Detection of metabolites of the new synthetic cannabinoid CUMYL‐4CN‐BINACA in authentic urine samples and human liver microsomes using high‐resolution mass spectrometry

Self Cite

5F‐ADB (methyl 2‐{[1‐(5‐fluoropentyl)‐1H‐indazole‐3‐carbonyl] amino}‐3,3‐dimethylbutanoate) is a frequently abused new synthetic cannabinoid that has been sold since at least the end of 2014 on the drug market and has been classified as an illicit drug in most European countries, as well as Turkey, Japan, and the United States. In this study, the in vitro metabolism of 5F‐ADB was investigated by using pooled human liver microsomes (HLMs) assay and liquid chromatography‐high‐resolution mass spectrometry (LC–HRMS). 5F‐ADB (5 μmol/L) was incubated with HLMs for up to 3 hours, and the metabolites were identified using LC–HRMS and software‐assisted data mining. The in vivo metabolism was investigated by the analysis of 30 authentic urine samples and was compared to the data received from the in vitro metabolism study. Less than 3.3% of the 5F‐ADB parent compound remained after 1 hour of incubation, and no parent drug was detected after 3 hours. We identified 20 metabolites formed via ester hydrolysis, N‐dealkylation, oxidative defluorination, hydroxylation, dehydrogenation, further oxidation to N‐pentanoic acid and glucuronidation or a combination of these reactions in vitro. In 12 urine samples (n = 30), 5F‐ADB was detected as the parent drug. Three of the identified main metabolites 5F‐ADB carboxylic acid (M20), monohydroxypentyl‐5F‐ADB (M17), and carboxypentyl ADB carboxylic acid (M8) were suggested as suitable urinary markers. The screening of 8235 authentic urine samples for identified 5F‐ADB metabolites in vitro resulted in 3135 cases of confirmed 5F‐ADB consumption (38%).

Section: Methodsmentioning

confidence: 99%

Metabolic profiling of synthetic cannabinoid 5F‐ADB by human liver microsome incubations and urine samples using high‐resolution mass spectrometry

Yeter

Öztürk

2019

Self Cite

“…For CUMYL-4CN-BINACA nitrile hydrolysis and carboxylation was also found to be a major metabolic pathway in vivo according to Ozturk et al 12 For the fluorinated compounds, oxidative defluorination (M20) and carboxylation (M21), followed by further oxidation at pentyl, aryl or indazole moiety (M22-M25) were major metabolic pathways. Oxidative defluorination was already identified as common metabolic pathway for other fluorinated synthetic cannabinoids such as, for example, 5F-AB-PINACA, 5F-PB-22 and AM-2201 not only in vitro, but also in vivo.…”

Section: Metabolic Pathwaysmentioning

confidence: 95%

“…11 To date, no information on the metabolism of the 5 cumyl-derivatives in this study is available except for CUMYL-4CN-BINACA. 12 To our knowledge, no toxicological case reports involving CUMYL-4CN-B7AICA or 5F-CUMYL-P7AICA have been published. CUMYL-PINACA was identified in 3 cases of transdermal poisoning and 5F-CUMYL-PINACA in a case of hospital presentation; however, metabolism was not investigated in these cases.…”

mentioning

confidence: 99%

In vitro metabolism of the synthetic cannabinoids CUMYL‐PINACA, 5F–CUMYL‐PINACA, CUMYL‐4CN‐BINACA, 5F–CUMYL‐P7AICA and CUMYL‐4CN‐B7AICA

Staeheli

Poetzsch

Veloso

et al. 2017

Synthetic cannabinoid consumption trends underlie fast changes and provide several challenges to clinical and forensic toxicologists. Due to their extensive metabolism, parent compounds are hardly detectable in urine. Therefore, knowledge of the metabolism of synthetic cannabinoids is essential to allow their detection in biological matrices. The aim of the present study was the elucidation of the metabolism of CUMYL-PINACA, 5F-CUMYL-PINACA, CUMYL-4CN-BINACA, 5F-CUMYL-P7AICA, and CUMYL-4CN-B7AICA with a focus on the analytical and interpretational differentiation of the compounds. Microsomal assay mixtures containing cosubstrates, 10 μg/mL substrate and 1 mg/mL pooled human liver microsomes were incubated for 1 hour at 37°C. Investigation of the metabolites was performed on a Thermo Fischer Ultimate 3000 UHPLC system coupled to a Sciex 6600 QTOF System. Hydroxylation was observed to be a major biotransformation step for all 5 cumyl-derivatives, followed by dihydroxylation.For CUMYL-PINACA, a major metabolic pathway was hydroxylation at the pentyl moiety, followed by a second hydroxylation at that pentyl moiety or oxidation to ketone. A major metabolic pathway for the compounds containing a nitrile function was nitrile hydrolysis followed by carboxylation and further hydroxylation. For the fluorinated compounds, oxidative defluorination and carboxylation were abundant metabolic steps. Some of the metabolic transformations lead to structurally identical metabolites, which should not be used as marker for the intake of a particular parent compound. In addition, several constitutional isomers containing either an indazole or azaindole core structure were detected, which should be differentiated by retention time rather than by their mass spectra alone. KEYWORDSin vitro metabolism, new psychoactive substances NPS, synthetic cannabinoids

“…The metabolism of many synthetic cannabinoids has been determined via HLM incubations . The findings reported in the current study contributes to the body of knowledge available on indazole carboxamide synthetic cannabinoid metabolism via HLM incubations.…”

Section: Discussionmentioning

confidence: 66%

“…The in vitro and in vivo metabolic profiles of several indazole carboxamide synthetic cannabinoids have been reported . In this study the in vitro metabolic profile of indazole carboxamide synthetic cannabinoid, MDMB‐CHMINACA, was determined.…”

Section: Introductionmentioning

confidence: 99%

In vitro Phase I metabolism of indazole carboxamide synthetic cannabinoid MDMB‐CHMINACA via human liver microsome incubation and high‐resolution mass spectrometry

Presley

Logan

Jansen‐Varnum

2019

Synthetic cannabinoids have proliferated over the last decade and have become a major public health and analytical challenge, critically impacting the clinical and forensic communities. Indazole carboxamide class synthetic cannabinoids have been particularly rampant, and exhibit severe toxic effects upon consumption due to their high binding affinity and potency at the cannabinoid receptors (CB1 and CB2). MDMB‐CHMINACA, methyl 2‐[1‐(cyclohexylmethyl)‐1H‐indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate, a compound of this chemical class, has been identified in forensic casework and is structurally related to several other synthetic cannabinoids. This study presents the first extensive report on the Phase I metabolic profile of MDMB‐CHMINACA, a potent synthetic cannabinoid. The in vitro metabolism of MDMB‐CHMINACA was determined via incubation with human liver microsomes and high‐resolution mass spectrometry. The accurate masses of precursor and fragments, mass error (ppm), and chemical formula were obtained for each metabolite. Twenty‐seven metabolites were identified, encompassing twelve metabolite types. The major biotransformations observed were hydroxylation and ester hydrolysis. Hydroxylations were located predominantly on the cyclohexylmethyl (CHM) moiety. Ester hydrolysis was followed by additional biotransformations, including dehydrogenation; mono‐ and dihydroxylation and ketone formation, each with dehydrogenation. Minor metabolites were identified and reported. The authors propose that CHM‐monohydroxylated metabolites specific to MDMB‐CHMINACA are the most suitable candidates for implementation into bioanalytical assays to demonstrate consumption of this synthetic cannabinoid. Due to the structural similarity of MDMB‐CHMINACA and currently trending synthetic cannabinoids whose metabolic profiles have not been reported, the results of this study can be used as a guide to predict their metabolic pathways.