In the field of forensic toxicology, several challenges exist with quantification analysis of cocaine and metabolites in post mortem samples. Cocaine can prove difficult to detect and quantify in blood, urine, and soft tissues following extensive decomposition. Alternative matrices, such as hair, nails, and bone could prove useful in detecting chronic drug use in post-mortem toxicology cases. Detection and quantification of drugs in complex matrices is difficult to accomplish due to time-consuming extraction processes, and inability to detect an analyte at trace levels. Further, analysis of drugs in hard tissues, such as hair and bone, has only been attempted in recent years. Even fewer studies have investigated detection of drugs following decomposition of remains, specifically outdoor decomposition. The objective of this study was to develop a robust extraction and clean up methodology, in which a homogenization step precedes, to efficiently extract drugs from complex matrices, reach a target limit of detection (LOD) and to maintain instrument performance using multidimensional chromatography. Multi-dimension chromatography platform such as two dimensional liquid chromatography tandem mass spectrometry ( 2D-LC/MS/MS,) offers options not compatible with single dimension v units. With large volume injection capabilities of aqueous and organic extracts, the analytical process be reduced from multiple hours to minutes. All rat specimens used for this study fell under an Institutional Animal Care and Use Committee (IACUC) protocol. The rodents underwent a 10-12 weeks chronic intravenous self-administration of cocaine. This was followed by a six-week period of abstinence, followed again by a three-week period of cocaine self-administration before being euthanized. Average daily dosages for each rat fell within a range of 13-19 mg/kg. A total of 14 cocaine positive rats were placed outside and above ground in the Boston University Forensic Anthropology Outdoor Research Facility (Holliston, MA, U.S.A) for a period of 12 months. All recoverable skeletal samples were collected for testing. Drug free control rat bones were also acquired by placing drug-free rats outdoors, above ground, until full decomposition occurred. In this study, a method analyzing cocaine and its major metabolites benzoylecgonine and ecgonine methyl ester was developed. After homogenization of whole bones, the extraction process was performed using a mixed mode reversed-phase/ion exchange sorbent. The use of a 2D LC/MS/MS technology eliminates the need for a lengthy evaporation step in the extraction method. The chosen 2D LC/MS/MS used in this application was identified using a 6x6 automated method development protocol. The manual extraction of the bone samples was completed in less than an hour. The analysis was performed using 100μL of the final organic solvent (MeOH) extracts. vi The limit of quantitation (LOQ) for cocaine and benzoylecgonine was measured at 0.05ng/g (0.05ng/mL or 50pg/g) of sample material and the LOQ for ecgonine methyl ester was measured at 0.1ng/g (0.1 ng/mL or 100pg/g). The extraction method for cocaine proved to give a linear dynamic range of 2.5 orders of magnitude (0.05 ng/g to 10ng/g with an R2 = 0.998. The micro extraction protocol combined with a multi-dimension chromatography used in this study decreased sample preparation time without sacrificing the quality seen with current single dimension chromatography techniques. The procedure developed in this study can be utilized on bone and completed in less than an hour before injection into the 2D-LC/MS/MS system.

Organic aerosol is a major constituent of atmospheric fine particulates (PM2.5), which adversely affect human health and change the Earth’s radiative energy balance. Primary organic aerosol is directly emitted from sources and secondary organic aerosol (SOA) is formed in the atmosphere following oxidation of volatile organic compounds (VOC) from anthropogenic and biogenic sources. Biogenic SOA is enhanced by anthropogenic pollutants such as sulfate and NOx that mainly come from fossil fuel combustion. However, the extent to which the anthropogenic pollutants enhance biogenic SOA in different environments is unknown. The central hypothesis of this thesis is that organosulfates, organic compounds containing a sulfate ester group, are useful as tracers for anthropogenically-influenced biogenic SOA. This research aims to provide a better understanding of the sources of PM2.5 organic carbon (OC), particularly secondary organic carbon (SOC), through the inclusion of organosulfates in an organic tracer-based source apportionment model. The specific objectives of this research include 1) development of a highly sensitive and accurate method to quantify highly polar organosulfates in atmospheric aerosols, 2) identification and quantification of major organosulfate species in the ambient air, and 3) determination of anthropogenic and biogenic sources and their contributions to PM2.5 OC using an organic tracer-based positive matrix factorization (PMF) model. A highly sensitive and accurate method was developed and validated for the quantification of highly polar organosulfates using hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The developed method shows excellent retention of carboxylic acid and hydroxyl containing organosulfates. The HILIC-MS/MS method was applied to PM2.5 samples collected in summer 2013 at a rural site in Centreville, AL. Quantified organosulfates accounted for approximately 0.3% of PM2.5 OC. Other major organosulfates, for which standards are not available, were monitored by their fragmentation to the bisulfate anion and/ or sulfate ion radical. The major organosulfates were determined to be 2-methyltetrol sulfate and other isoprene-derived organosulfates. Eight sources of the PM2.5 OC in Centreville, AL were identified using PMF model through the application of organosulfates and commonly used organic tracers measured in samples collected during the daytime and nighttime: vehicle emissions (8%), prescribed burning (11%), isoprene SOC formed under low-NOx (13%) and high-NOx conditions (11%), SOC formed by photochemical reactions (9%), oxidatively aged biogenic SOC (6%), sulfuric acid-influenced SOC (21%), and monoterpene SOC formed under high-NOx conditions (21%). The organosulfates enabled organic tracer-based PMF to resolve sulfuric acid-influenced SOC, while the daytime and nighttime measurements enabled organic tracer-based PMF to resolve SOC formation pathways with diurnal variations (e.g. SOC formed by photochemical reactions). The PM2.5 OC in Centreville was mainly secondary in origin (81%) and was influenced by NOx, ozone (a product of photochemical reactions of NOx and VOC), and sulfuric acid. Together, primary and secondary OC influenced by the fossil fuel use was 76%. Thus, the majority of the PM2.5 OC in Centreville during summer can be controlled by the reduction of fossil fuel use. The HILIC-MS/MS method was also applied to daily PM2.5 samples collected from an urban site in Atlanta, GA during August 2015. The major organosulfate species identified in Atlanta were dominated by 2-methyltetrol sulfate and other isoprene-derived organosulfates, similar to Centreville. They contributed 16% of PM2.5 OC and accounted for the majority of the isoprene-derived SOA that had not previously been identified at the molecular level. The concentrations of the major isoprene-derived organosulfates in Atlanta were two to six times higher than in Centreville. The greatest enhancement was obtained for 2-methylglyceric acid sulfate, a known isoprene SOA tracer formed under high-NOx conditions, reflecting the 15 times higher average NOx concentration in Atlanta during August 2015 compared to Centreville in summer 2013. These results indicate that NOx had a stronger influence on isoprene-derived organosulfate formation in urban Atlanta compared to rural Centreville. Overall, these results indicate that organosulfates are useful tracers for anthropogenically-influenced biogenic SOA. Thus, it is important to quantify them for use in organic tracer-based PMF modeling to determine the anthropogenically-influenced biogenic SOC in PM2.5 OC.

Antibiotics are used as prophylactic agents to promote growth and for treating infections in animals. However, the irrational use of antibiotics in livestock management is a significant cause of the development of antibioticresistant genes in the environment. Each year 2 million people suffer from the infections caused by bacteria which are resistant to antibiotics and 23,000 of these people are estimated to die because of antibiotic resistance. New drugs are continually coming into the market but are at the risk of developing resistance. Thus, there is a need for the development of analytical methods which can be used to monitor these antibiotic concentrations in environmental samples. This research is focused on developing and validating a Solid Phase Extraction (SPE) procedure and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying tylosin antibiotic in cattle waste. Tylosin was extracted from cattle waste samples using Strata polymeric weak cation cartridges by adding a sodium-EDTA buffer solution and methanol. Chemical analysis of the extracted tylosin was performed using a Varian 212-LC HPLC and Agilent 500 Ion Trap mass spectrometric detector. The concentrations of tylosin in study group animals were compared with respect to the date of sampling and cattle body weight with a control group and results are presented.

The purpose of the studies included in this thesis is to develop an effective an efficient method to study the proteome using separation and detection of peptides, when only a limited amount of sample, 10 micrograms of total protein or less, is available. The analysis will be applied to multiple myeloma cancer cells using ultra high-performance liquid chromatography-mass spectrometry for expression proteomics to illustrate utility. To detect low abundance peptides in a complex proteome, we use different strategies, including basic pH reversed-phase liquid chromatography (bRPLC), mass-to-charge fractionation in the mass spectrometer, and various liquid chromatography gradients to increase peptide separation to improve opportunities for detection and quantification. The different methods are optimized and compared by the number of peptides detected. Step-wise elution of bRP spin columns proved to yield more than 36,000 peptides using only 10 μg of protein. Mass-to-charge (m/z) fractionation was tested in mass analyzer Q-Exactive Plus (Thermo Scientific). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of an unfractionated sample was analyzed 4 times at different mass ranges, each mass range width of 150 m/z, resulting from 4 spectra combined, 31,732 peptides representing 3,967 proteins. Showingcomparable results to those form high pH reversed phase fractionation spin columns 5 fractions. Establishing a benchmark where the LC-MS/MS analysis of 600 μg of 10plex TMT-labeled peptides fractionated with bRPLC into 24 fractions yielded over 74,000 peptides from 7,700 proteins, we compared those results with analysis of 10 μg of total TMT-labeled peptides fractionated by bRP spin columns into 5 fractions, which produced 14,019 peptides from 3,538 proteins. These experiments were used to relatively quantify protein expression in naïve and drug resistant multiple myeloma cells lines as an example application in cancer research.

The field of proteomics encompasses the study of identities, interactions, and dynamics of all proteins expressed by a living system. Research in this dissertation blends biochemical and quantitative proteomics techniques to increase the latitude of biological applications for the bottom-up mass spectrometry proteomics approach. Together, isolation of selected protein “targets,” such as multiprotein complexes, and quantitative characterization yields information essential for more detailed understanding of microbial cell function. Often, a challenging aspect of characterizing a variety of biochemically enriched samples is limited protein yield. This dissertation describes an enzymatic proteolysis protocol employing an organic/aqueous solvent that alleviates excessive handling steps to reduce losses during sample preparation for small quantities of protein samples. Presence of artifactual, non-specific proteins in enriched protein complex isolates complicates biological interpretation of specific protein interactions. Heterologous expression of affinity-tagged bait proteins may also cause unintended collateral effects. A series of local and global protein isotope ratio measurements were performed to differentiate authentic interactions from artifactual interactions among affinity-isolated complexes and assess collateral effects, respectively. Protein localization provides clues regarding protein function. To infer protein localization, quantitative proteomics techniques were used to estimate protein enrichment of cold osmotic shock periplasmic isolates. Protein isotope ratios indicating enrichment, combined with identification of amino-terminal signal peptide cleavages, increase confidence of periplasmic localization. Collectively, this dissertation provides a framework for tailoring biochemical and quantitative techniques for targeted characterization of microbial protein isolates.

Antimicrobials administered to livestock can be excreted up to 80% in the feces and urine. Liquid swine manure from confined animal feeding operations is generally retained in lagoon storage until it is applied as a nutrient source to cropland. Thus, the applied manure becomes a possible source of antimicrobials to aquatic ecosystems. Veterinary antimicrobials have been detected in surface and ground waters in Canada, the United States and Europe, however, their environmental fate is not well known. Lincomycin and spectinomycin are two antimicrobials administered as a mixture to swine in the prairie region of Canada for the prevention of post-weaning diarrhea. In order to assess the potential for contamination of prairie wetlands, concentrations of both antimicrobials were monitored in the liquid manure from the nursery area of a commercial-scale barn during a 5-week study, and their persistence during simulated manure storage investigated. The potential for transport of lincomycin and spectinomycin to surface waters via surface runoff and to leach to groundwater was also assessed. This was achieved by monitoring manure-amended soil, simulated rainfall runoff, snow melt runoff and groundwater over a two-year period at two study sites in Saskatchewan, Canada following fall application of liquid swine manure from two commercial barns to crop and pasture land. Liquid chromatography coupled with tandem mass spectrometry was used to quantitate these antimicrobials in all matrix extracts. <p>In the nursery area of a commercial-scale barn, concentrations of lincomycin and spectinomycin in the cumulating liquid manure at the end of the study were equivalent to 32 and 3.0%, respectively, of doses administered in the feed. In a laboratory study, using fortified liquid manure, concentrations of both antimicrobials showed a rapid initial decrease during simulated lagoon storage, followed by a slower dissipation over a period of 5 months. The average time required for 50% dissipation of lincomycin was greater than one year (365 d) and was approximately 90 d for spectinomycin. <p>Lincomycin concentrations in soil (46.3 to 117 µg kg-1) collected immediately after fall manure application, decreased to non-detectable levels by mid-summer the following year. Lincomycin was present in simulated rainfall runoff (0.1 to 2.7 µg L-1) immediately after manure application with similar concentrations present in snow meltrunoff the following spring. Concentrations in groundwater were generally <0.005 µg L-1. Spectinomycin was not detected in the manure applied at the study sites nor in soil, runoff water or groundwater samples. This study confirms that some antimicrobials, including lincomycin, may be present in lagoon manure. Thus, the management practice of utilizing livestock manure from confined animal feeding operations as a plant nutrient source on cropland may result in antimicrobial transport to surface and ground waters.

When exposed to them, isocyanates can induce serious injuries in the respiratory tract and irritation on the skin and in the eyes. They are therefore interesting from the point of view of occupational health. The purpose of this thesis was to collect isocyanates in air with impinger-filter samplers and solvent free samplers. Furthermore the isocyanates were to be analyzed with liquid chromatography-mass spectrometry. The solvent free sampler consists of a polypropylene tube and filter holder fitted with glass fiber filters impregnated with derivatization reagent, coupled with a pump. The impinger-filter sampler was made out of an impinger flask, containing a derivatization solution, coupled in series with a filter holder and a pump. Di-n-butylamine was used as derivatization reagent in both samplers to stabilize the reactive isocyanates and to enable mass spectrometric detection. The solvent free sampler is highly advantageous because of its user friendliness during field measurements, as opposed to the impinger-filter method. An exposure chamber, equipped with two interior fans to ensure good circulation, was used to generate an atmosphere containing isocyanates. Analyzing the isocyanate-DBA derivates with LC-MS/MS worked very well and the method made it possible to detect isocyanate levels below 1 ng/mL. During quantification of isocyanates a standard curve with concentrations from 1 to 1000 ng/mL was used. Detection of isocyanate levels as low as one fifth of the limit value of some isocyanates was found to be possible, which would indicate that the methods are sensitive. Even though the solvent free sampler worked, the impinger-filter sampler was found to be more effective in collecting isocyanates. The coefficient of variation calculated from concentrations of isocyanates from the solvent free sampler varied between 0-35 %. The reason for this might be due to the fact that an optimized extraction method had not been tried out within the time limit of this project. Questions, identified during this thesis work, need to be answered before being able to obtain reliable results from field measurements with the solvent free sampler.

Antimicrobials administered to livestock can be excreted up to 80% in the feces and urine. Liquid swine manure from confined animal feeding operations is generally retained in lagoon storage until it is applied as a nutrient source to cropland. Thus, the applied manure becomes a possible source of antimicrobials to aquatic ecosystems. Veterinary antimicrobials have been detected in surface and ground waters in Canada, the United States and Europe, however, their environmental fate is not well known. Lincomycin and spectinomycin are two antimicrobials administered as a mixture to swine in the prairie region of Canada for the prevention of post-weaning diarrhea. In order to assess the potential for contamination of prairie wetlands, concentrations of both antimicrobials were monitored in the liquid manure from the nursery area of a commercial-scale barn during a 5-week study, and their persistence during simulated manure storage investigated. The potential for transport of lincomycin and spectinomycin to surface waters via surface runoff and to leach to groundwater was also assessed. This was achieved by monitoring manure-amended soil, simulated rainfall runoff, snow melt runoff and groundwater over a two-year period at two study sites in Saskatchewan, Canada following fall application of liquid swine manure from two commercial barns to crop and pasture land. Liquid chromatography coupled with tandem mass spectrometry was used to quantitate these antimicrobials in all matrix extracts. <p>In the nursery area of a commercial-scale barn, concentrations of lincomycin and spectinomycin in the cumulating liquid manure at the end of the study were equivalent to 32 and 3.0%, respectively, of doses administered in the feed. In a laboratory study, using fortified liquid manure, concentrations of both antimicrobials showed a rapid initial decrease during simulated lagoon storage, followed by a slower dissipation over a period of 5 months. The average time required for 50% dissipation of lincomycin was greater than one year (365 d) and was approximately 90 d for spectinomycin. <p>Lincomycin concentrations in soil (46.3 to 117 µg kg-1) collected immediately after fall manure application, decreased to non-detectable levels by mid-summer the following year. Lincomycin was present in simulated rainfall runoff (0.1 to 2.7 µg L-1) immediately after manure application with similar concentrations present in snow meltrunoff the following spring. Concentrations in groundwater were generally <0.005 µg L-1. Spectinomycin was not detected in the manure applied at the study sites nor in soil, runoff water or groundwater samples. This study confirms that some antimicrobials, including lincomycin, may be present in lagoon manure. Thus, the management practice of utilizing livestock manure from confined animal feeding operations as a plant nutrient source on cropland may result in antimicrobial transport to surface and ground waters.

Clopidogrel is an antiplatelet substance that prevents blood coagulation in the arteries. It is an inactive pro drug that becomes activated after first-pass metabolism by the liver. The active metabolite of clopidogrel is 2-oxoclopidogrel, which is unstable therefore pharmacokinetic data is obtained by measuring the inactive metabolite clopidogrel acid in plasma. Clopidogrel is taken orally in tablet form. The aim of this project was to develop a LC-MS/MS method for quantification of clopidogrel and its metabolite in plasma.   The method has been developed by optimizing the sample preparation. Different extraction procedures and extraction columns were tested, for example, by changing the extraction column from a C8 silica sorbent to Oasis HLB (a polymer sorbent). Different internal standards were evaluated as a result of discovering the signal suppression of the previous internal standard clopidogrel acid.  Flupentixol was found to be the best candidate.

Alkylphenol polyethoxylates are one of the most importantclasses of nonionic surfactants. They are widely used in the household detergents and dyeing industry. Indeed, alkylphenols have been estimated that they are persistence organic pollutions which are not easily biodegraded by microorganisms and demonstrated as endocrine disruptors. Therefore alkylphenols regard as environmental hormones. The purpose in this research is developing the method which can be used for detection and quantification of alkylphenols and alkylphenol polyethoxylates in waters and sediments. LC-MS/MS was used as the analytical instrument which can analyze alkylphenols combined with long ethoxylate chain with improved sensitivity and selectivity. The extraction is liquid-liquid extraction. The mobile phase consisting of acetonitrile, water and methanol(43/14/43%, v/v/v) with 20mM ammonium acetate added was used for HPLC separation. The recoveries of alkylphenols and alkylphenol polyethoxylates from water and sediments were 83.0~95.1% and 84.8~94.6%. Decision limit(CC£\) and detection capability(CC£]) of water were 0.10~1.65 ng/L and 0.13~2.03 ng/L. CC£\ and CC£] of sediments were 0.05~0.95 ng/g and 0.06~1.18 ng/g.