Besides the parent drug, the main metabolite of 6-APB was 4-carboxymethyl-3-hydroxy amphetamine and the main metabolites of 6-MAPB were 6-APB (N-demethyl metabolite) and 4-carboxymethyl-3-hydroxy methamphetamine. The cytochrome P450 (CYP) isoenzymes involved in the 6-MAPB N-demethylation were CYP1A2, CYP2D6, and CYP3A4. An intake of a common users' dose of 6-APB or 6-MAPB could be confirmed in rat urine using the authors' GC-MS and the LC-MS(n) standard urine screening approaches with the corresponding parent drugs as major target allowing their differentiation. Furthermore, a differentiation of 6-APB and 6-MAPB in urine from their positional isomers 5-APB and 5-MAPB was successfully performed after solid phase extraction and heptafluorobutyrylation by GC-MS via their retention times.

N,N-dimethyl-2-phenylpropan-1-amine - new designer agent found in athlete urine and nutritional supplement.

Reports of new designer agents banned in sport being detected in supplements widely available for athletes are constantly emerging. The task of anti-doping laboratories is to control athletes for the presence of substances listed by the World Anti-Doping Agency (WADA) and those that are structurally/biologically similar to them. Recently, a new designer stimulant, N,N-dimethyl-2-phenylpropan-1-amine (NN-DMPPA), was detected by the WADA accredited anti-doping laboratory in Warsaw during routine anti-doping control. The urine samples from four athletes were analyzed in the screening method for stimulants and narcotics and the presence of NN-DMPPA was detected. The identity of NN-DMPPA was confirmed by gas chromatography-mass spectrometry using a synthesized reference standard. The measured concentrations of NN-DMPPA were between 0.51 and 6.51 µg/mL. The presence of the NN-DMPPA compound has been detected in the 'nutritional supplement' NOXPUMP that had been purchased in a store in Poland. NN-DMPPA at 121.7 µg/g was indicated in the investigated supplement together with another banned stimulant ß-methylphenethylamine. The presence of this new stimulant was not indicated on the labelling of the supplement, a situation which is not unusual within this market. Thus, it is important to make athletes aware of the risk related to the use of supplements. Moreover, specific legistation dealing with the commercialization of drugs banned for sport should be undertaken.

Acute psychosis associated with recreational use of benzofuran 6-(2-aminopropyl)benzofuran (6-APB) and cannabis.

INTRODUCTION: There is evidence from around Europe of the availability and use of 6-(2-aminopropyl)benzofuran (6-APB) as a recreational drug. However, there is currently limited information on the acute toxicity of this compound. We describe here a case of acute toxicity associated with recreational use of legal high (6-APB) and cannabis, in which the comprehensive toxicological analysis confirmed the presence of a significant amount of 6-APB together with metabolites of both tetrahydrocannabinol and the synthetic cannabinoid receptor agonist (JWH-122). CASE REPORT: A 21-year-old gentleman with no previous medical and psychiatric history was brought to the emergency department (ED) after he had developed agitation and paranoid behaviour following the use of 6-APB purchased over the Internet. There was no obvious medical cause for his acute psychosis. He required diazepam to control his agitation and was subsequently transferred to a psychiatric hospital for ongoing management of his psychosis. Toxicological screening of a urine sample collected after presentation to the ED detected 6-APB, with an estimated urinary concentration of 2,000 ng/ml; other drugs were also detected, but at lower concentrations including metabolites of the synthetic cannabinoid receptor agonist JWH-122 and tetrahydrocannabinol. CONCLUSION: This is the first case of analytically confirmed acute toxicity associated with the detection of 6-APB which will provide some information on acute toxicity of this drug to help clinicians with the management of such patients and legislative authorities in their consideration for the need of its control.

Determination of atomoxetine and its metabolites in conventional and non-conventional biological matrices by liquid chromatography-tandem mass spectrometry.

A procedure based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described for determination of atomoxetine (ATX) and its metabolites 4-hydroxyatomoxetine (4-OH-ATX) and N-des-methylatomoxetine (N-des-ATX) in plasma, urine, oral fluid and sweat using duloxetine as internal standard. Analytes were extracted from 0.5 mL biological fluids and sweat patch with 2 mL aliquots of tert-butyl methyl ether. The organic layer was evaporated and redissolved in mobile phase. Chromatographic separation was carried out on reverse-phase column and an isocratic mobile phase formed by 40% water and 60% 5mM ammonium acetate, 47.2 mM formic acid, 4 mM trifluoroacetic acid in acetonitrile-water (85:15, v/v) at a flow rate of 0.5 mL/min. Separated analytes were identified and quantified by positive electrospray ionization tandem mass spectrometry and in multiple reaction monitoring acquisition mode. Limits of quantifications for the three analytes were 0.5 ng/mL plasma and oral fluid, 10 ng/mL urine and 1 ng/patch using 0.5 mL biological fluids or one sweat-patch per assay. Calibration curves were linear over the calibration ranges with r²>0.99. At three concentrations spanning the linear dynamic range of the assay, mean recoveries in different biological matrices were always higher than 65%. This method was applied to therapeutic monitoring of ATX and its metabolites 4-OH-ATX and N-des-ATX in conventional and non-conventional biological matrices from individuals in drug treatment.

A drug toxicity death involving propylhexedrine and mitragynine.

A death involving abuse of propylhexedrine and mitragynine is reported. Propylhexedrine is a potent α-adrenergic sympathomimetic amine found in nasal decongestant inhalers. The decedent was found dead in his living quarters with no signs of physical trauma. Analysis of his computer showed information on kratom, a plant that contains mitragynine, which produces opiumlike effects at high doses and stimulant effects at low doses, and a procedure to concentrate propylhexedrine from over-the-counter inhalers. Toxicology results revealed the presence of 1.7 mg/L propylhexedrine and 0.39 mg/L mitragynine in his blood. Both drugs, as well as acetaminophen, morphine, and promethazine, were detected in the urine. Quantitative results were achieved by gas chromatography-mass spectrometry monitoring selected ions for the propylhexedrine heptafluorobutyryl derivative. Liquid chromatography-tandem mass spectrometry in multiple reactions monitoring mode was used to obtain quantitative results for mitragynine. The cause of death was ruled propylhexedrine toxicity, and the manner of death was ruled accidental. Mitragynine may have contributed as well, but as there are no published data for drug concentrations, the medical examiner did not include mitragynine toxicity in the cause of death. This is the first known publication of a case report involving propylhexedrine and mitragynine.

Simultaneous quantification of atomoxetine as well as its primary oxidative and O-glucuronide metabolites in human plasma and urine using liquid chromatography tandem mass spectrometry (LC/MS/MS).

A sensitive and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the determination of atomoxetine and its metabolites (4-hydroxyatomoxetine, N-des-methylatomoxetine, and 4-hydroxyatomoxetine-O-glucuronide) has been developed for human plasma and urine. Using stable-labeled internal standards, the method proved to be accurate and precise for the analytes in all species, resulting in inter-batch accuracy (percent relative error, %RE) within 100+/-13% and inter-batch precision (relative standard deviation, %RSD) within 11%. Stability was demonstrated for the analytes in neat solutions and the reconstitution solvent, as well as plasma and urine (with or without the deconjugation reagent). The method was simple, robust (utilized for the analysis of several hundred clinical study samples), and amenable to high sample throughput.

Effect of hepatic impairment on the pharmacokinetics of atomoxetine and its metabolites.

BACKGROUND AND OBJECTIVES: Atomoxetine is a treatment for attention-deficit/hyperactivity disorder and is primarily eliminated via cytochrome P4502D6 (CYP2D6). The pharmacokinetics of atomoxetine and its primary metabolites were investigated in 10 adults with hepatic impairment (6 moderate, 4 severe) and 10 age- and sex-matched control subjects, all being genotyped as CYP2D6 extensive metabolizers. METHODS: A single oral 20-mg dose of atomoxetine was given. Multiple blood samples were collected for 48 hours in healthy subjects and for 120 hours in patients. Urine was collected up to 24 hours. Before atomoxetine administration (10-20 days), sorbitol clearance and debrisoquin (INN, debrisoquine) metabolic ratio were determined as markers of hepatic blood flow and CYP2D6 activity, respectively. RESULTS: The systemic clearance of atomoxetine was significantly reduced in those with hepatic impairment compared with controls, thereby resulting in increased exposure (area under the concentration-time curve from time 0 to infinity, 1.58 versus 0.85 microg. h(-1). mL(-1); P =.035) but no change in maximum concentration. Mean 4-hydroxyatomoxetine area under the concentration-time curve from time 0 to time t and maximum concentration were increased approximately 7-fold and 2-fold, respectively (P =.0001 and P =.0056, respectively). For the glucuronide conjugate of 4-hydroxyatomoxetine, the mean half-life was longer and the mean area under the concentration-time curve from time 0 to infinity and the maximum concentration were lower (P =.0028, P =.003, and P =.0001, respectively). The sorbitol clearance was lower and the debrisoquin metabolic ratio was higher, reflecting reduced hepatic blood flow and decreased CYP2D6 activity, respectively. Decreased atomoxetine clearance in patients with hepatic impairment was clearly correlated with decreased CYP2D6 activity and decreased hepatic blood flow. Mean atomoxetine plasma protein binding was lower in patients with hepatic impairment compared with controls (96.5% versus 98.7%, P =.0008). Atomoxetine was well tolerated in the 2 populations. CONCLUSION: For patients with attention-deficit/hyperactivity disorder who have hepatic impairment, dosage adjustment is recommended. Initial target doses should be reduced to 25% and 50% of the normal dose for patients with severe and moderate hepatic impairment, respectively.

Disposition and metabolic fate of atomoxetine hydrochloride: pharmacokinetics, metabolism, and excretion in the Fischer 344 rat and beagle dog.

These studies were designed to characterize the disposition and metabolism of atomoxetine hydrochloride [(-)-N-methyl-gamma-(2-methylphenoxy)benzenepropanamine hydrochloride; formerly know as tomoxetine hydrochloride] in Fischer 344 rats and beagle dogs. Atomoxetine was well absorbed from the gastrointestinal tract and cleared primarily by metabolism with the majority of its metabolites being excreted into the urine, 66% of the total dose in the rat and 48% in the dog. Fecal excretion, 32% of the total dose in the rat and 42% in the dog, appears to be due to biliary elimination and not due to unabsorbed dose. Nearly the entire dose was excreted within 24 h in both species. In the rat, low oral bioavailability was observed (F = 4%) compared with the high oral bioavailability in dog (F = 74%). These differences appear to be almost purely mediated by the efficient first-pass hepatic clearance of atomoxetine in rat. The biotransformation of atomoxetine was similar in the rat and dog, undergoing aromatic ring hydroxylation, benzylic oxidation (rat only), and N-demethylation. The primary oxidative metabolite of atomoxetine was 4-hydroxyatomoxetine, which was subsequently conjugated forming O-glucuronide and O-sulfate (dog only) metabolites. Although subtle differences were observed in the excretion and biotransformation of atomoxetine in rats and dogs, the primary difference observed between these species was the extent of first-pass metabolism and the degree of systemic exposure to atomoxetine and its metabolites.

Disposition and metabolic fate of atomoxetine hydrochloride: the role of CYP2D6 in human disposition and metabolism.

The role of the polymorphic cytochrome p450 2D6 (CYP2D6) in the pharmacokinetics of atomoxetine hydrochloride [(-)-N-methyl-gamma-(2-methylphenoxy)benzenepropanamine hydrochloride; LY139603] has been documented following both single and multiple doses of the drug. In this study, the influence of the CYP2D6 polymorphism on the overall disposition and metabolism of a 20-mg dose of (14)C-atomoxetine was evaluated in CYP2D6 extensive metabolizer (EM; n = 4) and poor metabolizer (PM; n = 3) subjects under steady-state conditions. Atomoxetine was well absorbed from the gastrointestinal tract and cleared primarily by metabolism with the preponderance of radioactivity being excreted into the urine. In EM subjects, the majority of the radioactive dose was excreted within 24 h, whereas in PM subjects the majority of the dose was excreted by 72 h. The biotransformation of atomoxetine was similar in all subjects undergoing aromatic ring hydroxylation, benzylic oxidation, and N-demethylation with no CYP2D6 phenotype-specific metabolites. The primary oxidative metabolite of atomoxetine was 4-hydroxyatomoxetine, which was subsequently conjugated forming 4-hydroxyatomoxetine-O-glucuronide. Due to the absence of CYP2D6 activity, the systemic exposure to radioactivity was prolonged in PM subjects (t(1/2) = 62 h) compared with EM subjects (t(1/2) = 18 h). In EM subjects, atomoxetine (t(1/2) = 5 h) and 4-hydroxyatomoxetine-O-glucuronide (t(1/2) = 7 h) were the principle circulating species, whereas atomoxetine (t(1/2) = 20 h) and N-desmethylatomoxetine (t(1/2) = 33 h) were the principle circulating species in PM subjects. Although differences were observed in the excretion and relative amounts of metabolites formed, the primary difference observed between EM and PM subjects was the rate at which atomoxetine was biotransformed to 4-hydroxyatomoxetine.

Excretion and metabolism of the antihypertensive agent, RWJ-26240 (McN-5691) in dogs.

The excretion and metabolism of a 2-ethynylbenzenealkanamine analog, antihypertensive RWJ-26240 (McN-5691), in beagle dogs was investigated. Recoveries of total radioactivity in urine and feces in the 7 days after oral administration of 14C-RWJ-26240 (6 mg/kg dose) were 2.8% and 96.8% of the radioactive dose, respectively. Representative plasma, urine, and fecal samples were pooled and purified for metabolite profiling, isolation, and identification. Unchanged RWJ-26240 (<19% of the dose) plus 12 metabolites were isolated and identified from these samples using chromatography (TLC, HPLC), spectroscopy (NMR, MS), and derivatization techniques. Unchanged RWJ-26240 plus identified metabolites accounted for >75% of the sample radioactivity in plasma and feces. The formation of RWJ-26240 metabolites can be depicted by the following proposed pathways: 1) N-demethylation, 2) O-demethylation, 3) phenyl hydroxylation, and 4) N-dealkylation. The first three pathways appeared to be quantitatively important steps which led to the production of four major metabolites (each >5% of the sample radioactivity). RWJ-26240 was extensively metabolized in the dog, and fecal excretion was the major route of elimination of RWJ-26240 and its metabolites.

Determination of amphetamine and related compounds in urine using on-line derivatization in octadecyl silica columns with 9-fluorenylmethyl chloroformate and liquid chromatography.

A method for the determination of amphetamine and related compounds in urine based on on-line derivatization with 9-fluorenylmethyl chloroformate (FMOC) and high-performance liquid chromatography is described. Derivatization is performed in a 20 x 2.1 mm I.D. column packed with a Hypersil ODS C18, 30 micron stationary phase, which is also used for sample clean-up and enrichment of the analytes. Next, the derivatized analytes are transferred to a LiChrospher 100 RP-C18 (5 micron, 125 x 4 mm I.D.) analytical column for their separation and quantification, using reversed-phase conditions and fluorescence detection. The described assay was applied to the determination of norephedrine, ephedrine, pseudoephedrine, amphetamine, phenylpropylamine and methamphetamine at concentrations of 0.5-10.0 micrograms/ml. Analyte conversions were about 55-96% of those obtained by the off-line derivatization mode under similar conditions, resulting in limits of detection in the 5-25 ng/ml range.

Selective determination of secondary amines as their N-diethylthiophosphoryl derivatives by gas chromatography with flame photometric detection.

A selective and sensitive method has been developed for the determination of secondary amines by gas chromatography (GC). After removal of primary amines by the reaction with o-phthaldialdehyde, secondary amines were converted into their N-diethylthiophosphoryl derivatives and then measured by GC with flame photometric detection using a DB-1701 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of secondary amines were ca. 0.05-0.2 pmol per injection. N-Methylcyclohexylamine was used as an internal standard. The calibration curves for secondary amines in the range 1-20 nmol were linear and sufficiently reproducible for quantitative determination. This method was successfully applied to small urine samples without prior clean-up. Overall recoveries of secondary amines added to urine samples were 91-105%. By using this method, secondary amines in urine samples could be analysed without any influence from primary amines and other coexisting substances. The analytical results of secondary amine content in urine samples of normal subjects are presented.

High-performance liquid chromatographic method for the simultaneous determination of pentisomide and its major metabolite N-desisopropylpentisomide in plasma, urine and tissues using solid-phase extraction.

A rapid, sensitive and selective high-performance liquid chromatographic method for the simultaneous determination of pentisomide and its major metabolite desisopropylpentisomide in plasma, urine and tissues has been developed. The method for plasma samples, urine samples and tissue samples, after homogenizing with 50% ethanol, involves extraction of samples via activated Bond-Elut C8 disposable columns with methanol at pH 10 after addition of internal standard, and initially on column washing of samples at pH 10 with water and acetonitrile. The obtained methanolic extract is evaporated to dryness under nitrogen at 25 degrees C; the sample residue is then reconstituted in mobile phase and an aliquot of this solution is injected into the liquid chromatograph. Separation is performed using a Nova-Pak C18 4 microns particle size column operating in combination with radial compression separation unit and a methanol-water-di-sec.-butylamine-phosphoric acid (40:60:0.5:0.2, v/v) pH 3.5 mobile phase with ultraviolet detection at 258 nm. Endogenous substances or a variety of drugs concomitantly used in pentisomide therapy, with the exception of disopyramide, do not interfere with the assay. The mean recovery of pentisomide and desisopropylpentisomide from plasma and urine and from tissues is more than 91 and 86%, respectively. The limit of detection of the assay is 10 ng/ml for both drugs. The intra- and inter-day coefficient of variation for replicate analyses of spiked plasma samples is less than 7 and 8%, respectively. Mean steady-state plasma levels of pentisomide and desisopropylpentisomide in patients on chronic oral therapy are reported.

HPLC measurement of penticainide and desalkylpenticainide in biological fluids.

A simple HPLC method has been developed to measure the antiarrhythmic agent penticainide and its N-desalkyl metabolite in biological fluids. Solvent extraction of a small (200 microL) sample volume with direct analysis of the extract is used to measure the plasma and urinary concentrations of these compounds attained during chronic therapy, although a larger sample volume (1.0 mL) and prior concentration of the extract are required for single oral dose work. In each case chromatographic analysis is performed using a microparticulate (5 microns) silica column and methanolic ammonium perchlorate (10 mM, pH 6.7) as eluent with UV detection (260 nm). No endogenous sources of interference have been encountered and potential interference from other drugs is minimal.

Identification of urinary metabolites of penticainide in the rat, dog, baboon and man.

[14C]-Penticainide, 2-[2-(diisopropylamino) ethyl]-4-methyl-2-(2-pyridyl)pentanamide, a new antiarrhythmic agent, was administered as a single oral dose to rats, dogs, baboons (30 mg kg-1) and to healthy, informed volunteers (300 mg). Excretion of radioactivity was followed for 3 days in urine and faeces. In man, about 95% of the administered radioactivity was eliminated in the urine and levels ranging from 56 to 86% were observed in animals. The radioactivity that did not appear in the urine was almost quantitatively recovered in the faeces. Metabolites in urine were isolated by thin-layer chromatography and identified by mass spectrometry and nuclear magnetic resonance. In addition to the unchanged drug, nine metabolites and an artifact compound resulting from the partial degradation of one metabolite, were identified among the thirteen radioactive compounds detected. The major metabolites resulted from N-dealkylation of the diisopropyl moiety, oxidation of the isobutyl side-chain and hydrolytic cleavage of the amide. Comparison of the excretion and metabolic patterns of animals with those of man revealed that the dog should be a most suitable model for predicting the pharmacological and toxicological effects of penticainide in man.

Determination of tiapamil and of its two main metabolites in plasma and in urine by high-performance liquid chromatography.

Selective high-performance liquid chromatographic methods for the determination of tiapamil and its two main metabolites in plasma and urine are described. Tiapamil together with its metabolites is extracted at alkaline pH into dichloromethane. Separation is carried out using normal-phase high-performance liquid chromatography with ultraviolet detection (278 nm). The unchanged drug and the desmethyl metabolite are analysed simultaneously. The second metabolite is analysed separately under more polar conditions. The sensitivity limits are 50 ng/ml for tiapamil, 100 ng/ml for the desmethyl metabolite and 75 ng/ml for the second metabolite, using 0.5 ml of plasma. The sensitivity limits in urine are 100 ng/ml for all three compounds using a 0.5 ml specimen. The method has been applied to the analysis of human plasma and urine after intravenous (70 mg) and oral (400 mg) administration of tiapamil.

High-performance liquid chromatographic determination of citalopram and four of its metabolites in plasma and urine samples from psychiatric patients.

A high-performance liquid chromatographic method is used for the determination of citalopram [1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-5-phthalancarbonitrile+ ++] and four of its metabolites (the methylamino, amino, propionic acid and N-oxide derivatives) in plasma and urine. The plasma samples were extracted with diethyl ether at pH 10 and pH 4. Filtered urine samples could be injected directly on to the column. Steady-state drug and metabolite levels were investigated in fifteen psychiatric patients. In urine, 12 +/- 5% (mean +/- S.D.) of a given dose of citalopram was excreted in unchanged form. The propionic acid derivative was further conjugated, possibly to glucuronic acid. Mean steady-state plasma levels and metabolites in 24-h urine are given as percentages of the dose.