Simultaneous determination of synthetic cannabinoids and their metabolites in human hair using LC-MS/MS and application to human hair.

Hair is one of the key samples for judging drug abuse in the field of forensic science. However, few studies have examined synthetic cannabinoids and their metabolites in human hair. Synthetic cannabinoids are a class of chemicals that bind to cannabinoid receptors, but they differ structurally from the cannabinoids found in cannabis. They have been sold sprayed on dried, shredded plant material under brand names such as "Spice" since the 2000s. In South Korea, synthetic cannabinoids have been widely distributed since 2009 and many types detected up to now. Unlike traditional drugs such as methamphetamine and cannabis, the abuse trends of synthetic cannabinoids were variable by regions and changed according to the times. If new types of synthetic cannabinoids become popular which has been altered in some structures, it becomes difficult to identify using exist analytical method. Therefore, it is important to develop a new analytical method for synthetic cannabinoids currently being abused in society. In this study, we developed simultaneous analytical methods for the detection of 18 synthetic cannabinoids and 41 of their metabolites in authentic human hair samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Selectivity, linearity, limits of detection (LODs), limits of quantification (LOQs), precision, accuracy, matrix effect, recovery, and process efficiency were evaluated, and all results were acceptable. Additionally, the distribution of synthetic cannabinoids in the head hair of Korean drug abusers from 2016 to 2018 was investigated. Hair samples from 43 individuals suspected of synthetic cannabinoid use were provided by law enforcement agencies. The drugs detected most prevalently in the head hair of Korean drug abusers were AB-CHMINACA and JWH-210.

Detection of 11-nor-9-carboxy-tetrahydrocannabinol in the hair of drug abusers by LC-MS/MS analysis.

Cannabis is the second most commonly abused illicit drug after methamphetamine in South Korea. To prove cannabis consumption, 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH), the metabolite of tetrahydrocannabinol (THC), was screened for in a hair analysis. In this study, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis method, which was used to analyze authentic hair samples in 2017. Possible contaminants on the surface of hair samples were eliminated by washing twice each with 2mL of methanol and distilled water. After adding an internal standard (THC-COOH-d3), the hair samples (about 20mg each) were digested with 1M NaOH, extracted twice with mixed organic solvents (n-hexane:ethyl acetate), and analyzed by an LC-MS/MS system. Identification and quantification of THC-COOH and THC-COOH-d3 were performed using a multiple reaction monitoring (MRM) mode at m/z 245 and 191 and m/z 248, respectively (quantifier ions are underlined). The following validation parameters were evaluated: selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect, and recovery. The LOD and LOQ of the method was 0.1pg/mg. Good linearity was achieved for THC-COOH in the range from 0.1 to 20pg/mg. The method showed an acceptable precision and accuracy, both of which were less than 15% at the three concentrations of THC-COOH (0.2, 1, and 10pg/mg). THC-COOH showed ion suppression at these three concentrations. The concentrations of THC-COOH in the authentic hair samples ranged from 0.10 to 27.30pg/mg (total 586 cases), and its concentrations were classified as low, medium, or high ranges, i.e., 0.10-0.39pg/mg, 0.39-1.99pg/mg, or 1.99-27.30pg/mg, respectively, according to statistical evaluation. This method showed the possibility of replacing the existing gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis. However, further development of our LC-MS/MS method is necessary in order to meet the recommended 0.05pg/mg cut-off.

Determination of AB-CHMINACA and its metabolites in human hair and their deposition in hair of abusers.

Despite global efforts to control the abuse of synthetic cannabinoids, the high-level of turnover from the market impedes regulation, endangering public health. N-[(1S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA) is the most popular synthetic cannabinoid in South Korea since its introduction in 2014. Nonetheless, few studies have been carried out on AB-CHMINACA and its metabolites, and its deposition in human hair. The purpose of this study was to develop and validate an analytical method for detection of AB-CHMINACA and its six metabolites in hair using a liquid chromatography tandem mass spectrometry (LC-MS/MS) system, for forensic applications. The methanol extracts of hair samples were evaporated, filtered, and analyzed by LC-MS/MS with electrospray ionization in positive ion mode. The limits of detection and quantification ranged from 0.5 to 10pg/mg and 2 to 50pg/mg, respectively. Good linearity was achieved within the range of 5-1000pg/mg or 10-1000pg/mg depending on the analyte. Intra- and inter-assay precision and accuracy values were below 15%. No significant variation was observed using different sources of hair matrices. These validation results proved the selectivity, accuracy and reproducibility of the method. The established method was applied to 37 authentic samples from suspected synthetic cannabinoid users. AB-CHMINACA and its two metabolites, AB-CHMINACA M2 and AB-CHMINACA M4, were detected. The concentration of the parent drug was much higher than those of its metabolites, and the amount of AB-CHMINACA M2 was greater than that of AB-CHMINACA M4 in all samples. No other metabolites were detected in the samples.

Novel approach to study the cardiovascular effects and mechanism of action of urban particulate matter using lung epithelial-endothelial tetra-culture system.

In vitro models have become increasingly sophisticated, and their usefulness in supporting toxicity testing is well established. The present study was designed to establish a novel in vitro model that mimics the cellular network surrounding airways and pulmonary blood vessels, to study the cardiovascular toxic effects of particulate matter (PM). Transwell culture method was used to develop a novel tetra-culture system consisting of tri-cultures (one lung epithelial and two immune cell lines) in the apical chamber and endothelial cells in the basolateral chamber. Tri-cultures were exposed to standard reference material (SRM) 1648a, an urban PM. SRM 1648a did not show cytotoxic effects; however, it increased IL-6 level in apical and basolateral chambers. The cells in the basolateral chamber showed increased monocyte adhesion. Furthermore, exposure of tri-cultured cells to SRM 1648a in the apical chamber induced ICAM-1 expression in endothelial cells in the basolateral chamber by activating the IL-6/STAT3 pathway. In conclusion, a tetra-culture system was established to facilitate the identification of cellular adhesion molecule expression induced by the interaction between pulmonary epithelial and endothelial cells. The tetra-culture system will contribute to elucidation of the relationships between inhalable PM and cardiovascular diseases.