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Sleeping aids are often abused in the commission of drug-facilitated crimes. Generally, there is little evidence that a victim ingested a spiked drink unknowingly because the unconscious victim cannot report the situation to the police immediately after the crime occurred. Although conventional segmental hair analysis can estimate the number of months since a targeted drug was ingested, this analysis cannot determine the specific day of ingestion. We recently developed a method of micro-segmental hair analysis using internal temporal markers (ITMs) to estimate the day of drug ingestion. This method was based on volunteer ingestion of ITMs to determine a timescale within individual hair strands, by segmenting a single hair strand at 0.4-mm intervals, corresponding to daily hair growth. This study assessed the ability of this method to estimate the day of ingestion of an over-the-counter sleeping aid, diphenhydramine, which can be easily abused. To model ingestion of a diphenhydramine-spiked drink unknowingly, each subject ingested a dose of diphenhydramine, followed by ingestion of two doses of the ITM, chlorpheniramine, 14days apart. Several hair strands were collected from each subject's scalp several weeks after the second ITM ingestion. Diphenhydramine and ITM were detected at specific regions within individual hair strands. The day of diphenhydramine ingestion was estimated from the distances between the regions and the days of ITM ingestion. The error between estimated and actual ingestion day ranged from -0.1 to 1.9days regardless of subjects and hair collection times. The total time required for micro-segmental analysis of 96 hair segments (hair length: 3.84cm) was approximately 2days and the cost was almost the same as in general drug analysis. This procedure may be applicable to the investigation of crimes facilitated by various drugs.

Methods to quickly purify methamphetamine hydrochloride from the cutting agent dimethyl sulfone for subsequent identification of confiscated crystalline samples using infrared absorption spectroscopy were compared and evaluated. Although sequential solvation and reprecipitation methods were simple, spectral contamination from dimethyl sulfone was inevitable and might affect the interpretation of the spectra. In addition, methamphetamine hydrochloride and dimethyl sulfone could form a solid solution because of solvation of both crystals into a single solution layer. By contrast, sublimation was an effective method for separation of methamphetamine hydrochloride and dimethyl sulfone. Sublimation combined with infrared absorption spectroscopy enabled rapid identification of crystalline methamphetamine hydrochloride.

We developed a novel immunoassay for herbal cannabis based on a new immunoassay principle that uses Ultra Quenchbody ("UQ-body"), a recombinant antibody Fab fragment fluorolabeled at the N-terminal regions. When the antigen binds to anti-Δ(9)-tetrahydrocannabinol (THC) UQ-body, the fluorescence intensity (FI) decreases. The analytical conditions of the immunoassay were optimized based on the FI reduction rate (FIRR). Following are the steps in the final analytical procedure: (1) 10mg of samples were extracted with 1ml of a 60:40 mixture of methanol and phosphate-buffered saline (PBS); (2) the extract was filtered through a centrifugal 0.2-μm polytetrafluoroethylene membrane filter; (3) the filtrate was diluted 100 times with extraction solvent; (4) 6-μl diluted solution was mixed with 19-μl PBS and 75-μl UQ-body solution; and (5) FIRR was measured under 275-mV excitation light. Herbal cannabis samples containing ≥4.0-mg/g THC gave FIRRs of ≥5.2%. FIRRs of negative samples (cigarette, tea, spice, and so-called "synthetic marijuana") were ≤3.1%. When setting the FIRR threshold to 5.0%, cannabis samples containing ≥4.0-mg/g THC were correctly judged as positive without being affected by false positives caused by the negative samples. This detection limit was lower than total THC level (10-200mg/g) in most herbal cannabis samples seized in Japan. In seven of the 10 cannabis samples, the results of the UQ-body test were comparable with those of the Duquenois-Levine test. Thus, the UQ-body-based immunoassay is presumed to be an effective and objective drug screening method for herbal cannabis; however, to show the true usefulness, it is necessary to test a number of real case samples in the field situation.

In recent years, the need for analyzing cannabis DNA has increased in order to accommodate the various types of cannabis samples encountered in forensic investigation. This study was aimed to establish a simple and accurate cannabis DNA detection system using DNA chromatography. Two chromatography chip systems with different features were successfully developed. One system (the "four-line version") involves tetraplex PCR amplification, which could be used to detect cannabis DNA and distinguish between drug-type and fiber-type cannabis using the tetrahydrocannabinolic acid synthase gene sequence. The other system was the "three-line version" with triplex amplification, which was specialized to distinguish cannabis from other plants, and had a sensitivity (10fg DNA/reaction) that was 100 times greater than the four-line version. In both versions, no false positives were observed for 60 medicinal plants, and accurate detection could be performed for several simulated forensic samples such as cannabis leaves, buds, stems, roots, seeds, resin, and cannabis leaves blended 1/100 in tobacco. Detection could be performed by the naked eye and only a thermal cycler was required for operation. Thus, DNA chromatography systems for cannabis detection are expected to contribute to the analysis of cannabis DNA in forensic chemistry laboratories without extensive equipment.

A portable near infrared spectrometer was applied to the presumptive identification of psychotropic drugs based on library searching. Data-treatment methods (mathematical pretreatment and library search algorithm) were examined on the basis of differentiation ability. The optimized mathematical pretreatment was a standard normal variate followed by the 2nd derivative. The correlation coefficient showed the best differentiation ability in the library search algorithms. Optimized data-treatment was effective for minimizing the effect of particle size on identification. The optimized data-treatment methods were validated by the spectra of psychotropic substances (n=120). Identification criteria for the psychotropic drugs were decided on the basis of the results of the validation. As a consequence, 8 out of 11 forensic samples containing psychoactive substances were able to be positively identified. Thus, the portable near infrared spectrometer with optimized data-treatment processing is a useful tool for rapid screening and presumptive identification of seized materials.

α-Pyrrolidinopentiophenone (α-PVP) is a popular recreational drug in Japan. This drug easily undergoes thermal decomposition during gas chromatography/mass spectrometry analysis. We evaluated three factors involved in the decomposition, namely the injection method (splitless or split, split ratio), injector temperature, and surface activity on the inlet liner. Splitless injection of α-PVP using a used deactivated split/splitless liner at an injector temperature of 250°C caused thermal decomposition. This decomposition was inhibited by split injection. A higher split ratio resulted in greater prevention. Based on the mass spectrum of deuterated α-PVP, the decomposition product was presumed to be an enamine whose double bond was located in the alkyl chain. Lowering the injection temperature from 250°C to 200°C did not prevent decomposition. New glass liners, both deactivated and non-deactivated, were compared. The use of a new deactivated liner minimized thermal decomposition, even for splitless injection, while the non-deactivated liner generated an increase in the amount of the decomposition product. These results showed that the injection method and the surface activity on the inlet liner were involved in the thermal decomposition of α-PVP.

A method for determining the geographical origin of unidentified cadavers by determining the genotype of Helicobacter pylori, which is latent in one-half of the world's population, was developed. In the first stage, DNA was extracted from samplings at 5 points in the gastric mucosa of 177 individuals randomly selected from cadavers undergoing medico-legal autopsy. 16S-rDNA of H. pylori DNA was detected by polymerase chain reaction (PCR) in 101 cadavers (57.0%); by sex, 74 of 123 (60.1%) males and 28 of 54 (46.4%) females were positive. There were no significant differences in H. pylori detection rate among the 5 sampling points of the gastric mucosa, cause of death, or age. In the second stage, amplified fragments of H. pylori vacA regions s and m from 17 individuals with the following ethnic backgrounds were sequenced: Japanese, 10; Chinese, 2; South Korean, 1; Taiwanese, 1; Thai, 1; Afghan, 1; and Filipino, 1. A phylogenetic tree constructed with these and 28 previously reported H. pylori strain sequences revealed 3 major gene clusters consisting of East Asian type I (Japanese, South Korean and Chinese), Western type II, and Southeast Asia type III. The Taiwanese and Filipino samples deviated from the clusters type III to which they typically belong. The ultimate aim of the present study was to develop a more accurate method of determining of geographic origin of unidentified cadavers through the combination of the present method with other, virus-based methods H. pylori DNA was detected from over half of the cadavers tested and vacA genotypes showed specificity to geographical origin. Therefore, these results suggest that the H. pylori genome provides valuable additional information for tracing the geographical origin of unidentified cadavers.

The enantiomers of methamphetamine were differentiated by supercritical fluid chromatography (SFC) with an enantioselective cellulose-based packed column. The optimization of the chromatographic conditions was achieved by changing column temperature, co-solvent proportion, additive concentration, flow rate and back pressure. In particular, the additive concentration crucially changed the resolution between the enantiomers. After determining the optimized conditions, the enantiomers of methamphetamine were successfully separated. The analytical precision, accuracy and limit of detection were checked by using the authentic standard and seized real samples. We believe that chiral SFC is a promising method for enantioseparation of forensic samples.

The Scott test, widely used as the field test for cocaine, is performed in three steps. If a sample contains cocaine, blue precipitates appear in step 1, the precipitates are dissolved and the solution turns pink in step 2, and the lower layer turns blue in step 3. However, some pyrrolidine-type cathinones produce cocaine-like results when tested, necessitating modification of the test procedure. Filtration of the second-step mixture weakened the blue color in step 3; however, the blue color did not completely disappear. Adding the Chen-Kao reagent to the test procedure enhanced the differentiation: when the reagent was added to cocaine, the solution was initially turbid, but then became clear over time; its addition to cathinones resulted in turquoise or light sky-blue precipitation. These results indicated that the Chen-Kao test was useful for exclusion of cathinones. A combination of the modified Scott test and the Chen-Kao test was successfully applied to the forensic samples containing cocaine or pyrrolidine-type cathinones. In conclusion, a combination of these tests will be the useful field-test procedure for cocaine.

Analogs of methcathinone (MC), a psychoactive stimulant, are in circulation all over the world. These analogs have been assumed to be unstable in alkaline solutions, as is MC itself. The aims of this study were: (i) to identify the degradation products of 4-methylmethcathinone (4-MMC), a typical MC analog, in solution at pH 12 and to determine the degradation pathway, (ii) to investigate the effects of antioxidants such as l-ascorbic acid and sodium sulfite on the degradation of 4-MMC, and (iii) to investigate the stability of seven MC analogs (4-MMC, 4-, 3-, or 2-fluoromethcathinone, 4-methoxymethcathinone, N-ethylcathinone, and N,N-dimethylcathinone) in solutions at different pHs.1-(4-Methylphenyl)-1,2-propanedione (MPPD), 4-methylbenzoic acid (MBA), N,4-dimethylbenzamide (DMBA), and N-acetyl-4-MMC (N-Ac-4-MMC) were identified as the degradation products of 4-MMC in pH 12 solution by gas chromatography-mass spectrometry. There are two degradation pathways for 4-MMC as follows: (a) 4-MMC→MPPD→MBA→DMBA and (b) 4-MMC→N-Ac-4-MMC. Oxidants such as dissolved oxygen were presumed to be involved in this degradation based on the suppressive effects generated by the addition of antioxidants. All of the seven MC analogs tested were stable in acidic (pH 4) solution but degraded in neutral-to-basic solutions. Their degradation rates increased with increasing pH, and varied with their chemical structures. These findings will be very useful for not only forensic analysis but also future pharmacokinetic analysis.

Impurities in 48 methamphetamine (MA) samples were analyzed by liquid–liquid extraction (LLE) and headspace solid-phase microextraction (HS-SPME) methods. MPS-2 autosampler was used to improve reproducibility of SPME method, and nonadecane (C 19 ) diluted with potassium bromide (KBr) powder was used as an internal standard for standardizing retention time. Impurities identified by SPME method showed different patterns compared with LLE method. Non-volatile impurities like methamphetamine dimer were not identified by SPME method, but some volatile impurities like diphenylketone, caprolactam and lots of unknowns were identified only by SPME method. 1-Phenyl-2-propanone (P2P), 1-phenyl-2-propanol and benzylcyanide peaks could be discriminated clearly by SPME method without interference of amphetamine, an artifact originates from MA degradation. Differences in the impurity patterns resulted in different clustering results. When 48 MA samples were classified into 5 LLE and 5 SPME clusters, cross-matching of the clusters resulted in 8 sub-clusters. It shows that combination of the different extraction methods can distinguish the differences which cannot be distinguished by LLE or SPME method alone, and can improve reliability of the profiling results.

An automated full-range quantitation method for identifying d-methamphetamine in human hair using a microchip-based ELISA system (microELISA) in combination with a micropulverized extraction method for sample preparation has been developed. An antibody and a peroxidase-linked methamphetamine, both are commercially available, were used for the competitive ELISA assay. Method validation was carried out using doped hair samples, and segmental analyses of real-case specimens were carried out by both microELISA and LC/MS/MS to verify the reliability and applicability of this new method. Due to the small size of the system and the lack of an evaporation process, sample preparation and quantitation can be accomplished easily and quickly (less than 30 min) in small-scale contamination-free environments.

We previously developed a simple and highly sensitive analytical method for 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) in spiked hair using micropulverized extraction (MPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Using this method, we were able to quantify THC-COOH at 0.2pg/mg, which is the cut-off level recommended by the Society of Hair Testing. However, it was impossible to prove the validity of the method and the presence of THC-COOH conjugated with glucuronide in hair because we did not have authentic hair containing THC-COOH at the cut-off levels at that time. In this study, the previously developed method was verified using recently obtained hair from a cannabis user. The concentrations of THC-COOH quantified using the method were 0.36±0.01pg/mg without hydrolyzation for glucuronide and 0.49±0.05pg/mg with hydrolyzation after MPE, whereas the concentration quantified using the conventional alkaline dissolution and gas chromatography/tandem mass spectrometry with negative ion chemical ionization was 0.50±0.02pg/mg. The results proved that THC-COOH could be extracted completely from authentic hair containing THC-COOH at the recommended cut-off level using MPE. In addition, MPE with and without hydrolyzation, unlike alkaline dissolution of hair, enabled the measurement of the percentage of the conjugate form in total THC-COOH. The percentage of conjugated THC-COOH in hair measured using the MPE was approximately 26%, which was greatly different from previously reported data (>75%). The discrimination between conjugated and unconjugated compounds in hair is important to understand the mechanism of drug uptakes into hair. More data obtained with our simple and highly sensitive method from the hair of cannabis users would help to understand the relationship of concentrations between THC-COOH and its conjugate in hair.

Amphetamine-type stimulants (ATSs) are often abused orally in the form of tablets for recreational purposes. The ATS tablets contain one or more active ingredients such as 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine (MA), ketamine (KA), and caffeine (CF). The aim of this work is to determine whether such components in tablets interact with each other in intestinal absorption. The interactions between MDMA, MA, KA, and CF in the uptake and permeation by human intestinal epithelial Caco-2 cell line were investigated in monolayer cultures. MDMA, MA, and KA mutually inhibited the uptakes by Caco-2 cells. The inhibition of MA uptake by KA was the greatest of all combinations (72.6% inhibition). Similarly, MDMA, MA, and KA mutually inhibited the permeation from the apical to the basolateral side through Caco-2 cells. Although CF did not affect the uptakes of MDMA, MA, and KA, CF enhanced the permeation of MDMA in comparison to MDMA alone. In addition, the interaction of MA with KA and that of MDMA with CF in intestinal absorption were investigated by oral administration to rats. The area under the plasma concentration-time curve of MA significantly decreased by co-administration with KA in comparison to MA alone, while that of MDMA significantly increased by co-administration with CF in comparison to MDMA alone. The results in rats were similar to those in Caco-2 cells. These findings suggest that the intestinal absorption of similar compounds with amine moieties such as MDMA, MA, and KA are mediated by a common transport system, and that CF affects the absorption of MDMA in a different way from the transport system. In human, intakes of ATS tablets mixed with such components might result in similar interactions in intestinal absorption to those in Caco-2 cells and rats.

The constituents of seven mushrooms sold as Amanita muscaria or Amanita pantherina (five A. muscaria and two A. pantherina) and four "extracts purported to contain A. muscaria" products that are currently circulated in Japan were determined. All mushroom samples were identified as A. muscaria or A. pantherina by macroscopic and microscopic observation. The dissociative constituents, ibotenic acid (IBO) and muscimol (MUS), were extracted with 70% methanol twice and determined by gas chromatography/mass spectrometry. The IBO (as the hydrate)/MUS contents were in the range of

The applicability of capillary electrophoresis (CE) with a UV detector using highly sulfated γ-cyclodextrin as a chiral selector was examined for analysis of impurities in seized methamphetamine. Samples of methamphetamine-hydrochloride dissolved in water at a high concentration (20 mg/mL) were analyzed. Electrokinetic injection has an advantage over hydrodynamic injection for improving the detection of trace impurities. Small peaks of the precursor impurities, such as (1 R ,2 S )-(−)-ephedrine and (1 S ,2 S )-(+)-pseudoephedrine, were detected and quantified without extraction. The seized drugs could be classified into three groups based on the contents of the two impurities.

The metabolism of methamphetamine (MA) and 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in freshly isolated rat hepatocytes was investigated, and compared with in vivo results. A suspended hepatocyte culture, established from male Wistar rats using a collagenase perfusion technique, was incubated in the presence of MA or 2C-B. After enzymatic hydrolysis of the conjugated forms, the metabolites were extracted by liquid-liquid partition and analyzed by gas chromatography/mass spectrometry (GC/MS). Amphetamine, p-hydroxymethamphetamine and p-hydroxyamphetamine were detected in the culture fluids of the rat hepatocytes inoculated with MA. The alcohol derivative, carboxylic acid derivative, 2-O-desmethyl-2C-B, 2-O-desmethyl-N-acetyl-2C-B and 5-O-desmethyl-N-acetyl-2C-B were detected in the case of 2C-B. The major metabolite of MA in rat hepatocytes was p-hydroxymethamphetamine. This is in good agreement with the urinary excretion profile for rats that were fed MA. 2-O-Desmethyl-2C-B and the carboxylic acid derivative were the major recovered metabolites of 2C-B in the rat hepatocyte culture, a slight deviation from the in vivo findings, in which 5-O-desmethyl-N-acetyl-2C-B was found to be the main component. Metabolites with a hydroxy group were largely present in their conjugated forms in the culture fluids, except for 2-O-desmethyl-2C-B. Taking these results into consideration, a primary hepatocyte culture system has the potential to provide a quick and handy method for estimating the in vivo metabolic fate of abused drugs.

A B S T R A C T We observed the decomposition of the hydrochloride salt of a-pyrrolidinoheptanophenone (a-PHPPHCl), a newly distributed pyrrolidine-type cathinone derivative when 2.5 ng of this substance was placed in glass test tubes and stored in a refrigerator for 3 days. To further investigate this phenomenon, we studied the (i) time course of the residual ratios of a-PHPP-HCl when a small amount (10 mg) of a-PHPPHCl was stored in glass vials in air at room temperature; (ii) identification of the decomposition products of a-PHPP-HCl; (iii) effect of air on the decomposition process; (iv) effect of the added amounts of aPHPP-HCl on its decomposition; and (v) comparison of the stability between various cathinone derivatives and their decomposition products. The decomposition of a-PHPP-HCl occurred in air and increased with time. Two possible decomposition products, a-(2 00-oxopyrrolidino)heptanophenone and a-PHPP-N-oxide, were identified. These products were formed by oxygen in air because the yield significantly decreased by storing them in a vacuum desiccator. With the decrease in the amount of aPHPP-HCl, the residual ratios decreased and amount of the decomposition products increased. This indicates that the decomposition of a-PHPP-HCl occurred on the upper surface of the samples. The hydrochloride salts of other cathinone derivatives were also unstable in air, and the residual ratios observed were different depending on the compounds. The pyrrolidine-type cathinone derivatives afforded two types of decomposition products, which were presumed to be 2 00-oxo and N-oxide derivatives, similar to a-PHPP-HCl. In contrast, secondary amine-type cathinone derivatives showed different decomposition patterns, possibly including the dealkylated derivative. These findings may be very useful for the future toxicological analysis of cathinone derivatives.

Chiral capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) using a chemically modified capillary containing sulfonated groups was developed for the following 8 amphetamine-type stimulants (ATS): amphetamine, methamphetamine (MA), norephedrine, norpseudoephedrine, ephedrine (EP), pseudoephedrine (pEP), dimethylamphetamine and methylephedrine. The running buffer was 10 mM formic acid containing 20 mM highly sulfated γ -cyclodextrin (pH 2.5) as the chiral selector. All 16 enantiomers were well resolved within 60 min, and precisely identified due to their characteristic mass spectra. Further, the RSDs of the migration times of the analytes were no more than 0.3% without any standardization. (1 R ,2 S )-(−)-EP and (1 S ,2 S )-(+)-pEP, which are important ATS impurities originating in the precursors, were added to a highly concentrated MA solution (1 mg/mL) and analyzed as mock samples for MA impurity analysis. Acceptable repeatability of the migration times of (−)-EP and (+)-pEP (ca. 0.3% RSDs) was still observed without interference from the large amount of MA. The limits of detection (LOD) of (−)-EP and (+)-pEP were approximately 2 μg/mL, therefore, their LOD as the impurity concentrations were calculated at about 0.2%. Seized MA samples were dissolved in water at a high concentration (1 mg/mL) and analyzed by this method. (−)-EP and (+)-pEP were clearly detected as impurities. Although these compounds had similar migration times and mass spectral patterns, the fine repeatability allowed easy identification of the impurities by a simple comparison of the absolute migration times of the specimens and those of authentic standards. This study is the first to report the use of a chemically modified capillary for the impurity profiling on CE/MS/MS.

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis™ HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic–lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 m×0.32 mm I.D., film thickness 0.25 μm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5–500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.

A 24-year-old male was found dead in a car left in a river for about 3 months. The cadaver was almost adipoceratous and autopsy findings revealed that there were neither remarkable injuries nor lethal diseases. Toluene, ethanol, 1-propanol, 2-propanol, 1-butanol, dimethyl sulfide, dimethyl disulfide, isovaleraldehyde and n-butyl n-butyrate were detected in the specimens collected at the autopsy by head space gas chromatography/mass spectrometry (GC/MS). The toluene concentrations (micrograms/g) were 31.0 in brain, 10.6 in liver, 5.4 in kidney, 15.0 in skeletal muscle and 187.1 in adipose tissue. The presence of diatom in lung, liver and kidney suggested that death was caused by drowning. So far as we know, this is the first report of detection of toluene in an adipoceratous body.

We report a case of seized crystalline methamphetamine (MA) samples showing unique drug profiles. The samples were mainly composed of (S)-(+)-MA, with each containing a slight amount of (R)-(-)-MA (enantiomeric excess: 99.2-99.4%). 1-Phenyl-2-propanol and N-methyl-2-phenylacetamide were detected as characteristic impurities. These analytical results suggested that the samples were synthesized as racemic MA by reductive amination of 1-phenyl-2-propanone, which was synthesized from phenylacetic acid, putatively prepared from phenylacetic acid ester, and then the racemic MA was optically resolved to the (+)-form-rich product. This proposed preparation route was in accordance with recent reports of seizures worldwide of the raw materials of MA such as phenylacetic acid derivatives, methylamine, and tartaric acid (optical resolving reagent).

We examined the applicability of chemically modified capillaries on the chiral capillary electrophoresis of essential compounds for methamphetamine (MA) profiling (MA, amphetamine, ephedrine, pseudoephedrine, norephedrine, and norpseudoephedrine) using highly sulfated γ-cyclodextrin as a chiral selector. Four types of chemically modified capillaries, namely, FunCap-CE/Type D (possessing diol groups), Type A (amino groups), Type C (carboxyl groups), and Type S (sulfate groups), were evaluated. Repeatability, speed, and good chiral resolution sufficient for routine MA profiling were achieved with the Type S capillary.

Methamphetamine (MA) is one of the most frequently abused drugs worldwide. The aim of this study is to improve the analytical method for profiling MA impurity in order to compare and classify MA crystals seized in different countries and to investigate the relationships between seizures. To compare MA samples seized in Japan and Thailand, the following analytical method was adopted. A 50mg sample of MA.HCl was dissolved in 1ml of buffer solution (four parts 0.1M phosphate buffer, pH 7.0, and one part 10% Na(2)CO(3)), impurities were extracted with 0.5ml of ethyl acetate containing four internal standards (n-decane, n-pentadecane, n-eicosane and n-octacosane) and analyzed by gas chromatography with a flame ionization detector on a DB-5 capillary column (0.32mm i.d.x30m, film thickness 1.0mum). Fourteen characteristic peaks on chromatograms were selected for the comparison and classification of samples, and the data were evaluated by the Euclidean distance of the relative peak areas after logarithmic transformation. Sixty-nine samples seized in Japan and 42 seized in Thailand were analyzed. The samples were classified into four groups roughly by cluster analysis. In addition, when it was difficult to compare samples that had fewer impurities on chromatograms obtained from liquid-liquid extraction (LLE), solid-phase microextraction (SPME) was effective. Because many characteristic peaks were detected using SPME, SPME made it easy to compare samples of high purity. The combination of LLE and SPME was useful for impurity profiling of MA samples seized in different countries.

Impurity profiling of methamphetamine (MA) using thermal desorption (TD) and gas chromatography–mass spectrometry (GC–MS) was examined. Using TD/GC–MS, impurities were extracted and separated under various conditions. Optimal chromatograms were obtained when a 20 mg MA sample was extracted at 120 °C for 3 min using a TD instrument, followed by separation of the extracts using a non-polar capillary column coated with (5%phenyl)-methylpolysiloxane. MA samples from nine different batches were analyzed under optimized conditions. Compounds related to the structure of MA, such as benzaldehyde, benzyl alcohol, amphetamine, cis - and trans -1,2-dimethyl-3-phenylaziridine, dimethylamphetamine, and N -acetylephedrine, were detected in the chromatograms without any laborious extraction procedure. Compounds such as ethanol, diethyl ether, and acetic acid, which are considered reagents and solvents for MA synthesis, were also detected in some of the chromatograms. The numbers and intensities of the peaks detected were different among the samples. Impurity profiling of MA using TD was compared with that using liquid–liquid extraction (LLE). Better reproducibility of peak areas was obtained using LLE, whereas higher intensities and numbers of peaks were detected using TD. Solvents were extracted more effectively using TD. The nine batches of MA were classified using both extraction procedures. The nine batches were divided roughly into two groups using data from LLE. Subsequently, the groups were classified in detail using data from TD. TD can be used to provide supplemental information for LLE, and the combination of these extraction methods can be helpful for impurity profiling of MA.

The profiling of impurities in methamphetamine (MA) using headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) is described. The extraction of the impurities with an SPME fiber was examined under varying conditions. Optimal chromatograms were obtained when a 50 mg MA sample at 85 degrees C for 30 min was extracted using a fiber coated with divinylbenzene/carboxen/polydimethylsiloxane. MA samples from nine different origins were analyzed under optimized extraction conditions. Compounds related to MA such as benzaldehyde, benzyl alcohol, amphetamine, benzyl methyl ketone, cis- and trans-1,2-dimethyl-3-phenylaziridine, dimethylamphetamine, N-acetylamphetamine, N-acetylmethamphetamine and N-formylmethamphetamine were detected in the chromatograms. Trace amounts of ethanol, diethyl ether and acetic acid were also detected in some of the chromatograms. The numbers and intensities of the peaks detected were different, depending on the sample. After the areas of the eight principal peaks were converted to their square root and logarithm, similarities among the samples were evaluated by Euclidian distance, cosine distance and correlation coefficient. The results showed that a combination of logarithmic conversion and cosine distance was the most suitable for discriminating and classifying the samples. HS-SPME/GC-MS is a simple and effective method for the extraction and identification of impurities. The present method, in combination with an appropriate statistical analysis, would be useful for developing a profile of impurities in MA.

A screening method for the detection of sedative-hypnotics in serum is described. The target drugs, which include practically all the sedative-hypnotics distributed in Japan, consisted of 5 barbiturates, 30 benzodiazepine-related drugs and 11 other sedative-hypnotics (i.e., apronalide, bromisovalum, chloral hydrate, triclofos, chlorpromazine, promethazine, diphenhydramine, hydroxyzine, zopiclone, zolpidem and tandospirone). Thirty-nine analytes, selected in terms of the pharmacokinetics of the target drugs, in human serum were screened using a combination of mixed-mode solid-phase extraction and liquid chromatography/electrospray-ionization single-quadrupole mass spectrometry. The detection limits (non-basic analytes, 1–50 ng/ml; basic analytes, 0.1–5 ng/ml) were sufficient to permit the screening of a single therapeutic administration of a target drug.

Morphological and toxicological analyses were performed on hallucinogenic mushrooms that are currently circulated in Japan. Scanning electron microscope (SEM) indicated a three-dimensional microstructures in the mushrooms. The complementary use of SEM with an optical microscope was effective for observing characteristic tissues, such as basidiomycetes, spores, cystidia and basidia. Hallucinogenic alkaloids were extracted with methanol and determined by high performance liquid chromatography (HPLC) with a UV detector set at 220nm. The psilocin/psilocybin contents in Psilocybe cubensis were in the range of 0.14–0.42%/0.37–1.30% in the whole mushroom (0.17–0.78%/0.44–1.35% in the cap and 0.09–0.30%/0.05–1.27% in the stem), respectively. The hallucinogenic alkaloids in Copelandia were 0.43–0.76%/0.08–0.22% in the whole mushroom (0.64–0.74%/0.02–0.22% in the cap and 0.31–0.78%/0.01–0.39% in the stem). It thus appears that P. cubensis is psilocybin-rich, whereas Copelandia is psilocin-rich.

Classification of seized methamphetamine by impurity profiling can provide very useful information in criminal investigations of drug traffic routes, sources of supply and relationships between seizures. The aim of this study is to improve and develop an analytical method for detecting impurities such as starting materials and by-products in illegally prepared methamphetamine·HCl samples. A 50 mg sample of methamphetamine·HCl was dissolved in 1 ml of buffer solution (four parts 0.1 M phosphate buffer pH 7.0 and one part 10% Na2CO3). Impurities were extracted with 0.5 ml of ethyl acetate containing four internal standards (ISs) (n-decane, n-pentadecane, n-nonadecane and n-hexacosane) and analyzed by gas chromatography (GC) using a flame ionization detector (FID) on a DB-5 capillary column ( 0.32 mm i.d. ×30 m, film thickness 1.0 μm). The use of a middle-bore column offered better separation of the impurity peaks. The correction of the retention times of impurity peaks with four ISs made peak identification very accurate for subsequent data processing. Twenty-four characteristic peaks were selected for comparison and similarity and/or dissimilarity between samples, and the data were evaluated by the Euclidean distance of the relative peak areas after logarithmic transformation. The results indicate that the present method would be useful for methamphetamine impurity profiling.

Morphological alterations occasionally found in the myocardium of methamphetamine (MAP) abusers include hypertrophy, atrophy, disarrangement of myofibrils and fibrosis. These cardiac alterations have been thought to be due to an indirect action of MAP via catecholamines released by MAP administration. However, the direct effect of MAP on cardiomyocytes is not clear. In previous studies, we showed that cell size of isolated adult rat ventricular cardiomyocytes (ARCs) exposed to MAP was larger than that of untreated cells in culture supplemented with 10% fetal calf serum (FCS). In this study, to determine further the direct effect of MAP on cardiomyocytes, cultured ARCs were exposed to 0.05, 0.1 and 0.5 mM MAP for 7 days in culture medium without FCS following 6-day normal culture in medium containing FCS. Myocyte size was measured and microtubular (MT) structures which were associated with functional disorder of hearts were immunohistochemically observed using confocal microscopy. The size in treated ARCs significantly increased time- and dose-dependently as compared with untreated cells, but it decreased 7 days after exposure to 0.5 mM MAP. The increases in cell size, however, were lower than that in serum-supplemented cultures. MT structures in intact ARCs appeared as a filamentous network throughout the cytoplasm and around the nucleus. MAP exposure for 3 days promoted MT assembly, but in 7-day treated cells, MT and actin structures were injured. These results suggested that MAP directly induced cellular hypertrophy and might lead to cardiac functional disorder.

The applicability of a new marker for estimation of bloodstain age by reverse-phase high performance liquid chromatography (HPLC) is described. Using a microBondasphere C18 column with a two step linear gradient of 10.5-46.25% acetonitrile in 0.1% trifluoroacetic acid, an intriguing peak (unidentified) at a retention time of about 5 min was observed on chromatograms from human adult bloodstains and designated as 'X'. The area of this peak, which could be detected in extracts of bloodstains, but not in their fresh whole blood, increased with time. The ratios of the X area to heme area in bloodstains stored at room temperature and 4 degrees C for up to 52 weeks old linearly correlated with stain age by plotting on a double logarithmic scale. In bloodstains exposed to fluorescent light at room temperature, the regression equation calculated from the ratios (Rx) and the ages of stains in weeks (W) is ln(1000.Rx) = 1.1084 + 0.3937.ln(7.W), and the coefficient of correlation (r) is 0.9776 (n = 144, P < 0.001). When stains were stored at 37 degrees C, the ratio transformed into logarithms correlated linearly with stain age. The regression equation describing the relationship in bloodstains exposed to fluorescent light at 37 degrees C is ln(1000.Rx) = 2.4477 + 0.0866.W (r = 0.9826, n = 144, P < 0.001). The results of the present study suggest that the HPLC method may be applicable to the estimation of bloodstain age.

The anaphylatoxin C3a or its desArg form ( C3a desArg ) generated during complement activation could be detected in the vicinity of incised skin wounds of guinea pigs using immunoblotting methods. The C3a desArg peptides were detectable immediately after injury in local sites up to 3 mm from the wound edge. In subsequent determinations of up to at least 3-day-old antemortem wounds, the maximum concentration of these peptides was largely localized up to 6 mm from the wound edge at 2 h after injury. In postmortem wounds, however, these peptides were undetectable. When they were released in antemortem wounded tissues they could be detected up to 1 day at 22°C after death. These results suggest that the detection of C3a desArg in wounds using immunoblotting methods can be useful for distinguishing ante- from postmortem wounds.

In order to examine the vital reaction in wounds, catecholamines, serotonin (5-HT) and their metabolites in the incised skin wounds of guinea pigs were analyzed simultaneously by high-performance liquid chromatography (HPLC) using electrochemical detection (ECD). The principal changes in the levels of these compounds in vital wounds were as follows: a considerable decrease in norepinephrine (NE) content was observed 12-24 h after injury which persisted to at least 7 days. 3,4-Dihydroxyphenylacetic acid (DOPAC) decreased slightly for up to 30 min and then showed no significant difference compared with postmortem levels. Epinephrine and dopamine were barely detected by the HPLC-ECD method employed. 5-HT concentrations which showed an increase up to 24 h showed maximum levels 800 microns from the wound edge at 10 and 30 min after injury. 5-Hydroxyindoleacetic acid (5-HIAA) was significantly higher than the postmortem level over almost the entire period of these experiments. A 5-HIAA content of at least twice the postmortem level was observed 800 microns from the wound edge of a 10-min-old vital wound. Therefore, 5-HIAA is a likely candidate as a new marker for evaluating the vital reaction in wounds. The vital characteristics of NE, DOPAC, 5-HIAA and 5-HT in 10-min-old wounds persisted for up to 12 h at room temperature after death. These results suggest that the HPLC-ECD method used here is very useful for simultaneous examination of the vital reaction in wounds from the earliest to the later stages of the wound-healing period.

This paper describes a modified method of simultaneous quantitative determination of histamine and polyamines (putrescine, cadaverine, spermidine and spermine) in incised skin wounds of rats using high-performance liquid chromatography (HPLC) with a strong-cation exchange column, shim-pack ISC-05/S0504P, employing a linear gradient of 0.7-2.5 M sodium chloride. There was scarcely any change in the histamine content of the vital wounds throughout the one week experimental period, although content decreased slightly in 1-h-old wounds. On the other hand, concentrations of putrescine and spermidine increased suddenly 12 h after wounding, and spermine content increased slightly in 4-day-old wounds. There was no post-mortem change in the histamine, spermidine and spermine contents of vital wounds allowed to stand at 25 degrees C up to 24 h after death. Based on these results, this HPLC method may be useful for the determination of wound age during the period of healing.