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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.

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.

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.

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.

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.

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.

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.

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.

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 vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas.Multiphoton images of the corneas were obtained from nonlinear signals of coherent anti-Stokes Raman scattering, third-order sum frequency generation, and second-harmonic generation.The localizations of the adhesion complex-containing basement membrane and Bowman layer were clearly visible in the third-order sum frequency generation images. The fine structure of type I collagen was observed in the corneal stroma in the second-harmonic generation images. The localizations of lipids, proteins, and nucleic acids (DNA/RNA) was obtained in the coherent anti-Stokes Raman scattering images.Imaging technologies have progressed significantly and been applied in medical fields. Optical coherence tomography and confocal microscopy are widely used but do not provide information on the molecular structure of the cornea. By contrast, multiphoton microscopy provides information on the molecular structure of living tissues. Using this technique, we successfully visualized the localizations of various biomolecules including lipids, proteins, and nucleic acids in the cornea. We speculate that multiphoton microscopy will provide essential information on the physiological and pathological conditions of the cornea, as well as molecular localizations in tissues without pretreatment.

Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining. Transfection of cells with Rootletin fragments revealed that the SHG signal can be ascribed to filaments assembled from the R234 domain, but not to cross-striations assembled from the R123 domain. Consistent with this, Rootletin-depleted cells lacked SHG signal expected as centrosome linker. As a proof of concept, we confirmed that similar fibrous SHG was observed even in unicellular ciliates. These findings have potential for broad applications in clinical diagnosis and biophysical experiments with various organisms.