Structure Elucidation of Urinary Metabolites of Fentanyl and Five Fentanyl Analogs using LC-QTOF-MS, Hepatocyte Incubations and Synthesized Reference Standards

Abstract

Fentanyl analogs constitute a particularly dangerous group of new psychoactive compounds responsible for many deaths around the world. Little is known about their metabolism, and studies utilizing liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis of hepatocyte incubations and/or authentic urine samples do not allow for determination of the exact metabolite structures, especially when it comes to hydroxylated metabolites. In this study, seven motifs (2-, 3-, 4- and β-OH as well as 3,4-diOH, 4-OH-3-OMe and 3-OH-4-OMe) of fentanyl and five fentanyl analogs, acetylfentanyl, acrylfentanyl, cyclopropylfentanyl, isobutyrylfentanyl and 4F-isobutyrylfentanyl were synthesized. The reference standards were analyzed by LC-QTOF-MS, which enabled identification of the major metabolites formed in hepatocyte incubations of the studied fentanyls. By comparison with our previous data sets, major urinary metabolites could tentatively be identified. For all analogs, β-OH, 4-OH and 4-OH-3-OMe were identified after hepatocyte incubation. β-OH was the major hydroxylated metabolite for all studied fentanyls, except for acetylfentanyl where 4-OH was more abundant. However, the ratio 4-OH/β-OH was higher in urine samples than in hepatocyte incubations for all studied fentanyls. Also, 3-OH-4-OMe was not detected in any hepatocyte samples, indicating a clear preference for the 4-OH-3-OMe, which was also found to be more abundant in urine compared to hepatocytes. The patterns appear to be consistent across all studied fentanyls and could serve as a starting point in the development of methods and synthesis of reference standards of novel fentanyl analogs where nothing is known about the metabolism.

Circumstances, Postmortem Findings, Blood Concentrations and Metabolism in a Series of Methoxyacetylfentanyl-Related Deaths

“…This is also true for most metabolites of methoxyacetylfentanyl. However, methoxyacetylfentanyl is structurally similar to acetyl-, acryl-, cyclopropyl- and 4-fluoro-isobutyrylfentanyl previously studied by our group using reference materials synthesized in-house and behaved very similar when comparing positional preference on the phenethyl moiety during metabolism ( 25 ). Assuming the same is true for methoxyacetylfentanyl, we tentatively assigned positional isomers on the phenethyl ring for many metabolites, but the reader should bear in mind that this was not confirmed.…”

“…It is postulated that M20 and M13 are hydroxylated in the 4-phenethyl position based on the known preference for this configuration as shown by Wallgren et al. ( 25 ). Two monohydroxylated and demethylated glucuronides (M04 and M09) were identified.…”

Untargeted Metabolic Profiling of 4-Fluoro-Furanylfentanyl and Isobutyrylfentanyl in Mouse Hepatocytes and Urine by Means of LC-HRMS

“…Other minor metabolites were formed by carbonylation (M21_iBF), oxidative Ndealkylation (M4_iBF), dihydroxylation of iBF (M9_iBF and M19_iBF) and subsequent methylation; phase II glucuronidation was observed for these metabolites, producing M5_iBF and M13_iBF respectively. This is the first report of iBF's complete metabolic profile; however, it should be pointed out that in a study conducted by Wallgren et al [28] iBF was included among the investigated drugs, and some hydroxylated metabolites isomers were characterized and quantified in hepatocyte samples. In addition, iBF-related fentanyl analogues, i.e., butyrylfentanyl [17,22,23] and 4F-iBF [19], were previously studied by other authors, both in vitro and in vivo.…”

“…Being two new derivatives, to the best of our knowledge their metabolic profile has not been characterized to date, and the pharmacological effects are also unknown; however, dose-dependent increases in locomotion and antinociception were reported for iBF [27]. For iBF, different isomers of the hydroxylated metabolites were characterized in hepatocyte samples; however, the complete metabolic profile was not investigated [28].…”

Targeted and untargeted detection of fentanyl analogues and their metabolites in hair by means of UHPLC-QTOF-HRMS

“…Very recently, a case of toxicity from intentional therapeutic use of β-hydroxyfentanyl (possibly mistaken as fentanyl) was reported [28]. Furthermore, β-hydroxyfentanyl had been detected in biological fluids as a metabolic product after fentanyl administration [29,30], but it has never been reported in hair before.…”