Analysis of benzphetamine and its metabolites in rat urine by liquid chromatography-electrospray ionization mass spectrometry.

An analytical method to identify and determine benzphetamine (BMA) and its five metabolites in urine was developed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using the solid-phase extraction column Bond Elut SCX. Deuterium-labeled compounds, used as internal standards, were separated chromatographically from each corresponding unlabeled compound in the alkaline mobile phase with an alkaline-resistant ODS column. This method was applied to the identification and determination of BMA and its metabolites in rat urine collected after oral administration of BMA. Under the selected ion monitoring mode, the limit of quantitation (signal-to-noise ratio 10) for BMA, N-benzylamphetamine (BAM), p-hydroxybenzphetamine (p-HBMA), p-hydroxy-N-benzylamphetamine (p-HBAM), methamphetamine (MA) and amphetamine (AM) was 700 pg, 300 pg, 500 pg, 1.4 ng, 6 ng and 10 ng in 1 ml of urine, respectively. This analytical method for p-HBMA, structurally closer to the unchanged drug of all the metabolites, was very sensitive, making this a viable metabolite for discriminating the ingestion of BMA longer than the parent drug or other metabolites in rat.

Development of a method for the quantitation of benzphetamine metabolites in human urine by high-performance liquid chromatography.

We developed a high-performance liquid chromatography (HPLC) method for quantitating p-hydroxy-N-benzylamphetamine glucuronide (pHBAG) and p-hydroxy-benzphetamine glucuronide (pHBZG), which are urinary metabolites of benzphetamine, in humans. Urine samples were hydrolysed with beta-glucuronidase (EC 3.2.1.31) at 37 degrees C overnight and the treated urine was applied to a solid phase extraction column. After washing the column with water, 0.01 mol/L acetic acid and methanol, pHBA and pHBZ were eluted with dichloromethane:isopropanol:28% ammonium hydroxide (78.4:19.6:2.0 v/v). The eluate was evaporated and the residue was dissolved in acetonitrile: 5 mmol/L 1-pentane sulphonic acid (5:95 v/v) and analysed by HPLC with gradient elution. The amounts of urinary pHBAG and pHBZG excreted by two human subjects after oral administration of 10 mg benzphetamine hydrochloride were determined. About 10-15% of benzphetamine was found to be excreted as pHBAG and pHBZG, and almost all of these metabolites were excreted within 24 h. Urine samples should be collected as early as possible after ingestion of benzphetamine to detect pHBAG and pHBZG.

Simultaneous analysis of benzphetamine and its metabolites, and quantitation of urinary p-hydroxy-N-benzylamphetamine by micellar electrokinetic chromatography.

We developed a method for simultaneous analysis of benzphetamine (BZ) and its metabolites, p-hydroxy-N-benzylamphetamine (pHBA), p-hydroxybenzphetamine (pHBZ), amphetamine (AP), methamphetamine and p-hydroxymethamphetamine by micellar electrokinetic chromatography (MEKC). Urine samples from 0-15 h (3-h intervals) after oral administration of BZ (10 mg) were hydrolyzed with beta-glucuronidase (EC 3.2.1.31) at 37 degrees C overnight. The treated urine was applied to a solid phase extraction column Bond Elut Certify. After sequentially washing the column with water, 0.1 mol/l acetic acid and methanol, the samples were eluted with dichloromethane:isopropanol:28% ammonium hydroxide=78.4:19.6:2.0 (v/v %). The eluate was evaporated and the residue dissolved in running buffer was analyzed by MEKC. In urine from 0-3 h, AP, pHBZ and pHBA were detected. After that, only pHBA, which is one of the major metabolites of BZ in human urine, could be detected in the urine by the present method. A method for quantitation of pHBA by MEKC is described here. The effects of acetonitrile and sodium dodecyl sulfate in the running buffer of MEKC on the separation of BZ and its metabolites are also reported.

alpha-Benzyl-N-methylphenethylamine (BNMPA), an impurity of illicit methamphetamine synthesis: II. Metabolism and urinary excretion (human).

Methamphetamine is a popular drug of abuse, which is readily synthesized in clandestine laboratories. Illicit synthesis results in the formation of various contaminants. Few impurities have been studied in vivo, and their metabolic fate is unknown. One such impurity is alpha-benzyl-N-methylphenethylamine (BNMPA). The detection of BNMPA or its metabolites in urine samples may provide a marker of use of illicitly synthesized methamphetamine. Benzphetamine is structurally similar to BNMPA. Based on metabolic studies of benzphetamine, we predicted the four major metabolites of BNMPA to be the N-demethyl compound, diphenyl-2-propanone (DP2P), p-hydroxy-N-demethyl BNMPA, and p-hydroxy-BNMPA. One male volunteer ingested 5 mg BNMPA. Seventeen urine specimens were collected over 50 h post ingestion. These specimens were analyzed for BNMPA and its four predicted major metabolites by gas chromatography-mass spectrometry following beta-glucuronidase hydrolysis or acid hydrolysis, liquid-liquid extraction, and derivatization with heptafluorobutyric anhydride. Specimens were also analyzed without hydrolysis to determine the abundance of nonconjugated ("free") metabolites. Only trace amounts of BNMPA and its N-demethyl metabolites were detected, and maximum excretion was from 2 to 4 h post ingestion. In the nonhydrolyzed samples, the phenyl-OH metabolites were also present in only trace amounts. Maximum excretion of DP2P was at 2 h. Following either hydrolysis procedure, phenyl-OH-BNMPA and phenyl-OH-N-demethyl BNMPA were the major metabolites detected. Maximum excretion of these two metabolites occurred at 4 h. With the exception of the parent compound and the N-demethyl metabolite, excretion of metabolites was greater than the limit of detection of this procedure (2.5 ng/mL) up to 21 h post ingestion. Metabolites were detectable in sufficient quantities to serve as an adequate marker of illicit methamphetamine consumption within the preceding 24 h.

Localization of the epitope in methamphetamine and its antibody use for the detection of methamphetamine and benzphetamine by polarization fluoroimmunoassay.

An antibody was prepared, using a four carbon-bridged methamphetamine molecule as an immunogen in order to develop a polarization fluoroimmunoassay for urine screening of methamphetamine and benzphetamine. Also, its binding characteristics were investigated to locate epitope sites of methamphetamine. The study showed that the antibody was highly capable of eliciting a polarization fluoroimmunoassay response. However, the detection limit was much greater for benzphetamine (0.05 ppm) than for methamphetamine (0.2 ppm) and weakly antibody binding was found with methamphetamine. This difference in sensitivity may reflect the similarity of benzphetamine to the immunogen used to produce the antibody. Both benzphetamine and the immunogen have a tertiary amine attached to a carbon bridges whereas methamphetamine has only a secondary amine and amphetamine has a primary amine group. The difference of cross-reactivity data between phenylethylamine drugs and beta-hydroxyl phenylethylamine drugs indicates that the beta-carbon position have a major influence on the antibody interaction. Thus, the substitution of hydroxyl group on beta-carbon resulted in virtually no antibody affinity, even if a tertiary amine or secondary amine group was present in the molecule. This suggests that the beta-carbon chain plays a primary role as the epitope site with cooperative binding site of tertiary amine or secondary amine in alpha-carbon position. A hydroxyl group at the beta-carbon position plays an important inhibitory role to the antibody binding.

[Specific detection of urinary sympathomimetic amines for control of anti-doping by gas chromatography-mass spectroscopy]. / Détection spécifique par chromatographie gazeuse-spectrométrie de masse des amines sympathomimétiques urinaires dans le cadre des contrôles antidopage.

A specific, sensitive and reliable gas chromatography-mass spectrometry (GC-MS) technique for detection of sympathomimetic amines following urinary extraction is proposed. Amphetamine, phentermine, ephedrine, mephenorex, methylphenidate, benzphetamine, clobenzorex and internal standard (fenfluramine) are extracted from urines at pH 7.0 using elution by chloroform-isopropanol on C18 cartridges. Derivatization followed by GC-MS analysis allows identification of these drugs founded on relative retention times and mass spectra. The quantitation limit for derivatizable drugs was found to be 200 ng/ml and 500 ng/ml for underivatizable drugs.

The metabolism of 1-phenyl-2-(N-methyl-N-benzylamino)propane (benzphetamine) and 1-phenyl-2-(N-methyl-N-furfurylamino)propane (furfenorex) in man.

The metabolic fate of 1-phenyl-2-(N-methyl-N-benzylamino)propane (benzphetamine) and 1-phenyl-2-(N-methyl-N-furfurylamino)propane (furfenorex) in healthy volunteers has been investigated. Nine metabolites with traces of the unchanged drug were detected in human urine after oral administration of benzphetamine, and five metabolites were found following administration of furfenorex. The major metabolites were 1-(p-hydroxyphenyl)-2-(N-benzylamino)propane for benzphetamine and 1-phenyl-2-(N-methyl-N-gamma-valerolactonylamino)propane for furfenorex. In both cases, methamphetamine, amphetamine and their hydroxylated metabolites were also excreted as minor metabolites. Identified metabolites excreted in three days after administration of benzphetamine accounted for 30-44% of the dose and those excreted after administration of furfenorex, 31-46%.

The metabolism of 1-phenyl-2-(N-methyl-N-benzylamino)propane (benzphetamine) in vivo in the rat.

1. The metabolism of 1-phenyl-2-(N-methyl-N-benzylamino)propane (benzphetamine) was studied in vivo in the rat. 2. Nine metabolites were obtained from urine after oral administration of benzphetamine to rats. The major metabolite, identified as 1-(p-hydroxyphenyl)-2-(N-benzylamino)propane, was formed by aromatic hydroxylation and N-demethylation. One of the minor metabolites was methamphetamine, formed by N-debenzylation. 3. Metabolites excreted in three days after administration of the drug amounted to about 40% of the dose.