In vitro metabolic profile of the new synthetic opioid bucinnazine by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)

Abstract

The aim of this study is to develop an LC-MS/MS method that can be used to identify the in vitro metabolites of bucinnazine. The method validation was evaluated according to the ASB Standards for Method Validation in Forensic Toxicology. A calibration model, bias calculations, precision calculations, a carryover study, and calculation of the limit of detection and limit of quantitation were evaluated for method validation. Bucinnazine hydrochloride was incubated at 37 °C with Sprague-Dawley rat microsomes (Gibco, New York, USA) for up to three hours for metabolite formation. Analyses were carried out using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). For identification of the metabolites, LC-MS/MS analysis was performed on a Shimadzu LC-MS/MS 8050 system (Shimadzu Corp., Kyoto, Japan) equipped with a Poroshell HPH C18 column with a gradient mobile phase consisting of 5 mM ammonium formate and 0.1% formic acid in water as mobile phase A, and 100% methanol as mobile phase B. Fentanyl-d5 was used as the internal standard. The ion transitions for bucinnazine and fentanyl-d5 were 273 m/z -> 117 m/z and 273 m/z -> 91 m/z, and 342 m/z -> 105 m/z and 342 m/z - > 188 m/z, respectively. Method validation was successfully performed, allowing the method to be used for sample analysis. Metabolite formation was observed over a time period of 0 minutes, 30 minutes, 90 minutes, and 180 minutes. Metabolites were identified by observing increasing and decreasing peaks in the chromatograms of the samples. Peaks observed in both the samples and the controls were excluded from the analysis. Given the increase in illicit bucinnazine cases in the United States, a deeper understanding of its metabolites as biomarkers is important for the development of reliable identification strategies to detect bucinnazine misuse cases. At least two metabolites of bucinnazine were identified using LC-MS/MS. Hydroxylation and dealkylation of the parent compound was observed, and an initial metabolic pathway of bucinnazine was described. Further work to identify other metabolites, and to identify the specific enzymes responsible for metabolite formation will be completed.