The method of detection ADB-BUTINACA in urine and application in forensic cases

Abstract

ADB-BUTINACA (ADB-BINACA) is a new synthetic cannabinoid that was first found in Europe in 2019. This novel drug was then rapidly increased in prevalence from early 2021. Although National Forensic Service (NFS) also identified it in authentic seizures from early last year, it was unable to test biological samples due to the unavailability of metabolic data with the absence of reference standards. NFS finally established the method of detection in biological samples. The possible metabolites were first found using LC-QTOF. Amongst the metabolites, ADB-BUTINACA and its metabolites, ADB-BUTINACA-N-butanoic acid and ADB-BUTINACA-N1hydroxybutyl, were purchased from Cayman chemicals. Methanol, ethyl acetate, sodium hydroxide, acetic acid, hydrochloric acid and ammonium formate were obtained from SIGALD. JWH-18-N-OH-D5 from Sigma aldrich was used as an internal standard (IS). Three of authentic urine samples were tested for confirmation of the method. For sample preparation, 1 mL of urine and 3 mL of 0.1 phosphate buffer were mixed with IS and then it was hydrolyzed using β-glucronidase ( E. coli ) for an hour. The prepared samples including reference blank and standard were then extracted using SPE, Rapid Trace. Those were analyzed by LC 1260 with 4500 QTRAP® mass spectrometer (AB SCIEX) in column, Zorbax Eclipse XDB-C18. Two mobile phases, 2 mM ammonium formate/0.2% formic acid in distilled water and 2 mM ammonium formate/0.2% formic acid in methanol, were used for gradient elution. The multiple reaction monitoring (MRM) data were obtained in positive ions [M + H] mode. The method was further validated by admittable specificity, linearity, LOD, LOQ, accuracy, recovery and interference. ADB-BUTINACA metabolites were expected using the data obtained from the LC-QTOF, which major candidates were corresponding to previous studies. Due to the absent of all those metabolites in market, two metabolites were only used to analyze the samples. ADB-BUTINACA (m/z 331.1), ADB-BUTINACA-N-butanoic acid (m/z347.2) and ADB-BUTINACA-N-hydroxybutyl (m/z 361.2) all were clearly shown in data by the method described above. The results from the authentic urine samples also confirmed both the two of metabolites while little parent compound was shown. It was found that ADB-BUTINACA-N-butanoic acid was predominant when comparing with the other metabolite, ADB-BUTINACA-N-hydroxybutyl. The metabolism pathways were recently reported using the human hepatocytes in authentic urine and blood samples in a few studies. Those studies suggested possible metabolism pathway and candidate metabolites using LC-QTOF. Twenty metabolites were proposed with the assumption that the parent structure is modified by the metabolism pathways such as mono-and dihydroxylation and hydroxylation of the N-butyl side chain. Currently only two biomarkers are available in market, ADB-BUTINACA-N-butanoic acid and ADB-BUTINACA-N-hydroxybutyl. ADB-BUTINACA was found in a few seizures from early 2021 but it was unable to detect in authentic biological samples in NFS until the method was established in later 2021. It was found in the seizures from nineteen cases in NFS, Wonju, last year. Amongst, a few urine samples from the cases were then reserved in freezer for further research and it was shown that one of the cases involved ADB-BUTINACA metabolites. This is an example obviously presenting that the forensic science is behind its emerging speed. It is now possible to test this new psychoactive substance as the detection method has developed. Unfortunately, new synthetic cannabinoids are continuously coming out every year but there is no alternative due to its unpredictability and rapid replacement. Therefore, global co-operation and communication seems to be only possible solutions to response promptly to this challenge.