Determination of designer doping agent--2-ethylamino-1-phenylbutane--in dietary supplements and excretion study following single oral supplement dose.

The quantitative analysis of a new designer doping agent, 2-ethylamino-1-phenylbutane (EAPB) and its metabolite, 2-amino-1-phenylbutane (APB) in urine samples, and the determination of EAPB in dietary supplement samples, have been presented. The main purpose of the present study was to develop simple and reliable gas chromatography-mass spectrometry method (GC-MS) for excretion study following a single oral administration of dietary supplements containing EAPB. Three analytical methods for the determination of EAPB in urine and supplement samples, and APB in urine samples using the GC-MS system, have been validated. The method of the determination of EAPB in supplement samples was applied to analyze seventeen dietary supplements, CRAZE and DETONATE. Two other methods were used to determine the urinary excretion profile of EAPB and APB in the case of three healthy volunteers and, on further investigation, it was applied to the anti-doping control in sport. Quantification was obtained on the basis of the ions at m/z 86, 58 and 169, monitored for EAPB, APB and diphenylamine (used as an internal standard), respectively. The limits of detection and quantification were 2.4 and 7.3µg/g for EAPB in the case of supplement analysis, 2.9 and 8.8ng/mL for EAPB in the case of urine analysis, and 3.2 and 9.7ng/mL for APB. The other validation parameters as linearity, precision and trueness have been also investigated with the acceptable results. The extraction yield of all presented methods was above 69%. EAPB was detected in fourteen analyzed supplements (not included EAPB in their labels) and its content varied between 1.8 and 16.1mg/g. Following oral administration of three supplements with EAPB to one male and two female volunteers, the parent compound of EAPB and its metabolite were monitored and the excretion parameters as the maximum concentration of the analyte in urine (2.2-4.2µg/mL for EAPB; 1.1-5.1µg/mL for APB) and the time for the maximum height of the excretion peak (2-8h and 22h in one case for EAPB; 20-22h and 4h in one case for APB) have been indicated. EAPB and APB were detected at the level above 50ng/mL (50% of the minimum required performance level for stimulants in the anti-doping control in-competition in sport) in the urine up to 46-106h and 58-120h, respectively. Additionally, the result of the anti-doping control during swimming competition of one athlete, whose urine sample was analyzed for stimulants and narcotics, has been presented. The qualitative and quantitative analyses of new designer agents in urine samples and the excretion studies of these substances are of a great importance in the anti-doping control in sport. Moreover, the presentation of detection examples of these agents in supplements that haven't got included an information about them in the labeling, make athletes (and other supplement customers) more and more aware of the risk of the supplement use and possible health and doping consequences.

Targeting misuse of 2-amino-N-ethyl-1-phenylbutane in urine samples: in vitro-in vivo correlation of metabolic profiles and development of LC-TOF-MS method.

A phenyethylamine derivative, 2-amino-N-ethyl-1-phenylbutane (2-AEPB), has recently been detected in doping control and drugs-of-abuse samples, and identified as a non-labelled ingredient in a dietary supplement. To facilitate efficient control of this substance we have studied the in vitro metabolic behaviour of 2-AEPB with human liver preparation, compared these results with in vivo pathways in human, and finally propose an analytical strategy to target the potential misuse of 2-AEPB for toxicological, forensic and doping control purposes. The major in vitro formed metabolites originated from desethylation (M1) and monohydroxylation (M2). A minor metabolite with hydroxylation/N-oxidation was also observed (M3). In vitro-in vivo correlation was studied in an excretion study with a single, oral dose of 2-AEPB-containing supplement. An unmodified substance was the most abundant target compound and detected until the last point of sample collection (72 h), and the detection of M1 (40 h) and M2 (27 h) demonstrated good correlation to in vitro results. In the study with authentic cases (n = 6), 2-AEPB and M1 were mainly found in free urinary fraction, whereas higher inter-individual variability was observed for M2. It was predominantly conjugated and already within this limited number of cases, the ratio between glucuronide- and sulpho-conjugated fractions varied significantly. As a conclusion, hydrolysis is not mandatory in the routine sample preparation, and as the separation can be based on either gas chromatography or liquid chromatography, this study verifies that routine mass spectrometric detection methods targeted to amphetamine derivatives can be easily extended to control the misuse of 2-AEPB.

Designer phenethylamines routinely found in human urine: 2-ethylamino-1-phenylbutane and 2-amino-1-phenylbutane.

2-Ethylamino-1-phenylbutane (EAPB) and 2-amino-1-phenylbutane (APB) were identified by gas chromatography-mass spectrometry in multiple urine samples submitted for stimulant drug testing and screened positive for amphetamines by enzyme immunoassay. Forty-two samples from all over the USA were found, containing both analytes during a 3-month period May-July 2013. A sports dietary supplement 'CRAZE' has been determined to be one of the sources of EAPB supply. EAPB along with its suggested metabolite APB were detected in a urine sample, obtained from a person known to use 'CRAZE'.

HPLC determination of ITF 188 and its metabolite ITF 1078 in urine after intranasal administration of new heparin salt ITF 1300 to dogs.

Heparin salt ITF 1300 in which low molecular weight heparin is salified with a new counterion, di-[3-(N,N-dibutylamino)]propyl carbonate (ITF 188), was selected for the pharmacological development. A specific, sensitive and reproducible HPLC method for the determination of ITF 188 and its alcoholic metabolite ITF 1078 in urine was developed. The method was employed for the study of urinary excretion of the counterion after intranasal administration of ITF 1300 to dogs. The total amount of ITF 188 and ITF 1078 found in urine within 72 hours after the nasal instillation of ITF 1300 accounts for about 5% of the administered dose.

A labetalol metabolite with analytical characteristics resembling amphetamines.

A metabolite of labetalol that is responsible for previous reports of false-positive assays for amphetamines by thin-layer chromatography and immunoassay has been identified. The compound, 3-amino-1-phenylbutane (APB), an oxidative metabolite of labetalol, was initially identified in a patient's urine by gas chromatography-mass spectrometry and confirmed by analysis of the pure material. Gas chromatography showed a single major peak with relative retention time indistinguishable from that of methamphetamine, both underivatized and as the pentafluoropropionyl derivative. By classical thin-layer chromatography, APB migrated identically to methamphetamine but showed ninhydrin color development characteristic of amphetamine. APB was shown to cross-react approximately 2% with the Abbott TDx amphetamine/methamphetamine II kit, 10% with the Syva EMIT d.a.u. polyclonal amphetamine class kit, and 3% with the Syva EMIT d.a.u. monoclonal amphetamine kit. This degree of cross-reactivity is sufficient to cause false-positive immunoassays when cutoffs of 300 ng/mL are used.

Novel carbamate metabolites of mofegiline, a primary amine monoamine oxidase B inhibitor, in dogs and humans.

Mofegiline or MDL 72,974A ((E)-4-fluoro-beta-fluoromethylene benzene butanamine hydrochloride) is a selective enzyme-activated irreversible inhibitor of monoamine oxidase B, which is under development for use in the treatment of Parkinson's disease. Male beagle dogs were given single p.o. (20 mg/kg) and i.v. (5 mg/kg) doses of [14C]-Mofegiline. Total radioactivity excreted in urine and feces over 96 hr was, respectively, 75.5 +/- 3.8 and 6.3 +/- 3.4% of the dose after p.o. and 67.9 +/- 0.5 and 3.9 +/- 2.4% after i.v. administration. Unchanged drug in urine represented 3% of the dose after po and less than 1% after i.v. administration. Mofegiline was thus extensively metabolized in dogs, and urinary excretion was the major route of elimination of metabolites. HPLC, with on-line radioactivity detection, showed the presence of four major peaks (M1, M2, M3, and M4), representing respectively 50, 9, 5, and 0.5% of the administered dose excreted in 0-24 hr urine. TSP-LC-MS, FAB-MS, and NMR spectra of the purified metabolites were obtained. M1, the major metabolite in dogs, was shown to have undergone defluorination of the beta-fluoromethylene moiety, and one carbon addition. Its structure was confirmed to be a cyclic carbamate. M2 was a N-carbamoyl O-beta-D-glucuronide conjugate of parent drug. The formation of M1 and M2 is likely to involve initial reversible addition of CO2 to the primary amine function. M3 was a N-succinyl conjugate of the parent drug. M4 had also undergone defluorination to yield a urea adduct of an unsaturated alpha, beta aldehyde. Structures of M1 and M3 were further confirmed by comparing their MS and NMR spectra with those of authentic reference compounds. TSP-LC-MS ion chromatograms of human urine, obtained from two male volunteers after p.o. administration of 24 mg of drug, showed selected molecular ion peaks with the same retention time as the metabolites identified in dogs. In humans, these common metabolites represented a similar percentage of the administered dose to that in dogs. The present study demonstrates that NMR, TSP-LC-MS are complementary analytical techniques, which allow structural identification of unhydrolyzed drug conjugates. The formation of carbamates of amine-containing drugs may be more common than previously reported.

Bioavailability and disposition of terodiline in man.

Terodiline was concomitantly administered intravenously (12.5 mg) and orally ([2H]terodiline, 12.5 mg) to 10 healthy volunteers. In four of the subjects, a tracer dose of the intravenously given terodiline was 3H-labeled. In a separate study, six subjects were given [3H]terodiline orally. Estimated pharmacokinetic parameters were as follows: systemic clearance, 93 mL/min; renal clearance, 14 mL/min; volume of distribution at steady-state, 407 L; terminal half-life, 54 h; and mean residence time, 77 h. After intravenous infusion, a rapid distribution phase (half-life, 4.5 min) could be observed. The maximum serum concentration after the oral dose was 29 micrograms/L and the time to maximum concentration was 5 h (estimated by noncompartmental analysis). Absorption commenced within the first hour and by deconvolution the maximum rate of absorption was determined to occur between 1 and 3 h, and by 3.4 h 90% of the available dose had been absorbed. Calculation of bioavailability by noncompartmental AUC, two-compartmental analysis, urinary excretion, and 24-h oral/intravenous concentration ratio gave similar results (ANOVA test, not significant). About 75% and 25% of administered radioactivity could be recovered in urine and feces, respectively. Intact terodiline in feces accounted for about 1% of the dose. p-Hydroxyterodiline was quantitated in feces and accounted for about 5% of the dose. Another metabolite, 3,4-dihydroxyterodiline, which has not previously been detected in urine or serum, was also identified.

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.

Identification and quantitation of an oxidative metabolite of labetalol in sheep: pharmacokinetic and metabolic implications.

A sensitive and selective assay has been developed for the identification and quantitation of 3-amino-1-phenyl butane (3-APB), a metabolite of labetalol, in biological fluids using electron impact gas chromatography/mass-selective detection. Samples were extracted with n-hexane, derivatized with heptafluorobutyric anhydride and chromatographed on a cross-linked fused-silica capillary column. A positive EI spectrum was obtained using a mass-selective detector. Identification of the metabolite was accomplished using an authentic standard; quantitation was performed in the selected ion monitoring mode using ions m/z 345 (M+) and 132. The assay was linear over the calibration range of 0.5-1000 ng of the analyte and the intra-sample coefficients of variation were less than 12% in all cases. The absolute recovery of 3-APB following extraction from urine and bile was found to be 102.9 +/- 4.9% and 98.3 +/- 1.45% (mean +/- SEM) respectively. The minimum quantitation limit of the assay was 0.5 ng ml-1 (approximately 2 pg injected). Application of the assay in a pharmacokinetic-pharmacodynamic study of labetalol in sheep is demonstrated. The metabolite was detected in urine and bile samples obtained from adult non-pregnant sheep following labetalol administration. The cumulative amount of 3-APB excreted in urine over 24 h was found to be 71.55 micrograms in one animal following a 100 mg dose of labetalol. Evidence for biliary excretion, glucuronidation and sulfation of 3-APB was also found.

Stereospecific gas chromatographic/mass spectrometric assay of the chiral labetalol metabolite 3-amino-1-phenylbutane.

We previously identified 3-amino-1-phenylbutane (APB) as an oxidative N-dealkylated, metabolite of the antihypertensive agent labetalol. Labetalol has two asymmetric centers and is used clinically as a mixture of the four possible stereoisomers; APB has one asymmetric center. We now report an enantiospecific gas chromatographic/mass spectrometric assay for APB in urine. After adding the internal standard 1-methyl-2-phenoxyethylamine and alkalinizing, the urine samples were extracted with ether. The extracts were derivatized with the optically active acid chloride prepared from (S)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid. The derivatives were separated by capillary gas chromatography and detected by electron capture negative ion chemical ionization mass spectrometry with selected ion monitoring. The derivative of the R enantiomer eluted first, and the [M--32]- ions were monitored for both the drug and the internal standard. The method was linear in the 0.05-2.5 micrograms enantiomer-1 ml-1 range and had inter-assay and intra-assay coefficients of variation of less than 6%. The assay was used in the analysis of urine samples from a patient in labetalol therapy and no interference was found. Further studies are needed to elucidate the oxidative metabolism of labetalol and its stereochemical aspects.

Determination of an irreversible inhibitor of monoamine oxidase B (MDL 72974A) in human plasma and urine by gas chromatography-positive-ion chemical ionization mass spectrometry.

A sensitive and specific assay has been developed for the quantitative measurement in human plasma and urine of the irreversible inhibitor of monoamine oxidase B [(E)-4-fluoro-beta-fluoromethylenebenzene-butanamine HCl salt] (MDL 72974A) (I). This assay is based on gas chromatography-mass spectrometry with ammonia as the chemical ionization reagent gas. After addition of 1-fluoro-2-(4-chlorobenzene)-ethanamine HCl salt (MDL 71946A) as the internal standard, plasma (1 ml) and urine (100 microliter) samples were extracted using an automated solid-liquid extraction procedure on CN columns. The eluent was dried with a stream of nitrogen, and the residue was derivatized with pentafluoropropionic anhydride. Selected-ion monitoring of the [MNH4]+ ions m/z 361 (I) and 351 (internal standard) was used for quantification. The method yielded a linear response over the concentration range 0.25-100 pmol/ml in plasma with a limit of quantitation of 0.25 pmol/ml. The within-day reproducibility at a concentration of 5 pmol/ml was 4.6% and at a concentration of 50 pmol/ml was 1.3%. The day-to-day reproducibility was 5.2 and 7.0% at concentrations of 10 and 30 pmol/ml, respectively. The method was applied to the quantification of I in plasma and urine after the administration of 12-mg doses of I to a healthy male volunteer.

Identification of puberty-accelerating pheromones in male mouse urine.

Gas chromatography-mass spectrometry was employed to identify the two volatile amines in male mouse urine. These amines were much less concentrated in urine of castrated males. The identified amines, isobutylamine and isoamylamine, were assayed for the potential of puberty acceleration in postweaning female mice. A total of 105 young female mice were exposed to one of the following five odors: distilled water (control), 0.1 M isobutylamine, 0.1 M isoamylamine, a mixture of 0.05 M isobutylamine and 0.05 M isoamylamine, or fresh male mouse urine. The mixture of these amines accelerated the vaginal opening of young females. Except for the control, all experimental odors accelerated the first vaginal estrus in ICR strain mice.

An apparent fatal overdose of terodiline.

The results of a forensic toxicological investigation on a young man with an unknown cause of death are reported here. Analysis revealed the presence of a possibly fatal level of terodiline in blood and urine. No other drugs were detected. Terodiline was detected by thin-layer and gas-liquid chromatography and identified by gas chromatography/mass spectrometry. Quantification was carried out by a mass fragmentographic procedure using the m/z 100 from terodiline for selective ion monitoring (SIM). The blood and urine concentrations were found to be greater than 10 mg/L, whereas therapeutic concentrations in serum are usually not more than 1 mg/L. Support and confirmation of the laboratory results was provided at the subsequent inquest. It was revealed that the deceased had died from the inhalation of vomit due to an oral overdose of terodiline. To the best of our knowledge this is the first reported death due to fatal poisoning with terodiline in the United Kingdom.

Labetalol is metabolized oxidatively in humans.

Previous studies on the metabolic fate of antihypertensive agent labetalol in humans identified only conjugated metabolites of the drug and accounted for only a portion of the dose. In this study, urine samples obtained from three patients on chronic labetalol therapy for hypertension were analyzed initially by thin layer chromatography for the presence of other metabolites. All three urine samples were found to contain 3-amino-1-phenylbutane. The identify of this metabolite in one of the urine samples was confirmed by electron capture negative-ion chemical ionization mass spectrometry. The mass spectrometry experiments also identified the presence in the urine sample of the D-hydroxy derivative of 3-amino-1-phenylbutane. The two metabolites are the result of oxidative biotransformations of labetalol. 3-Amino-1-phenylbutane has been reported to be a potent sympathomimetic agent, and the question arises whether the newly identified metabolites of labetalol contribute to its pharmacological effects.

Urinary metabolites of N-nitrosodibutylamine and N-nitrodibutylamine in the rat: identification of N-acetyl-S-alkyl-L-cysteines.

N-Acetyl-S-butyl-L-cysteine, N-acetyl-S-3-oxobutyl-L-cysteine and N-acetyl-S-3-hydroxybutyl-L-cysteine have been isolated and identified (as their methyl esters) from the urine of rats given N-nitrosodibutylamine (NDBA), N-nitrodibutylamine (NO2DBA) and their 1-acetoxy derivatives. Greater amounts of these N-acetyl-S-alkyl-L-cysteines were detected in the urine after administration of NDBA than of NO2DBA, and greater urinary excretion of the three N-acetyl-S-alkyl-L-cysteines was observed with 1-acetoxy NDBA than with 1-acetoxy NO2DBA. This suggests that the markedly different biological activities of NDBA and NO2DBA might be due, in part, to a difference in their alkylating abilities in vivo.

Biotransformation of terodiline I. Identification of metabolites in dog urine by mass spectrometry.

Nine metabolites of terodiline (N-tert-butyl-4,4-diphenyl-2-butylamine) have been identified in dog urine by various chromatographic techniques and mass spectrometry. The main metabolic pathway is aromatic hydroxylation, leading to the quantitatively most important metabolite, N-tert-butyl-4-(4-hydroxyphenyl)-4-phenyl-2-butylamine, and to two dihydroxylated metabolites, one mono substituted in both rings (N-tert-butyl-4,4'-bis(4-hydroxyphenyl)-2-butylamine), and one disubstituted in one ring (N-tert-butyl-4-(3,4-dihydroxyphenyl)-4-phenyl-2-butylamine). The latter is further metabolized by methylation, forming N-tert-butyl-4-(4-hydroxy-3-methoxyphenyl)-4-phenyl-2-butylamine, the second most abundant metabolite. Still another metabolite is formed by hydroxylation in the tert-butyl group to N-(2-hydroxymethyl-2-propyl)-4,4-diphenyl-2-butylamine. A very minor dihydroxylated metabolite results from oxidation both in an aromatic ring and in the tert-butyl group, giving N-(2-hydroxymethyl-2-propyl)-4-(4-hydroxyphenyl)-4-phenyl-2-butylamine. Oxidation of the carbon adjacent to the nitrogen and subsequent deamination gives the two ketones 4-(4-hydroxyphenyl)-4-phenyl-2-butanone and 4-(4-hydroxy-3-methoxyphenyl)-4-phenyl-2-butanone. Reduction of the carbonyl function in the former yields the corresponding alcohol, 4-(4-hydroxyphenyl)-4-phenyl-2-butanol. Some unchanged terodiline is also present. All metabolites formed by functionalization appear to be extensively conjugated, presumably with glucuronic acid.