Methamphetamine Metabolites Research Articles

Identification of Methamphetamine and Amphetamine in Drug Abuse Urine Sample using Multi-drug Rapid Test and Gas Chromatography- Mass Spectrometry (GC-MS) at the Centre of Drug Testing Laboratory, National Narcotics Board Republic of Indonesia

Introduction: Drug abuse is a real threat that threatens the Indonesian nation in the form of non-military threats. The second most abused illicit substance in the world is methamphetamine (MA). Most of the methamphetamine users in the world live in East and Southeast Asia. Methamphetamine is a powerful stimulant of the central nervous system (CNS) that can make someone feel an increase in sympathomimetic. Methods: First, the preliminary test was conducted using a BioCare multi-drug rapid test and a specific methamphetamine rapid test that works based on the Lateral Flow Assay (LFA) principle. Second, the sample preparation used Liquid-Liquid Extraction (LLE). Third, GC-MS confirmed the test using GCMS Shimadzu-QP2020NX-GC-2030 (HP-5MS column, Length 30 m, diameter 0.25 m, film 0.25). Result and Discussion: The Preliminary test shows that the urine is positive and contains methamphetamine and amphetamine. Based on the sample chromatogram, There are 2 peaks, which are methamphetamine and amphetamine compounds it can be seen by the retention time is 5.560 minutes (methamphetamine) a similarity index of 98% to methamphetamine compounds, and 4.885 minutes (amphetamine) with a similarity index of 96% to amphetamine compounds. Conclusion: The urine samples with code 210DGB from methamphetamine users were detected due to the presence of methamphetamine and amphetamine, which is the metabolite of methamphetamine.

Illicit drug consumption estimated using wastewater analysis and compared by settlement size in New Zealand

Estimation of consumption of illicit drugs by wastewater-based epidemiology provides estimates of community drug-use patterns. This study describes monitoring data of three illicit drugs in New Zealand using wastewater-based epidemiology.Wastewater samples were collected at monthly intervals for larger (population ~ 50,000+) cities or in smaller towns where more data was required by authorities. In other smaller towns, samples were collected every 2 months. Samples were extracted and analysed for parent compounds and metabolites of methamphetamine, MDMA, cocaine, heroin and fentanyl consumption using solid-phase extraction followed by liquid chromatography coupled with tandem-mass spectrometry (LC-MS/MS) detection. Back calculations were performed to estimate the consumption of each drug in each catchment area.Methamphetamine was the drug measured with the highest estimated mean consumption rates (724 mg/1000 people per day) in New Zealand. North Island small urban settlements had the highest estimated mean methamphetamine consumption rates (1259 mg/1000 people/day).Cocaine had the lowest estimated consumption rates (9.4 mg/1000 people/day). The highest estimated mean cocaine consumption rate was in North Island major urban settlements (24.4 mg/1000 people/day).Major urban settlements had the highest estimated mean MDMA (420 mg/1000 people/day) and cocaine consumption rates (18.8 mg/1000 people/day). South Island medium urban settlements had unexpectedly high estimated mean consumption rates of MDMA (533 mg/1000 people/day) and cocaine (17.0 mg/1000 people/day). The higher-than-expected estimated cocaine consumption was from one medium urban settlement that is also a popular tourist destination in the South Island.Heroin biomarkers were not detected at any locations, and fentanyl was detected around or below the limit of reporting.This research provides information for appropriate responses for improved social and health investment to support social services associated with illicit drug consumption.

Pholedrine is a marker of direct disposal of methamphetamine

Consumption of methamphetamine has primarily been estimated in wastewater-based epidemiology by measuring the parent compound. However, this could lead to overestimation when methamphetamine is directly disposed into the sewer system. In this respect, it would be advantageous to measure a specific metabolite of methamphetamine instead. We identified 4-hydroxymethamphetamine (pholedrine) as a potential marker. Stability experiments were performed in both filtered and unfiltered wastewater. Correlations with relative loads in wastewater were used to establish its potential as a marker of direct disposal of methamphetamine, or even as a wastewater-based epidemiology biomarker of methamphetamine consumption. This study then investigated the use of pholedrine in combination with methamphetamine to better detect direct disposal events and its potential as a marker of methamphetamine consumption. Examples from both South Australia and New Zealand exemplify the use of pholedrine to identify potential instances of direct disposal of methamphetamine.

Accidental death via intravaginal absorption of methamphetamine

In this paper a drug fatality that involved an unintended drug delivery route is described. The decedent, a 23-year-old female in custody in a county jail on suspicion of a felony drug offense, was discovered in a holding cell unconscious and unresponsive. Following unsuccessful cardiopulmonary resuscitation attempts she was pronounced dead at the scene. At autopsy a wad of multiple small loosely wrapped plastic packages held together with another layer of clear plastic was found in the decedent's vagina. The smaller plastic packages contained an off-white pasty substance that was later identified as methamphetamine. Toxicological testing of specimens collected during autopsy revealed methamphetamine in the decedent's subclavian blood, vitreous fluid, and urine at extremely high concentrations (42.6, 20.1, and 771mg/L, respectively). Amphetamine, the active metabolite of methamphetamine, was also present in the subclavian blood, vitreous fluid, and urine at significant concentrations (1.3, 0.5, and 20.4mg/L, respectively). The cause of death was attributed to toxic effects of methamphetamine and the manner of death was ruled accidental. This report suggests that lethal concentrations of methamphetamine may be distributed to the systemic circulation via intravaginal absorption.

175 Structure-based engineering to generate high-affinity immunotherapy for the drug of abuse

Methamphetamine (METH) abuse is a major threat in the USA and worldwide without any FDA approved medications. Anti-METH antibody antagonists block or slow the rate of METH entry into the brain and have shown efficacy in preclinical studies (Peterson, Laurenzana, Atchley, Hendrickson, & Owens, 2008). A key determinant of the antibody’s efficacy is its affinity for METH and we attempted to enhance the efficacy by designing mutations to alter the shape or the electrostatic character of the binding pocket. Towards this goal, we developed a single chain anti-METH antibody fragment (scFv6H4) from a parent IgG (1). The crystal structure of scFv-6H4 in complex with METH was determined (Celikel, Peterson, Owens, & Varughese, 2009). Based on its elucidated binding interactions, we designed point mutations in the binding pocket to improve its affinity for METH and amphetamine (AMP), the active metabolite of METH. The mutants, scFv-S93T,-I37 M and -Y34 M were cloned, expressed in yeast and tested for affinity against METH and AMP. Two mutants showed enhanced binding affinity for METH: scFv-I37 M by 1.3-fold and scFv-S93T by 2.6-fold. Additionally, all the mutants showed increase in affinity for AMP: scFv-I37 M by 56-fold, scFv-S93T by 17-fold and scFvY34 M by 5-fold. Crystal structure for one of the high-affinity mutant, scFv-S93T, in complex with METH was determined (Figure 1). Binding pocket of the mutant is more hydrophobic in comparison with the wild type. ScFv-6H4 binds METH in a deep pocket containing two water molecules. The substitution of a serine residue by a threonine leads to the expulsion of a water molecule (Figure 2), relieving some unfavorable contacts between the hydrocarbon atoms of METH and the water molecule and increasing the affinity to sub-nanomolar range. Therefore, the present study shows that efficacy could be enhanced by altering the hydrophobicity or the shape of the binding pocket.

Anticancer and Anti-inflammatory Effects of Cysteine Metabolites of the Green Tea Polyphenol, (−)-Epigallocatechin-3-gallate

(-)-Epigallocatechin-3-gallate (EGCG) has been shown to have cancer preventive activity in vitro and in vivo. We have previously shown that EGCG can undergo conjugation to cysteine to form 2'-cysteinyl-EGCG and 2''-cysteinyl-EGCG. Studies of thiol-conjugated metabolites of methamphetamine indicate that such metabolites are not detoxified but retain biological activity. Here, we examined the growth inhibitory, pro-oxidant, and anti-inflammatory activities of the cysteine metabolites of EGCG. Both compounds dose-dependently inhibited the growth of colon cancer and intestinal cell lines. Both metabolites prevented aberrant arachidonic acid release and nitric oxide production by lipopolysaccharide-stimulated RAW264.7 cells. Under cell culture conditions, 2''-cysteinyl-EGCG produced H2O2 at a faster rate than EGCG. The results of the present study show that cysteine conjugates of EGCG retain the growth inhibitory, anti-inflammatory, and pro-oxidant activities of EGCG in vitro and may play a role in disease prevention in vivo. These results remain to be confirmed in vivo.

Determination of 4-Hydroxy-3-methoxymethamphetamine as a Metabolite of Methamphetamine in Rats and Human Liver Microsomes Using Gas Chromatography-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry

The aim of this study was to determine whether methamphetamine (MA) is metabolized to 4-hydroxy-3-methoxymethamphetamine (HMMA), which is known as the main metabolite of 3,4-methylenedioxymethamphetamine (MDMA). After MA was intravenously administered to rats, the plasma, urine, and bile were collected periodically. HMMA together with MA and its main metabolites, amphetamine and 4-hydroxymethamphetamine, were detected in the rat plasma, urine, and bile by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. In addition, HMMA was produced when MA was incubated with human liver microsomes. HMMA may be produced as a metabolite of MA when humans have consumed MA, although the amount of HMMA would be small compared with that of MA, amphetamine, or 4-hydroxymethamphetamine. The results of the present study will be helpful in determining the type of drug used.

Direct Analysis of Conjugate Metabolites of Methamphetamine, 3,4-Methylenedioxymethamphetamine, and Their Designer Drugs in Biological Fluids

This review summarizes technical improvements in the forensic analysis for conjugate metabolites of methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA) and their designer drugs. The improvements include the establishments of liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) in combination with the pretreatment methods. The direct determination techniques for the conjugates proved to be effective in rapidly and accurately examining many biological specimens without annoying hydrolysis. The metabolism of MA and MDMA, with attention to the phase II metabolism o ft he major hydroxy metabolites, is also discussed based on concentration data in biological specimens obtained from drug abusers.

Development of a micropulverized extraction method for rapid toxicological analysis of methamphetamine in hair

We developed a rapid sample preparation method for the toxicological analysis of methamphetamine and amphetamine (the major metabolite of methamphetamine) in human hair by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), to facilitate fast screening and quantitation. Two milligrams of hair were mechanically micropulverized for 5 min in a 2-ml plastic tube together with 100 μl of an aqueous solvent containing 10% acetonitrile, 100 mM trifluoroacetic acid and the corresponding deuterium analogues as internal standards. The pulverizing highly disintegrated the hair components, simultaneously allowing the extraction of any drugs present in the hair. After filtering the suspension with a membrane-filter unit, the clear filtrate was directly analyzed by HPLC–MS/MS. No evaporation processes were required for sample preparation. Method optimization and validation study were carried out using real-case specimens and fortified samples in which the drugs had been artificially absorbed, respectively. Concentration ranges for quantitation were 0.040–125 and 0.040–25 ng/mg for methamphetamine and amphetamine, respectively. Real-case specimens were analyzed by the method presented here and by conventional ones to verify the applicability of our method to real-world analysis. Our method took less than 30 min for a set of chromatograms to be obtained from a washed hair sample.

メタンフェタミン摂取者尿中のｐ‐ヒドロキシメタンフェタミン抱合体濃度およびその加水分解条件の検討

The urinary concentrations of the main metabolites of methamphetamine (MA), specifically p-hydroxymethamphetamine-sulfate (p-OHMA-Sul) and p-hydroxymethamphetamine-glucuronide (p-OHMA-Gul) have been directly measured in MA users using an optimized LC-ESI MS method. The concentrations of the main metabolites and unchanged MA in 50 MA users' urine ranged from 0.02 to 21.7 μg/ml for p-OHMA-Sul, from <0.02 to 2.43 μg/ml for p-OHMA-Glu, from <0.005 to 1.33 μg/ml for p-hydroxymethamphetamine (p-OHMA), from 0.01 to 6.79 μg/ml for amphetamine (AP) and from 0.04 to 133 μg/ml for MA, and the ratios of sulfate to glucuronide (S/G ratios) ranged from 2.2 to 37.1 (13.8±8.1). These results demonstrate that the majority of p-OHMA is eliminated as its conjugate and the sulfation is quantitatively more important than glucuronidation for the conjugation of p-OHMA in humans. The urinary concentration time-dependency in two MA users have also revealed that the conjugates were mostly excreted in urine within 3 days post-intake. Both the acid and enzymatic hydrolysis conditions for p-OHMA conjugates were also optimized using the authentic standards of p-OHMA-Sul and p-OHMA-Gul. In the acid hydrolysis, the hydrolysis rate of p-OHMA-Sul was faster than p-OHMA-Glu, and a 3-h incubation with 1.2 mol/l HCl at 98 degrees Celsius gave complete hydrolysis of p-OHMA-Sul and p-OHMA-Glu. On the other hand, the enzymatic hydrolysis of two conjugates with Sulfatase Type H-1 from Helix pomatia required 6 hours for completion.

A public health response to the methamphetamine epidemic: the implementation of contingency management to treat methamphetamine dependence

BackgroundIn response to increases in methamphatemine-associated sexually transmitted diseases, the San Francisco Department of Public Health implemented a contingency management (CM) field program called the Positive Reinforcement Opportunity Project (PROP).MethodsMethamphetamine-using men who have sex with men (MSM) in San Francisco qualified for PROP following expressed interest in the program, provision of an observed urine sample that tested positive for methamphetamine metabolites and self-report of recent methamphetamine use. For 12 weeks, PROP participants provided observed urine samples on Mondays, Wednesdays and Fridays and received vouchers of increasing value for each consecutive sample that tested negative to metabolites of methamphetamine. Vouchers were exchanged for goods and services that promoted a healthy lifestyle. No cash was provided. Primary outcomes included acceptability (number of enrollments/time), impact (clinical response to treatment and cost-effectiveness as cost per patient treated).ResultsEnrollment in PROP was brisk indicating its acceptability. During the first 10 months of operation, 143 men sought treatment and of these 77.6% were HIV-infected. Of those screened, 111 began CM treatment and averaged 15 (42%) methamphetamine-free urine samples out of a possible 36 samples during the 12-week treatment period; 60% completed 4 weeks of treatment; 48% 8 weeks and 30% 12 weeks. Across all participants, an average of $159 (SD = $165) in vouchers or 35.1% of the maximum possible ($453) was provided for these participants. The average cost per participant of the 143 treated was $800.ConclusionClinical responses to CM in PROP were similar to CM delivered in drug treatment programs, supporting the adaptability and effectiveness of CM to non-traditional drug treatment settings. Costs were reasonable and less than or comparable to other methamphetamine outpatient treatment programs. Further expansion of programs like PROP could address the increasing need for acceptable, feasible and cost-effective methamphetamine treatment in this group with exceptionally high rates of HIV-infection.

Urinary excretion of the main metabolites of methamphetamine, including p-hydroxymethamphetamine-sulfate and p-hydroxymethamphetamine-glucuronide, in humans and rats

The urinary concentrations of the main metabolites of methamphetamine (MA), specifically p-hydroxymethamphetamine-sulfate ( p-OHMA-Sul) and p-hydroxymethamphetamine-glucuronide ( p-OHMA-Glu), were directly measured in MA users and rats using an optimized LC-ESI MS method. The concentrations of the two conjugates in 50 MA human users’ urine ranged from 0.09 to 88.6 µM (0.02–21.7 µg ml−1) for p-OHMA-Sul and from <0.05 to 7.13 µM (<0.02–2.43 µg ml−1) for p-OHMA-Glu; the ratios of sulfate to glucuronide (S/G ratios) ranged from 2.2 to 37.1 (13.8 ± 8.1). The results demonstrate that the sulfation is quantitatively more important than glucuronidation for the conjugation of p-OHMA in humans. The urinary concentration time-dependency in two MA users also revealed that the conjugates were mostly excreted in urine within 3 days post-intake. In contrast, in rat, almost all of the conjugated p-OHMA (>99%) was excreted as the glucuronide in urine. These findings confirm that a large species variation exists in the conjugation of p-OHMA between humans and rats.

Behavioral treatment approaches for methamphetamine dependence and HIV-related sexual risk behaviors among urban gay and bisexual men

Background: Methamphetamine-dependent gay and bisexual men (GBM) are at high risk for HIV transmission, largely due to drug-associated sexual risk behaviors. This project evaluated the efficacy of four behavioral drug abuse treatments for reducing methamphetamine use and sexual risk behaviors among this population. Methods: In this randomized controlled trial, 162 methamphetamine-dependent (SCID-verified) GBM in Los Angeles County were randomly assigned to one of four treatment conditions for 16 weeks: standard cognitive behavioral therapy (CBT, n = 40), contingency management (CM, n = 42), combined cognitive behavioral therapy and contingency management (CBT + CM, n = 40), and a culturally tailored cognitive behavioral therapy (GCBT, n = 40). Stimulant use was assessed thrice-weekly during treatment using urine drug screens (48 measures). Sexual risk behaviors were monitored monthly (four measures). Follow-up assessments were conducted at 6 (80.0%) and 12 months (79.9%). Results: Statistically significant differences in retention ( F(3,158) = 3.78, p < .02), in longest period of consecutive urine samples negative for methamphetamine metabolites ( F(3,158) = 11.80, p < .001), and in the Treatment Effectiveness Score were observed by condition during treatment ( F(3,158) = 7.35, p < .001) with post hoc analyses showing the CM and CBT + CM conditions to perform better than standard CBT. GEE modeling results showed GCBT significantly reduced unprotected receptive anal intercourse (URAI) during the first 4 weeks of treatment ( X 2 = 6.75, p < .01). During treatment between-group differences disappeared at follow-up with overall reductions in outcomes sustained to 1-year. Conclusions: Among high-risk methamphetamine-dependent GBM, drug abuse treatments produced significant reductions in methamphetamine use and sexual risk behaviors. Drug abuse treatments merit consideration as a primary HIV prevention strategy for this population.

The use of cyclohexanone as a “derivatizing” reagent for the GC–MS detection of amphetamines and ephedrines in seizures and the urine

A GC–MS method has been developed for the detection of amphetamine, methamphetamine, and the ephedrines, in seizures and the urine, based on on-GC condensation (derivatization) with cyclohexanone. The method is simple: the dried seizure material or the urine extract was mixed with cyclohexanone and injected into the GC–MS. The method was found to be superior to the methods based on acyl and trimethylsilyl (TMS) derivatization. Unlike for the acyl and TMS derivatives, the molecular and fragment ions of the cyclohexanone condensation products (cyclohexanone derivatives) were of substantial abundance, a useful property in unambiguous compound characterization. Furthermore, the high stability of the “derivatizing” reagent, cyclohexanone, compared with acyl and TMS derivatizing reagents, is a useful property in method development. The present method has proved selective and, tentatively, sensitive enough in the following areas (where methods based on acyl and TMS derivatization, as tested in this laboratory, have failed): (a) detection of amphetamine as a metabolite of methamphetamine; (b) detection of norpseudoephedrine as a metabolite of pseudoephedrine; (c) detection of amphetamine as an impurity of methamphetamine; (d) detection of cathine (norephedrine) as a constituent of Khat leaves; and (e) differentiation of Khat use from phenylpropanolamine use.

Genetic toxicity of methamphetamine in vitro and in human abusers.

Methamphetamine (METH) is a widely abused psychomotor stimulant. Although numerous studies have examined METH-induced neurotoxicity, its ability to produce genotoxic effects has not been evaluated. In this article, we report on the genotoxicity of METH in vitro and in human METH abusers. METH induced his(+) revertants in Salmonella typhimurium strains TA98 and TA100, and increased the frequency of hprt mutants, micronuclei, and sister chromatid exchange (SCE) in cultured Chinese hamster ovary K1 (CHO-K1) cells. These METH-induced genotoxic effects were eliminated if METH exposure was conducted in the presence of rat liver S9, indicating that the genotoxicity was caused by METH, and not by metabolites of METH. In addition, reactive oxygen species (ROS) scavengers inhibited the METH-induced micronuclei in CHO-K1 cells. Further investigation with 76 human long-term METH abusers and 98 unexposed controls demonstrated that total METH exposure correlated with micronucleus and SCE frequencies in cultured lymphocytes. The results of this study indicate that METH is a genotoxic agent and that ROS may play a role in METH-induced genotoxicity.

Methamphetamine concentrations in fetal and maternal brain following prenatal exposure

Levels of methamphetamine in maternal striatum and whole fetal mouse brain were assessed at 0.5, 1, 2, and 4 h postinjection on gestational day 14 (GD14) following a single, subcutaneous injection of 40 mg/kg (+)-methamphetamine hydrochloride to pregnant mice. In the dams, striatal concentrations of methamphetamine peaked at 1 h postinjection, reaching levels of approximately 510 ng/mg protein. Amphetamine, the primary metabolite of methamphetamine, increased to 77 ng/mg protein at 2 h and remained elevated by 4 h postinjection. In the fetal brain, peak methamphetamine concentrations of approximately 122 ng/mg protein were attained at 1 h. Amphetamine was only detectable in fetal brain at 2 and 4 h postinjection. Regional analysis of methamphetamine levels in fetal striatum, cortex, and brainstem revealed that the drug was not uniformly distributed. Maternal administration of methamphetamine results in fetal brain drug concentrations, which approximate those reported in human infants whose mother abused methamphetamine. This dosage regimen, therefore, serves as an appropriate animal model for assessing the potential risks to human offspring exposed to methamphetamine in utero.

MDA-MDMA concentrations in urine specimens.

Urine specimens collected from active-duty U.S. Army personnel were submitted for analysis to the Tripler Army Medical Center, Forensic Toxicology Drug Testing Laboratory as part of the random drug testing program. During an 18-month drug-screening period, 34 specimens tested positive for amphetamines with the Roche Abuscreen Radioimmunoassay for Methamphetamine (High Specificity); based on gas chromatographic-mass spectrometric (GC-MS) analysis, the presence of 3,4-methylenedioxymethamphetamine (MDMA) was suspected. These samples were subsequently submitted to the Division of Forensic Toxicology, Office of the Armed Forces Medical Examiner, Armed Forces Institute of Pathology for further testing. All 34 samples screened positive using both the Abbott TDx Amphetamine/ Methamphetamine II assay and the Amphetamine class assay. Confirmation and quantitation by GC-MS revealed the presence of both MDMA and 3,4-methylenedioxyamphetamine (MDA) in all samples. The MDMA concentrations ranged from 0.38 to 96.2 mg/L (mean, 13.4 mg/L) and the MDA concentrations ranged from 0.15 to 8.6 mg/L (mean, 1.6 mg/L). The mean ratio of MDA, the N-demethylation metabolite of MDMA, to MDMA was 0.15, similar to the ratio of amphetamine, the N-demethylation metabolite of methamphetamine, to methamphetamine of 0.10. The presence of MDA in urine specimens at a concentration approximately 10-15% that of the MDMA present is consistent with MDMA metabolism, which may be indicative of the use of MDMA only, as compared with the combined use of both drugs.

Modification of behavioral responses to methamphetamine evoked by the stimulant's metabolite p-hydroxynorephedrine in rats.

The central effect of p-hydroxynorephedrine (OH-NE), one of the p-hydroxylated metabolites of methamphetamine (MAP) and amphetamine (AMP), was investigated in rats. Locomotion and stereotypy were examined after SC injections of 0.5-5 mg/kg of MAP or 0.02-0.5 mg/kg of apomorphine (APO) in animals treated with either saline or 5-50 mg/kg of OH-NE IP 20 hr before behavioral assessment. The locomotor stimulating effect of both 0.5-2 mg/kg of MAP and 0.2 mg/kg of APO was enhanced by 5 mg/kg of OH-NE. On the other hand, 30 mg/kg of OH-NE severely suppressed the stimulating effect of MAP but had no influence on that induced by 0.2 mg/kg of APO. The stereotypy induced by 5 mg/kg of MAP or 0.5 mg/kg of APO was enhanced and prolonged in the OH-NE-treated rats. Subsequently, examinations were performed to determine whether OH-NE had any effect on the dopaminergic mechanism. Hypomotility induced by 0.02 mg/kg of APO was alleviated by 5 mg/kg of OH-NE, but was aggravated by 30 mg/kg. These results suggest that OH-NE administered prior to SC injections of MAP or APO influences their behavioral effects via the dopaminergic mechanism. The possibility that other neural mechanisms may be involved in this OH-NE-induced behavioral modification is also discussed.

Simultaneous quantitative analysis of methamphetamine and 4-hydroxymethamphetamine in body fluids by gas chromatography/mass spectrometry.

Pentodeuterated 4-hydroxymethamphetamine (HMAMP-d5), 1-(4-hydroxyphenyl)-2-(methyl-d3-amino)-propane-1,2-d2 of high quality was prepared and proved to be a most useful internal standard for quantitative analyses of 4-hydroxymethamphetamine (HMAMP), a main metabolite of methamphetamine (MAMP). Highly reliable results were obtained by gas chromatography/mass spectrometry. Simultaneously we determined also MAMP, amphetamine (AMP), 4-hydroxyamphetamine (HAMP), and HMAMP in body fluids, using two internal standards, HMAMP-d5 for HMAMP and HAMP and pentodeuterated methamphetamine, 1-phenyl-2-(methyl-d3-amino)-propane-1,2-d2, for MAMP and AMP.

Reversible inhibition of aromatic hydroxylation of methamphetamine in rat liver microsomal preparations pretreated with methamphetamine

The effects of a single and repeated administration of methamphetamine (MP) in vivo in rats on its own metabolism in vitro were investigated. In both cases, the p-hydroxylation of MP to p-hydroxymethamphetamine by a microsomal fraction from rat liver was inhibited for a period of 16 hr after the last injection of MP. This inhibition was diminished by dialysis of the microsomal preparations. In contrast, the reduced level of cytochrome P-450 in hepatic microsomes from rats pretreated with the SKF 525-A did not revert to the control value after dialysis. When microsomes were preincubated with N-hydroxymethamphetamine, which is the metabolite of MP and a potent substrate for the formation of a metabolic intermediate (MI) complex with cytochrome P-450, the content of the MI was increased and the MP-hydroxylation activity decreased in direct proportion to the length of the preincubation. These results suggest that the inhibition of MP-hydroxylation may be due to reduction of the level of cytochrome P-450 that accompanies the formation of the MI complex. Furthermore, it appears that the complex can be dissociated by dialysis.