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.

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 identification of Aconitum plants based on loop-mediated isothermal amplification assay.

OBJECTIVE: Aconitum plants (Ranunculaceae) exhibit toxicity, and accidental ingestion of the plants has been reported in Japan. Identifying the cause of poisoning is important for emergency medical treatment, and a rapid and simple detection technique is required for the identification of poisoning cause. In the present study, we developed a rapid and simple method for detecting Aconitum plant DNA using a loop-mediated isothermal amplification (LAMP) assay. RESULTS: Specific LAMP primers for Aconitum plants were designed based on the trnL-trnF intergenic spacer region. Using the LAMP primers, the LAMP assay included an initiation reaction of 10 min followed by amplification for 20 min at the isothermal reaction temperature of 65 °C. The LAMP reaction was demonstrated to be specific and highly sensitive to Aconitum plants, given that the assay can be used for 1 pg of purified DNA. Using raw extracted DNA as template, the entire detection procedure from DNA extraction to final detection required only 30 min. Moreover, the protocol identified samples containing approximately 5 mg of Aconitum plants cooked and digested with artificial gastric juice. The currently proposed protocol exhibits good potential as a screening method of Aconitum plant poisoning for emergency medical care.

Development of simple and accurate detection systems for Cannabis sativa using DNA chromatography.

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

Development of a novel immunoassay for herbal cannabis using a new fluorescent antibody probe, "Ultra Quenchbody".

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.

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.