Metabolic profiling of synthetic cannabinoid 5F‐ADB and identification of metabolites in authentic human blood samples via human liver microsome incubation and ultra‐high‐performance liquid chromatography/high‐resolution mass spectrometry

Abstract

Indazole carboxamide synthetic cannabinoids, a prevalent class of recreational drugs, are a major clinical, forensic and public health challenge. One such compound, 5F-ADB, has been implicated in fatalities worldwide. Understanding its metabolism and distribution facilitates the development of laboratory assays to substantiate its consumption. Synthetic cannabinoid metabolites have been extensively studied in urine; studies identifying metabolites in blood are limited and no data on the metabolic stability (half-life, clearance and extraction ratio) of 5F-ADB have been published prior to this report. Methods: The in vitro metabolism of 5F-ADB was elucidated via incubation with human liver microsomes for 2 h at 37 C. Samples were collected at multiple time points to determine its metabolic stability. Upon identification of metabolites, authentic forensic human blood samples underwent liquid-liquid extraction and were screened for metabolites. Extracts were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS) operated in positive electrospray ionization mode. Results: Seven metabolites were identified including oxidative defluorination (M1); carboxypentyl (M2); monohydroxylation of the fluoropentyl chain (M3.1/M3.2) and indazole ring system (M4); ester hydrolysis (M5); and ester hydrolysis with oxidative defluorination (M6). The half-life (3.1 min), intrinsic clearance (256.2 mL min −1 kg −1), hepatic clearance (18.6 mL min −1 kg −1) and extraction ratio (0.93) were determined for the first time. In blood, M1 was present in each sample as the most abundant substance; two samples contained M5; one contained 5F-ADB, M1 and M5. Conclusions: 5F-ADB is rapidly metabolized in HLM. 5F-ADB, M1 and M5 are pharmacologically active at the cannabinoid receptors (CB 1 /CB 2) and M1 and M5 may contribute to a user's impairment profile. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for pharmacologically active metabolites, especially for drugs with short half-lives. The authors propose that M1 and M5 are appropriate markers to include in laboratory blood tests screening for 5F-ADB.

Analytical findings in a non-fatal intoxication with the synthetic cannabinoid 5F-ADB (5F-MDMB-PINACA): a case report

“…5F-ADB undergoes extensive hepatic metabolism. Results of metabolism studies with pooled human liver microsomes (pHLM) as well as results of both blood and urine screenings have been previously published [ 22 , 53 , 63 , 68 , 69 ]. According to the literature, the metabolism of 5F-ADB is dominated by ester hydrolysis, defluorination (revealing the N -(5-OH-pentyl) metabolite) and monohydroxylations.…”

Identification of the metabolites of methylophiopogonanone A by ultra‐high‐performance liquid chromatography combined with high‐resolution mass spectrometry

“…Identification of the metabolites is very challenging as the concentrations of the metabolites are very low and some of them are unpredictable. The development of mass spectrometry, especially high‐resolution mass spectrometry, has facilitated the identification of the metabolites through the measurements of the exact masses, elemental compositions and the product ions 15–17 …”

“…The development of mass spectrometry, especially high-resolution mass spectrometry, has facilitated the identification of the metabolites through the measurements of the exact masses, elemental compositions and the product ions. [15][16][17] Therefore, the goal of the present study was to identify the metabolites of MOA produced by rat, monkey and human liver microsomes and hepatocytes by using ultra-high-performance liquid chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry (UPLC/HRMS) and to propose the metabolic pathways. Acetonitrile (HPLC grade) was obtained from Merck (Darmstadt, Germany).…”

Identification of the metabolite of ophiopogonanone A by liquid chromatography/quadrupole time‐of‐flight mass spectrometry

“…Liver microsomes and hepatocytes derived from the liver tissue are widely used for drug metabolism study as they retain most of the drug‐metabolizing enzymes, such cytochrome P450 enzymes and glucuronosyltransferases 17,18 . In addition, it has been previously demonstrated that high‐resolution mass spectrometry is a powerful technique for metabolite identification due to its high resolution, sensitivity, and high throughput, which facilitates the identification of drug metabolites through the measurements of accurate mass, elemental composition, and the product ions 19,20 …”