Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on December 17, 2009. Major professor: David Clague, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in biomedical Engineering." "June 2009." Also available on microfiche.

Forensic toxicology is a branch of science that involves the analysis of drugs and other substances in biological fluids and tissues such as blood, urine, and oral fluid to aid medical or legal investigation of death, poisoning, and drug use. Due to the various components of different matrices, efficient and effective sample preparation techniques are necessary for reliable and accurate analysis. Following sample clean-up, a sensitive, specific, and robust method is ideal for consistent detection, identification, and quantitation of analytes. With the rise of drug abuse, there is a growing need to develop a single method that can target multiple classes of drugs quickly and effectively. This study validated two different sample preparation techniques for the detection and quantitation of six drug classes comprised of twenty-three drugs and metabolites in oral fluid. The drug classes were as follows: amphetamines, local anesthetics, opioids, hallucinogens, antidepressants, and novel psychoactive substances (NPS). Amphetamines used were amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA). Local anesthetics contained benzoylecgonine (BZE), cocaine, and lidocaine. Opioids included codeine, methadone, morphine, 6-monoacetylmorphine (6-MAM), fentanyl, and oxycodone. Hallucinogens included lysergic acid diethylamide (LSD) and phencyclidine (PCP). Antidepressants were amitriptyline, citalopram, fluoxetine, and trazodone. Lastly, NPS included ethylone, α-pyrrolidinopentiophenone (α-PVP), and 2,5-dimethoxy-4-iodophenethylamine N-(2-methoxybenzyl) (25I-NBOMe). Supported liquid extraction (SLE) and solid phase extraction (SPE) were assessed followed by confirmatory analysis by liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Both methods were validated according to guidelines in the Standard Practices for Method Validation in Forensic Toxicology set by the American Academy of Forensic Science (AAFS) Standards Board (ASB). Parameters assessed include calibration model, bias, precision, limit of detection (LOD), limit of quantitation (LOQ), dilution integrity, ion suppression/enhancement, interference studies, and stability. Matrix recovery was added as another parameter. All calibration models were 0.99 or greater and all compounds were stable for at least 72 hours. Bias, precision, LOD, LOQ, dilution integrity, and interferences were similar between both methods. SLE yielded slightly better LOD and LOQ values. SLE had greater values of matrix recovery as well as lower levels of ionization suppression/enhancement. Overall, SLE was determined to be the better method of sample preparation for this panel of drugs in oral fluid. Not only did it yield higher values for several of the parameters assessed but it also was more efficient (1 hour versus 2 hours) while using less solvent.

It is common practice to add oxygenates, such as ethers or alcohols, to gasoline in areas suffering from ozone or smog problems in order to reduce pollution. The most commonly used oxygenates are ethanol (EtOH) and methyl tert-butyl ether (MTBE). However, MTBE is now forbidden by the environmental protection agency (EPA) because of the possibility of ground water contamination. The current trend is to use EtOH, therefore this work focuses on the analysis and quantification of EtOH in gasoline by solid phase microextraction (SPME). The major problem in quantifying EtOH in gasoline is the coelution of hydrocarbons with EtOH. There have been several approaches to solve this problem; among the chromatographic ones, three major types have been proposed: (1) the first one uses a detector selective for oxygen containing compounds; (2) the second one uses two or more columns; (3) and the third one uses an extraction step prior to GC analysis. In this work an extraction step with water is used prior to a solid phase microextraction (SPME) sample preparation coupled to a gas chromatographic (GC) analysis. Solid phase microextraction is a recent technique, invented by Pawliszyn in 1989, and available commercially since 1994. A fiber is used to extract small amounts (ppm, ppb, ppt) of analytes from a solution, usually water. The fiber is beneficial in concentrating analytes. Most work using SPME has been done with hydrophobic (non polar) analytes, extracted using a polydimethylsiloxane (PDMS; non polar) coating on a fused silica fiber. Since very little work has been done with polar analytes, the novel approach of this work is the extraction of EtOH. Since EtOH is the analyte of interest, a polar fiber, carboxen/polydimethyl siloxane (Car/PDMS) is used. Two methods are used for quantification of EtOH in gasoline: the method of a standard calibration curve, and the method of standard addition. They are both successful in quantifying the amount of EtOH in gasoline. The relative errors, with the method of standard addition, vary from 5.3% to 14%, while the ones with the method of calibration curve vary from 1.6% to 7.2%. Moreover, some extraction time studies for both direct and headspace sampling are performed. Direct sampling shows the presence of an equilibrium condition for the carboxen/PDMS fiber, for which no extraction theory is available. Conversely, headspace sampling shows no equilibrium state; after a sampling time of one hour, the amount of EtOH extracted decreases with sampling time. This is probably due to displacement of EtOH by other compounds in the fiber. / Master of Science

Electrokinetics based microfluidic systems are potentially promising for lab-on-a-chip applications due to their effectiveness in manipulating nanoscale and biological objects, label-free operation, simple fabrication processes, small voltage requirements, and most importantly simple system integration strategy. Among various electrokinetics techniques, AC electrothermal flow (ACEF) is the most promising technique in microfluidic manipulation toward biomedical applications due to its effectiveness in high conductivity biological and physiological fluids. As relatively little is known about the ACEF induced fluid motion at highly conductive samples, the characteristics of electrothermal manipulation of fluid samples with different conductivities were investigated systematically. For low conductivity sample (below 1 S/m), the characteristics of the electrothermal fluid motion was in quantitative agreement with the theory. For high conductivity samples (greater than 1 S/m), the fluid motion appeared to deviate from the model as a result of electrochemical reactions and the temperature effect. Here, a universal electrode approach which directly implements ACEF-induced sample preparation on a SAM based electrochemical sensor for point-of-care diagnostics of urinary tract infections has also been demonstrated. Using uropathogenic E. coli clinical isolates as model systems, we demonstrate that "on-chip" ACEF-induced sample preparation can improve the sensor performance without complicated system integration strategy and presents a pathway for implementing truly lab on a chip, instead of chip in a lab. Finally an integrated chip approach has been proposed for transforming electrochemical sensing system from laboratory research into point-of-care diagnostics with multiple microelectrodes.

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Increasing consumer awareness of food safety issues requires the development of highly sophisticated techniques for the authentication of food commodities. The food products targeted for falsification are either products of high commercial value or those produced in large quantities. For this reason, the present investigation is directed toward the characterization of coffee samples according to geographical origin attributes. In addition, the current examination is focused on the identification of particular marker compounds that compose the volatile and semivolatile aroma fraction of flavoured and dessert coffees. The conducted research involved the development of a rapid headspace solid phase microextraction (HS-SPME) – gas chromatography – time-of-flight mass spectrometry (GC-TOFMS) method for the verification of geographical origin traceability of coffee samples. As opposed to the utilization of traditional univariate optimization methods, the current study employs the application of multivariate experimental designs to the optimization of extraction-influencing parameters. Hence, the two-level full factorial first-order design aided in the identification of two influential variables: extraction time and sample temperature. The optimum set of conditions for the two variables was 12 min and 55 oC, respectively, as directed by utilization of the Doehlert matrix and response surface methodology. The high-throughput automated SPME procedure was completed under optimized conditions by implementing a single divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) metal fiber with excellent properties of durability, which ensured the complete analysis of coffee samples in sequence. The coffee sample originating from an authentic Brazilian coffee producing region and characterized by rich volatile and semivolatile chromatographic profiles was selected as a reference starting point for data evaluation. The combination of the retention index (RI) system using C8-C40 alkanes and the mass spectral library search was utilized for the confirmation of analyte identity in this reference sample. Twenty-nine volatile and semivolatile compounds selected across the wide range of GC chromatogram were then evaluated in terms of chromatographic peak areas for all samples that are to be submitted to this classification study. The semiquantitative results were submitted to statistical evaluation, namely principal component analysis (PCA) for the establishment of corresponding geographical origin discriminations.

This thesis examines some of the challenges involved with using FTIR spectroscopy for cancer detection including sample preparation and correcting for distortion from cell scattering. Sample preparation affects the spectra differently depending on the cell type, and can lead to significant changes in cancer biomarkers for a given cell type. Biomarkers derived from specific cancer types under one sample preparation are not reliable for other cancer types, and may not be suitable for the same cancer type using a different sample preparation. Cell scattering can also significantly affect the cell spectra, and as a result, correcting for the cell scattering distortion leads to changes in the biomarkers. For reliable cancer detection controlling variability is critical, especially in the complex spectra of biological samples. Standard sample preparation methods and scattering correction post-processing could improve comparison of cancer detection methods.

Increasing consumer awareness of food safety issues requires the development of highly sophisticated techniques for the authentication of food commodities. The food products targeted for falsification are either products of high commercial value or those produced in large quantities. For this reason, the present investigation is directed toward the characterization of coffee samples according to geographical origin attributes. In addition, the current examination is focused on the identification of particular marker compounds that compose the volatile and semivolatile aroma fraction of flavoured and dessert coffees. The conducted research involved the development of a rapid headspace solid phase microextraction (HS-SPME) – gas chromatography – time-of-flight mass spectrometry (GC-TOFMS) method for the verification of geographical origin traceability of coffee samples. As opposed to the utilization of traditional univariate optimization methods, the current study employs the application of multivariate experimental designs to the optimization of extraction-influencing parameters. Hence, the two-level full factorial first-order design aided in the identification of two influential variables: extraction time and sample temperature. The optimum set of conditions for the two variables was 12 min and 55 oC, respectively, as directed by utilization of the Doehlert matrix and response surface methodology. The high-throughput automated SPME procedure was completed under optimized conditions by implementing a single divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) metal fiber with excellent properties of durability, which ensured the complete analysis of coffee samples in sequence. The coffee sample originating from an authentic Brazilian coffee producing region and characterized by rich volatile and semivolatile chromatographic profiles was selected as a reference starting point for data evaluation. The combination of the retention index (RI) system using C8-C40 alkanes and the mass spectral library search was utilized for the confirmation of analyte identity in this reference sample. Twenty-nine volatile and semivolatile compounds selected across the wide range of GC chromatogram were then evaluated in terms of chromatographic peak areas for all samples that are to be submitted to this classification study. The semiquantitative results were submitted to statistical evaluation, namely principal component analysis (PCA) for the establishment of corresponding geographical origin discriminations.

Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on October 14, 2009. Major professor: David Clague, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering, with specializations in Biomedical Engineering." "September 2009." Includes bibliographical references (p. 125-127).

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