Extraction and Quantification of Fentanyl and Metabolites from Complex Biological Matrices to Support Medicolegal Death Investigations

Opioids addiction is a severe national crisis that affects public health, social and economic welfare. The number of fentanyl overdose-related deaths are rising at alarming rates. The interpretation of toxicological findings in these cases is a crucial component in medicolegal investigations. However, when working with biological specimens, the analyst is prone to deal with tissues that are in various stages of decomposition that can impose several analytical challenges. This research project aims to develop a comprehensive analytical approach for the extraction and detection of fentanyl and its major metabolites, norfentanyl and opionyl fentanyl (4-ANPP) from highly complex biological matrices. The development of a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction protocol is presented in this study, as an essential analytical scheme which facilitates and improves the examination of these drugs from liver and relevant insects. Initially, the focus of this research will be centered to respond to the opioid epidemic crisis, by offering an improved extraction method and a fully validated protocol to be ready to use in any forensic toxicology laboratory. The partnership with the Office of the Chief Medical Examiner in the state of West Virginia was strategically designed to perform a validation study using de-identified casework specimens. Also, investigation of the effects of designer opioids on the development of Lucilia sericata (blowfly) larvae will be carried out by allowing the insects to feed on fentanyl-spiked tissues. QuEChERS extraction and liquid chromatography with tandem mass spectrometry (LC-MS-MS) analysis will be conducted on larvae, pupae, pupa casings, adults, and tissues used as the feeding media. Growth effects will be analyzed through evaluation of larval mass, percent of insects during each development stage, adult emergence rate, and time intervals for pupation and emergence. This data will be used to assess the effect of fentanyl on insect growth and any implications on the post-mortem interval estimation on opioid-overdose cases. One-way ANOVA (analyis of varianc), Tukey HSD (honestly significant difference) test, and Dunnet test will be used for the evaluation of significant differences between control and treatment groups. This study will provide the forensic toxicology community with a more straightforward and cost-effective extraction I technique for fentanyl in complex matrices that is important to postmortem and law enforcement investigations. Also, a significant goal of extracting fentanyl and metabolites from the insect's life stages provides a tool with great utility for death investigations involving corpses experiencing advanced decomposition.

Note: This project contains a research and/or development component, as defined in applicable law," and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). ca/ncf