Development of an Extraction Method for the analysis of Synthetic Opioids in Bone Samples using the Bead Ruptor

Abstract

<b>Abstract ID 23216</b> <b>Poster Board 462</b> Synthetic opioids have been the leading cause of overdose deaths in recent years. Many of these deaths are attributed to novel synthetic opioids (NSO). Detecting NSOs in toxicological matrices can prove difficult due to their high potencies leading to low concentrations in the body and a general lack of literature on these substances. In the case of extreme decomposition or significant scavenging, the only toxicological matrix that may be available in bone tissue. In these instances, establishing blood:bone drug concentration correlation is important to help determine the cause of death. To achieve this, correlation studies of animal models are necessary, and a careful choice of a suitable animal model is required. Rabbits, as opposed to rodents, are the most appropriate choice, due to their exhibition of spontaneous cortical bone remodeling that is not present in rodents. Previous research has not yet been able to establish such a correlation using rodents, leading to studies with a new choice of animal model for appropriate correlation data. The aim of this work is to develop a fast and suitable method for the extraction of synthetic opioids from bone samples. This method will be applied to establish blood:bone drug correlation using rabbits as the chosen animal model. The method homogenizes bone samples and extracts the analyte simultaneously utilizing the Bead Ruptor. For the development of the extraction protocol commercial drug-free rabbit bone samples that were fortified with the following synthetic opioid standards: fentanyl, norfentanyl, bucinnazine, and AP-238 in a concentration range from 1 to 1000 ng/g of bone. Fentanyl-d5 and norfentanyl-d5 were included as internal standards. For the fortification process, bones were spiked with 25 μL of the analytes solution and left drying naturally. 500 mg of fortified bone samples were homogenized in 4 mL methanol using the Omni International Bead Ruptor Elite. The homogenized samples were centrifuged, and the supernatant was collected. The supernatants were filtered through a 0.45 μm syringe filter and then diluted to 15 mL with pH 10 deionized water to prepare for SPE, where a 3 cc flangeless Oasis MCX cartridge was utilized. After loading, the samples were first washed with 2 mL 0.1 M HCl, followed by a 2% formic acid in methanol solution. The analytes were eluted with a 5% ammonium hydroxide in methanol solution, which was evaporated and reconstituted with 50 μL ethyl acetate for GC-MS analysis. A GC-MS method was developed and validated to identify and quantitate the selected NSOs. Calibration curves were prepared using the standard addition method. Yields for all analytes varied from 29% to 89%, with norfentanyl having the lowest yield and bucinnazine having the highest. The use of the Bead Ruptor for the extraction during the homogenization process showed to be efficient with less time and cost spent, when compared to the traditional methods for bone analysis that consist of drying the samples, gridding them, and letting them soak in solvent for 24 hours. The GC-MS method showed sufficient linearity for all analytes at a linear range of 1-1000 ng/g with R2 &gt;0.9984 for all analytes. Both bias and precision were less than 20% for all analytes. The developed extraction and GC-MS methods were capable of extracting, detecting, and quantitating the target opioids. Once animal experiments begin, these methods can be applied to the bone samples obtained from the rabbits for the determination of the blood:bone correlation of synthetic opioids. This work was supported by the VCU Quest Fund 2022.