The study of metabolite-to-parent drug ratios of methamphetamine and methylenedioxymethamphetamine in hair

The metabolite-to-parent drug ratios were determined in the hair of 2444 methamphetamine (MA) abusers who had produced MA-positive hair results from 2001 to May 2005 and in the hair of 53 ecstasy abusers who had produced positive methylenedioxymethamphetamine (MDMA) hair results from 2002 to May 2005. For the hair analyses, hair strands were washed, cut into small pieces and extracted for 20 h in 1 mL methanol containing 1% HCl. Drugs in the extract were determined by gas chromatography-mass spectrometry (GC-MS) using selective ion monitoring after derivatization with trifluoroacetic anhydride. The six range groups were divided as follows on the basis of MA concentrations in hair (n = 2389): 0.5-5 ng/mg (n = 950), 5-10 ng/mg (n = 582), 10-20 ng/mg (n = 503), 20-30 ng/mg (n = 160), 30-40 ng/mg (n = 80), more than 40 ng/mg (n = 114) to assess the correlations between MA concentrations and metabolite-to-parent drug ratios. In groups of higher MA concentrations, lower ratios of AP/MA were found, and there was a statistically significant difference among six range groups. Comparisons of age groups (tens, twenties, thirties, forties, fifties, and sixties) and male and female subjects for the ratios of AP/MA showed a statistically significant difference. The detection of metabolites and the parent drug with reasonable ratios was found to be a useful indicator for distinguishing internal drug incorporation from external contamination. In our study, MA users can produce 0.4-116% (mean = 9%) of amphetamine (AP) concentrations in hair, and ecstasy users 1-110% (mean = 12%) of methylenedioxyamphetamine (MDA) in appropriately washed hair samples.     

Distribution of methamphetamine and amphetamine in drug abusers’ head hair

In order to aid the interpretation of hair results from methamphetamine (MA) abusers the MA and amphetamine (AP) concentrations in 2070 hair samples were statistically evaluated. The MA and AP concentrations in hair were put into three groups arbitrarily representing low, medium and high ranges and the metabolite-to-parent drug ratios of each group were examined. The concentration ranges proposed here were also applied to the interpretation of five authentic cases. The low, medium and high ranges of MA were 0.5–4.2, 4.2–24.5 and 24.5–608.9 ng/mg and those of AP were 0.1–0.4, 0.4–1.7 and 1.7–41.4 ng/mg. The AP-to-MA ratios showed large variation but a tendency that it decreased as the MA ranges increased. This evaluation was very useful to presume the severity of individuals’ MA abuse and to provide law enforcement agencies more understandable information. It could also facilitate the court's decision regarding specific circumstances surrounding the drug-related crimes.     

Preparation and application of a fortified hair reference material for the determination of methamphetamine and amphetamine

Quality assurance is one of the major issues in forensic analytical laboratories, where the need for a reference material (RM) has rapidly increased. RMs are very useful for method development and validation, internal quality control or proficiency tests. In the present study, we prepared a RM using drug-free hair for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) according to the recommendations of ISO Guide 35. The concentrations of MA and AP were determined using two extraction methods, agitation with 1% HCl in methanol at 38 degrees C and ultrasonication with methanol/5M HCl (20:1), followed by gas chromatography/mass spectrometry (GC/MS) after derivatization with trifluoroacetic anhydride (TFAA). The assignment of values was conducted through the homogeneity study and characterization of the material. Furthermore, an internal proficiency test was performed with the prepared RM, of which the results were compared with those of the authentic hair RM prepared in our previous study. As a result, a hair RM containing MA and AP was prepared at the level of 4.86+/-0.69 ng/mg and 4.63+/-0.44 ng/mg, respectively. Most participants showed satisfactory performances in the internal proficiency test with the both RMs. The hair RM prepared in this study demonstrated its suitability for quality assurance in forensic laboratories.     

Hair analysis to document non-fatal pesticide intoxication cases

We reported two non-fatal cases of intoxication with pesticides namely alachlor and carbofuran. Hair stand samples were collected from two men approximately 1 year after alachlor intoxication for case 1, and 14 days after the last exposure for case 2. Hair analysis was performed using a liquid chromatography-tandem mass spectrometry method. In case 1, alachlor was detected in the 5 analysed hair segments (concentrations between 12 and 136 pg/mg) and its metabolites were not detected. In case 2, carbofuran and its main metabolite (3-hydroxycarbofuran) were detected in the hair strand (global analysis) at the concentrations of 207 and 164 pg/mg, respectively. However, additional data are required in order to interpret such results.     

A comparative study on the concentrations of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in head and pubic hair

In this study, the concentrations of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in pubic, axillary and beard hair were measured and the correlation between the concentrations of THCCOOH in head and pubic hair from same cannabis users were evaluated. The papers on body hair analysis for THCCOOH were rarely found although police officers submit body hair as a complimentary specimen to forensic laboratories in case cannabis users had no hair. Head, pubic, axillary, and beard hair were collected. All hair samples were cut into 0.5mm segments and decontaminated with methanol, digested with 1 mL of 1M NaOH at 85 °C for 30 min and extracted in 2 mL of n-hexane:ethyl acetate (9:1) two times after adding 1 mL of 0.1N sodium acetate buffer (pH = 4.5) and 200 μL of acetic acid followed by derivatization with 50 μL of PFPA and 25 μL of PFPOH for 30 min at 70 °C. The extracts were analyzed using gas chromatography tandem mass spectrometry operating in negative chemical ionization mode (GC/MS/MS-NCI). We determined the concentrations of THCCOOH in both pubic and head hair. The concentrations of THCCOOH in pubic hair were higher than those in head hair. We also evaluated the concentrations of THCCOOH in body hair (pubic, axillary and beard hair) and head hair according to the positive/negative urine test results. There was no statistically significant difference in the concentrations of THCCOOH in head and body hair according to urine results.     

Hair analysis: self-reported use of "speed" and "ecstasy" compared with laboratory findings

Drug use histories were collected from 100 subjects recruited from the "dance scene" in and around Glasgow, Scotland. In addition, each subject donated a hair sample which was analyzed by gas chromatography/mass spectrometry (GC/MS) for amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MD MA) and 3,4-methylenedioxyethylamphetamine (MDEA). The hair samples were analyzed in two 6 cm segments or in full, ranging from 1.5 to 12 cm depending on the length of the hair. Approximately 10 mg of hair was ground to a fine powder before treatment with beta-glucuronidase/aryl sulfatase. A solid-phase extraction procedure was carried out followed by derivatization with pentafluoropropionic anhydride (PFPA). All extracts were analyzed by gas chromatography/mass spectrometry (GC/MS). Of the 139 segments analyzed, 77 (52.5%) were positive for at least one of the five amphetamines. The drug concentrations found in the hair were compared with the self-reported drug histories. A concordance of greater than 50% was found between the self-report data and levels detected in hair. However, no correlation was found between the reported number of "ecstasy" tablets consumed and the drug levels detected in hair. An increase in the average drug levels measured was observed from low to high use (number of "ecstasy" tablets/month). A large number of false negatives and a low number of false positives were observed.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS. Application to the determination of MDMA after low ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C(18) 5 microm, 2.1 mm x 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 degrees C in NaOH 1M before liquid-liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1-50 ng/mL in blood and urine; in the range 5-500 pg/mg for MA, MDMA, MDEA and MBDB, and 20-500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T+12h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D+8) and scalp hair at day 60 (D+60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Hair testing and self-report of cocaine use

Hair analysis is a useful tool in both clinical and forensic fields: it allows information about drugs of abuse (DOA) consumption to be obtained. However, in spite of analytical results, sometimes patients continue to deny using drugs or, on the contrary, insist on describing themselves as severe drug addicts; indeed there are often considerable difficulties in getting truthful statements about the real amount of drugs used. In this study we have tried to compare cocaine concentration in hair samples with self-reported drug intake. We enrolled 113 subjects (61 Africans, 52 Caucasians) who had been recently sent to jail. They were asked to tell about their use of illicit drugs during the last three months and then submitted to hair analysis. Hair segments (3 cm) were analyzed by GC-MS for amphetamines, cocaine and opiates. Useful data was obtained from 82 subjects, separated into two main groups on account of ethnic origin (African or Caucasian) and divided further into daily, weekly and monthly users. The results showed qualitative results and self-reported consumption to be in good agreement, although the correlation between frequency of consumption and concentration in hair revealed sometimes higher concentrations in contrast with the admission of low consumption. There was a definite separation between occasional and daily use (especially in Caucasian people), while concentrations found where weekly use was reported were more variable. Concentrations of cocaine measured in Africans' hair were much higher than in Caucasians'. Even if this study is exclusively based on self-report, it provides some interesting information in order to differentiate the frequency of consumption, and especially underlines the great importance of ethnic bias on hair analysis.     

Analysis of pain management drugs, specifically fentanyl, in hair: application to forensic specimens

This article discusses the immunoassay screening of pain management drugs, and the mass spectrometric confirmation of fentanyl in human hair. Hair specimens were screened for fentanyl, opiates (including oxycodone), tramadol, propoxyphene, carisoprodol, methadone, and benzodiazepines and any positive results were confirmed using gas chromatography or liquid chromatography with mass spectral detection. The specific focus of the work was the determination of fentanyl in hair, since autopsy specimens were also available for comparison with hair concentrations. Using two-dimensional gas chromatography with electron impact mass spectrometric detection, fentanyl was confirmed in four of nine hair specimens collected at autopsy. The accuracy of the assay at 10 pg/mg was 95.17% and the inter-day and intra-day precision was 5.04 and 13.24%, respectively (n=5). The assay was linear over the range 5-200 pg/mg with a correlation of r(2)>0.99. The equation of the calibration curve forced through the origin was y=0.0053x and the limit of quantitation of the assay was 5 pg/mg. The fentanyl concentrations detected were 12, 17, 490, and 1930 pg/mg and the results were compared with toxicology from routine post-mortem analysis. The screening of pain management drugs in hair is useful in cases where other matrices may not be available, and in routine testing of hair for abused drugs.     

Windows of detection of zolpidem in urine and hair: application to two drug facilitated sexual assaults

A LC-MS/MS method for the detection of zolpidem in hair was developed to detect this drug after a single dose in possible drug facilitated sexual assaults. To determine the window of detection of zolpidem in both urine and hair, three volunteers received a 10 mg dose. Urine specimens were collected each 12 h for 144 h. Hair was sampled 3-5 weeks after exposure. Hair and urine extracts were separated on a Xterra MS C18 column using a gradient of acetonitrile and formate buffer. For each compound, detection was related to two daughter ions. Zolpidem was detected for up to 60 h in urine with peak concentrations obtained at 12 h. A single exposure to zolpidem was detected in hair at concentrations ranging from 1.8 to 9.8 pg/mg. Hair analysis was applied to two possible criminal cases. In the first case, zolpidem tested positive in the corresponding hair segment at 4.4 pg/mg. In the other case, zolpidem was detected in all the segments analyzed, demonstrating likely previous drug use in addition to recent exposure associated with a positive blood result.     

Correlation of the incidence of cocaine and cocaethylene in hair and postmortem biologic samples

Hair samples are useful as a matrix for drug testing because drugs can be detected in hair for longer periods than in blood or urine. The authors report a prospective comparison of the detection of cocaine and cocaethylene in routine postmortem biologic specimens to the detection of cocaine and cocaethylene in hair. The authors collected hair samples from various areas of the head in 53 autopsy cases, prepared them, and analyzed them by gas chromatography/mass spectrometry (GC/MS) for cocaine and cocaethylene. The authors compared the results of hair analysis with the results of toxicologic analysis performed on routine postmortem samples by enzyme multiplied immunoassay technique and GC/MS. Cocaine was found in either biologic fluids or in hair in 16 of 53 samples tested. Nine samples were positive for cocaine in both biologic fluids and hair. Five samples contained cocaine only in biologic fluids, and two contained cocaine only in hair. Cocaethylene was present in two cases. Drug screening of hair provides additional information in some autopsy cases, but the authors have not made hair analysis a routine practice. It may prove useful to save hair samples in all cases for later analysis if warranted by additional history or autopsy findings.     

Evaluation of cocaine, amphetamines and cannabis use in university students through hair analysis: preliminary results

The evaluation of drug abuse in a defined population was performed through toxicological hair analysis. Hair samples from university students ranging from 18 to 25 years of age were anonymously collected and screened for cocaine, amphetamines and cannabinoids by radioimmunoassay (RIA). Positive results (cut-off values adopted were 2 ng/mg for cocaine and amphetamines and 0.5 ng/mg for cannabinoids) were confirmed by GC/MS. Preliminary results showed 19% of positive results for cocaine on 200 samples analysed. No confirmed positive results were obtained for amphetamine analysis. RIA technique demonstrated its unsuitability for cannabinoids preliminary screening on hair, giving a high percent of false positive results.     

Analysis of hair after contamination with blood containing 6-acetylmorphine and blood containing morphine

The study was carried out to investigate external contamination of hair by blood in heroin-related post-mortem cases. Solutions were prepared containing 0.05, 0.1, 0.2, 0.5 and 3.0μg/mL of 6-monoacetylmorphine (6-AM) only or morphine only in human blood. Samples of approximately 3.2g of drug-free hair were contaminated by soaking in the blood solutions for 5min. They were then removed and left at room temperature. Approximately 0.5g of hair was collected from each of the blood soaked hair samples at 6h, 1, 2, 4 and 7 days after contamination. As each hair sample was collected it was shampoo-washed to prevent further drug absorption. Hair samples were analysed in triplicate using a fully validated method described previously. 6-AM broke down to morphine in all samples. In hair contaminated with blood containing 0.05, 0.1 and 0.2μg/mL 6-AM or morphine drug was either not detected or was detected below the limit of quantitation (0.2ng/mg hair) at all contamination times. In hair contaminated with blood spiked with 0.5μg/mL morphine, the concentration in hair ranged from 0.54 to 0.91ng/mg and in hair contaminated with blood spiked with 3.0μg/mL, from 3.25 to 5.77ng/mg. The concentrations of 6-AM ranged from 0.65 to 1.11ng/mg and morphine from 0.34 to 0.80ng/mg in hair contaminated with 0.5μg/mL 6-AM in blood. 6-AM ranged from 2.12 to 3.67ng/mg and morphine from 0.84 to 2.05ng/mg in hair contaminated with 3μg/mL 6-AM in blood. For 6-AM and morphine ANOVA statistical evaluation showed no significant difference among the concentrations over time.     

Detection of thiopental and pentobarbital in head and pubic hair in a case of drug-facilitated sexual assault

The quali-quantitative determination of two barbiturates, thiopental and its metabolite pentobarbital, in head and pubic hair samples of a woman who had been sexually assaulted during hospitalisation, is reported. Hair was analysed by means of solid-phase microextraction (SPME) and gas chromatography-multiple mass spectrometry (GC-MS-MS), in chemical ionisation conditions. Thiopental and pentobarbital were found in three proximal head hair segments (sample 1A: 0.30 and 0.40 ng/mg; sample 1B: 0.20 and 0.20 ng/mg; sample 3: 0.15 and 0.20 ng/mg) and pubic hair sample. Two distal head hair segments were negative for both barbiturates. Despite the lack of collection and toxicological analysis of blood or urine samples within the hospital setting, analytical findings from hair revealed the use of the anaesthetic agent thiopental to sedate the victim quickly and deeply and commit sexual assault.     

Miniaturized sample preparation method for determination of amphetamines in urine

A simple and miniaturized sample preparation method for determination of amphetamines in urine was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). Urine was directly applied to the extraction column that was pre-packed with Extrelut and sodium carbonate. Amphetamine (AP) and methamphetamine (MA) in urine were adsorbed on the surface of Extrelut. AP and MA were then converted to a free base and derivatized to N-propoxycarbonyl derivatives using propylchloroformate on the column. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 100 and 102%, respectively. The calibration curves showed linearity in the range of 0.50-50 microg/mL for AP and MA in urine. When urine samples containing two different concentrations (0.50 and 5.0 microg/mL) of AP and MA were determined, the intra-day and inter-day coefficients of variation were 1.4-7.7%. This method was applied to 14 medico-legal cases of MA intoxication. The results were compared and a good agreement was obtained with a HPLC method.     

Diethyl phosphates accumulation in rabbits' hair as an indicator of long term exposure to diazinon and chlorpyrifos

Long term exposure to organophosphate pesticides can be evaluated by quantitative analysis of their non-specific metabolites in hair matrix. The aim of this study was to determine whether these metabolites can be internally incorporated into the hair of rabbits exposed to diazinon and chlorpyrifos. The influence of dose and dose duration of each pesticide dosage were investigated. Three groups of rabbits were exposed to different dosages of diazinon (3.0 and 6.0mg/kg/day) and chlorpyrifos (18.0mg/kg/day) via drinking water. Hair samples were collected every month and analyzed for diethyl phosphate (DEP) and diethyl thiophosphate (DETP) by gas chromatography-mass spectrometry (GC-MS). The mean concentrations of the low-dose treated group, ranged from 112 to 257pg/mg for DEP and from 295 to 515pg/mg for DETP in hair. The high-dose treated group demonstrated a range of mean concentrations from 142 to 585pg/mg for DEP and from 406 to 988pg/mg for DETP in hair. For the chlorpyrifos treated group, the concentrations ranged from 138 to 1070 for DEP and from 554 to 886pg/mg for DETP. Analysis revealed the incorporation of these metabolites into the rabbit hair in a dosage and dose duration-dependent manner. These data confirms the ability of using hair analysis for diethyl phosphates to assess long-term OP exposure.     

Evaluation of the catalytic decomposition of H2O2 through use of organo-metallic complexes--a potential link to the luminol presumptive blood test

Hair testing for drugs of abuse is performed in Lombardy by eleven analytical laboratories accredited for forensic purposes, the most frequent purposes being driving license regranting and workplace drug testing. Individuals undergoing hair testing for these purposes can choose the laboratory in which the analyses have to be carried out. The aim of our study was to perform an interlaboratory exercise in order to verify the level of standardization of hair testing for drugs of abuse in these accredited laboratories; nine out of the eleven laboratories participated in this exercise. Sixteen hair strands coming from different subjects were longitudinally divided in 3-4 aliquots and distributed to participating laboratories, which were requested to apply their routine methods. All the participants analyzed opiates (morphine and 6-acetylmorphine) and cocainics (cocaine and benzoylecgonine) while only six analyzed methadone and amphetamines (amphetamine, methamphetamine, MDMA, MDA and MDEA) and five Δ(9)-tetrahydrocannabinol (THC). The majority of the participants (seven labs) performed acidic hydrolysis to extract the drugs from the hair and analysis by GC-MS, while two labs used LC-MS/MS. Eight laboratories performed initial screening tests by Enzyme Multiplied Immunoassay Technique (EMIT), Enzyme-linked Immunosorbent Assay (ELISA) or Cloned Enzyme Donor Immunoassay (CEDIA). Results demonstrated a good qualitative performance for all the participants, since no false positive results were reported by any of them. Quantitative data were quite scattered, but less in samples with low concentrations of analytes than in those with higher concentrations. Results from this first regional interlaboratory exercise show that, on the one hand, individuals undergoing hair testing would have obtained the same qualitative results in any of the nine laboratories. On the other hand, the scatter in quantitative results could cause some inequalities if any interpretation of the data is required.     

Differentiation of Morphologically Similar Human Head Hairs from Two Demographically Similar Individuals Using Amino Acid Ratios,

Human hair is frequently encountered as forensic evidence and can contribute valuable information to investigators. Conventional forensic hair analyses include microscopic hair comparison (MHC) and DNA analysis. However, MHC is not supported by statistics and DNA analysis cannot always be performed. Recent studies have demonstrated that evaluation of differences in the hair proteins may offer an alternate method to these analyses. In this study, an evaluation of the amino acids present in hair was investigated as an approach to differentiate morphologically indistinguishable hair samples from two demographically similar individuals. Proteins in the hair were digested using hydrochloric acid, and the resulting amino acids were derivatized with N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) for analysis using gas chromatography-mass spectrometry (GC-MS). Eight derivatized amino acids were detected and quantified relative to an internal standard, L-norvaline, and used to construct twenty-eight amino acid ratios. Hair samples were collected from four areas of the head on various days over the course of one month, and no significant differences in amino acid ratios (p-value > 0.05) were observed among the areas of the head, and the ratios were consistent over the time period of this study. Additionally, fifteen of these amino acid ratios were found to be significantly different between the two individuals when compared using a two-sample t-test (p-value ≤ 0.05). These data indicate that amino acid analysis was able to differentiate two morphologically similar hair samples from different individuals and demonstrates the applicability of this method to distinguish similar hair samples when DNA analysis cannot be performed.     

Evaluation of nicotine and cotinine in human hair.

To validate data on tobacco use, the authors investigated the use of hair samples for quantifying nicotine and cotinine by gas chromatography/mass spectrometry. Hair was taken from 22 nonsmokers and 42 smokers, cut close to the scalp at the back of the head. The hair (about 100 mg from each subject) was incubated in 3 mL of 1N NaOH at 100 degrees C for 1 h. After this, the samples were extracted by diethyl ether. The drugs were separated on a 12-m BP-5 capillary column and detected using selected ion monitoring (nicotine, m/z 84; cotinine, m/z 98). Hair from nonsmokers and smokers contains nicotine and cotinine. Although it is difficult to determine an absolute cutoff level, an amount greater than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from nonsmokers. In the population of nonsmokers, the influence of environmental smoke exposure was noted.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS: Application to the determination of MDMA after low Ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C₁₈ 5 μm, 2.1 mm × 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A^® (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 °C in NaOH 1 M before liquid–liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1–50 ng/mL in blood and urine; in the range 5–500 pg/mg for MA, MDMA, MDEA and MBDB, and 20–500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T + 12 h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D + 8) and scalp hair at day 60 (D + 60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.