Trial analysis of drug profiling by liquid chromatography/mass spectrometry and inductively coupled plasma mass spectrometry to establish the origin of ephedrines used as precursors for illicit production of methamphetamine.

RATIONALE: We investigated whether chemical information on the origin of ephedrine and pseudoephedrine (ephedrines) can be acquired by liquid chromatography/mass spectrometry (LC/MS) as a substitute method for stable isotope ratio mass spectrometry (IRMS), which is not routinely available in forensic laboratories. We examined the characteristic inorganic elemental contaminants of ephedrines as a preliminary study. METHODS: The stable isotope ratios measured by IRMS analysis are expressed relative to the stable isotope ratios of conventional standards. Referring to the method using validated standard samples in IRMS, we selected a standard sample for acquiring stable isotopic ratio by LC/MS. The abundance ratio of the [M + 2H]+ ion to the [M + H]+ ion was measured by means of selected ion monitoring. We carried out qualitative analysis of inorganic elements contained in ephedrines produced by different manufacturing methods with ICPMS. RESULTS: We found that the ratio of stable isotope ion to molecular ion (stable isotope ratio) of ephedrines could be measured with LC/MS. The stable isotope ratio of ephedrines determined by LC/MS were confirmed to show relatively good correlations with the carbon and hydrogen stable isotope ratios found by IR-MS. We identified strontium as a characteristic inorganic element contained in ephedrines prepared by the semisynthetic method from molasses, or in the biosynthetic method from ephedra plants. CONCLUSIONS: Our results suggest that useful chemical information can be obtained by LC/MS, which is easy to carry out, and is generally available in forensic laboratories. It would be worthwhile to investigate the usefulness of stable isotope ratio measurements of Sr in the future.

Simple HPLC method for detection of trace ephedrine and pseudoephedrine in high-purity methamphetamine.

A simple and sensitive HPLC technique was developed for the qualitative determination of ephedrine and pseudoephedrine (ephedrines), used as precursors of clandestine d-methamphetamine hydrochloride of high purity. Good separation of ephedrines from bulk d-methamphetamine was achieved, without any extraction or derivatization procedure on a CAPCELLPACK C18 MGII (250 × 4.6 mm) column. The mobile phase consisted of 50 mM KH2 PO4-acetonitrile (94:6 v/v %) using an isocratic pump system within 20 min for detecting two analytes. One run took about 50 min as it was necessary to wash out overloaded methamphetamine for column conditioning. The analytes were detected by UV absorbance measurement at 210 nm. A sample (20 mg) was simply dissolved in 1 mL of water, and a 50 µL aliquot of the solution was injected into the HPLC. The detection limits for ephedrine and pseudoephedrine in bulk d-methamphetamine were as low as 3 ppm each. This analytical separation technique made it possible to detect ephedrine and/or pseudoephedrine in seven samples of high-purity d-methamphetamine hydrochloride seized in Japan. The presence of trace ephedrines in illicit methamphetamine may strongly indicate a synthetic route via ephedrine in methamphetamine profiling. This method is simple and sensitive, requiring only commonly available equipment, and should be useful for high-purity methamphetamine profiling.

Effective injection in pulsed splitless mode for impurity profiling of methamphetamine crystal by GC or GC/MS.

Gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) are commonly used for the impurity profiling of illegal drugs. For the impurity profiling of methamphetamine, it is very important to obtain information about impurities related to the manufacturing route and the precursor chemicals [B. Remberg, A.H. Stead, Drug characterization/impurity profiling, with special focus on methamphetamine: recent work of the United Nations International Drug Control Programme, Bull. Narcotics LI (1999) 97-117 ]. There are many artifact impurities arising from the preparation of samples and conditions of GC. Moreover, some impurities pose a barrier to the statistical processing of methamphetamine profiling. We investigated capillary GC analysis using pulsed splitless (PS/L) injection to minimize the thermal decomposition of impurities at the injection port and improve the transfer of samples into the column. We confirmed that the optimal conditions of PS/L-mode are 230 degrees C (injection temperature), 50 psi (pulsed pressure) and 1.1 min (pulsed time) for the methamphetamine profiling. Based on the impurity profiles of 48 methamphetamine crystals in PS/L-mode, we can achieve very easy handling and obtained, good results.

Phylogenetic relationship of psychoactive fungi based on rRNA gene for a large subunit and their identification using the TaqMan assay (II).

"Magic mushroom (MM)" is the name most commonly given to psychoactive fungi containing the hallucinogenic components: psilocin (1) and psilocybin (2). We investigated the rRNA gene (internal transcribed spacer (ITS) and large subunit (LSU)) of two Panaeolus species and four Psilocybe species fungi (of these, two are non-psilocybin species). On the basis of sequence alignment, we improved the identification system developed in our previous study. In this paper, we describe the new system capable of distinguishing MMs from non-psilocybin Psilocybe species, its application data and the phylogeny of MM species.

[DNA and chemical analyses of commercial fly agaric-related products].

Since June 6, 2002, psilocin and psilocybin-containing fungi (commonly called "magic mushrooms") have been regulated by the Narcotics and Psychotropics Control Law in Japan. However, various fly agaric-related products are now entering the Japanese market via the internet. In this study, fly agaric-related products available in this way were investigated for raw materials by DNA analysis and for additives by chemical analysis. Nucleotide sequence analysis of the mitochondrial 12S rDNA region suggested that these fly agaric-related products originate from A. muscaria or A. muscaria var. persicina. Furthermore, they were classified into three strains based on the ITS2-LSU nucleotide sequence. Harmine derivatives and/or tryptamine derivatives were detected in some of these products by LC/MS analysis. In accordance with this, the matK gene of Peganum harmala was found in all of the harmine derivative-containing samples.

17α-estradiol is generated locally in the male rat brain and can regulate GAD65 expression and anxiety.

Increasing evidence suggests that 17ß-estradiol, a sex hormone, is synthesized by neurons. In addition, 17α-estradiol, the stereoisomer of 17ß-estradiol, is reported to be the dominant form in the male mouse brain. However, probably because the method to detect these isomers requires unusual and precise experimental design, the presence of this endogenous 17α-estradiol has not been reported subsequently and the actual role is therefore not well elucidated. We first quantified the estradiol level in hippocampal extracts using gas chromatography/mass spectrometry. As a result, 17α-estradiol was found in all of the male rats tested, while that of 17ß-estradiol was detected only in a certain subset. The estrogen-biosynthesis inhibitor letrozole decreased the expression of the major presynaptic GABA synthesizing enzyme GAD65 in cultured neurons and the effect was abrogated by exogenously supplied 17α-estradiol. Next, injection of the inhibitor into the brain reduced the 17α-estradiol level, indicating its biogenesis in the brain. Under the same conditions, immuno-staining of GAD65 was also decreased. Furthermore, the inhibitor treatment increased anxiety index of rats in the open field and this was ameliorated by the addition of 17α-estradiol. We showed that 17α-estradiol was generated in the brain and acted as a regulator of inhibitory neurotransmission as well as behavior. These results may have implications for a variety of diseases, such as the menopausal depression and Alzheimer's disease that have been reported to be related to estrogen levels.

Evaluation of rewarding effect of toluene by the conditioned place preference procedure in mice.

Toluene and many toluene-containing products are abused via inhalation. Previous investigations have used the place preference paradigm to evaluate the rewarding effects of commonly abused drugs such as morphine, cocaine, and amphetamine. A conditioning paradigm of toluene inhalation was developed in order to estimate the rewarding effect in mice. Conditioning sessions (five for toluene, five for air) were conducted twice daily for 5 days using a newly developed airtight inhalation shuttlebox (15x30x15 cm: wxlxh), which was divided into two compartments of equal size. One compartment was white with a textured floor, and the other was black with a smooth floor. All conditioning sessions were 20 min in duration, with a minimum of 7 h between sessions. Test sessions were carried out 1 day after the final training session with mice in a drug-free state. The time spent in each compartment during a 20-min session was measured using a digital video camera. Exposure to toluene vapors (700-3200 ppm) produced a significant conditioned place preference in mice. These results suggest that the conditioned place preference procedure using the newly developed airtight inhalation shuttlebox constitutes an important tool for studying the rewarding effect of abused solvents.

Impurity profiling of ephedrines in methamphetamine by high-performance liquid chromatography.

Separation of the enantiomers and diastereomers of ephedrines was investigated for impurity profiling of methamphetamine. We describe a method for the analysis of (1S,2R)-(+)-ephedrine, (1R,2S)-(-)-ephedrine, (1S,2S)-(+)-pseudoephedrine and racemic methylephedrine in bulk methamphetamine by HPLC using two different columns: a phenyl-beta-cyclodextrin-type column and an ODS-type column. The analytes were detected by UV absorbance measurement at 210 nm. As little as 0.05% of each ephedrine in bulk methamphetamine could be determined. In the impurity profiling of methamphetamine, the identification of ephedrines may provide valuable information about the precursor. This method was confirmed to be sufficiently sensitive to identify trace amounts of (1R,2S)-(-)-ephedrine and (1S,2S)-(+)-pseudoephedrine in bulk methamphetamine synthesized by the Emde method.

[Discrimination of psychoactive fungi (commonly called "magic mushrooms") based on the DNA sequence of the internal transcribed spacer region].

'Magic mushrooms' (MMs) are psychoactive fungi containing the hallucinogenic compounds, psilocin (1) and psilocybin (2). Since June 6, 2002, these fungi have been regulated by the Narcotics and Psychotropics Control Law in Japan. Because there are many kinds of MMs and they are sold even as dry powders in local markets, it is very difficult to identify the original species of the MMs by morphological observation. Therefore, we investigated the internal transcribed spacer (ITS) region in the ribosomal RNA gene of MMs obtained in Japanese markets to classify them by a genetic approach. Based on the size and nucleotide sequence of the ITS region amplified by PCR, tested MMs were classified into 6 groups. Furthermore, a comparison of the DNA sequences of the MMs with those of authentic samples or with those found in the databases (GenBank, EMBL and DDBJ) made it possible to identify the species of tested MMs. Analysis by LC revealed that psilocin (1) was contained at the highest level in Panaeolus cyanescens among the MMs, but was absent in the Amanita species.

Growth characteristics of Cannabis sativa L. cultivated in a phytotron and in the field.

Growth characteristics of Cannabis saliva L. are indispensable factors to verify the statements by the criminals of illegal cannabis cultivation. To investigate growth characteristics of C. sativa, two varieties, cannabidiolic acid (CBDA)-rich (CBDA-type) which being cultivated for fiber production and delta9-tetrahydrocannabinolic acid (THCA)-rich (THCA-type) which is used for drug abuse, were cultivated from seeds under the same growth environment in a phytotron. THCA-type showed high germination rate (100%) whereas only 39% of the CBDA-type seeds germinated 6 days after sowing. Plant height, number of true leaves, number of nodes, number of axillary buds and flowering of these two varieties were periodically observed. THCA-type grew more rapidly (plant height: 125.8 cm for THCA-type, 84.7 cm for CBDA-type, 75 days after cultivation) demonstrating vigorous axillary bud formation and earlier male-flowering (63 days for THCA-type, 106 days for CBDA-type, after sowing). Propagation of THCA-type was tested using the axillary shoot cuttings of female plants either with or without the main stem. All the cuttings with the main stem rooted after 21 days and grew healthily in a phytotron. However, all the newly developed leaves were single instead of palmate. In the field, THCA-type male-flowered after 155 days of cultivation after sowing on March 31. The height of the field-cultivated plants reached 260.9 cm 163 days after sowing. Despite the great differences in final plant heights, the increases of plant height per day during the vegetative growth stage were similar in the field and in the phytotron. Thus estimating the starting time of illegal cannabis cultivation might be possible if the plant is in the vegetative growth stage.

Use of stable isotope ratios for profiling of industrial ephedrine samples: application of hydrogen isotope ratios in combination with carbon and nitrogen.

The utility of hydrogen stable isotope ratio measurement by IR-MS for establishing the origin of ephedrine and pseudoephedrine (ephedrines), precursors of methamphetamine, was evaluated. There are two kinds of commercial semisynthetic ephedrines, one produced from molasses and the other from pyruvic acid. While the semisynthetic ephedrines derived from pyruvic acid cannot be discriminated from biosynthetic ephedrines and synthetic ephedrines based on delta(13)C and delta(15)N values, they could be identified from the delta(2)H values. The low deuterium content of biosynthetic ephedrines (delta(2)H: -193 to -151 per thousand) allows a clear distinction from synthetic ephedrines (delta(2)H: -73 to -30 per thousand), semisynthetic ephedrines derived from pyruvic acid (delta(2)H: +75 to +148 per thousand) and semisynthetic ephedrines derived from molasses (delta(2)H: -74 to +243 per thousand). The wide range of delta(2)H values of semisynthetic ephedrines is therefore very useful for the detailed classification of ephedrines, in combination with the measurement of delta(13)C and delta(15)N values as described in our previous work. This study was carried out on a limited number of samples reflecting the various routes of ephedrines manufacture. But it has become apparent that this stable-isotope analysis is an appropriate means by which to screen for manufacturing process of ephedrines. This approach should be useful for worldwide precursor control of methamphetamine.

Evaluation of characteristic deuterium distributions of ephedrines and methamphetamines by NMR spectroscopy for drug profiling.

We have established a method for quantitative analysis of the deuterium contents (D/H) at the phenyl, methine, benzyl, N-methyl and methyl groups of l-ephedrine/HCl, d-pseudoephedrine/HCl and methamphetamine/HCl by 2H NMR spectroscopy. Comparison of the 5 position-specific D/H values of l-ephedrine/HCl and d-pseudoephedrine/HCl prepared by three methods (chemical synthesis, semichemical synthesis, and biosynthesis) showed that chemically synthesized ephedrines and semisynthetic ephedrines have highly specific distributions of deuterium at the methine position and at the benzyl position, compared with the other positions. The classification of several methamphetamine samples seized in Japan in terms of the D/H values at these two positions clearly showed that the methamphetamine samples had been synthesized from ephedrines extracted from Ephedra plants or semisynthetic ephedrines but not from synthetic ephedrine. This isotope ratio analysis method should be useful to trace the origins of seized methamphetamine in Southeast Asia.

A rapid and reliable solid-phase extraction method for high-performance liquid chromatographic analysis of opium alkaloids from papaver plants.

A rapid and reliable solid-phase extraction method for HPLC analysis of opium alkaloids from Papaver plants was established. Fifty mg of dried and powdered plant sample was extracted with 5 ml of 5% acetic acid for 30 min under sonication. After centrifugation, 3 ml of the supernatant was loaded on a reversed-phase cation-exchange solid-phase extraction cartridge. After seriate washings with 0.1 M hydrochloric acid and methanol, alkaloids were eluted with a mixture of 28% ammonia and methanol (1:19). The eluate was concentrated under nitrogen stream at 40 degrees C and the residue was dissolved in 50% aqueous methanol for high performance liquid chromatographic analysis. With this solid-phase extraction method, the recovery of morphine, codeine, oripavine, thebaine, papaverine, noscapine and sanguinarine was from 99.94 to 112.18% when the standard alkaloids were added to the plant samples. Opium alkaloids of a variety of genus Papaver plants cultivated in a field and phytotron were analyzed by this method.

Phylogenetic relationship of psychoactive fungi based on the rRNA gene for a large subunit and their identification using the TaqMan assay.

"Magic mushrooms" (MMs) are psychoactive fungi containing the and Psychotropics Control Law in Japan. Because there are many kinds of MMs and they are often sold even as dry powders in local markets, it is very difficult to identify the original species of the MMs by morphological observation. Therefore, we investigated the rRNA gene for a large subunit (LSU) of several MMs to classify them by a genetic approach. In this paper, we described the phylogeny of species of MMs based on the partial sequence (about 970 bp) of the LSU and the rapid identification of MMs using the TaqMan PCR assay.

DNA fingerprinting of Cannabis sativa using inter-simple sequence repeat (ISSR) amplification.

Chemical analysis of cannabinoid, and Inter-Simple Sequence Repeat (ISSR) fingerprinting of DNA were used to identify different samples of Cannabis sativa L. for forensic purposes. Three samples were classified into two types, tetrahydrocannabinol (THC) and cannabidiol (CBD) chemo-types, by high performance liquid chromatography (HPLC). The two samples of the CBD type were not distinguished by their HPLC patterns. ISSR fingerprinting identified polymorphic DNA patterns between these samples. ISSR fingerprinting clearly differentiated between cannabis samples that could not be achieved by HPLC analysis.

Determination of origin of ephedrine used as precursor for illicit methamphetamine by carbon and nitrogen stable isotope ratio analysis.

The sale of ephedrine, one of the precursors of methamphetamine, is strictly controlled and monitored in various countries to prevent the production of illicit methamphetamine. There are three kinds of production scheme for ephedrine manufacture, and it is very useful for precursor control to investigate the origin of ephedrine used for the synthesis of illicit methamphetamine. By means of stable isotope ratio mass spectrometry (IR-MS), we investigated the origin of ephedrine based on the delta(13)C and delta(15)N values. The various origins of ephedrine (biosynthetic, semisynthetic, or synthetic) could be discriminated clearly by using these values. The delta(15)N values of synthetic ephedrine were more negative than those of ephedrine from other sources. By the repeated distillation of methylamine in our laboratory, we confirmed that this could be due to isotope separation during distillation for the purification of methylamine used for ephedrine synthesis. The values for ephedrine used as the precursor were well-correlated with those for methamphetamine synthesized from it. This drug characterization analysis should be useful to illuminate the origin of the precursors used for clandestine methamphetamine and to trace the diversion of medicinal ephedrine for illicit manufacture of methamphetamine.

Application of revised version of neural independent component analysis to classification problems of confiscated methamphetamine.

Recently, profiling the chemical substances in illegally distributed drugs has been needed in order to reveal the drug channels. However, this kind of profiling is often difficult because such drugs contain various kinds of impurities and the quantity of these impurities changes. Due to these circumstances, several methods, including a slightly revised ICA (Independent Component Analysis) by a Hebbian learning artificial neural network, were applied for profiling illegally distributed methamphetamine. Eventually, better classification results with the ICA than with other methods were obtained. These results show that ICA could make it easier to profile illegally distributed methamphetamine.