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In postmortem examinations, the drug analysis of hair is effective for revealing drug-use history. Additionally, a method to estimate the day of death using hair was previously developed by analyzing a single hair strand segmented at 0.4-mm intervals (micro-segmental hair analysis). However, for drowned bodies, drugs in the hair may be washed out due to soaking in water for extended periods. To evaluate the possibility of measuring drug distribution in the hair of drowned bodies, drug stability in hair samples soaked in various aqueous solutions was examined. First, reference hair strands of drug users containing specific drugs consistently along the hair shaft were prepared. The participants ingested 4 hay-fever medicines (fexofenadine, epinastine, cetirizine, and loratadine) every day for approximately 4 months before hair collection. Each reference strand was divided into regions, and each region was soaked in different solutions containing various solutes for extended periods up to approximately 2 months. In solutions without divalent ions (Ca

Four nitazenes (metonitazene, etonitazene, protonitazene, and isotonitazene), highly potent benzimidazole synthetic opioids, and their four nitro group positional isomers (isonitazenes) were synthesized and analyzed using infrared (IR) spectroscopy, gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry (LC/MS). In addition, the agonistic activity of all compounds at the human μ-opioid receptor was measured using a cell-based assay system. In the IR spectra, characteristic peaks for nitazenes and isonitazenes were observed. In GC/MS, all compounds were well separated on the chromatogram, although distinguishing nitazenes from the corresponding isonitazenes by electron ionization mass spectra was difficult. In LC/MS, all compounds were detected in both positive and negative modes of electrospray ionization. Characteristic fragment ions were observed in the product ion spectra of isonitazenes, enabling nitazenes to be distinguished from isonitazenes. All nitazenes tested demonstrated higher agonistic activity at the human μ-opioid receptors than the synthetic opioid fentanyl. The agonistic activities of isonitazenes were 11-35 times lower than those of the corresponding nitazenes. However, iso-etonitazene and iso-isotonitazene showed moderate activity similar to that of fentanyl, indicating that these drugs could cause poisoning at a comparable level as fentanyl, if these drugs are abused in the future.

Methyl 3-oxo-2-phenylbutyrate (MAPA) is a recently circulating precursor of phenylacetone (P2P), a precursor of amphetamine and methamphetamine. MAPA has a hybrid chemical structure of acetoacetic acid ester and P2P. Acetoacetic acid ester is de-esterified and decarboxylated to give the ketone by heating under acidic conditions; therefore, MAPA is presumed to be converted to P2P by such treatment. Considering that ethyl 3-oxo-2-phenylbutyrate (EAPA), methyl 3-oxo-4-phenylbutyrate (MGPA), and ethyl 3-oxo-4-phenylbutyrate (EGPA) have the same chemical features as MAPA, these three compounds are potential P2P precursors. The authors examined the analysis of these compounds by gas chromatography-mass spectrometry (GC-MS) and their conversion to P2P by heating under acidic and basic conditions. These compounds were remarkably decomposed into P2P during GC-MS analysis regardless of the injection method and injector temperature. EAPA and EGPA also caused ester exchange to methyl ester by injection of methanol solution. P2P production and transesterification were almost prevented by methoxime derivatization. These compounds were converted to P2P by heating under acidic conditions. The reaction of MGPA and EGPA proceeded quicker than that of EAPA. The important by-product associated with the reaction was phenylacetylcarbinol (formed from EAPA and MGPA), which will be converted to (pseudo)ephedrine, important methamphetamine impurities. By heating under basic conditions, MGPA and EGPA were converted to P2P but EAPA was mainly converted to phenylacetic acid. In the future, when these compounds are in circulation, our study will be useful for identifying and elucidating the synthetic method of P2P.

The original article described difference of total Δ

1-Phenyl-2-propanone (P2P) is a main precursor of methamphetamine. The authors investigated the formation of P2P degradation products during long-term storage and the factors that affected P2P degradation. Samples of neat P2P, 1 mg/mL P2P in methanol and in ethyl acetate were prepared. These samples were stored at room temperature or 4 °C for 1, 3, and 6 months; then the samples were analyzed by gas chromatography–mass spectrometry. A similar experiment (but stored only for 1 month) was also performed for phenylacetylcarbinol. Neat P2P stored at room temperature gave degradation products after 3-month storage, and the degradation proceeded over the next 3 months. Benzaldehyde, benzoic acid, benzyl acetate, 1-phenyl-1,2-propanedione, phenylacetylcarbinol, 1-acetoxy-1-phenyl-2-propanone, and 1,1-diphenylacetone were identified as degradation products after 6-month storage. The degradation was prevented incompletely by storage at 4 °C and almost completely by storage in the organic solvents. Neat phenylacetylcarbinol stored at room temperature was remarkably decomposed. Benzaldehyde, benzoic acid, 1-phenyl-1,2-propanedione, and 1-acetoxy-1-phenyl-2-propanone were regarded as the degradation products. The degradation was prevented incompletely by storage at 4 °C and almost completely by storage in the organic solvents. These results suggested that P2P and phenylacetylcarbinol were oxidized by oxygen in air and that the organic solvents inhibited the oxidation. P2P was presumed to be initially oxidized to phenylacetylcarbinol, then it was converted to benzaldehyde, benzoic acid, 1-phenyl-1,2-propanedione, and 1-acetoxy-1-phenyl-2-propanone. Production of phenylacetylcarbinol from P2P is useful information for methamphetamine impurity profiling because phenylacetylcarbinol is a precursor of ephedrines, the other methamphetamine precursors.

To prove drug-related crimes, it is important to estimate the date on which a specific drug was ingested. Previously, we developed a method, "micro-segmental hair analysis," to estimate the day of ingestion of a single-dose drug by segmenting a hair strand into 0.4-mm segments, which correspond to daily hair growth. In this study, the method was improved to estimate the days of continuous drug ingestion. The subjects ingested four hay-fever medicines (fexofenadine, epinastine, cetirizine, and loratadine) continuously (1-18 days) and chlorpheniramine as a single dose at intervals of several weeks as an internal temporal marker (ITM). The hair strands of the subjects were collected and subjected to a micro-segmental analysis. The distribution curves of each hay-fever medicine in a hair strand had broad peaks reflecting the number of days of drug ingestion. The positions on the curves corresponding to the first and final ingestion days of hay-fever medicines were identified using the ITM. The positions were near the hair segments on both ends of full width at half maximum (W2 ) of the broad peak. When the first and final days of continuous ingestion were estimated using W2 , independent of peak shape, the absolute average error from the actual ingestion days was approximately 2 days. Overall, we established a method to estimate the days of both single-dose and continuous drug ingestions. Furthermore, the method would be useful to investigate drug ingestion history in various scenes such as drug-related crimes and therapeutic drug monitoring.

Cannabis is the most abused drug in the world and molecular biology techniques for the analysis of cannabis have attracted attention in recent years. We report a case of three persons conspiring to possess cannabis-like leaf pieces for profit. We present the results of DNA testing by a simple kit on suspected cannabis samples with exceptionally shaped leaves. DNA was obtained from the samples of cannabis leaves by a simple and rapid procedure using a cannabis DNA detection kit or commercially available purified plant DNA extraction kit. Undiluted or diluted DNA solution was used as templates for amplification of cannabis-specific sequences by triplex PCR. The presence or absence of PCR amplicon was visually observed by a DNA chromatography detection system to determine whether the sample was cannabis or not. When the DNA was extracted by the simple procedure and used without dilution, PCR failed to identify cannabis samples. However, when purified DNA was used, PCR amplification was successful in identifying all cannabis samples. Notably, in case of DNA extracted by the simple procedure, 10 to 50-fold dilution of the solution reduced PCR inhibition and PCR amplification was successful. Although the quality of the DNA used as PCR template needs to be considered, simple DNA testing with a kit is very effective for identifying suspected cannabis samples with exceptional morphology.

Hair can be the only available specimen to prove drug-facilitated crimes (DFCs). However, it is difficult to identify drugs abused in DFCs, particularly benzodiazepines ingested in a single dose, using the conventional segmental hair analysis. In this study, an analytical procedure to identify a trace amount of drug in hair based on micro-segmental analysis was developed. Hair was donated from a subject who had been administered midazolam in a single dose. First, tens of hair strands were analyzed according to our routine procedure. Drug screening and identification were performed using a quadrupole-Orbitrap liquid chromatography–tandem mass spectrometry (LC–MS/MS) instrument. Next, two hair strands were segmented at 0.4-mm intervals and multi-target selected reaction monitoring of each segment (micro-segmental analysis) was performed using a tandem quadrupole LC–MS/MS instrument. After distribution curves of midazolam-derived ions in each hair strand were constructed, the residual extracts from segments corresponding to drug-containing regions were collected selectively into one tube. The extract was concentrated using solid-phase extraction and then injected into the LC–MS/MS instrument for drug identification. We failed to identify drugs using our routine procedure, although a slight peak, predicted to arise from midazolam, was detected, while the peak detected in the concentrated extract from drug-containing hair regions (named “selective concentration”) was identified as midazolam. The “selective concentration” enabled the identification of a trace amount of drug using several hair strands. The method would be helpful in proving DFCs even after it was impossible to identify drugs using conventional segmental hair analysis.

Purpose Recently, the opioid epidemic has become a serious problem, particularly in North America and Europe. The aim of this study was to clarifyQuery the metabolic fate of a new synthetic opioid furanylfentanyl. Methods The metabolism of furanylfentanyl was investigated by incubating fresh human hepatocytes with 10 µM furanylfentanyl at 37 °C for 48 h in an atmosphere of 5% CO2. After incubation, the culture medium was deproteinized and analyzed by liquid chromatography/mass spectrometry. Results On the chromatogram, four metabolites of furanylfentanyl were presumably detected: 4´-hydroxy-furanylfentanyl, β-hydroxy-furanylfentanyl, 4´-hydroxy-3´-methoxy-furanylfentanyl, and a ring-opened carboxylic acid metabolite. These newly found metabolites of furanylfentanyl were then definitely identified using chemically synthesized authentic standards. Conclusions Four metabolites of furanylfentanyl were newly identified. Although it has been proposed over recent years that a dihydrodiol metabolite, which has the same molecular weight as the ring-opened carboxylic acid metabolite, is formed from furanylfentanyl, this study demonstrated that the ring-opened carboxylic acid metabolite, rather than the dihydrodiol metabolite, is formed from furanylfentanyl.

The metabolism of a new synthetic opioid tetrahydrofuranylfentanyl (THF-fentanyl) was investigated using fresh human hepatocytes. Fourteen metabolites of THF-fentanyl, such as tetrahydrofuran ring-opened metabolites, desphenethylated metabolites, hydroxylated metabolites, and hydroxylated and methoxylated metabolites and their glucuronides, were detected in the culture medium of hepatocytes incubated with THF-fentanyl. Six metabolites, i.e. desphenethylated metabolite, 4'-hydroxy-THF-fentanyl, β-hydroxy-THF-fentanyl, 4'-hydroxy-3'-methoxy-THF-fentanyl, ring-opened alcohol metabolite, and ring-opened carboxylic acid metabolite, were identified via chemically synthesized authentic standards. A ring-opened alcohol metabolite and a ring-opened carboxylic acid metabolite are thought to be formed by reduction or oxidation of the intermediate aldehyde, which was formed by ring-opening of the metabolite hydroxylated at the carbon atom adjacent to the oxygen atom of the tetrahydrofuran ring. A ring-opened carboxylic acid metabolite was the main metabolite of THF-fentanyl based on the peak intensity.

On the analysis of cannabidiol (CBD) e-liquid by gas chromatography-mass spectrometry, we experienced suspected thermal decomposition of CBD to Δ

Purpose Since the 1980s, the detection sensitivity of mass spectrometers has increased by improving the analysis of drugs in hair. Accordingly, the number of hair strands required for the analysis has decreased. The length of the hair segment used in the analysis has also shortened. In 2016, micro-segmental hair analysis (MSA), which cuts a single hair strand at a 0.4-mm interval corresponding to a hair growth length of approximately one day, was developed. The advantage of MSA is that the analytical results provide powerful evidence of drug use in the investigation of drug-related crimes and detailed information about the mechanism of drug uptake into hair. This review article focuses on the MSA technique and its applications in forensic toxicology. Methods Multiple databases, such as SciFinder, PubMed, and Google, were utilized to collect relevant reports referring to MSA and drug analysis in hair. The experiences of our research group on the MSA were also included in this review. Results The analytical results provide a detailed drug distribution profile in a hair strand, which is useful for examining the mechanism of drug uptake into hair in detail. Additionally, the analytical method has been used for various scenarios in forensic toxicology, such as the estimation of days of drug consumption and death. Conclusions The detailed procedures are summarized so that beginners can use the analytical method in their laboratories. Moreover, some application examples are presented, and the limitations of the current analytical method and future perspectives are described.

This study aims to expose the toxicity of fentanyl analogs and their metabolites by measuring the agonistic activity of these compounds on opioid receptors. The agonistic activity of fentanyl, four analogs of fentanyl (acetylfentanyl, butyrylfentanyl, tetrahydrofuranylfentanyl, and furanylfentanyl), and their metabolites were evaluated using a cell-based assay system, which measured the cellular cAMP level after the reaction of a test compound with cells expressing opioid receptor. Fentanyl and its four analogs showed agonistic activity on μ-opioid receptor at

The trans- and cis-forms of 3,4-dimethyl-5-phenyl-2-oxazolidone (DPO) are impurities found in methamphetamine, but their production mechanism has been unclear. Here, the authors examined whether DPOs were produced by the reaction of ephedrines (ephedrine and pseudoephedrine) with deteriorated chloroform containing phosgene. Two sources of chloroform were used in this experiment: amylene-stabilized chloroform, which had been stored on a laboratory table after opening the bottle, and ethanol-stabilized chloroform, which had been stored without opening the bottle. Samples 1–4 were prepared and analyzed by gas chromatography/mass spectrometry. Sample 1 was prepared by extracting hydrochloride salts of ephedrines with chloroform under basic conditions. Sample 2 represented a chloroform solution of pseudoephedrine hydrochloride. Sample 3 was a methanol solution of pseudoephedrine hydrochloride, which had been exposed to chloroform prior to dissolution. Sample 4 was prepared by extracting pseudoephedrine hydrochloride, which had been exposed to chloroform prior to the extraction, with ethyl acetate under basic conditions. Phosgene was detected in amylene-stabilized chloroform but not ethanol-stabilized chloroform. The amylene-stabilized chloroform almost completely converted ephedrines to DPOs in samples 1 and 2, and partially converted pseudoephedrine to trans-DPO in samples 3 and 4. In contrast, the ethanol-stabilized chloroform did not give DPOs in any sample. The experimental results indicated that DPOs were produced by the reaction of ephedrines with deteriorated chloroform containing phosgene and that the reaction proceeds regardless of chemical forms, even at room temperature. DPOs are useful markers of contact between ephedrines and deteriorated chloroform.

Purpose Drug distribution in scalp hair can provide historical information about drug use, such as the date and frequency of drug ingestion. We previously developed micro-segmental hair analysis, which visualizes drug distribution at 0.4-mm intervals in individual hairs. The present study examines whether the distribution profiles of drugs can be markers for the administration or external contamination of the drugs using scalp, axillary, and pubic hairs. Methods A single dose of anti-itch ointment containing diphenhydramine (DP) and lidocaine (LD) was topically applied to the axillary or pubic areas of two volunteers; DP was also orally administered; and LD was intra-gingivally injected. Scalp, axillary, and pubic hairs were assessed using our micro-segmental analysis. Results The localization of DP and LD differed within individual scalp hair strands, implying DP and LD were predominantly incorporated into scalp hair via the bloodstream and via sweat/sebum, respectively, showing double-peak profiles. However, DP and LD were distributed along the shafts of axillary and pubic hairs without appearance of the double-peak profiles when the ointment had been applied to the axillary and pubic areas. The distributions of DP and LD in scalp hairs did not significantly differ according to administration routes, such as oral administration, gingival injection, and topical application. Conclusions Micro-segmental analysis revealed differences in the distribution profiles of drugs in hairs, and distinguished hairs with and without external contamination. These findings will be useful for understanding of the mechanism of drug uptake into hair and for estimating the circumstances for a drug use.

The agonistic activity of fluorinated and nonfluorinated fentanyl analogs on µ-opioid receptor was investigated using a cell-based assay system. Based on the activity, fentanyl analogs were ranked as follows: fentanyl > isobutyrylfentanyl ≈ butyrylfentanyl ≈ methoxyacetylfentanyl > acetylfentanyl. However, among the fentanyl analogs fluorinated on the N-phenyl ring, 2-fluoro analogs and 3-fluoro analogs showed the strongest and weakest activities, respectively. These results suggest that the 2-fluorinated isomers of fentanyl analogs are more likely to cause poisoning.

Cannabis is classified into two types: drug-type cannabis, which is abused worldwide, and fiber-type cannabis, which is used for industrial purposes. The two types are a result of differences in the sequences of tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes. In the present study, we aimed to establish a PCR-based method to distinguish between drug-type and fiber-type cannabis by detecting the differences in the sequences of THCAS and CBDAS. We constructed a single-plex PCR targeting active THCAS, and observed drug-type cannabis-specific amplification when using 10pg to 1ng of DNA; however, amplification was also observed in fiber-type cannabis when the DNA content reached 10ng. Similarly, single-plex PCR targeting active CBDAS showed fiber-type cannabis-specific amplification in 100pg of DNA, as well as in >1ng of drug-type cannabis DNA. Therefore, when an allele-specific duplex PCR system was constructed, in which both primer sets were mixed at an appropriate ratio, unintended nonspecific amplification was suppressed and amplicons of different sizes were observed between the drug-type and fiber-type cannabis, using DNA samples in the range of 1pg to 10ng. When the constructed duplex PCR was performed on DNA extracted from various cannabis seed samples, it was possible to distinguish between the drug-type and the fiber-type as well as detect a hybrid-type with both active THCAS and active CBDAS and a special type with neither. The identification method developed in the present study can quickly and accurately distinguish between drug-type and fiber-type cannabis, and is expected to be used for various purposes such as the detection of genetic contamination of industrial hemp as well as forensic examination of cannabis-related cases.

Synthetic cannabinoids (SCs) are a major category of new psychoactive substances that are frequently distributed after addition to plants. To date, various SCs with small differences in their chemical structures have prevailed in the illegal drug market. Thus, the development of a method for rapid detection with high discrimination capability is critically important for the forensic field. Vibrational spectroscopy is a possible analytical technique for this purpose because it can sensitively reflect differences among chemical structures. In this study, we applied surface-enhanced Raman scattering (SERS) with gold nanoparticle co-aggregation in a wet system to plant samples containing SCs. The experimental protocol used was simple and involved only mixing of the sample with several other solutions. It was possible to detect SERS spectra from various stock solutions of SCs by this method. The method was then applied to street samples containing SCs. Some of the plant samples containing SCs did not produce significant SERS signals even though stock solutions of the same SCs did produce SERS spectra. We investigated the reason for this discrepancy and speculated that the solubility in aqueous solutions was a factor determining whether a significant SERS signal could be detected or not. According to this hypothesis, minimal sample pre-treatment methods were applied. This allowed for the detection of SERS spectra from the examined plant samples. The developed approach is a powerful method for screening analysis of SCs in plant fragments.

Ingested drugs can be taken up into nails and hairs from the bloodstream and these drugs can be stably retained over several months or more. Free edges of nails can often be used as a specimen to prove drug intake. However, the mechanism of drug uptake into the nails is unclear. Although it is presumed that there are horizontal uptake routes at the nail root and vertical uptake routes at the nail bed against the direction of growth, three-dimensional distribution analysis is required to verify this phenomenon. Herein, we first developed a method to measure the three-dimensional distribution of drugs in the nails using the combination of micro-segmentation of nails (0.2 × 1.5 × 0.06 mm size) and highly sensitive quantification of drugs by LC-MS/MS. Carbinoxamine was administered as a model compound to a subject in a single dose, and then the free edges of big toenails were collected every several weeks over a year. The three-dimensional distribution of carbinoxamine in each free edge was visualized and arranged along the collection period. Carbinoxamine was localized in the lower layer during the early collection period (up to 78 days after drug ingestion) and in the upper and middle layers later (134 days or later). The changes in the drug distribution in the nails along the collection period implied that two-way drug uptake takes place in the whole nail through both the nail bed and the nail root simultaneously. The developed analytical method could be useful to elucidate the mechanism of drug uptake into the nails.

Impurity profiling of seized illicit methamphetamine (MA) provides information on MA manufacturing methods in clandestine laboratories, and this drug intelligence supports formulation of strategies to control MA abuse. In the present study, we developed a simultaneous chiral analysis method for MA and its precursors using supercritical fluid chromatography–tandem mass spectrometry equipped with an enantioselective stationary phase. Chromatographic conditions were optimized by systematic investigation of the flow rate, temperature, back pressure, co-solvent, additive, and mobile phase composition. The ability of the developed method was evaluated using standard and authentic illicit MA. The use of a chiral selector in the stationary phase allowed for simultaneous chiral differentiation of MA and its precursors including ephedrine, norephedrine, chloropseudoephedrine, methylephedrine, dimethylamphetamine, and amphetamine. Sufficient limit of detection, repeatability of retention time, and linearity were achieved. A switching valve interfacing a chromatograph and a mass spectrometer enabled analyzing large amounts of MA directly. The application to the authentic illicit MA samples was achieved and revealed the existence of impurities, which was not detected by conventional gas chromatography–mass spectrometry. The developed supercritical fluid chromatography–tandem mass spectrometry method could be a powerful analytical tool for MA impurity profiling.

During investigations of unnatural death, the time of death is generally estimated using anatomical examinations. However, it can be difficult to accurately determine the day of death, because postmortem changes in the body tissues can be greatly affected by the circumstances of the location of the corpse. We recently developed a method to estimate the day of drug ingestion, using micro-segmental hair analysis based on internal temporal markers (ITMs). In this method, ITMs are ingested at a specific time interval before hair collection to mark timescales within individual hair strands. A single hair strand is segmented at 0.4-mm intervals, corresponding to average daily hair growth. The day of drug ingestion is eventually estimated by calculating the distances between segments containing the drug and ITMs in a hair strand. In the present study, the method was applied to estimate the day of death. A corpse was discovered with a documented medical history of lidocaine administration for surgery 57 days before the discovery. Micro-segmental analysis of a hair plucked from the corpse was performed using lidocaine as an ITM. Lidocaine was detected at specific regions in the hair strands. The day of death was estimated using the known surgery day, the distance from the hair root to the lidocaine peak in the hair strand, and the average hair growth rate. The novel estimation method using a hair enabled us to narrow the estimated time range of death up to the day of death, unlike the conventional anatomical examination. The micro-segmental hair analysis based on drug use history can be extremely helpful in determining the time of an unnatural death.

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.

Purpose The usefulness of hepatocytes isolated from a liver-humanized mouse (PXB-cells) as a model in vitro system for the prediction of the in vivo metabolism of new drugs of abuse was evaluated. Methods For the drug metabolism study, fentanyl, a powerful synthetic opioid, and acetylfentanyl, an N-acetyl analog of fentanyl, were selected as model drugs. PXB-cells were cultured with the drug for 24–48 h and then the media were collected and analyzed by liquid chromatography/mass spectrometry after deproteinization with acetonitrile. Results The main metabolite formed from fentanyl by PXB-cells was the desphenethylated metabolite (nor-fentanyl), and the other major metabolites formed were 4′-hydroxy-fentanyl, β-hydroxy-fentanyl and (ω-1)-hydroxy-fentanyl. ω-Hydroxy-fentanyl and 4′-hydroxy-3′-methoxy-fentanyl were the minor metabolites. Similar results were obtained for acetylfentanyl. The metabolite profile of fentanyl in PXB-cells was consistent with the in vivo metabolite profile of fentanyl reported previously. Most of the 4′-hydroxy- and 4′-hydroxy-3′-methoxy-metabolites of fentanyl and acetylfentanyl were conjugated in PXB-cells, indicating that PXB-cells had high conjugation enzyme activities. From experiments using human liver microsomes and anti-CYP antibodies, it was revealed that CYP3A4 was involved in the production of nor-fentanyl, β-hydroxy-fentanyl and (ω-1)-hydroxy-fentanyl, while CYP2D6 was partially involved in the production of 4′-hydroxy-fentanyl. Conclusions Our results indicated that PXB-cells have high activities of phase I and phase II drug-metabolizing-enzymes, can be stably supplied, and are easy to use; thus, PXB-cells are highly useful for the prediction of the in vivo metabolism of drugs of abuse.

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.

The cis and trans isomers of 3-methylfentanyl and its three analogs were chemically synthesized, and these compounds were characterized and differentiated by gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), and nuclear magnetic resonance (NMR) spectroscopy. The cis and trans isomers of the 3-methylfentanyl analogs were completely separated by GC/MS. Although the high temperature of the GC injection port caused thermal degradation of β-hydroxy-3-methylfentanyl, the degradation was completely suppressed by trimethylsilyl derivatization. The isomers were also well separated by LC/MS on an octadecylsilyl column with 10 mM ammonium acetate and methanol as the mobile phase. The proton NMR signals were split when the hydrochloride salts of the 3-methylfentanyl analogs were dissolved in deuterated chloroform because stereoisomers were formed by the coordination of the hydrochloride proton to the nitrogen of the piperidine ring of the 3-methylfentanyl analogs.

Pyrrolidinophenones (PPs) are synthetic cathinones containing a pyrrolidine ring that are used recreationally worldwide. Recently, many studies on the metabolism and cytotoxicity of PPs have been published. Here, we focus on new designer drug containing an indan skeleton, 1-(2,3-dihydro-1H-inden-5-yl)-2-(pyrrolidine-1-yl)butan-1-one (5-PPDI), because there have been no reports to date regarding the metabolism of indan-type cathinones. The identification of 5-PPDI phase I metabolites in human urine enables us to determine whether a person has taken 5-PPDI. This metabolite detection approach plays a very important role in the field of forensic science. We synthesized analytical standards of 5-PPDI and four proposed metabolites. A urine sample was prepared by salting-out assisted liquid-liquid extraction with acetonitrile. Analyses of all standards and the urine sample were performed by liquid chromatography high resolution tandem mass spectrometry. As a result, we were able to detect 5-PPDI and its metabolites in the urine specimen. Two diastereomers of synthesized 1-OH metabolites were successfully separated, and only one diastereomer was observed in the urine specimen. To the best of our knowledge, this is the first report on the stereoselective reduction of PPs in humans. Further, we performed quantitative analyses of 5-PPDI and its metabolites in the urine. We identified three characteristic features of 5-PPDI phase I metabolism: (1) hydroxylation at the indan skeleton, (2) stereoselective reduction of the carbonyl group, and (3) hydroxylation of the indan skeleton possibly proceeding more preferentially than any other metabolization. In addition, several structural isomers and diastereomers of 2'-OH metabolites were detected. Based on these data, we propose phase I metabolic pathways of 5-PPDI, which will be essential in understanding the metabolism of other PPs with an indan skeleton.

The metabolism of butyrylfentanyl, a new designer drug, was investigated using fresh human hepatocytes isolated from a liver-humanized mouse model. In the culture medium of hepatocytes incubated with butyrylfentanyl, the desphenethylated metabolite (nor-butyrylfentanyl), ω-hydroxy-butyrylfentanyl, (ω-1)-hydroxy-butyrylfentanyl, 4'-hydroxy-butyrylfentanyl, β-hydroxy-butyrylfentanyl, 4'-hydroxy-3'-methoxy-butyrylfentanyl, and ω-carboxy-fentanyl were identified as the metabolites of butyrylfentanyl. Each metabolite was definitively identified by comparing the analytical data with those of authentic standards. The amount of the main metabolite, nor-butyrylfentanyl, reached 37% of the initial amount of butyrylfentanyl at 48 h. ω-Hydroxy-butyrylfentanyl and (ω-1)-hydroxy-butyrylfentanyl, formed by hydroxylation at the N-butyryl group of butyrylfentanyl, were the second and third largest metabolites, respectively. The majority of 4'-hydroxy-butyrylfentanyl and 4'-hydroxy-3'-methoxy-butyrylfentanyl was considered to be conjugated. CYP reaction phenotyping for butyrylfentanyl using human liver microsomes and various anti-CYP antibodies revealed that CYP3A4 was involved in the formation of nor-butyrylfentanyl, (ω-1)-hydroxy-butyrylfentanyl, and β-hydroxy-butyrylfentanyl. In contrast, CYP2D6 was involved in the formation of ω-hydroxy-butyrylfentanyl.

Sensitive detection of drugs using a method with high qualification capability is important for forensic drug analysis. Vibrational spectroscopy is a powerful screening technique because it can provide detailed structural information of the compounds included in samples with simple experimental protocols. Among various spectroscopic techniques, surface enhanced Raman scattering (SERS) spectroscopy has attracted enormous attention owing to its ultra-high sensitivity. In this study, we developed a method for rapid detection of hypnotics using SERS with gold nanoparticle co-aggregation in a wet system. The developed method required a simple analytical protocol. This enabled rapid analysis with high stability and repeatability. We analyzed various hypnotics (19 types including benzodiazepines and nonbenzodiazepines) to investigate the structure-spectrum relationship. As a proof of concept for application to real crime samples, simulated spiked beverages containing one hypnotic (etizolam, flunitrazepam, zolpidem, or zopiclone) were analyzed. Diluting the beverage samples decreased the matrix effect and allowed for detection of these hypnotics. Except for flunitrazepam, strong signals were observed for all hypnotics, and the estimated lower limit of detection was 50 ppm in apple drink. The developed approach is a rapid method for screening analysis of hypnotics with low sample requirements.

In the study reported here, two glucuronic acid-conjugated metabolites of 4-bromo-2,5-dimethoxyphenethylamine (2C-B)-a ring-substituted psychoactive phenethylamine-were chemically synthesized for the first time and a method for analyzing them in urine was developed. β-D-Glucuronide of 4-bromo-2,5-dimethoxyphenylethylalcohol was successfully synthesized using methyl 2,3,4-tri-Ο-acetyl-1-O-(trichloroacetimidoyl)-α-D-glucuronate as a glucuronyl donor and boron trifluoride diethylether complex as a Lewis acid catalyst. β-D-Glucuronide of 4-bromo-2,5-dimethoxyphenylacetic acid was synthesized by condensing 4-bromo-2,5-dimethoxyphenylacetic acid and benzyl D-glucuronate followed by benzyl group deprotection based on catalytic hydrogenation. Two glucuronic acid-conjugated metabolites of 2C-B in urine were qualitatively and semiquantitatively evaluated via direct liquid chromatography/mass spectrometry (LC/MS) analysis of a diluted urine sample. The simple method proposed is expected to be useful for studying the metabolic fate of 2C-B.

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.

Chromatographic differentiation of the ring-substituted regioisomers of amphetamine (AMP) and methamphetamine (MA) was performed by supercritical fluid chromatography (SFC). The behaviour of the retention against the changes of column temperature and co-solvent proportion was studied. The obtained information facilitated the optimization of the each regioisomer. As a result, 2-, 3-, and 4-ring-substituted analogues of AMP and MA with methyl, methoxy, fluoro, chloro, and bromo groups were separated, generally within 6 min. In addition, we found that the separation pattern of the examined regioisomers was classified into two, which depended on the electron donating/withdrawing effect of the substituent. Our results indicate that SFC could be used in forensic drug analysis for fast, reliable identification of structurally similar drugs of abuse. Copyright © 2016 John Wiley & Sons, Ltd.

Hair and nails are often used to prove long-term intake of drugs in forensic drug testing. The aim of this study was to evaluate the effectiveness of drug testing using hair and nails and the feasibility of determining when drugs were ingested by measuring the time-courses of drug concentrations in hair and toenails after single administrations of various drugs. Healthy subjects ingested four pharmaceutical products containing eight active ingredients in single doses. Hair and toenails were collected at predetermined intervals, and drug concentrations in hair and nails were measured for 12 months. The administered drugs and their main metabolites were extracted using micropulverized extraction with a stainless steel bullet and were analyzed using liquid chromatography/tandem mass spectrometry. Acidic compounds such as ibuprofen and its metabolites were not detected in both specimens. Acetaminophen, a weakly acidic compound, was detected in nails more frequently than in hair. The maximum concentration of allyl isopropyl acetylurea, a neutral compound, in nails was significantly higher than in hair. Nails are an effective specimen to detect neutral and weakly acidic compounds. For fexofenadine, a zwitterionic compound, and for most basic compounds, the maximum concentrations in hair segments tended to be higher than those in nails. The hair segments showing the maximum concentrations varied between drugs, samples, and subjects. Drug concentrations in hair segments greatly depended on the selection of the hair. Careful interpretation of analytical results is required to predict the time of drug intake. Copyright © 2016 John Wiley & Sons, Ltd.

In forensic science, drug profiling is clarifying the identity of seized drugs of abuse based on their physicochemical properties and it is applied to various drugs, including crystalline methamphetamine. Impurity analysis is particularly important in drug profiling because the impurities can be a measure for speculating how the methamphetamine was synthesized in the clandestine laboratories. However, developments in scientific techniques have allowed the synthesis of high-purity, homogeneous crystalline methamphetamine, and thus new techniques to characterize methamphetamine are needed. In this study, we developed a method for chiral separation of ephedrine and its stereoisomers by supercritical fluid chromatography. Ephedrine is a common starting compound for methamphetamine synthesis. It possesses two chiral center carbon atoms and has four stereoisomers, (1R,2S)-(-)-ephedrine, (1S,2R)-(+)-ephedrine, (1S,2S)-(+)-pseudoephedrine, and (1R,2R)-(-)-pseudoephedrine. Because the stereostructure of ephedrines contained in methamphetamine seizure reflects the starting materials and the synthetic pathways, the stereoisomer ratio will provide additional information for drug profiling. The developed method achieved rapid separation of four isomers in about 11min with low limits of detection (1pg on column). Due to a switching valve connecting a chromatograph to a mass spectrometer, dense methamphetamine sample solutions containing small amount of ephedrines could be analyzed directly with a simple pre-treatment. Using multivariate analysis, 44 real samples were objectively grouped based on stereoisomer ratio. Our method is expected to improve the profiling of crystalline methamphetamine.