Date of Award

2016

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

College of Arts and Sciences

First Advisor

Stephen L. Morgan

Abstract

Efficiently hydrolyzing glucuronide metabolites is an important step in the analysis of drugs in urine. Amitriptyline and cyclobenzaprine are both tricyclic compounds with tertiary aliphatic amine groups that are subsequently metabolized to a less common form of glucuronide metabolite, quaternary ammonium linked glucuronides (N+- glucuronide). A collaborative study was conducted to investigate discrepancies in recoveries of these two commonly prescribed compounds in patient urine samples when hydrolyzed with different enzyme from four different commercially available β-glucuronidase sources.

Similarly, there is potential for 6-monoacetylmorphine to be converted to morphine during β-glucuronidase hydrolysis of urine samples. 6-monoacetylmorphine is a unique metabolite of heroin and thus a marker of heroin use. A collaborative study was performed to analyze the degree of 6-monoacetylmorphine (6-MAM) conversion by the same four commercially available β-glucuronidase enzymes in drug free spiked urine and patient urine samples.

Meconium is an important biological matrix in determining drug exposure of a newborn. A novel method for the quantitation of ten commonly prescribed benzodiazepines and/or their metabolites (7-aminoclonazepam, clonazepam, α-hydroxyalprazolam, alprazolam, nordiazepam, diazepam, midazolam, oxazepam, lorazepam, and temazepam) in meconium was developed using enzymatic hydrolysis, WAX-S dispersive pipette extraction (DPX) tips, and LC-MS/MS. The proposed method minimizes sample volume and sample preparation time. A successful blind study with a corresponding laboratory verified the effectiveness of the method.

As marijuana continues to be decriminalized, the need for analyses to determine marijuana impairment for driving under the influence cases rises. An analytical procedure was developed and validated for the analysis of Δ9-tetrahydrocannabinol (THC) and its metabolites, 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy- Δ9-tetrahydrocannabinol (THC-COOH), in whole blood using liquid chromatography tandem mass spectrometry (LC-MS/MS). An automated DPX method was employed on a Hamilton NIMBUS96 platform to extract the analytes of interest. A full well plate (96) of samples could be extracted in less than three minutes. The method was fully validated according to the Scientific Working Group of Forensic Toxicology (SWGTOX) guidelines. This method resulted in a successful patient sample comparison with a local forensic toxicology lab, South Carolina Law Enforcement Division.

The measurement of free catecholamines and metanephrines in the clinical laboratory is important for the diagnosis of tumors, such as pheochromocytoma. We have developed an automated, high throughput DPX sample preparation procedure for the selective extraction of norepinephrine, epinephrine, dopamine, normetanephrine, and metanephrine in urine. The method was assessed for linearity, sensitivity, precision, accuracy, carryover, matrix effects and recovery. Using diphenyl borinic acid for complexation and styrene divinyl benzene for extraction enabled high recoveries and reduced ion suppression. The simple and high throughput nature of this method would be ideal for clinical laboratories experiencing high demand for catecholamine and metanephrine urine analysis.

Determining the limit of detection (LOD) for a toxicological analytical method is an important component of method validation. However, with growing applications in an increasing variety of applications, the recommendation for determining an LOD from relevant data have evolved in varying directions. Specifically, guidelines often do not clarify the ambiguities and the effects of different choices associated with the estimate of uncertainty (standard deviation) for signal-to-noise LOD criteria. Further, LOD is based on fitting calibration relationship by the method of least squares which require assumptions (e.g., constant variance) that are rarely addressed. In addition to clarifying these ambiguities, the application of tolerance intervals for limits of detection determinations is discussed for forensic dye analysis.

Rights

© 2016, Kaylee R. Mastrianni

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