The disposition into hair of new designer drugs; methylone, MBDB and methcathinone

The disposition into hair of methylone and other new designer drugs, methcathinone and MBDB, was studied with the animal model. Moreover, the incorporation rates of these drugs were compared with those of their related eight compounds previously studied in order to evaluate their incorporation tendency into hair and the usefulness of hair specimens for the retrospective confirmation of the use of these drugs. When the ratio of hair concentration to AUC in plasma ([Hair]/AUC) was represented as an index of the incorporation rate of drugs into hair, the [Hair]/AUC of methylone was 14 times higher than that of methcathinone. It might support earlier findings that the methylenedioxy group on the benzene ring leads to considerably higher incorporation rates. However, [Hair]/AUC of methylone was five-sevenths times lower in comparison with that of MDMA. This suggested that the beta-carbonyl group leads to lower incorporation rates. Although methylone has both groups in its structure, the positive effect of the methylenedioxy group may be stronger than the negative effect of the beta-carbonyl group. On the other hand, the [Hair]/AUC of MBDB, which has methylenedioxyphenyl-2-butanamine structure, was higher than that of MDMA while that of methcathinone, having beta-ketone in its structure, was extremely low. In conclusion, as with MA and MDMA, the incorporation tendency of methylone and MBDB (except for methcathinone) into hair is relatively high, and a hair sample would be a good specimen for the confirmation of retrospective use of these drugs.     

The dependence of the incorporation of methamphetamine into rat hair on dose,frequency of administration and hair pigmentation

In this paper, the incorporation of methamphetamine (MA) into rat hair was studied. The main purpose of this study was to investigate whether MA can be detected or positive hair results can be obtained in hair of rats administered a single dose of MA. The relationship between dose and frequency of administration and the concentrations of MA and its metabolite, amphetamine (AP), in rat hair were evaluated and the MA and AP concentrations in white and pigmented hair were compared. MA was administered to rats as follows: low dose (0.5 mg/kg/day), medium dose (2 mg/kg/day) and high dose (10 mg/kg/day). The frequency of administration was one time per day for 1, 2, 3, 4, 5, 15 and 30 days. Hair and urine samples were collected from rats and analyzed by gas chromatography/mass spectrometry (GC/MS). MA could be identified in pigmented rat hair when MA was administered for 4 or more days at low daily dose and on day 1 following administration of medium and high daily doses. Positive results for MA were obtained from pigmented rat hair when MA was administered for 30 days at low daily dose, for 4 or more days at medium daily dose, or for 2 or more days at high daily dose. The concentrations of MA and AP found in rat hair were proportional to the dose and frequency of administration. The concentrations of MA and AP in pigmented rat hair were 2–10 times higher than those in white rat hair. The results of this study on the incorporation of MA into rat hair can serve as a model to better understand the incorporation of MA into human hair even though there are differences between animal models and human hair.     

Characterization of [3H]flunitrazepam binding to melanin.

In both clinical and forensic toxicology, the analysis of hair for drugs is an important tool to determine drug use in the past or to verify abstinence from illegal drugs during extended periods. Melanin is proposed as one of the factors that influences drug incorporation to hair and we have characterized the binding of the drug flunitrazepam to melanin in vitro. The drug was 3H labeled and melanin granules from cuttlefish, Sepia officinalis, were used according to the suggested standard for melanin studies. We observed a rapid Langmuir-like binding followed by a slower diffusion-limited binding that may be interpreted as an initial surface binding followed by deeper bulk binding. From three concentrations of melanin, with a 60-min incubation time, a mean saturation value of 180 +/- 20 pmol/mg was calculated. The binding of a group of benzodiazepines and tranquilizers was compared to the binding of [3H]flunitrazepam by means of displacement experiments. These drugs showed binding characteristics similar to [3H]flunitrazepam except phenobarbital, which had a lower affinity to melanin. The method presented in this study allowed measurements with low melanin and drug concentrations and it has the strength of directly measuring the amount of drug bound to melanin, in contrast to previous indirect methods.     

Relationship of melanin degradation products to actual melanin content: application to human hair

Methods not only for characterizing but also for quantitating melanin subtypes from the two types of melanin found in hair--eumelanin and pheomelanin--have been established. In relation to testing for drugs of abuse in hair, these methods will allow for correction of drug binding to specific melanin subtypes and will serve to improve drug measurement in hair. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) make up the majority of the eumelanin polymer while benzothiazene units derived from 2-cysteinyl-S-Dopa (2-CysDopa) and 5-cysteinyl-S-Dopa (5-CysDopa) compose the majority of the pheomelanin polymer. Our results show that: (1) pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), markers for DHI and DHICA units, respectively, are produced in 0.37 and 4.8% yields, respectively, when melanins are subjected to alkaline hydrogen peroxide degradation, (2) 3-aminotyrosine (3AT) and 4-amino-3-hydroxyphenylalanine (AHP), markers for 2-CysDopa and 5-CysDopa, respectively, are produced in 16 and 23% yield, respectively, when subjected to hydriodic acid hydrolysis, and (3) that black human hair contains approximately 99% eumelanin and 1% pheomelanin, brown and blond hair contain 95% eumelanin and 5% pheomelanin; and red hair contains 67% eumelanin and 33% pheomelanin. These data will allow deeper investigation into the relationship between melanin composition and drug incorporation into hair.     

Metabolic denitrosation of N-nitroso-N-methylaniline: detection of amine-metabolites

The enzymatic denitrosation of N-nitroso-N-methylaniline (NMA) was investigated by measuring the resulting amine metabolites when NMA was incubated with liver microsomes of PB-pretreated mice. Aniline was found to be the main amine metabolite. Small amounts of the secondary amine, N-methylaniline (MA) and its metabolite, p-methylaminophenol (p-MAP), could also be detected. Incubation of MA resulted in the formation of aniline and p-MAP. The velocity of the metabolism of MA was somewhat faster than that of NMA. On the basis of the measured Vmax values the formation of aniline from MA or from NMA proceeded at nearly identical rates. The dissociation constants as a measure of binding affinity to cytochrome (cyt.) P-450 were determined by measuring the binding spectra. NMA has one Ks of 3.1 mM, whereas MA shows two apparent Ks values, 650 microM and 25 mM, respectively. The results are discussed in relation to the enzymatic mechanism of denitrosation of NMA.     

Simultaneous determination of dimethylamphetamine and its metabolites in rat hair by gas chromatography–mass spectrometry

In order to study the disposition of dimethylamphetamine (DMAP) and its metabolites, DMAP N-oxide, methamphetamine (MA) and amphetamine (AP), from plasma to hair in rats, a simultaneous determination method for these compounds in biological samples using gas chromatography–mass spectrometry with selected ion monitoring (GC–MS-SIM) was developed. As DMAP N-oxide partially degrades to DMAP and MA during GC–MS analysis, it was necessary to avoid conditions which co-extract the N-oxide in the sample preparation so as to assure no contribution of artifactual products from DMAP N-oxide in the detection of the other compounds. For confirmation of the satisfactory separation of DMAP N-oxide from the others, the internal standards used for quantification were labeled with different numbers of deuterium atoms. Determination of unchanged DMAP was performed without any derivatization, that of DMAP N-oxide was carried out after conversion into trifluoroacetyl-MA by reaction with trifluoroacetic anhydride, and MA and AP were quantified after trifluoroacetyl-derivatization.After intraperitoneal administration of DMAP HCl to pigmented hairy rats (5 mg kg⁻¹ day⁻¹, 10 days, n=3), concentrations of DMAP and its metabolites in urine, plasma and hair were measured by GC–MS-SIM. The area under the concentration versus time curves (AUCs) of DMAP, DMAP N-oxide, MA and AP in the plasma were 397.2±97.5, 279.7±68.3, 18.4±1.2 and 15.9±2.2 μg min ml⁻¹, while their concentrations in the hair newly grown for 4 weeks after administration were 4.82±0.67. 0.45±0.09, 3.25±0.36 and 0.89±0.05 ng mg⁻¹, respectively. This fact suggested that the incorporation tendency of DMAP N-oxide from plasma into hair was distinctly low in comparison with the other compounds.     

Application of ion mobility spectrometry to the rapid screening of methamphetamine incorporated in hair

Using ion mobility spectrometry (IMS), a simple, sensitive and rapid screening for methamphetamine (MA) incorporated in user's hair has been developed. To completely unbind MA from hair matrix and to achieve its effective vaporization for the IMS detection, the hair sample was digested in 5 M NaOH (methanol-water, 4:1, v/v) solution prior to IMS measurement. MA in hair was semi-quantitatively detected by monitoring the digested hair sample employing dibenzylamine (DBA) as internal standard. The minimum amount of hair sample required was 2 mg and its digested sample was ample for four IMS measurements. Teh detection limit of MA in hair was 0.5 ng mg⁻¹. This proposed method was applicable to the semi-quantitative detection of MA in users' hair samples, and to the sectional analysis for MA in a limited amount of user's hair. The IMS results obtained were in good agreement with their GC-MS determination.     

Analysis of inducible contractile rings suggests a role for protein kinase C in embryonic cytokinesis and wound healing.

A semi-in vitro system derived from Xenopus oocytes which allows induction of contractile ring (CR) formation and closure is described and exploited to elucidate regulatory and structural features of cytokinesis. The inducible CRs (ICRs) are composed of actin filaments and closure is actin filament-dependent as is cytokinesis in vivo. ICR closure in this system is calcium-dependent and pH-sensitive, as is cytokinesis in permeabilized cells (Cande: Journal of Cell Biology 87:326, 1980). Closure of ICRs proceeds at a rate and with a kinetic pattern similar to embryonic cytokinesis. Collectively, these data demonstrate that this system is a faithful mimic of cytokinesis in vivo. ICR formation and closure is protein kinase C (PKC)-dependent and neomycin-sensitive, indicating that the PKC branch of the polyphosphoinositide pathway regulates formation of the actomyosin ring which is the effector of cytokinesis. Kinetic measurements show that the rate of ICR closure reaches a peak of 4-8 microns/sec. Since the maximum measured velocity of actin filament translocation by vertebrate, non-muscle myosins is 0.04 micron/sec, the later observations support a model in which the CR is segmented, containing multiple sites where filaments overlap in a "sliding filament" fashion. Because the rate decreases after reaching a peak, the results also suggest that the number of overlap sites decrease with time.     

Simultaneous determination of amphetamine,methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in human hair by gas chromatography-mass spectrometry

A procedure is presented for the simultaneous identification and quantification of amphetamine (AP), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) in human hair. The method involves decontamination of hair with dichloromethane and warm water, heat-alkaline hydrolysis in the presence of deuterated internal standards, liquid-liquid extraction and gas chromatography-mass spectrometry after derivatization with pentafluoropropionic anhydride-pentafluoropropanol. The limit of detection for AP, MA and MDA was 0.05 ng/mg using a 50-mg hair sample; for MDMA it was 0.1 ng/mg. Coefficients of variation ranged from 7 to 18%. This assay has been successfully utilized in the evaluation of the deposition of the drugs in hair obtained from various parts of the anatomy of a stimulant abuser.     

Development of a reference material using methamphetamine abusers' hair samples for the determination of methamphetamine and amphetamine in hair

In the present study, we developed a reference material (RM) using authentic hair samples for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) in human hair. MA abusers' hair samples were collected, homogenized and finally bottled. The concentration of each bottle was 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). Both analytical procedures were fully validated and their extraction efficiency was compared. The homogeneity of analytes was evaluated and their property values were determined with their uncertainties. The two methods were acceptable to analyze MA and AP in human hair through the validation and comparative studies using spiked and authentic hair samples as well as NIST SRM 2379 certified reference material. Satisfying homogeneity was reached for MA and AP in the prepared RM. Finally, a human hair RM containing MA and AP is prepared at the level of 7.64+/-1.24 and 0.54+/-0.07 ng/mg, respectively. This material can be useful in forensic laboratories for internal quality control and external quality assurance.     

Hair analysis for drugs of abuse XIX. Determination of ephedrine and its homologs in rat hair and human hair

A sensitive GC-MS method was developed for the quantitative analysis of ephedrine (EP), phenylpropanolamine (PPA) and methylephedrine (ME) in animal and human hair. After washing with 0.1% sodium dodecyl sulfate, hair samples (10 mg) were added with deuterated internal standards, extracted by 1-h sonication and over night soaking in 2 ml of 5 M HCl-methanol (1:20) at room temperature. Following evaporation of the liquid phase, the residue was dissolved in phosphate buffer solution (pH 6.0) and purified using a solid-phase extraction procedure with Bond Elut Certify columns. Two types of derivatization were compared - using trifluoroacetic anhydride (TFAA) and pentafluoropropionic anhydride (PFPA) - for discrimination of EP and methamphetamine (MA). Derivatized extracts were analyzed by GC-MS in the EI mode using a capillary column (OV-1 equivalent). From the results comparing three GC-MS conditions, PFP-derivatives separated with a temperature gradient of 20°C/min from 60°C to 280°C gave the best resolution between EP and MA. ME was analyzed as a trimethylsilyl derivative using N,O-bis-trimethylsilyl acetamide at the above GC condition. The assay was linear from 0.5 to 50 ng/mg (r=0.998) and capable of detecting less than 50 pg of derivatized EP, PPA and ME on-column. Intra-assay precision was characterized by C.V. values from 5 to 16% in the concentration range of 1–10 ng/mg hair. The method was used for the quantitative determination of EP, PPA and ME in the hair obtained from three rats with dark brown hair after ten intraperitoneal injections (5 mg/kg/day) of the three drugs and from three male and one female volunteers with black hair after an oral dose of 50 mg/day of EP-HCl for three days. Hair samples were collected by shaving from the back of rats and cutting from the scalp of humans 28 days after the first dose. The incorporation rates of EP, PPA and ME into hair (the ratios of [hair concentration] to [AUC]) obtained from the animal experiment were 0.10, 0.07 and 0.03, respectively, which are a little lower than those (0.14, 0.10 and 0.04) of their desoxy-compounds, MA, amphetamine and dimethylamphetamine. EP was detected at an average of 2.25 ng/mg (n=4) in human scalp hair and at a range of 1–29 ng/mg (n=3) in human beard hair until day 14, but its metabolite (PPA) was at a trace level in the hair of the four subjects. The method was successfully used for detection of ME and EP in the hair of a neonate and its mother who was abusing Bron syrup containing ME during the pregnancy.     

Simultaneous enantioselective determination of amphetamine and congeners in hair specimens by negative chemical ionization gas chromatography-mass spectrometry

Enantioselective quantification of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers in hair using gas chromatography-mass spectrometry (GC-MS) is described. Hair specimens were digested with 1M sodium hydroxide at 100 degrees C for 30 min and extracted by a solid phase procedure using Cleanscreen ZSDAU020. Extracted analytes were derivatised with (S)-heptafluorobutyrylprolyl chloride and the resulting diastereoisomers were quantified by GC-MS operating in the negative chemical ionization mode. Extraction yields were between 73.0 and 97.9%. Limits of detection varied in the range of 2.1-45.9 pg/mg hair, whereas the lowest limits of quantification varied between 4.3 and 91.8 pg/mg hair. Intra- and inter-assay precision and respective accuracy were acceptable. The enantiomeric ratios (R versus S) of AM, MA, MDA, MDMA and MDEA were determined in hair from suspected amphetamine abusers. Only MA and AM enantiomers were detectable in this collective and the quantification data showed in most cases higher concentrations of (R)-MA and (R)-AM than those of the corresponding (S)-enantiomers.     

Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX.     

Antisense noncoding RNA promoter regulates the timing of de novo methylation of an imprinting control region

Epigenetic marks at cis acting imprinting control regions (ICRs) regulate parent of origin-specific expression of multiple genes in imprinted gene clusters. Epigenetic marks are acquired during gametogenesis and maintained faithfully thereafter. However, the mechanism by which differential epigenetic marks are established and maintained at ICRs is currently unclear. By using Kcnq1 ICR as a model system, we have investigated the functional role of genetic signatures in the acquisition and maintenance of epigenetic marks. Kcnq1 ICR is methylated on the maternal chromosome but remains unmethylated on the paternal chromosome. Here, we show that a paternal allele of Kcnq1 ICR lacking the Kcnq1ot1 promoter remains unmethylated during spermatogenesis; however, it becomes methylated specifically during pre-implantation development. Analysis of the chromatin structure at the paternal ICR in spermatogenic cells and in E13.5 embryonic tissues revealed that the ICRs of both wild type and mutant mice are enriched with H3K4me2 in spermatiogenic cells of the testicular compartment, but the mutant ICR lost H3K4me2 specifically in epididymal sperm and an increase in repressive marks was observed in embryonic tissues. Interestingly, we also detected a decrease in nucleosomal histone levels at the mutant ICR in comparison to the wild-type ICR in epididymal sperm. Taken together, these observations suggest that the Kcnq1ot1 promoter plays a critical role in establishing an epigenetic memory in the male germline by ensuring that the paternal allele remains in an unmethylated state during pre-implantation development.     

Single hair analysis of methamphetamine and amphetamine by solid phase microextraction coupled with in matrix derivatization

A sensitive method for detection of methamphetamine (MA) and amphetamine (AP) in human hair was developed using solid phase microextraction (SPME) and one-pot derivatization. MA and AP were directly derivatized to N-propoxycarbonyl derivatives in an aqueous solution by propylchloroformate in a one-pot reaction before extraction by SPME. The derivatives were extracted to a coating of SPME from a headspace of the vial. The adsorbed derivatives were thermally desorbed in the injection port of a gas chromatograph. Pentadeuterated MA was used as an internal standard. The absolute recoveries of MA and AP from the spiked hair were 2.80-17.5%, respectively. The calibration curves showed linearity in the range of 0.05-20 ng/0.08 mg/vial for MA and 0.1-20 ng/0.08 mg/vial for AP in hair. Detection limits (S/N = 3) of MA and AP were 0.02 and 0.05 ng/0.08 mg/vial. The coefficients of variation of intraday were 1.04-26.4%. Additionally, this proposed method was applied to segmental analysis in clinical and medico-legal cases of MA intoxication.     

Routine analysis of amphetamine and methamphetamine in biological materials by gas chromatography-mass spectrometry and on-column derivatization

A simple determination method of amphetamine (AP) and methamphetamine (MA) in biological materials was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). AP and MA in biological materials were adsorbed on the surface of Extrelut and then extracted and derivatized simultaneously on the Extrelut column. AP and MA were derivatized to the N-propoxycarbonyl derivatives using propylchloroformate. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 88.2 and 92.5%, and those from blood were 89.7 and 90.3%, respectively. The calibration curves showed linearity in the range of 12.5-2000 ng/ml (ng/g) for AP and MA in urine and blood, and 0.25-20 ng/mg in hair. When urine samples containing two different concentrations (200 and 1000 ng/ml) of AP and MA, blood samples containing two different concentrations (200 and 1000 ng/g) of AP and MA, hair samples containing two different concentrations (0.5 and 5.0 ng/mg) of AP and MA, the coefficients of variation of intra-day and inter-day were 0.68-3.60% in urine, 0.42-4.58% in blood, and 1.20-13.1% in hair. Furthermore, this proposed method was applied to a medico-legal case of MA intoxication.     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

Integration of GC/EI-MS and GC/NCI-MS for simultaneous quantitative determination of opiates, amphetamines, MDMA, ketamine, and metabolites in human hair

In this paper, the possibility of using a multiple ionization mode approach of GC/MS was developed for the simultaneous hair testing of common drugs of abuse in Asia, including amphetamines (amphetamine, AP; methamphetamine, MA; methylenedioxy amphetamine, MDA; methylenedioxy methamphetamine, MDMA; methylenedioxy ethylamphetamine, MDEA), ketamine (ketamine, K; norketamine, NK), and opiates (morphine, MOR; codeine, COD; 6-acetylmorphine, 6-AM). This strategy integrated the characteristics of gas chromatography-mass spectrometry (GC-MS) using electron impact ionization (EI) and negative chemical ionization (NCI). Hair samples (25 mg) were washed, cut, and incubated overnight at 25 degrees C in methanol-trifluoroacetic acid (methanol-TFA). The samples were extracted by solid phase extraction (SPE) procedure, derivatized using heptafluorobutyric acid anhydride (HFBA) at 70 degrees C for 30 min, and the derivatives analyzed by GC-MS with EI and NCI. The limit of detection (LOD) with GC/EI-MS analysis obtained were 0.03 ng/mg for AP, MA, MDA, MDMA, and MDEA; 0.05 ng/mg for K, NK, MOR, and COD; and 0.08 ng/mg for 6-AM. The LOD of GC/NCI-MS analysis was much lower than GC/EI-MS analysis. The LOD obtained were 30 pg/mg for AP and MDA in GC/EI-MS and 2 pg/mg in GC/NCI-MS. Therefore, the sensitivity of AP and MDA in GC/NCI-MS was improved from 15-fold compared with EI. The sensitivity of AP, MA, MDA, MDMA, MDEA, MOR, and COD was improved from 15- to 60-fold compared with EI. In addition, the sensitivity of 6-AM increased 8-fold through selection of m/z 197 for the quantitative ion. Moreover, K and NK could dramatically improve their sensitivity at 200- and 2000-fold. The integration of GC/EI-MS and GC/NCI-MS can obtain the high sensitivity and complementary results of drugs of abuse in hair. Six hair samples from known drug abusers were examined by this new strategy. These results show that integrating the characteristics of GC/EI-MS and GC/NCI-MS were not only enhancement of the sensitivity but also avoid wrong results and wrong interpretations of correct results.     

Sensitive, rapid and validated gas chromatography/negative ion chemical ionization-mass spectrometry assay including derivatisation with a novel chiral agent for the enantioselective quantification of amphetamine-type stimulants in hair

A novel chiral derivatisation agent, (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, was used for the indirect resolution of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers using gas chromatography coupled to mass spectrometry operating in the negative-ion chemical ionization mode (GC/MS-NICI). This new chiral derivatisation reagent was readily obtained in optically pure form after a simple two-step synthesis. Optimal derivatisation was accomplished in 15 min at room temperature in a carbonate buffer and the resulting diastereoisomers were base line separated by GC in 12 min only. No racemization was observed during the derivatisation. The method was applied and fully validated for the enantiomeric quantification of amphetamines and methylenedioxylated amphetamines in hair. The analyses of 24 hair specimens from suspected ATS abusers showed that 24 cases were positive for MA and/or AM enantiomers and that in most cases the concentrations of (S)-MA and (S)-AM exceeded those of the corresponding (R)-enantiomers. One hair specimen was tested positive for both enantiomers of MDMA and MDA.     

Hair analysis and self-report of methamphetamine use by methamphetamine dependent individuals

The questions of whether the dose of drug that is consumed corresponds to drug concentration levels in hair and how results of hair analyses can be interpreted are still debated. The aim of this study was to investigate (1) whether there is a correlation between doses of Methamphetamine (MA) use and MA concentration levels in hair and (2) whether results of hair analyses can be used to estimate dose, frequency, and patterns of MA use. In this study, segmental hair analysis was performed through consecutive 1cm as well as 1-4 cm (=3 cm) segmental hair lengths. MA dependent individuals (n=9) provided information on doses (0.25-4 g/day) of MA use as well as the frequency of MA use. The concentrations of MA and its metabolite amphetamine (AP) in hair were determined using gas chromatography/mass spectrometry (GC/MS). One-way analysis of variance (ANOVA) test was performed to evaluate whether MA and AP concentrations in consecutive 1cm length segmental hair were consistent with the history of MA use. The cumulative doses of MA use calculated from the daily dose and the frequency during 1-4 months were well correlated to the concentrations of MA and AP in 1-4 cm segmental hair length (correlation coefficient, r=0.87 for MA and r=0.77 for AP). The results from this study show the patterns and histories of MA use from MA dependent individuals and could assist in the interpretation of hair results in forensic toxicology as well as in rehabilitation and treatment programs.