Impurity profiling of dexamphetamine sulfate by cyclodextrin-modified microemulsion electrokinetic chromatography.

A CD-modified microemulsion electrokinetic chromatography method has been developed and validated for dexamphetamine sulfate which allows the simultaneous determination of charged and uncharged impurities of the drug including the levorotary (R)-enantiomer. The optimized background electrolyte consisted of 1.5% w/w SDS, 0.5% w/w ethyl acetate, 3.5% w/w 1-butanol, 2.5% w/w 2-propanol and 92% w/w 50 mM sodium phosphate buffer, pH 3.0, containing 5.5% w/w sulfated ß-CD. Separations were performed in a 50.2/40 cm, 50 µm id fused silica capillary at a temperature of 20°C and an applied voltage of -14 kV. Carbamazepine was used as internal standard. The assay was validated in the range of 0.1-1.0% for the related substances and 0.1-5.0% for levoamphetamine based on a concentration of 3 mg/mL of dexamphetamine sulfate. The LOD of all analytes ranged between 0.05 and 0.2%. In commercial samples of dexamphetamine sulfate, levoamphetamine was found at concentrations between 3.2 and 3.8%, whereas none of the other impurities could be detected.

Involvement of CYP2D6 in the in vitro metabolism of amphetamine, two N-alkylamphetamines and their 4-methoxylated derivatives.

1. Amphetamine (AM) and five amphetamine derivatives, N-ethylamphetamine (NEA), N-butylamphetamine (NBA), 4-methoxyamphetamine (M-AM), 4-methoxy-N-ethylamphetamine (M-NEA) and 4-methoxy-N-butylamphetamine (M-NBA) were incubated with microsomal preparations from cells expressing human CYP2D6 to determine whether the enzyme was capable of catalyzing the direct ring oxidation of all substrates; the N-dealkylation of NEA, NBA, M-NEA and M-NBA; and the O-demethylation of M-AM, M-NEA and M-NBA. 2. None of the six compounds examined was N-dealkylated to any extent. 3. The only metabolites produced from AM, NEA and NBA were the corresponding ring 4-hydroxylated compounds, and the rates of formation were low. 4. All ring 4-methoxylated substrates were efficiently O-demethylated by CYP2D6 to their corresponding phenols. The size of the N-alkyl group influenced the rates of formation of these phenolamines. In contrast to reported findings with 2- and 3-methoxyamphetamines, none of the 4-methoxyamphetamines was ring-oxidized in the CYP2D6 enzyme system to 2- or 3-hydroxy-4-methoxyamphetamines or to dihydroxyamphetamines.

Stimulant medications decrease energy expenditure and physical activity in children with attention-deficit/hyperactivity disorder.

OBJECTIVE: To determine the effect of stimulant medications used to treat children with attention-deficit/hyperactivity disorder (AD/HD) on energy expenditure, fuel utilization, and physical activity. STUDY DESIGN: Energy expenditure and physical activity were measured, respectively, by room respiration calorimetry and microwave motion detectors in 31 children with AD/HD (26 boys and 5 girls; ages 6 to 12 years) both while they were receiving their prescribed stimulant medication and after the medication had been discontinued for at least 24 hours. Fuel utilization was calculated from calorimetry data. RESULTS: Total and awake energy expenditure including energy expended while doing schoolwork, riding a stationary bicycle, resting, and watching a movie were from 4% to 8% lower when the children were receiving their prescribed stimulant medication. Total and awake activity were also lower while they were receiving medication (16% to 22%) and accounted for the lower rates of energy expenditure. Sleeping metabolic rate, basal metabolic rate, and fuel utilization were unaffected by medication. CONCLUSIONS: Stimulant medications decrease physical activity, and hence, decrease the activity component of total daily energy expenditure in children with AD/HD.

A convenient derivatization method for gas chromatography/mass spectrometric determination of phenmetrazine in urine using 2,2,2-trichloroethyl chloroformate.

Phenmetrazine is a central nervous system stimulant currently used as an anorectic agent. The drug is abused and is reported to cause death from overdose. We describe a new derivatization method for phenmetrazine using 2,2,2-trichloroethyl chloroformate. Quantitation of urinary phenmetrazine can be easily achieved by using N-propylamphetamine as an internal standard. The phenmetrazine 2,2,2-trichloroethyl carbamate showed a molecular ion isotope cluster at m/z 351, 353, 355, and 357 (isotope effect of three chlorine atoms in the derivatized molecule) and other peaks at m/z 247, 245, 204, 114, and 70 in the electron ionization mass spectrometry, thus aiding in unambiguous identification. The underivatized phenmetrazine showed a relatively weaker molecular ion at m/z 177 and a base peak at m/z 71. The N-propylamphetamine 2,2,2-trichloroethyl carbamate (internal standard) showed a very weak molecular ion at m/z 351 and a base peak at m/z 260. Another strong characteristic peak at m/z 91 was also observed. The retention time of derivatized phenmetrazine (9.5 min) was substantially longer than the retention time of the underivatized molecule (2.5 min). Moreover, underivatized phenmetrazine showed poor peak shape (substantial tailing) while derivatized phenmetrazine had excellent chromatographic property. The within-run and between-run precisions of the assay were 1.9% and 3.2% at a urinary phenmetrazine concentration of 20 micrograms/mL. The assay was linear for urinary phenmetrazine concentration of 1 microgram/mL to 100 micrograms/mL with a detection limit of 0.5 microgram/mL.

Time-lapse changes of d- and l-enantiomers of racemic (dl)-ethylamphetamine in human urine.

The time-lapse changes of d- and i-forms in urine specimens collected in the 24 h after the oral dosing of two adult male subjects with 20 mg of racemic (dl)-ethylamphetamine (EAMP)-HCl were examined by high-performance liquid chromatography. The percentage of the excreted dose of nonmetabolized l-EAMP was larger than that of d-EAMP, and the percentage of the excreted dose of metabolized l-amphetamine (AMP) was smaller than that of d-AMP in both subjects, A and B. These differences were observed 4.5-24 h after administration of the drug. The changes in the l/d ratio of EAMP and AMP were not similar between subjects A and B, but the change in the total l/d ratio was nearly the same. The regression line for subject A was as follows: y = 0.021323x + 0.98399. The regression line for subject B was as follows: y = 0.020947x + 0.94893. These two regression lines suggested that the time lapsed after the administration to humans could be predicted. The total percent of the excreted doses of EAMP and AMP was 47.46% (the d-forms, 20.73%; the l-forms, 26.73%) for subject A and was 31.43% (the d-forms, 14.14%; the l-forms, 17.29%) for subject B. The l/d ratio was 1.29 for subject A and 1.22 for subject B, which was somewhat higher than that (1.01) of the dl-EAMP-HCl used.

Aromatic ring oxidation of N-n-butylamphetamine is enhanced in the rat by prior treatment with quinidine.

Prior administration of quinidine is known to reduce aromatic oxidation of amphetamine and its analog methoxyphenamine by inhibiting the cytochrome P450IID6 results in isozyme. In contrast, it is now shown that prior administration of quinidine results in a significant increase in the aromatic oxidation of N-n-butylamphetamine in rat, suggesting that the P450IID6 isozyme is not involved in this metabolic reaction.

Cardiovascular effects of two potential beta-hydroxylase inhibitors.

The purpose of this study was to evaluate the cardiovascular effects of two potential beta-hydroxylase inhibitors, a dithiocarbamate and a thiourea derivative of d-amphetamine, 1 and 2, respectively. These compounds when given intravenously elicited a significant dose-dependent depressor effect in normotensive rats and dogs anesthetized with chloralose-urethane. The hypotensive effect of these compounds appears to be the result of a combination of two action components: (a) the major component, which can be blocked by atropine, exerted through the parasympathetic division and (b) a decreased availability of sympathetic neurotransmitter at the synaptic cleft. A CNS-stimulant effect of these compounds is suggested by their ability to reduce hexobarbital sleeping time in mice and their analeptic effect in all anesthetized animals employed.

Amphetamine in rat brain after intraperitoneal injection of N-alkylated analogues.

Three N-alkylated analogues of amphetamine were administered intraperitoneally to male Sprague-Dawley rats and whole brain levels of amphetamine (AM) and the N-alkyl analogue were determined one hour after injection of the N-alkylated compounds. The drugs administered were the N-2-cyanoethyl-(I) (fenproporex), the N-3-chloropropyl-(II) (mefenorex) and the N-n-propyl-(III) derivatives of AM: the first two of these are used clinically as anorexiants, and the latter has been used extensively to study aspects of metabolism of AM-like compounds. Analysis of AM, I, II and III was performed using electron-capture gas chromatography with a capillary column after reaction of compounds with pentafluorobenzoyl chloride under aqueous conditions. In a second comparative study, equimolar doses (0.05 mMole/kg) of I or AM were administered intraperitoneally to the rats and brain levels determined after one hour. Results indicate extensive N-dealkylation occurs for compounds I, II and III in the rat.

In vivo and in vitro o-methylation of 1-(3,4-dihydroxyphenyl)-2-(n-propyl-amino) propane - an intermediate in N-(N-propyl) amphetamine metabolism.

In vitro metabolism of 1-(3,4-dihydroxyphenyl)-2-(n-propylamino)-propane (Id) by rat liver cytosol in the presence of S-adenosyl-L-methyl-methionine produced almost quantitatively the corresponding 3-methoxy metabolite. 1-(4-Hydroxy-3-methoxyphenyl)-2-(n-propylamino) propane was also recovered from the urine of rats dosed with the catecholamine (Id).

Structure-activity relationships among some d-N-alkylated amphetamines.

d-N-Alkylated amphetamines were synthesized in a series up to and including d-N-butylamphetamine and potencies of these compounds were compared in (1) rhesus monkeys allowed to respond for intravenous infusions of the drugs, (2) rats allowed to drink a milk solution for 15 minutes each day and (3) isolated, spontaneously beating guinea-pig atria. In the self-administration procedure, d-amphetamine (A), d-N-methylamphetamine (NMA), and d-N-ethylamphetamine (NEA) were self-administered above saline levels at two or more doses by all animals. For these three drugs, maximal response rates were found at similar doses in all animals. However, maximal rates were generally higher in animals maintained on pentobarbital than in animals maintained on cocaine under control conditions. d-N-propylamphetamine (NPA) was self-administered above saline levels by three of four animals at one or more doses. Maximal response rates for NPA were about 1/2 of that of A, NMA and NEA, and the dose-response curve was shifted to the right of these compounds by about 4 times. d-N-butylamphetamine (NBA) maintained responding above saline levels at two doses in only one of three animals. In rats, all of the compounds decreased milk intake in a dose-related manner. A, NMA and NEA were equipotent in disrupting intake, while NPA and NBA were, respectively, 1/4 and 1/6 as potent as the shorter-chain compounds. With the exception of NBA, all compounds increased the rate of beating of the guinea-pig atrium over the range of concentrations tested. In general, for substituents larger than ethyl, potency of d-N-alkylated amphetamines was inversely related to N-alkyl length.

Long-term behavioral changes in the rhesus monkey after multiple daily injections of d-methylamphetamine.

The effects of multiple daily injections of d-methylamphetamine on spaced lever-press responding were investigated in the rhesus monkey. Responding was measured after single injections of d-mehtylamphetamine (0.0625-1.0 mg/kg) both before and after the monkeys were maintained on a multiple daily d-methylamphetamine injection regimen in doses ranging from 0.5 to 16 mg/kg/day. Tolerance developed to the suppressant effects of this drug on responding. As evidenced by a shift in the dose-response curve measuring number of responses and number of reinforcements per session, this tolerance persisted for at least three months after cessation of the multiple drug injection regimen. In order for tolerance to develop, it was necessary for the animals to perform the task while maintained on drug. When saline was substituted for d-methylamphetamine, no evidence of a withdrawal syndrome was seen.