Quantitative determination of amphetamines, cocaine, and opiates in human hair by gas chromatography/mass spectrometry

Hair of young subjects (N = 36) suspected for drug abuse was analysed for morphine, codeine, heroin, 6-acetylmorphine, cocaine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA). The analysis of morphine, codeine, heroin, 6-acetylmorphine, cocaine, and methadone in hair included incubation in methanol, solid-phase extraction, derivatisation by the mixture of propionic acid anhydride and pyridine, and gas chromatography/mass spectrometry (GC/MS). For amphetamine, methamphetamine, MDA, MDMA, and MDEA analysis, hair samples were incubated in 1M sodium hydroxide, extracted with ethyl acetate, derivatised with heptafluorobutyric acid anhydride (HFBA), and assayed by GC/MS. The methods were reproducible (R.S.D. = 5.0-16.1%), accurate (85.1-100.6%), and sensitive (LoD = 0.05-0.30ng/mg). The applied methods confirmed consumption of heroin in 18 subjects based on positive 6-acetylmorphine. Among these 18 heroin consumers, methadone was found in four, MDMA in two, and cocaine in two subjects. Cocaine only was present in two, methadone only in two, methamphetamine only in two, and MDMA only in seven of the 36 subjects. In two out of nine coloured and bleached hair samples, no drug was found. Despite the small number of subjects, this study has been able to indicate the trend in drug abuse among young people in Croatia.     

Detection of phentermine in hair samples from drug suspects

Phentermine (PT) has been widely used as an anti-obesity drug. This drug has to be used with caution due to its close resemblance with amphetamines in its structure and toxicity profile. Recently, PT is in distribution by illegal modes and is found to be available through sources such as the internet, thus their misuse and/or abuse is threatening to be a serious social issue. In the present study, 32 cases of drug suspects were observed for PT abuse, detected using hair samples for drug analysis. PT and other amphetamines, such as methamphetamine (MA), amphetamine (AP), 3,4-methylenedioxyamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA), were extracted using 1% HCl in methanol for 20 h at 38°C. The extracts were derivatized with trifluoroacetic anhydride (TFAA) and analyzed using gas chromatography/mass spectrometry (GC/MS). Among the 32 cases of PT abuse, MA and its main metabolite, AP were identified in seven cases and MDMA and its main metabolite, MDA were detected in two other cases.     

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.     

Time-resolved hair analysis of MDMA enantiomers by GC/MS-NCI

The aim of the study was to determine the enantioselective disposition of 3,4-methylenedioxymethamphetamine (MDMA) and other amphetamine-type stimulants (ATS) in segmented hair specimens of self-declared ecstasy abusers, who took part in a double-blind placebo-controlled six-way crossover study during approximately 7 weeks, during which they received a 75 and a 100 mg dose of racemic MDMA twice. Hair specimens were washed and cut into pieces of 2 cm length. After digestion and solid phase extraction, the enantiomers were derivatized with a chiral agent (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, developed at the authors laboratory and quantified by gas chromatography coupled to mass spectrometry operating in the negative chemical ionization mode. Most of the hair specimens that were tested positive for MDMA showed a predominance of the (R)-enantiomer. The R/S ratios of MDMA varied between 1.02 and 2.75 and total concentrations ranged from 0.1 to 20.1 ng/mg. The enantiomers of its metabolite 3,4-methylenedioxyamphetamine (MDA) were also quantified in most hair segments. The R/S ratios of MDA varied between 0.60 and 1.60, while the concentrations of the enantiomers ranged from 10 to 160 pg/mg hair. When segmental analysis was performed on single hair specimens, no inversion of the R versus S ratios of MDMA and MDA was observed. The predominance of (R)-MDMA in hair was in accordance with those already published for other matrices. Furthermore, both enantiomers of amphetamine (AM) were also detected in hair segments of four volunteers and the R/S ratios ranged from 1.00 to 1.47.     

Evaluation of ephedrine, pseudoephedrine and phenylpropanolamine concentrations in human urine samples and a comparison of the specificity of DRI amphetamines and Abuscreen online (KIMS) amphetamines screening immunoassays

The purpose of this study was to evaluate the ability of two amphetamine class screening reagents to exclude ephedrine (EPH), pseudoephedrine (PSEPH), and phenylpropanolamine (PPA) from falsely producing positive immunoassay screening results. The study also sought to characterize the prevalence and concentration distributions of EPH, PSEPH, and PPA in samples that produced positive amphetamine screening results. Approximately 27,400 randomly collected human urine samples from Navy and Marine Corps members were simultaneously screened for amphetamines using the DRI and Abuscreen online immunoassays at a cutoff concentration of 500 ng/mL. All samples that screened positive were confirmed for amphetamine (AMP), methamphetamine (MTH), 3,4-Methylenedioxyamphetamine (MDA), 3,4-Methylenedioxymethamphetamine (MDMA), EPH, PSEPH, and PPA by gas chromatography/mass spectrometry (GC/MS). The DRI AMP immunoassay identified 1,104 presumptive amphetamine positive samples, of which only 1.99% confirmed positive for the presence of AMP, MTH, MDA, or MDMA. In contrast, the online AMP reagent identified 317 presumptive amphetamine positives with a confirmation rate for AMP, MTH, MDA, or MDMA of 7.94%. The presence of EPH, PSEPH, or PPA was confirmed in 833 of the 1,104 samples that failed to confirm positive for AMP, MTH, MDA, or MDMA; all of the 833 samples contained PSEPH. When compared to the entire screened sample set, PSEPH was present in approximately 3%, EPH in 0.9%, and PPA in 0.8% of the samples. The results indicate that cross reactivities for EPH, PSEPH, and PPA are greater than reported by the manufacturer of these reagents. The distribution of concentrations indicates that very large concentrations of EPH, PSEPH, and PPA are common.     

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.     

Characteristics and trends of 3,4-methylenedioxymethamphetamine (MDMA) tablets found in Taiwan from 2002 to February 2005

One hundred and eighty-one 3,4-methylenedioxymethamphetamine (MDMA) containing tablets were sampled from confiscated drugs received by the Taiwan National Bureau of Controlled Drugs for testing from 2002 to February 2005. Sample tablets demonstrated various colors and logos. The appearances, contents of MDMA and other components in these tablets were analyzed in order to understand the characteristics and trends of MDMA use. Samples were analyzed using GC-MS methodology. Deuterated internal standards were used for drug quantification. The MDMA contents varied from 16 to 193 mg/tablet. 66-71% of the tablets seized each year contained only MDMA, and the content of MDMA in MDMA only tablets varied from 89 to 133 mg/tablet. There was a decreasing trend in MDMA content in these tablets over time. Other components commonly found besides MDMA included caffeine (18%), methamphetamine (7%), 3,4-methylenedioxyethylamphetamine (MDEA) (7%) and amphetamine (4%). 3,4-Methylenedioxyamphetamine (MDA), ketamine, ephedrine, diazepam, chlorzoxazone and nicotinamide were also detected. During the study period, the number of other drugs found as well as the combinations of different drugs detected in these tablets increased.     

Estimation of gamma-hydroxybutyrate (GHB) co-consumption in serum samples of drivers positive for amphetamine or ecstasy

There is no toxicological analysis of gamma-hydroxybutyrate (GHB) applied routinely in cases of driving under influence (DUI); therefore the extent of consumption of this drug might be underestimated. Its consumption is described as occurring often concurrently with amphetamine or ecstasy. This study examines 196 serum samples which were collected by police during road side testing for GHB. The samples subject to this study have already been found to be positive for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and/or 3,4-methylenedioxyethamphetamine (MDEA). Analysis has been performed by LC/MS/MS in the multiple reaction monitoring (MRM) mode. Due to its polarity, chromatographic separation of GHB was achieved by a HILIC column. To differentiate endogenous and exogenous levels of GHB, a cut-off concentration of 4μg/ml was applied. Of the 196 samples, two have been found to be positive for GHB. Of these samples, one sample was also positive for amphetamine and one for MDMA. Whilst other amphetamine derivates were not detected in these samples, both samples were found to be positive for cannabinoids. These results suggest that co-consumption of GHB with amphetamine or ecstasy is relatively low (1%) for the collective of this study.     

Chemical profiling of 3,4-methylenedioxymethamphetamine (MDMA) tablets seized in Hong Kong

During 2000-2001, the Government Laboratory of Hong Kong received over 600,000 ecstasy tablets in more than 2,600 cases. Using GC-MS or FTIR, the major amphetamine-type stimulants were identified, and the samples were categorized into four groups containing: (1) 3,4-methylenedioxymethamphetamine (MDMA), (2) methamphetamine (MA), (3) 3,4-methylenedioxyamphetamine (MDA), or (4) amphetamine. Our study revealed that in Hong Kong MDMA tablets have made up 98 and 71% of the total ecstasy tablets examined in 2000 and 2001, respectively. Among the MDMA cases, 613 cases involving a total of 123,776 tablets in 2001 were randomly selected, and their active ingredients, minor ingredients, and/or impurities were studied using GC-MS and HPLC. Based on the chemical profiles, and irrespective of their different physical characteristics, tablets obtained in different seizures could be determined as to whether or not they could have come from a common origin. The impurities detected in the MDMA tablets also served as excellent chemical markers from which plausible synthetic route(s) of the MDMA were inferred. Our study revealed that 3,4-methylenedioxyphenyl-2-propanone (MDP2P), 3,4-methylenedioxyphenyl-2-propanol (MDP), 3,4-methylenedioxy-N-methylbenzylamine (MDB), piperonal and N-formyl-3,4-methylenedioxymethamphetamine (N-formyl-MDMA) were the most common impurities detected in MDMA tablets seized in Hong Kong. The finding of the phosphate salt of MDMA is intriguing. Based on a presumptive color test, spectroscopic data (FTIR/ESI-MS) and the percentage of MDMA content in a purified phosphate salt of MDMA, the ratio of the phosphate to MDMA was determined to be 1:1, suggesting that the compound is a dihydrogen phosphate salt [i.e. (HMDMA)H2PO4].     

Use of MDA (the "love drug") and methamphetamine in Toronto by unsuspecting users of ecstasy (MDMA)

It has recently been reported that purity of illicit tablets of ecstasy (MDMA) is now high. Our objective was to confirm whether hair of drug users, who request only ecstasy from their supplier, contains MDMA in the absence of other drugs. GC-MS analysis of scalp hair segments disclosed the presence of MDMA in 19 of 21 subjects and amphetamine/methamphetamine in eight subjects. Surprisingly, seven subjects had hair levels of the MDMA metabolite, MDA, equal to or greater than those of MDMA, suggesting use of MDA in addition to that of MDMA. These amphetamine derivatives might be included by clandestine laboratories to enhance effects of the drug cocktail or because of a perception that MDA synthesis might be simpler than that of MDMA. Drug users and investigators examining possible brain neurotoxic effects of MDMA need to consider that "ecstasy" tablets can contain MDA and methamphetamine despite no demand for the drugs.     

Prevalence of gamma-hydroxybutyrate (GHB) in serum samples of amphetamine, metamphetamine and ecstasy impaired drivers

Two hundred and forty-seven serum samples which have been collected by police during roadside testing and have been found positive for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and/or 3,4-methylenedioxyethamphetamine (MDE) were analyzed for gamma-hydroxybutyrate (GHB). Serum samples were spiked with deuterated GHB as internal standard and acetonitrile was added to achieve dilution and protein precipitation. Samples were analyzed with a LC-MS/MS system operated in the multiple reaction monitoring mode (MRM) using a TurboIonSpray source. Chromatographic separation was achieved using a Synergi Polar RP column applying a gradient elution with a runtime of 15 min. To differentiate between endogenous and exogenously administered GHB a cut-off concentration of 10 microg/mL was applied. Five samples exceeded this concentration and were found positive for GHB. These samples were only found positive for amphetamine but no other amphetamine derivatives were detected, while in three samples THC and in one sample cocaine, benzoylecgonine and ethanol were found.     

Interpretation of a 3,4-methylenedioxymethamphetamine (MDMA) blood level: discussion by means of a distribution study in two fatalities

The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy" is a currently used or abused designer drug and fatalities are frequently encountered in forensic practice. However, the question remains open whether an MDMA blood level can be toxic or even potentially lethal. In order to provide insight in the interpretation of a detected MDMA concentration, the distribution of MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) in various body fluids and tissues was studied and discussed in two different fatalities. Apart from peripheral blood samples (such as femoral and subclavian blood), various blood samples obtained centrally in the human body and several body fluids (such as vitreous humour) were examined. In addition, various tissues such as cardiac muscle, lungs, liver, kidneys, and brain lobes were analysed. In contrast to the peripheral blood levels, high MDMA and MDA levels were found in cardiac blood and the majority of the organs, except for the abdominal adipose tissue. The high concentrations observed in all lung lobes, the liver and stomach contents indicate that post-mortem redistribution of MDMA and MDA into cardiac blood can occur and, as a result, blood sampled centrally in the body should be avoided. Therefore, our data confirm that peripheral blood sampling remains "the golden standard". In addition, a distinct difference in peripheral blood MDMA concentrations in our two overdose cases was established (namely 0.271 and 13.508 microg/ml, respectively). Furthermore, our results suggest that, if a peripheral blood sample is not available and when putrefaction is not too pronounced, vitreous humour and iliopsoas muscle can be valuable specimens for toxicological analysis. Finally, referring to the various mechanisms of death following amphetamine intake, which can result in different survival times (e.g. cardiopulmonary complications versus hyperthermia), the anatomo-pathological findings and the toxicological results should be considered as a whole in arriving at a conclusion.     

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.     

尿中苯丙胺、甲基苯丙胺、MDA和MDMA的固相微萃取和GC/MS/SIM测定

目的研究固相微萃取(SPME)用于尿中苯丙胺(AMP)、甲基苯丙胺(MET)、3,4-亚甲二氧基苯丙胺(MDA)和3,4-亚甲二氧基甲基苯丙胺(MDMA)的提取。方法样品调节至碱性和用盐饱和后用顶空SPME,内标为MET-d5。萃取纤维为100μm聚二甲基硅氧烷(PDMS)。用气质联用选择离子检测(GC/MS/SIM)。结果0.2μg/ml加标尿样,AMP、MET、MDA和MDMA的富集倍数分别为22,60,13和47。检出限(S/N=3)为0.4～9.5ng/ml。线性范围为0.05～1μg/ml。0.2、0.5和1.0μg/ml加标尿样,相对回收率77.9%～112.4%,变异系数2.7%～18.0%(n=5)。用该方法分析5个案件样品,和常规液液萃取结果接近。结论顶空SPME法用于尿中AMP、MET、MDA和MDMA等化合物的分析,无需有机溶剂,富集效率高,提取-富集-进样一体化,简单方便实用。     

Availability of target odor compounds from seized ecstasy tablets for canine detection

The aim of this study was to compare seized samples of 3,4-methylenedioxy-N-methylamphetamine (MDMA) pills, used to train law enforcement detection canine teams, to determine what differences exist in the chemical makeup and headspace odor and their effect on detectability. MDMA solutions were analyzed by liquid chromatography-mass spectrometry. Analysis of these samples showed a wide variance of MDMA (8-25%). Headspace SPME-GC/MS analysis showed that several compounds such as 3,4-methylenedioxyphenylacetone and 1-(3,4-methylenedioxyphenyl)-2-propanol are common among these MDMA samples regardless of starting compound and synthesis procedure. However, differences, such as the level of the various methylenedioxy starting compounds, were shown to affect the overall outcome of canine detection, indicating the need for more than one MDMA training aid. Combinations of compounds such as the primary odor piperonal in conjunction with a secondary compound such as MDP-2-OH or isosafrole are recommended to maximize detection of different illicit MDMA samples.     

Validation of an ELISA-based screening assay for the detection of amphetamine, MDMA and MDA in blood and oral fluid

The use of amphetamine and 'ecstasy' (MDMA) has increased exponentially in many European countries since the late nineties, leading to a rapid growth in the number of clinical and forensic analyses. Therefore, a rapid screening procedure for these substances in biological specimens has become an important part of routine toxicological analysis in forensic laboratories. The objective of this study was to evaluate the Cozart amphetamine enzyme-linked immunosorbent assay (ELISA) for the screening of plasma samples and oral fluid samples (collected with the Intercept device). Authentic plasma samples from drivers (n=360) were screened, using an 1:5-fold dilution. True positive, true negative, false positive and false negative results were determined relative to the in-house routine GC-MS analysis. Samples consisted of 144 amphetamine-only positives, 141MDMA/MDA-only positives, and 74 negatives when using the limit of quantitation as the cut-off level for confirmation (10 ng/mL). Using these results, receiver operating characteristic (ROC) curves were generated and optimal cut-off values for the screening assay were calculated. Analysis showed that the ELISA is able to predict the presence of either amphetamine or *MDMA/MDA (*MDMA as its metabolite MDA) in plasma samples with 98.3% sensitivity and 100% specificity at a cut-off value of 66.5 ng/mL d-amphetamine equivalents. A similar analysis was conducted on 216 oral fluid specimens collected from a controlled double blind study. Subjects received placebo or a high (100 mg) or low (75 mg) dose of MDMA. Oral fluid samples were collected at 1.5 and 5.5h after administration. Combined results of the analysis of the high and low dose oral fluid samples indicated a screening cut-off of 51 ng/mL d-amphetamine equivalents with both a sensitivity and specificity of 98.6% (using a LC-MS/MS confirmation cut-off of 10 ng/mL). In conclusion, these data indicate that the Cozart AMP EIA plates constitute a fast and accurate screening technique for the identification of amphetamine and MDMA/MDA positive plasma samples and oral fluid specimens (collected with Intercept. It should be emphasized that method validation should be performed for each type of biological matrix.     

The use of supercritical fluid extraction for the determination of amphetamines in hair

A laboratory study interested in the analysis of human hair for drugs-of-abuse was conducted to determine if drugs could be detected and quantified from hair. Supercritical fluid extraction (SFE) techniques followed by GC-MS analysis were applied to extract amphetamines from hair. The group of amphetamines included methylenedioxyamphetamine (MDA), methylenedioxymetamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and internal standard mephentermine (MP). To validate information on amphetamine use in hair, powdered hair samples free from drugs were collected and soaked in a known amphetamine standard solution. Authentic fortified case hair samples taken from known drug users known to have consumed amphetamines were also analyzed for amphetamine. Results from this study show that amphetamine use can be detected in spiked and authentic fortified human hair using SFE techniques for qualitative and quantitative reproducible results.     

尿中MDMA及其代谢物的GC和GC／MS分析

考察MDMA在人体内的代谢以及建立尿中MDMA和体内主要代谢物MDA的分析方法。尿样水解后经液-液提取处理,用GC／MS（EI、PCI）和GC／FID法分析。人摄入MDMA后尿中MDA和原体MDMA比约为0．10～0．14。GC／MS／SIM和GC／FID法的最低检出限为2ng／ml和50ng／ml,回收率大于85％,变异系数小于10％。该法简便快速、灵敏度高、结果可靠,可用于MDMA滥用者的尿样鉴定。MDA／MDMA浓度比可作为评判毒分结果的参考指标。     

Simultaneous analysis of psychotropic phenylalkylamines in oral fluid by GC-MS with automated SPE and its application to legal cases

Phenylalkylamine derivatives, such as methamphetamine (MA), amphetamine (AM), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), phentermine (PT), fenfluramine (FFA) and phenmetrazine (PM), and ketamine (KT) are widely abused recreational or anorectic drugs in Korea and are regulated under the Controlled Substance Act in Korea. Phenylalkylamines and ketamine analysis is normally performed using both urine and hair samples but there is no established method for the simultaneous analysis of all these phenylalkylamines and ketamine in oral fluids. Oral fluid is easy to collect/handle and can provide an indication of recent drug abuse. In this study, to confirm the presence of phenylalkylamine derivatives and ketamine in oral fluid after screening with an immunoassay, an analytical method using automated solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) was developed and fully validated according to international guidelines. The applicability of the assay was demonstrated by analyzing of authentic oral fluid samples and the results of oral fluid analysis were compared with those in urine and hair to to evaluate the feasibility of oral fluid in forensic cases. The recovery of phenylalkylamines and ketamine from oral fluid collection devices was also assessed. Oral fluid specimens from 23 drug abuse suspects submitted by the police were collected using Salivette (Sarstedt, Nümbrecht, Germany), Quantisal (Immunalysis, Pomona, CA) or direct expectoration. The samples were screened using a biochip array analyzer (Evidence Investigator, Randox, Antrim, UK). For confirmation, the samples were analyzed by GC-MS in selected-ion monitoring (SIM) mode after extraction using automated SPE (RapidTrace, Zymark, MA, USA) with a mixed-mode cation exchange cartridge (CLEAN SCREEN, 130 mg/3 ml, UCT, PA, USA) and derivatization with trifluoroacetic anhydride (TFA). The results from the immunoassay were consistent with those from GC-MS. Twenty oral fluid samples gave positive results for MA, AM, PT and/or PM among the 23 cases, which gave positive results in urine and/or hair. Although large variations in the MA, AM, PT and PM concentrations were observed in three different specimens, the oral fluid specimen was useful for demonstrating phenylalkylamines and ketamine abuse as an alternative specimen for urine.     

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.