Date of Graduation
Eberly College of Arts and Sciences
Forensic and Investigative Science
Marina Galvez Peralta
Drug abuse within the United States is a dynamic and pervasive issue that affects the quality and life expectancy of our society. One of the major drug classes that are constantly utilized is opioids. Opioids are prescribed for pain management in patients suffering from cancer and other medical illnesses. Long-term use of opioids could lead to tolerance in humans, which poses a risk of reaching fatal overdose levels. Unfortunately, opioids are abused recreationally, and in some instances, individuals are unaware of the presence of dangerous synthetic drugs like fentanyl, hidden in materials such as heroin, cocaine, oxycodone, and methamphetamine. Recent drug reports highlight the increase in seized opioids, which is also reflected by the number of associated deaths, escalating to epidemic levels. According to the National Institute of Drug Abuse (NIDA), opioid overdose deaths amount to 68,630 in 2020 (1). Therefore, the incorporation of novel approaches that offer superior sample preparation schemes for specimens of toxicological interest is greatly needed.
To this end, this study aims to evaluate the presence and prevalence of a suite of synthetic opioids and other drugs in several postmortem biological specimens obtained from a cohort of individuals. Specifically, a sample preparation procedure called QuEChERs is used to assess its effectiveness when dealing with complex matrices like blood, liver, and brain. The comprehensive drug panel covers several drug entities, including fentanyl and other synthetic opioids, cathinones, D9-tetrahydrocanbinol (THC), amphetamine, PCP, and cocaine, to a maximum of thirty-five analytes and metabolites using a targeted LC-QQQ-MS system to quantify results. This study has two objectives:  to determine the drug distribution in multiple matrices from the same source individuals, and  to evaluate the utility of oral fluid in outpatient clinical settings for patients receiving addiction mediating medication. This research thesis explores the use of QuEChERs over a non-traditional toxicological matrix called oral cavity fluid as an initial effort to understand the advantages and limitations of this specimen in postmortem toxicology casework. Another goal is to correlate the drug or metabolites concentration across matched matrices from the same individual. Oral fluid was identified as an alternative matrix of interest in toxicology by the Organization for Scientific Area Committee (OSAC), and in response to this need, this project provides data that assist in filling this gap of knowledge. Additionally, a separate study will be conducted to evaluate the utility of oral fluid in outpatient clinical settings for patients receiving addiction mediating medications.
The third aspect of this research deals with the retrieval of analytical data obtained from single source biological endpoints to better understand the post-mortem redistribution of opioid- like materials. These authentic matrix-matched specimens were obtained from the Washington DC Office of the Chief Medical Examiner’s office from deidentified deceased individuals. Postmortem redistribution is also a priority area of research within OSAC due to the limited available published data on the distribution of synthetic opioids within the body compartments of a decedent, and major findings are discussed in this thesis work.
This project includes the method validation of a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method applied to postmortem oral cavity fluid and blood. This process involves a blood extraction with salts followed by a dispersive solid phase extraction clean-up and LC-MS/MS analysis for the drugs. A suite of drugs will be utilized for standardization and analysis for the presence of drugs within the blood as well as the oral fluid matrix. A complete method validation following the American Standard Board (ASB) 036 standard will be followed to optimize the method fitness of purpose for both proposed study matrices.
Oral fluid samples will be collected from deidentified individuals using a Quantisal device that collects 1 mL ±10% of oral fluid specimens. The Quantisal device is advantageous because of its ability to collect observed specimens for later detection of recent drug use from a wide panel of drugs with reproducible results. Once the oral fluid samples are collected, they will be put into storage at -20°C until analysis. During analysis of oral fluid an aliquot of 400 μL will be extracted using the QuEChERS extraction method and analyzed on the LC-MS/MS, for blood matrix, an aliquot of 100 μL from the collected sample will be extracted following the QuEChERS method and analyzed on the LC-MS/MS. For the analysis of the liver and brain 0.1g from each respective sample will be extracted utilizing the QuEChERS extraction method and will be analyzed on the LC-MS/MS.
The QuEChERS extraction is able to be used for postmortem matrices such as the blood, liver, brain, and oral cavity fluid. The distribution of fentanyl in this study was found in the fattier tissues such as the brain and the liver, as expected due to its lipophilicity, but it was also found in the central cavity and peripheral postmortem blood samples for all analyzed tissues from decedents. After the completion of this study into oral fluid drug concentrations and postmortem blood, liver and brain opioid concentrations in authentic samples, the implementation of this method could be used for clinical patients or forensic investigations.
Morris, Kylea M., "Assessment of QuEChERS extraction protocol for the LC-MS/MS detection of emerging fentanyl analogs in biological specimens for clinical and forensic purposes" (2022). Graduate Theses, Dissertations, and Problem Reports. 11351.