Mechanisms Of Drug Incorporation Research Articles

First evidence of the incorporation of daprodustat and other hypoxia-inducible factor stabilizers into equine hair by passive transfer based on segmental quantitative analysis

Daprodustat is a hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) inhibitor and is used as an erythropoiesis stimulant for the treatment of anemia in humans. In general, administering daprodustat to horses will result in a lifetime ban from both equestrian sports and horseracing by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. To control the misuse/abuse of daprodustat, we conducted nasoesophageal administration of daprodustat (100 mg/day for 3 days) to three thoroughbred mares and the post-administration hair samples collected from the three horses over 6 months were analyzed to demonstrate the potential longer-term detection of daprodustat and its metabolites in hair compared with the detection times of daprodustat of 1 and 2 weeks in plasma and urine respectively. The results of the quantitative 2-cm segmental analysis showed that daprodustat was primarily localized in the proximal region (0–2 cm) at 0.375–0.463 pg/mg at 1 month post-administration. These drug bands were gradually spread out along the hair shaft at a rate consistent with the reported growth rate of horse mane hair (approximately 2.5 cm/month) over the following 6 months. In addition, to attain deeper insight into the mechanism of drug incorporation into hair, a total of 11 relevant parameters, including the actual PK parameters and simulated physicochemical and biopharmaceutical parameters for three HIF stabilizers (i.e., daprodustat, vadadustat, and IOX4), were investigated after normalization of the z-scores of all these parameters. Multiple regression analysis indicated that the major factors contributing to the incorporation of the three drugs into hair were their maximum plasma concentrations and lipophilicities, strongly suggesting that the three HIF stabilizers permeated from the bloodstream into the hair bulb via passive transfer with concentration gradients. This work is the first reported evidence showing the incorporation of HIF stabilizers into hair via passive transfer. In addition, cross-species comparison of drug incorporations into hair between daprodustat in horse and roxadustat in human was made in order to have a better understanding of the interactive interpretations about the analysis results obtained from different species. The above findings are not only useful and beneficial for the purpose of doping control but also provide a better understanding of the mechanism of drug incorporation into horse hair.

High Spatial-Resolution Matrix-Assisted Laser Desorption/Ionization-Ion Trap-Time-of-Flight Tandem Mass Spectrometry Imaging for Depicting Longitudinal and Transverse Distribution of Drugs Incorporated into Hair.

This study aims to achieve high spatial-resolution tandem mass spectrometry (MS/MS) imaging for depicting longitudinal and transverse distribution of drugs in hair, which can provide indispensable information for the proper interpretation of hair test results, including the mechanism of drug incorporation into hair. Two types of hair samples were obtained and analyzed: User's Hair, sampled from a volunteer who took an over-the-counter medicine containing methoxyphenamine (MOP), a nonregulated analogue of methamphetamine; and Soaked Hair, prepared by soaking blank hair in MOP solution. Longitudinal and transverse-sectioning of single hair shafts was accomplished by freeze-sectioning using customized microtomes. Vapor deposition of α-cyano-4-hydroxycinnamic acid provided the finest matrix layer (resolution <1 μm, 0.7-μm thickness), although it provided less effective ionization of MOP compared to aerosol spraying or a combination of both. Matrix-assisted laser desorption/ionization (MALDI)-ion trap (IT)-time-of-flight (TOF) MS/MS permitted the imaging of trace-level MOP in hair with a MS/MS window setting of ±0.02 Da and a spatial resolution setting at 5 or 10 μm. For Soaked Hair, localization of MOP in the peripheral part was clearly depicted, but no such biased distribution was observed in the transverse sections of User's Hair. MOP-positive bands generated corresponding to the time periods of MOP intake could be observed on the longitudinal sections of User's Hair. This method can provide forensically crucial information regarding hair analysis for drugs: drug incorporation mechanism into hair, discrimination of undesired surface contamination from endogenous incorporation of ingested drugs, and precise elucidation of drug-use history.

CONTROL OF PHYSICO-CHEMICAL PROPERTIES OF A LIPID NANOCONTAINER FOR DELIVERY OF DRUGS WITH LOW BIOAVAILABILITY

We have used two ways to control the physicochemical properties of liposomal nanocontainers for the delivery of fluoroquinolones: variation of the lipid composition of liposomes and selection of a functionalizing polymer based on chitosan mannose derivatives. The presence of anionic phospholipid cardiolipin in the liposome composition increases the loading efficiency by 5-20%. The mechanism of drug incorporation into membranes was studied, and the main binding sites were determined. The functionalization of the liposomal surface was carried out by chitosan mannose derivatives of various molecular weights, potentially providing targeted delivery to alveolar macrophages.

Hair Testing for Drugs in the Field of Forensics

Hair testing for drugs has been used extensively in the field of forensics since the 1990s as a means of obtaining firm evidence of drug ingestion. In addition to its longer detection windows, hair is the only specimen that can provide chronological information on individual drug use. Illicit drugs and hypnotics account for the majority of substances involved in crimes; they are usually analyzed to prove an addictive use or an exposure to drugs in drug-facilitated crimes. The mechanism of drug incorporation into hair has been intensively investigated to properly interpret the results of hair analysis. However, the exact mechanism remains under much discussion, despite the growing application of hair tests. Recently, the authors have applied matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging and sectional hair analysis of 1-mm segments using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for single-strand hair, to investigate the incorporation pathways of drugs into hair. Time-course changes in drug distribution along single-strand hair suggest that the incorporation of drugs occurs in two regions of the hair root, the hair bulb and the upper part of hair root, and suggest that incorporation from the hair bulb continues for about 2 weeks. Distribution profiles of different drugs in hair additionally revealed that the main incorporation pathway varies (i.e., via the hair bulb or the upper part of hair root) depending on the properties of the drug/metabolite. These findings should be taken into account upon discussing individual drug-use history based on the results of hair analysis.

Toxicological hair analysis: Pre-analytical, analytical and interpretive aspects.

Background and aims Hair analysis for drug detection is one of the widely accepted imperative techniques in the field of forensic toxicology. The current study was designed to investigate the efficacy of chromatography for detection of drugs of abuse in hair. Method A comprehensive review of articles from last two decades on hair analyses via PubMed and similar resources was performed. Issues concerning collection, decontamination and analytical techniques are summarised. Physiochemical nature of hair, mechanism of drug incorporation and its stability in hair are briefly discussed. Furthermore, various factors affecting results and interpretation are elucidated. Result A hair sample is chosen over traditional biological samples such blood, urine, saliva or tissues due to its inimitable ability to provide a longer time frame for drug detection. Its collection is almost non-invasive, less cumbersome and does not involve any specialised training/expertise. Recent advances in analytical technology have resulted in better sensitivity, reproducibility and accuracy, thus providing a new arena of scientific understanding and test interpretation. Conclusion Though recent studies have yielded many insights into drug binding and drug incorporation in hair, the major challenge in hair analysis lies in the interpretation of results, which may be affected by external contamination and thus lead to false-positives. Therefore, there is a need for more sensitive and selective analysis methods to be developed in order to minimise factors that induce the effect of melanin, age and so on, and this would certainly provide a new dimension to hair analysis and its applications.

Nails in Forensic Toxicology: An Update.

The analysis of nails as a keratinized matrix to detect drugs or illicit substances has been increasingly used in forensic and clinical toxicology as a complementary test, especially for the specific characteristics of stably accumulating substances for long periods of time. This allows a retrospective investigation of chronic drug abuse, monitoring continuous drug or pharmaceutical use, reveal in utero drug exposure or environmental exposures. We herein review the recent literature investigating drug incorporation mechanisms and drug detection in nails for forensic toxicological purposes. Mechanisms of drug incorporation have not yet been fully elucidated. However, some research has lately contributed to a better understanding of how substances are incorporated into nails, suggesting three potential mechanisms of drug incorporation: contamination from sweat, incorporation from nail bed and incorporation from germinal matrix. In addition, numerous methods dealing with the determination of drugs of abuse, medications and alcohol biomarkers in nails have been reported in studies over the years. The latter methods could find application in clinical and forensic toxicology. The studies herein reviewed point out how important it is to standardize and harmonize the methodologies (either pre-analytical or analytical) for nails analysis and the optimization of sampling as well as the development of proficiency testing programs and the determination of cut-off values.

Enhanced and Selective Antiproliferative Activity of Methotrexate-Functionalized-Nanocapsules to Human Breast Cancer Cells (MCF-7).

Methotrexate is a folic acid antagonist and its incorporation into nanoformulations is a promising strategy to increase the drug antiproliferative effect on human breast cancer cells by overexpressing folate receptors. To evaluate the efficiency and selectivity of nanoformulations containing methotrexate and its diethyl ester derivative, using two mechanisms of drug incorporation (encapsulation and surface functionalization) in the in vitro cellular uptake and antiproliferative activity in non-tumoral immortalized human keratinocytes (HaCaT) and in human breast carcinoma cells (MCF-7). Methotrexate and its diethyl ester derivative were incorporated into multiwall lipid-core nanocapsules with hydrodynamic diameters lower than 160 nm and higher drug incorporation efficiency. The nanoformulations were applied to semiconfluent HaCaT or MCF-7 cells. After 24 h, the nanocapsules were internalized into HaCaT and MCF-7 cells; however, no significant difference was observed between the nanoformulations in HaCaT (low expression of folate receptors), while they showed significantly higher cellular uptakes than the blank-nanoformulation in MCF-7, which was the highest uptakes observed for the drug functionalized-nanocapsules. No antiproliferative activity was observed in HaCaT culture, whereas drug-containing nanoformulations showed antiproliferative activity against MCF-7 cells. The effect was higher for drug-surface functionalized nanocapsules. In conclusion, methotrexate-functionalized-nanocapsules showed enhanced and selective antiproliferative activity to human breast cancer cells (MCF-7) being promising products for further in vivo pre-clinical evaluations.

Finding cannabinoids in hair does not prove cannabis consumption.

Hair analysis for cannabinoids is extensively applied in workplace drug testing and in child protection cases, although valid data on incorporation of the main analytical targets, ∆9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-THC (THC-COOH), into human hair is widely missing. Furthermore, ∆9-tetrahydrocannabinolic acid A (THCA-A), the biogenetic precursor of THC, is found in the hair of persons who solely handled cannabis material. In the light of the serious consequences of positive test results the mechanisms of drug incorporation into hair urgently need scientific evaluation. Here we show that neither THC nor THCA-A are incorporated into human hair in relevant amounts after systemic uptake. THC-COOH, which is considered an incontestable proof of THC uptake according to the current scientific doctrine, was found in hair, but was also present in older hair segments, which already grew before the oral THC intake and in sebum/sweat samples. Our studies show that all three cannabinoids can be present in hair of non-consuming individuals because of transfer through cannabis consumers, via their hands, their sebum/sweat, or cannabis smoke. This is of concern for e.g. child-custody cases as cannabinoid findings in a child’s hair may be caused by close contact to cannabis consumers rather than by inhalation of side-stream smoke.

Hair as an Alternative Matrix in Bioanalysis

Alternative matrices are steadily gaining recognition as biological samples for toxicological analyses. Hair presents many advantages over traditional matrices, such as urine and blood, since it provides retrospective information regarding drug exposure, can distinguish between chronic and acute or recent drug use by segmental analysis, is easy to obtain, and has considerable stability for long periods of time. For this reason, it has been employed in a wide variety of contexts, namely to evaluate workplace drug exposure, drug-facilitated sexual assault, pre-natal drug exposure, anti-doping control, pharmacological monitoring and alcohol abuse. In this article, issues concerning hair structure, collection, storage and analysis are reviewed. The mechanisms of drug incorporation into hair are briefly discussed. Analytical techniques for simultaneous drug quantification in hair are addressed. Finally, representative examples of drug quantification using hair are summarized, emphasizing its potentialities and limitations as an alternative biological matrix for toxicological analyses.

Incorporation of methamphetamine and amphetamine in human hair following controlled oral methamphetamine administration

BackgroundAlthough hair testing is well established for the assessment of past drug exposure, uncertainties persist about mechanisms of drug incorporation into hair and interpretation of results. The aim of this study was to administer methamphetamine (MAMP) under controlled conditions as a model drug to investigate drug incorporation into human hair. Material and methodsSeven volunteers with a history of stimulant use received 4×10mg (low) doses of sustained release S-(+)-MAMP HCl within 1 week, with weekly head hair samples collected by shaving. 3 weeks later, 4 of them received 4×20mg (high) doses. After extensive isopropanol/phosphate buffer washing of the hair, MAMP and its metabolite amphetamine (AMP) concentrations were determined in all weekly hair samples by LC–MS–MS in selected reaction monitoring mode with the undeca- and deca-deuterated drugs, respectively, as internal standards (LLOQ, 0.005ngmg−1). ResultsMAMP Tmax occurred from 1 to 2 weeks after both doses, with Cmax ranging from 0.6 to 3.5ngmg−1 after the low and 1.2 to 5.3ngmg−1 after the high MAMP doses. AMP Cmax in hair was 0.1–0.3ngmg−1 and 0.2–0.5ngmg−1, respectively, for low and high doses. Highly dose-related concentrations within subjects, but large variability between subjects were observed. MAMP concentrations were above the 0.2ngmg−1 cut-off for at least 2 weeks following administration of both low and high doses. The overall AMP/MAMP ratio ranged from 0.07 to 0.37 with a mean value of 0.15±0.07, and a median of 0.13. The percentage of MAMP and AMP removed with the washing procedure decreased with time after administration. A strong correlation was found between area under the curve of MAMP (r2=0.90, p=0.00) and AMP (r2=0.94, p=0.00) concentrations calculated for the 3-week period following administration and the total melanin concentration in hair. Significant correlations were observed also between Cmax and melanin. ConclusionsThis study demonstrated that despite large inter-individual differences, the incorporation of MAMP and AMP into hair is dose-related with much of the observed scatter of MAMP and AMP concentrations explained by melanin concentration in hair.

Hair analysis for Δ9-tetrahydrocannabinolic acid A—New insights into the mechanism of drug incorporation of cannabinoids into hair

Differentiation between external contamination and incorporation of drugs or their metabolites from inside the body via blood, sweat or sebum is a general issue in hair analysis and of high concern when interpreting analytical results. In hair analysis for cannabinoids the most common target is Δ9-tetrahydrocannabinol (THC), sometimes cannabidiol (CBD) and cannabinol (CBN) are determined additionally. After repeated external contamination by cannabis smoke these analytes are known to be found in hair even after performing multiple washing steps. A widely accepted strategy to unequivocally prove active cannabis consumption is the analysis of hair extracts for the oxidative metabolite 11-nor-9-carboxy-THC (THC-COOH). Although the acidic nature of this metabolite suggests a lower rate of incorporation into the hair matrix compared to THC, it is not fully understood up to now why hair concentrations of THC-COOH are generally found to be much lower (mostly <10 pg/mg) than the corresponding THC concentrations. Δ9-Tetrahydrocannabinolic acid A (THCA A) is the preliminary end product of the THC biosynthesis in the cannabis plant. Unlike THC it is non-psychoactive and can be regarded as a ‘precursor’ of THC being largely decarboxylated when heated or smoked. The presented work shows for the first time that THCA A is not only detectable in blood and urine of cannabis consumers but also in THC positive hair samples. A pilot experiment performed within this study showed that after oral intake of THCA A on a regular basis no relevant incorporation into hair occurred. It can be concluded that THCA A in hair almost exclusively derives from external contamination e.g. by side stream smoke. Elevated temperatures during the analytical procedure, particularly under alkaline conditions, can lead to decarboxylation of THCA A and accordingly increase THC concentrations in hair. Additionally, it has to be kept in mind that in hair samples tested positive for THCA A at least a part of the ‘non-artefact’ THC probably derives from external contamination as well, because in condensate of cannabis smoke both THC and THCA A are present in relevant amounts. External contamination by side stream smoke could therefore explain the great differences in THC and THC-COOH hair concentrations commonly found in cannabis users.

Study of carbonyl iron/poly(butylcyanoacrylate) (core/shell) particles as anticancer drug delivery systems: Loading and release properties

The aim of this study is to develop a detailed investigation of the capabilities of carbonyl iron/poly(butylcyanoacrylate) (core/shell) particles for the loading and release of 5-Fluorouracil and Ftorafur. The anionic polymerization procedure, used to obtain poly(alkylcyanoacrylate) nanoparticles for drug delivery, was followed in the synthesis of the composite particles, except that the polymerization medium was a carbonyl iron suspension. The influence of the two mechanisms of drug incorporation (entrapment in the polymeric network and surface adsorption) on the drug loading and release profiles were investigated by means of spectrophotometric and electrophoretic measurements. The optimum loading conditions were ascertained and used to perform drug release evaluations. Among the factors affecting drug loading, both pH and drug concentration were found to be the main determining ones. For both drugs, the release profile was found to be biphasic, since the drug adsorbed on the surface was released rather rapidly (close to 100% in 1 h), whereas the drug incorporated in the polymer matrix required between 10 and 20 h to be fully released. The kinetics of the drug release from the core/shell particles was mainly controlled by the pH of the release medium, the type of drug incorporation, and the amount of drug loaded.

Ftorafur loading and controlled release from poly(ethyl-2-cyanoacrylate) and poly(butylcyanoacrylate) nanospheres

In the present work, a method is described to prepare polymeric colloidal nanospheres, consisting of poly(ethyl-2-cyanoacrylate) (PE-2-CA) or poly(butylcyanoacrylate) (PBCA), loaded with the anticancer drug ftorafur. The method is based on the anionic polymerization procedure, often used in the synthesis of poly(alkylcyanoacrylate) nanospheres for drug delivery. A detailed investigation of the capabilities of both polymeric nanoparticles to load this drug is shown. The effect of synthesis residuals and degradation products on the absorbance of supernatants was considered in the loading and release measurement methodologies, because of their potential perturbing influence on the determination of ftorafur concentration in solution. We found the existence of two mechanisms of drug incorporation: absorption or entrapment in the polymeric network, and surface adsorption, detectable by means of zeta potential and spectrophotometric measurements. Among the factors affecting the drug incorporation to the polymer network, the type of polymer, the pH and the drug concentration are the main determining ones. Moreover, the acidity of the medium needs to be controlled in order to avoid the formation of macroaggregates of solids. The optimum loading conditions were used to perform ftorafur release evaluations from polymeric particles, and the influence of the mechanism of drug incorporation, the amount of drug loaded, and the type of polymer on the drug release were studied.

New trends in hair analysis and scientific demands on validation and technical notes

This review focuses on basic aspects of method development and validation of hair testing procedures. Quality assurance is a major issue in drug testing in hair resulting in new recommendations, validation procedures and inter-laboratory comparisons. Furthermore recent trends in research concerning hair analysis are discussed, namely mechanisms of drug incorporation and retention, novel analytical procedures (especially ones using liquid chromatography–mass spectrometry (LC–MS) and alternative sample preparation techniques like solid-phase microextraction (SPME)), the determination of THC-COOH in hair samples, hair testing in drug-facilitated crimes, enantioselective hair testing procedures and the importance of hair analysis in clinical trials. Hair testing in analytical toxicology is still an area in need of further research.

Hair as a Biological Indicator of Drug Use, Drug Abuse or Chronic Exposure to Environmental Toxicants

In recent years hair has become a fundamental biological specimen, alternative to the usual samples blood and urine, for drug testing in the fields of forensic toxicology, clinical toxicology and clinical chemistry. Moreover, hair-testing is now extensively used in workplace testing, as well as, on legal cases, historical research etc. This article reviews methodological and practical issues related to the application of hair as a biological indicator of drug use/abuse or of chronic exposure to environmental toxicants. Hair structure and the mechanisms of drug incorporation into it are commented. The usual preparation and extraction methods as well as the analytical techniques of hair samples are presented and commented on. The outcomes of hair analysis have been reviewed for the following categories: drugs of abuse (opiates, cocaine and related, amphetamines, cannabinoids), benzodiazepines, prescribed drugs, pesticides and organic pollutants, doping agents and other drugs or substances. Finally, the specific purpose of the hair testing is discussed along with the interpretation of hair analysis results regarding the limitations of the applied procedures.

Hair analysis for veterinary drug monitoring in livestock production

This review summarizes the basic information and applications concerning the use of hair analysis for the detection of misuse of therapeutic and anabolic agents in livestock animals. Hair biology, hair-shaft structure and the mechanisms of drug incorporation are described, considering the different factors which can affect the deposition. Sampling and extraction methods are reviewed with special attention to the particularities of this matrix, while the use of different analytical techniques is discussed, taking into account the concentration and the sensitivity required for drug detection. Advantages, drawbacks, promising prospects and possible applications of this technique in the future are also discussed.

HAIR ANALYSIS FOR DRUGS OF ABUSE. PLAUSIBILITY OF INTERPRETATION

Over more than 20 years hair analysis for drugs has been gaining increasing attention and recognition in various toxicological fields as preemployment and employment screening, forensic sciences, doping control of banned substances, clinical diagnostics in health problems. Hair analysis for drugs can expand the toxicological examination of conventional materials and thus contribute with additional important information to the complex evaluation of a certain case. Hair is a unique material for the retrospective investigation of chronic drug consumption, intentional or unintentional chronic poisoning in criminal cases, gestational drug exposure or environmental exposure to pollutants and adulterants and with specific ultrasensitive procedures allow to demonstrate even a previous single dose administration in a very low amount. Assuming the ideal hair steady and uniform growth, segmental hair analysis can provide the information about the time course of the substance use or exposure. However, the physiological background of hair growth, mechanisms of drug incorporation are not simple, not yet understood in full details and need not be evaluated exactly in all cases. The hair sampling, storage, sample preparation, analytical performance themselves are also very important for final results. Different laboratory attitudes can produce different results. The full information on circumstances of the case examined must be taken into account during interpretation. The pitfalls in hair analysis should be known and avoided to assure the responsible and correct interpretation of laboratory results adequate to an individual case.

Hair Drug Testing Routine in Many Clinical and Forensic Applications

Hair Drug Testing Routine in Many Clinical and Forensic Applications

Codeine Concentration in Hair after Oral Administration Is Dependent on Melanin Content

Analysis of drugs in hair has been used on a qualitative basis to estimate earlier exposure to drugs. Clinical applications are rare because of the lack of dose-response relationships in the studies performed to date, and questions remain regarding the mechanisms of drug incorporation into hair. Several human studies have shown differences in drug accumulation between pigmented and nonpigmented hair. However, the melanin concentration in hair was not determined and correlated to the amount of drug incorporated. Nine human subjects were given codeine as a single oral dose, and plasma codeine concentrations were determined for 24 h, using gas chromatography-mass spectrometry. Hair samples were obtained weekly for a month. Total melanin, eumelanin, and codeine were measured quantitatively in hair samples by spectrophotometry, HPLC, and gas chromatography-mass spectrometry, respectively. There was an exponential relationship between codeine and melanin concentrations in hair, (r(2) = 0.95 with total melanin and r(2) = 0.83 with eumelanin). After normalizing the results by the area under the curve for codeine in plasma, we obtained r(2) = 0.86 for codeine vs total melanin and r(2) = 0.90 vs eumelanin. Our results stress the importance of melanin determination when measuring drugs in hair. We postulate that analysis of drug concentration in hair may be worthwhile in the monitoring of drug compliance if the results are normalized for melanin content.

Hair analysis for Drug Abuse XV. Disposition of 3,4-methylenedioxymethamphetamine (MDMA) and its related compounds into rat hair and application to hair analysis for MDMA abuse

In order to clarify the mechanism of drug incorporation into hair, disposition of 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), 3-methoxy-4,5-methylenedioxyamphetamine (MMDA) and metabolites of MDMA, 4-hydroxy-3-methoxyamphetamine (HMAP) and 4-hydroxy-3-methoxymethamphetamine (HMMA), into hair was investigated with an animal model. After the intraperitoneal administration of those six drugs to pigmented hairy rats (5 mg/kg/day, 10 days, n = 3), the parent compounds and their metabolites in the rat plasma (5, 15, 30, 60, 120, 360 min after administration) and in the newly grown rat hair for 4 weeks were determined by GC/MS-SIM. When the ratio of hair concentration to area under the concentration versus time curves (AUCs) in plasma was represented as an index of incorporation rate (ICR) of drugs into hair, the order of ICRs was HMAP < MDA < HMMA < MDMA < MDEA < MMDA. In the comparison between MDA, MDMA and MDEA, their ICRs increased according to the length of carbon branches from proton to ethyl at the N position. From the point of view that the ICRs of MMDA was 2.3 times as much as that of MDA, the methoxy group on the benzene ring seemed to serve as a positive factor for the ICR. However, the ICRs of 4-hydroxy-3-methoxy compounds, HMAP and HMMA, were lower in comparison with those of MDA and MDMA, respectively. On the other hand, the ICRs of MDA, MDMA and MDEA were 5.5–6.1 times larger than those of amphetamine, methamphetamine and ethylamphetamine, suggesting that the methylenedioxy group on the benzene ring raises their ICRs very positively. Moreover, in order to apply the results from the animal experiments to human cases, the scalp hair samples of seven MDMA abusers were analyzed. MDMA and its metabolites, MDA, were simultaneously detected in all the samples by GC/MS. In the two samples, MDEA was found in addition to MDMA and MDA. It was shown that a hair sample is a good specimen for the confirmation of retrospective use of methylenedioxyamphetamines.