Post-translational control of beige fat biogenesis by PRDM16 stabilization.

Compelling evidence shows that brown and beige adipose tissue are protective against metabolic diseases1,2. PR domain-containing 16 (PRDM16) is a dominant activator of the biogenesis of beige adipocytes by forming a complex with transcriptional and epigenetic factors and is therefore an attractive target for improving metabolic health3-8. However, a lack of knowledge surrounding the regulation of PRDM16 protein expression hampered us from selectively targeting this transcriptional pathway. Here we identify CUL2-APPBP2 as the ubiquitin E3 ligase that determines PRDM16 protein stability by catalysing its polyubiquitination. Inhibition of CUL2-APPBP2 sufficiently extended the half-life of PRDM16 protein and promoted beige adipocyte biogenesis. By contrast, elevated CUL2-APPBP2 expression was found in aged adipose tissues and repressed adipocyte thermogenesis by degrading PRDM16 protein. Importantly, extended PRDM16 protein stability by adipocyte-specific deletion of CUL2-APPBP2 counteracted diet-induced obesity, glucose intolerance, insulin resistance and dyslipidaemia in mice. These results offer a cell-autonomous route to selectively activate the PRDM16 pathway in adipose tissues.

Rubicon prevents autophagic degradation of GATA4 to promote Sertoli cell function.

Autophagy degrades unnecessary proteins or damaged organelles to maintain cellular function. Therefore, autophagy has a preventive role against various diseases including hepatic disorders, neurodegenerative diseases, and cancer. Although autophagy in germ cells or Sertoli cells is known to be required for spermatogenesis and male fertility, it remains poorly understood how autophagy participates in spermatogenesis. We found that systemic knockout mice of Rubicon, a negative regulator of autophagy, exhibited a substantial reduction in testicular weight, spermatogenesis, and male fertility, associated with upregulation of autophagy. Rubicon-null mice also had lower levels of mRNAs of Sertoli cell-related genes in testis. Importantly, Rubicon knockout in Sertoli cells, but not in germ cells, caused a defect in spermatogenesis and germline stem cell maintenance in mice, indicating a critical role of Rubicon in Sertoli cells. In mechanistic terms, genetic loss of Rubicon promoted autophagic degradation of GATA4, a transcription factor that is essential for Sertoli cell function. Furthermore, androgen antagonists caused a significant decrease in the levels of Rubicon and GATA4 in testis, accompanied by elevated autophagy. Collectively, we propose that Rubicon promotes Sertoli cell function by preventing autophagic degradation of GATA4, and that this mechanism could be regulated by androgens.

Possibility of drug-distribution measurement in the hair of drowned bodies: evaluation of drug stability in water-soaked hair using micro-segmental analysis.

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 (Ca2+ and Mg2+), the drug content in the hair decreased up to approximately 5 % with increasing salt concentration and soaking time. However, the decreased drug content was negligible in solutions containing divalent ions, implying that the divalent ions prevented drugs contained in hair from washing out. As natural river and sea waters contain divalent ions, the drugs in hair were hardly washed out even when the hair was soaked for 2 months. Thus, it was concluded that drug-distribution measurements using micro-segmental analysis can also be applied to the hairs of drowned bodies.

Evaluation of Agonistic Activity of Fluorinated and Nonfluorinated Fentanyl Analogs on µ-Opioid Receptor Using a Cell-Based Assay System.

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.

Estimation of day of death using micro-segmental hair analysis based on drug use history: a case of lidocaine use as a marker.

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.

Rapid detection of synthetic cannabinoids in herbal highs using surface-enhanced Raman scattering produced by gold nanoparticle co-aggregation in a wet system.

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.

Rapid detection of hypnotics using surface-enhanced Raman scattering based on gold nanoparticle co-aggregation in a wet system.

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.

Metabolism of Butyrylfentanyl in Fresh Human Hepatocytes: Chemical Synthesis of Authentic Metabolite Standards for Definitive Identification.

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, ß-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 ß-hydroxy-butyrylfentanyl. In contrast, CYP2D6 was involved in the formation of ω-hydroxy-butyrylfentanyl.

A case of necrotizing fasciitis following Edwardsiella tarda septicemia with gastroenteritis.

Edwardsiella tarda is an uncommon pathogen that causes gastroenteritis in humans and is found in the aquatic environment. In rare cases, it also causes fatal infections, including sepsis and necrotizing fasciitis. However, it remains unknown whether E. tarda gastroenteritis could lead to these lethal diseases via hematogenous spread. Here we have reported a previously healthy 64-year-old woman with necrotizing fasciitis consecutively caused by E. tarda septicemia with gastroenteritis. The patient was transferred to the emergency department due to disturbance of consciousness and hypotension after suffering from diarrhea for a month. As whole-body computed tomography (CT) revealed an edematous change in the small intestine, septic shock following gastroenteritis was suspected, and the patient was immediately started on empiric antibiotic therapy and provided critical care. Her general physical conditions gradually began improving, but, on day 7, rapidly appearing blisters on both the lower limbs were noted, and she was accordingly examined again by conducting a CT scan. Based on the results, she was diagnosed with necrotizing fasciitis in both lower extremities, and surgical debridement was rapidly performed. Microbiological analysis of the specimens revealed E. tarda bacteremia, which suggested that E. tarda caused a series of infections in this patient. Finally, she fully recovered and was discharged within 3 months. Cumulatively, we proposed that gastroenteritis by E. tarda could directly result in fatal infections through the blood stream.

Metabolism of Fentanyl and Acetylfentanyl in Human-Induced Pluripotent Stem Cell-Derived Hepatocytes.

To evaluate the capability of human-induced pluripotent stem cell-derived hepatocytes (h-iPS-HEP) in drug metabolism, the profiles of the metabolites of fentanyl, a powerful synthetic opioid, and acetylfentanyl, an N-acetyl analog of fentanyl, in the cells were determined and analyzed. Commercially available h-iPS-HEP were incubated with fentanyl or acetylfentanyl for 24 or 48 h. After enzymatic hydrolysis, the medium was deproteinized with acetonitrile, then analyzed by LC/MS. Desphenethylated metabolites and some hydroxylated metabolites, including 4'-hydroxy-fentanyl and ß-hydroxy-fentanyl, were detected as metabolites of fentanyl and acetylfentanyl in the medium. The main metabolite of fentanyl with h-iPS-HEP was the desphenethylated metabolite, which was in agreement with in vivo results. These results suggest that h-iPS-HEP may be useful as a tool for investigating drug metabolism.

A case of ectopic ACTH syndrome due to DDAVP-sensitive but V1b receptor-negative bronchial typical carcinoid with lymphatic metastasis and plasma ProGRP elevation.

Ectopic ACTH syndrome (EAS) is a potentially fatal endocrine disease that results from a variety of neuroendocrine tumors (NETs), such as small cell lung cancer (SCLC) and bronchial typical carcinoid. Typical carcinoid is usually slow growing, not associated with plasma progastrin releasing peptide (ProGRP) elevation. Here, we report a 47-year-old female smoker with progressive typical carcinoid and plasma ProGRP elevation. Several types of Cushingoid features were found on physical examination. In addition, laboratory examination showed elevated plasma ACTH and serum cortisol levels. These findings indicated ACTH-dependent Cushing's syndrome. Moreover, the serum cortisol level was not suppressed by overnight high-dose dexamethasone treatment, suggesting the presence of an extra-pituitary tumor. Contrast-enhanced brain MRI revealed no pituitary adenoma, which also supported the idea that EAS occurred in the present case. Strikingly, chest computed tomographic (CT) scan showed a single 18-mm peripheral nodule in the right middle lobe of the lung. Tumor marker analysis revealed an elevation in plasma ProGRP. These data suggested a possibility that SCLC secreted ACTH and caused EAS in this patient. Of note, the plasma ACTH level was increased (1.7 fold) in l-desamino-8-D-arginine vasopressin (DDAVP) test, also suggesting the specific clinical feature in this case. After additional imaging examinations, we performed surgical resection with the suspicion of limited SCLC. As a result, pathological examination revealed a vasopressin receptor Ib (V1b) receptor-negative bronchial typical carcinoid with ACTH production and mediastinal lymphatic metastasis. In summary, we present a case of EAS caused by progressive bronchial typical carcinoid with plasma ProGRP elevation. We propose a novel subtype of lung typical carcinoid.

Micro-pulverized extraction pretreatment for highly sensitive analysis of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol in hair by liquid chromatography/tandem mass spectrometry.

RATIONALE: A primary metabolite of Δ(9) -tetrahydrocannabinol, 11-nor-9-carboxytetrahydrocannabinol (THC-COOH), serves as an effective indicator for cannabis intake. According to the recommendations of the Society of Hair Testing, at least 0.2 pg/mg of THC-COOH (cut-off level) must be present in a hair sample to constitute a positive result in a drug test. Typically, hair is digested with an alkaline solution and is subjected to gas chromatography/tandem mass spectrometry (GC/MS/MS) with negative ion chemical ionization (NICI). METHODS: It is difficult to quantify THC-COOH at the cut-off level using liquid chromatography/tandem mass spectrometry (LC/MS/MS) without acquisition of second-generation product ions in triple quadrupole-ion trap mass spectrometers, because large amounts of matrix components in the low-mass range produced by digestion interfere with the THC-COOH peak. Using the typical pretreatment method (alkaline dissolution) and micro-pulverized extraction (MPE) with a stainless bullet, we compared the quantification of THC-COOH using GC/MS/MS and LC/MS/MS. RESULTS: MPE reduced the amount of matrix components in the low-mass range and enabled the quantification of THC-COOH at 0.2 pg/mg using a conventional triple quadrupole liquid chromatograph coupled to a mass spectrometer. On the other hand, the MPE pretreatment was unsuitable for GC/MS/MS, probably due to matrix components in the high-mass range. The proper combination of pretreatments and instrumental analyses was shown to be important for detecting trace amounts of THC-COOH in hair. CONCLUSIONS: In MPE, samples can be prepared rapidly, and LC/MS/MS is readily available, unlike GC/MS/MS with NICI. The combination of MPE and LC/MS/MS might therefore be used in the initial screening for THC-COOH in hair prior to confirmatory analysis using GC/MS/MS with NICI.

Utilization of matrix-assisted laser desorption/ionization imaging mass spectrometry to search for cannabis in herb mixtures.

Herb mixtures including cannabis among the other herbs have recently appeared. When cannabinoids from herb extracts are detected by chemical examinations such as gas chromatography/mass spectrometry, forensic analysts have to determine whether cannabis is actually in the mixture or the cannabinoids are spiked. Morphological examinations are time-consuming, since it is difficult to find several pieces of cannabis among a large number of herb pieces using a microscope. Here, we propose a procedure for efficiently searching for cannabis in herb mixtures using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI/IMS). Pieces of herb mixtures were spread on double-sided adhesive tape attached to a stainless steel plate. The pieces were then covered with a conductive sheet and pressed. After a solution containing a matrix reagent was sprayed, the distribution of cannabinoids in the sample was visualized by MALDI/IMS. Then, just the pieces with cannabinoids could be picked up selectively with tweezers and decolorized. Cystolith hairs and trichomes, which are characteristic of cannabis, were observed in most of these pieces using a biological microscope. This MALDI/IMS procedure enables cannabis to be found in herb mixtures without inefficient random sampling and microscopic morphological examination.

Identifying a suspect powder as a cannabis concentrate through chemical analysis and DNA testing.

PURPOSE: Cannabis is regulated in many countries, and cannabis products are diversifying, which can hinder identification. Here, we report the seizure of a powder sample with a cannabis-like odor in a spice bottle labeled "nutmeg" and identification of the sample by chemical testing and cannabis DNA testing. METHODS: The sample was observed under a microscope, extracted with methanol, and analyzed by gas chromatography-mass spectrometry (GC-MS). The chemical profile of the seized powder was compared with that of nutmeg samples. Gas chromatography-flame ionization detection was used to estimate the total Δ9-tetrahydrocannabinol (Δ9-THC) concentration in the sample. A commercially available cannabis DNA testing kit was used to confirm the presence of cannabis plant DNA in the seized sample. RESULTS: The characteristics of cannabis in the seized powder were difficult to determine through microscopic observation alone. GC-MS analysis identified ß-caryophyllene (an aromatic component of cannabis) and five cannabinoids unique to cannabis, including Δ9-THC. No common compounds were identified in the seized powder or nutmeg samples. The total Δ9-THC concentration in the sample was very high (approximately 47% by weight). Cannabis DNA testing confirmed that the seized powder contained cannabis. CONCLUSIONS: The seized powder was found to be a processed product made from a finely pulverized resin-like cannabis concentrate. Our results indicate that combined chemical and DNA analysis should help identify cannabis-related samples in various forms.

Effects of temperature, humidity, light, and soil on drug stability in hair: a preliminary study for estimating personal profiles using micro-segmental analysis of corpse hair.

PURPOSE: Micro-segmental hair analysis (MSA), which enables detailed measurement of the distribution of drugs in a single hair strand, is useful for examining the day of death and drug use history of a person. However, corpses are often found in severe environments, such as soil and freezers, which affect the drug contents in hair. Therefore, we examined the effects of temperature, humidity, light, and soil on drug stability in hair as a preliminary study to estimate personal profiles using MSA of corpse hair. METHODS: Four hay-fever medicines (fexofenadine, epinastine, cetirizine, and desloratadine) were used as model drugs to evaluate drug stability in hair. Reference hair strands consistently containing the four medicines along the hair shaft were collected from patients with hay-fever who ingested the medicines daily for 4 months. The hair strands were placed in chambers with controlled temperatures (- 30 to 60 °C) and relative humidities (ca. 18 % and > 90 %), exposed to light (sunlight and artificial lights) or buried in soil (natural soil and compost). RESULTS: Sunlight and soil greatly decomposed the hair surfaces and decreased the drug contents in hair (up to 37 %). However, all analytes were successfully detected along the hair shaft, reflecting the intake history, even when the hair was exposed to sunlight for 2 weeks and buried in the soil for 2 months. CONCLUSIONS: Although the exposure to sunlight and storage in soil for long times made drug-distribution analysis difficult, MSA could be applied even to hair strands collected from corpses left in severe environments.

Synthesis and analytical characterization of 1-(2-thienoyl)-6-allyl-nor-d-lysergic acid diethylamide (1T-AL-LAD).

Lysergic acid diethylamide (LSD) analogs have emerged as new psychoactive substances (NPS) since the mid-2010s, and new compounds continue to emerge for recreational use. Since the end of 2023, "1D-AL-LAD" appeared on X (formerly Twitter) and other websites. As for the compound "1D-LSD" (which also has "1D" in the name), several studies show that the ingredient of seized blotter paper printed "1D-LSD" was actually 1-(2-thienoyl)-LSD (1T-LSD). However, there are no reports of seizures of 1-(1,2-dimethylcyclobutanecarbonyl)-LSD (1D-LSD). Accordingly, it was considered that all or at least a certain percentage of "1D-AL-LAD (1-(1,2-dimethylcyclobutanecarbonyl)-6-allyl-nor-LSD)" is actually 1-(2-thienoyl)-6-allyl-nor-LSD (1T-AL-LAD). This compound is handled by a number of distributors as of April 2024; therefore, it should be characterized in advance if seized. In this study, 1T-AL-LAD was synthesized and characterized using nuclear magnetic resonance spectroscopy, Fourier transform-infrared spectroscopy, liquid chromatography/high-resolution mass spectrometry (LC/HRMS) and gas chromatography/MS (GC/MS). This compound was easily distinguished from previously reported lysergamides. There were some differences in the detectability of 1T-AL-LAD compared with other lysergamides using GC/MS and the fragmentation patterns in LC/HRMS. These differences can be reasonably explained. This information will be of help to determine this substance in seized materials should it emerge on the market.

Development of a method to evaluate the effects of external environments on drug stability in nails using micro-segmental analysis.

Nails can be used as an alternative to hair for examining past drug use. However, daily hand-and-nail care can eliminate the internal drugs. Therefore, we developed an evaluation method to examine the effects of the external environment on drug stability in nails using micro-segmental analysis. First, reference nails containing drugs were prepared by collecting fingernails from participants who had consumed hay-fever medicines continuously for 4 months. Next, the entire free edge of a reference nail was cut into halves at the centerline; one side was stored as an untreated block, and the other was treated with various hand/nail care products. Both nail blocks were washed and segmented at 0.5-mm intervals in the width direction. Each segment in the extraction solution was crushed with stainless-steel beads, sonicated, and soaked in the solution for 24 h. The analytes in extracts were quantified by LC-MS/MS, and the drug concentrations between the treated and untreated blocks were compared. The drug concentrations decreased slightly in nails treated with manicure and gel-nail products. The analytes in nails tended to be lower in water-rich products such as hand soap and hand cream than in oil-rich products such as nailcare oil and acetone-free remover. The developed method using micro-segmental analysis enabled the evaluation of the effects of various hand/nail care products on drug stability in a limited number of nails. This would also be useful for examining the effects of severe environments on drugs in nails collected from cases of unnatural death.

Metabolism of highly potent synthetic opioid nitazene analogs: N-ethyl-N-(1-glucuronyloxyethyl) metabolite formation and degradation to N-desethyl metabolites during enzymatic hydrolysis.

The metabolism of the highly potent synthetic opioids metonitazene, etonitazene, and protonitazene was investigated in fresh human hepatocytes. In the hydrolyzed culture medium, N-desethyl-, N,N-di-desethyl-, O-desalkyl-, N-desethyl-O-desalkyl-, N,N-di-desethyl-O-desalkyl-, and N-oxidated metabolites were detected as phase I metabolites, whereas in the unhydrolyzed culture medium, O-glucuronides of phase I metabolites with O-dealkylation were detected as phase II metabolites. The detected phase I metabolites were identified by comparing their analytical data with those of synthesized authentic standards. In contrast, phase II metabolites were identified by comparing their analytical data with those of the glucuronidated products formed by the incubation of the corresponding substrates with human liver microsomes in the presence of uridine diphosphate glucuronic acid. In addition to the aforementioned metabolites, some putative N-ethyl-N-(1-glucuronyloxyethyl) metabolites were detected in the unhydrolyzed culture medium. Purification and hydrolysis experiments revealed that N-ethyl-N-(1-glucuronyloxyethyl) metabolites formed the corresponding N-desethyl metabolites via unstable N-ethyl-N-(1-hydroxyethyl) metabolites during enzymatic hydrolysis.

Identification of 1-(thiophene-2-carbonyl)-LSD from blotter paper falsely labeled "1D-LSD".

PURPOSE: Since the mid-2010s, lysergic acid diethylamide (LSD) analogs made for substance abuse have periodically emerged. In this case, three pieces of blotter paper labeled "1D-LSD" and presumably impregnated with this LSD analog, were seized. Several websites indicate that 1D-LSD is 1-(1,2-dimethylcyclobutane-1-carbonyl)-LSD. Because this analog is much more difficult to synthesize than previously reported LSD analogs, we doubted that the blotter paper contained 1D-LSD. Herein, we determined the structure of the absorbed compound. METHODS: One of the seized specimens was extracted and analyzed using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), high-resolution mass spectrometry (HRMS), and nuclear magnetic resonance (NMR) spectroscopy to estimate the extract components. The estimated compound was then synthesized, yielding an authentic standard. The contents of the seized specimens were identified using authentic standard analysis with GC/MS, LC/MS, and NMR spectroscopy. RESULTS: Instrumental analyses confirmed the active compound to be 1-(thiophene-2-carbonyl)-LSD, which was inconsistent with the labeling on drug-infused blotter paper. CONCLUSION: As in this case, similar blotter paper analyses should consider the possibility of a mismatch between the label and ingredient. To the authors' knowledge, this is the first case report in which 1-(thiophene-2-carbonyl)-LSD was seized and the first seizure of an LSD analog in which an aromatic carboxylic acid had been condensed to LSD. This type of lysergamide may become prevalent in the near future, and we should remain alert for newly appearing lysergamides.

Analysis of highly potent synthetic opioid nitazene analogs and their positional isomers.

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.