Semester

Summer

Date of Graduation

2025

Document Type

Thesis (Campus Access)

Degree Type

MS

College

Eberly College of Arts and Sciences

Department

Forensic and Investigative Science

Committee Chair

Luis Arroyo

Committee Member

Tatiana Trejos

Committee Member

Prentiss Jones

Abstract

Drug abuse is a dynamic and widespread health issue that affects the quality and life expectancy of our society. Pain, an inherent aspect of life, is a complex phenomenon to treat humans that varies from patient to patient. The duress experienced by patients sometimes triggers the need to find alternative methods to cope with the pain, including alcohol, smoking, self-destructive behavior, or the abuse of recreational drugs. One of the most frequently prescribed medications for pain control is opioids. Investigating the prevalence of “hidden” drug entities in patients under rehabilitation therapy is challenging but can help in understanding adherence and abuse patterns. Long-term use of opioids may lead to tolerance in humans, which sometimes poses a risk of fatal overdoses. 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 on Drug Abuse (NIDA), 81,806 opioid-related overdose deaths occurred in 2022. Therefore, developing efficient and novel approaches that offer superior sample preparation schemes is highly desirable in clinical and toxicology scenarios. To this end, this study aims to evaluate novel toxicology extraction strategies applied to oral fluid and submandibular salivary gland tissue specimens analyzed by LC-MS/MS to detect a large suite of drugs. The drug panel includes fentanyl and other synthetic opioids, cathinones, amphetamines, cocaine, PCP, and Δ9-tetrahydrocannabinol (THC) for a total of thirty-five analytes.

The validation of the novel extraction methods followed the current American National Standards Institute/American Academy of Forensic Science Standards Board ANSI/ASB Standard 036 (ASB 036) for method validation in forensic toxicology. The validation was completed for quantitative analysis, which includes the parameters of bias, precision, carryover, limit of detection, limit of quantitation, interference studies, ionization suppression/enhancement, one to four dilution integrity, and room-temperature processed sample stability.

Two extraction methods were compared to determine the efficacy of the extraction of a large suite of drugs from oral fluid. The first extraction method includes a solid phase extraction with a mixed-mode cation exchange sorbent cartridge (MCX). The second extraction method includes protein precipitation (PP) followed by filtration through a Nanosep® centrifuge device. Extraction protocols were compared for the efficiency of the extraction in the terms of matrix effects, recovery, dilution integrity, sample stability, time, and cost of extraction.

The applicability of the extraction strategies was assessed through the analysis of twenty-five authentic antemortem oral fluid specimens collected from deidentified individuals in a drug rehabilitation care facility over an eight-month period. These specimens were first analyzed through a previously validated method, QuEChERS, which stands for Quick, Easy, Cheap, Effective, Rugged, and Safe. Results were compared with those obtained through MCX and PP filtration extraction of the same twenty-five case samples. Buprenorphine, naloxone, and methamphetamine were the most frequently detected analytes throughout this dataset.

Another aspect of this project involves applying a modified QuEChERS method to analyze postmortem submandibular gland tissue. Twenty-two submandibular gland tissue specimens were collected from deidentified individuals to assess the utility of the QuEChERS extraction method on this biological matrix. Fourteen matched submandibular gland and postmortem whole blood were analyzed for comparison and to assess the validity of the submandibular gland specimens as an alternative matrix. In the matched blood and submandibular gland tissue specimens, amphetamine and methamphetamine, were the most frequently detected analytes ranging from 25.3 to 315.6 ng/mL and 6.9 to 2857.6 ng/mL, respectively, for the whole blood and ranging from 3.6 to 227.3 µg/kg and 4.1 to 2072.9 µg/kg, respectively, for the submandibular gland. The benefits of the QuEChERS extraction method on two alternative biological matrices, oral fluid and submandibular gland tissue, are demonstrated in this study for utilization in toxicology casework.

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