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.

Hereditary Hemochromatosis (HH) is an inherited recessive autosomal disorder characterized by accumulation of excess iron. When iron binding proteins become saturated, concentrations of free, or non-transferrin-bound iron (NTBI) rise, a condition thought to be responsible for the adverse effects associated with HH. To investigate that disturbing iron homeostasis plays a role in free radical injury in HH, protein carbonyls were found to be 1-7 times higher in patients with HH than in controls, with the greatest increases being observed in untreated HH patients with high ferritin and >90% transferrin saturation with iron. An Unpaired t test revealed a P value of 0.0278 (P< 0.05), which is considered to be statistically significant. Our data showed a significant positive correlation (linear relationship) between the level of carbonyl content and ferritin concentration in plasma samples from patients with HH. In vitro oxidation of transferrin and ferritin standards with hydrogen peroxide and excess iron, followed by immobilized trypsin digestion (Poroszyme), high-resolution LC-MS/MS analysis (Q-TOF Ultima, Waters) and MS/MS data processing (PEAKS, Bioinformatics Solution), identified several tryptic peptides containing oxidized Met,Trp and His residues. Mapping of the oxidized ferritin residues showed them to be located on the inner face of each sub-unit, the face directed toward the ferritin core where iron is normally stored. Using the same methodology, oxidized residues were subsequently detected in ferritin and transferrin isolated from plasma samples of patients severely affected with HH. Comparing of MS/MS spectra of in vitro oxidized samples that have most fragment ion peaks in common with oxidized peptide MS/MS spectra from samples of patients with HH revealed a significant correlation between the two. These data show that elevated NTBI may be involved in oxidative modification of the iron binding proteins, ferritin and transferrin, and that such modifications may play a significant role in the pathophysiology of HH.

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.

Hereditary Hemochromatosis (HH) is an inherited recessive autosomal disorder characterized by accumulation of excess iron. When iron binding proteins become saturated, concentrations of free, or non-transferrin-bound iron (NTBI) rise, a condition thought to be responsible for the adverse effects associated with HH. To investigate that disturbing iron homeostasis plays a role in free radical injury in HH, protein carbonyls were found to be 1-7 times higher in patients with HH than in controls, with the greatest increases being observed in untreated HH patients with high ferritin and >90% transferrin saturation with iron. An Unpaired t test revealed a P value of 0.0278 (P< 0.05), which is considered to be statistically significant. Our data showed a significant positive correlation (linear relationship) between the level of carbonyl content and ferritin concentration in plasma samples from patients with HH. In vitro oxidation of transferrin and ferritin standards with hydrogen peroxide and excess iron, followed by immobilized trypsin digestion (Poroszyme), high-resolution LC-MS/MS analysis (Q-TOF Ultima, Waters) and MS/MS data processing (PEAKS, Bioinformatics Solution), identified several tryptic peptides containing oxidized Met,Trp and His residues. Mapping of the oxidized ferritin residues showed them to be located on the inner face of each sub-unit, the face directed toward the ferritin core where iron is normally stored. Using the same methodology, oxidized residues were subsequently detected in ferritin and transferrin isolated from plasma samples of patients severely affected with HH. Comparing of MS/MS spectra of in vitro oxidized samples that have most fragment ion peaks in common with oxidized peptide MS/MS spectra from samples of patients with HH revealed a significant correlation between the two. These data show that elevated NTBI may be involved in oxidative modification of the iron binding proteins, ferritin and transferrin, and that such modifications may play a significant role in the pathophysiology of HH.

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.

In this study, a LC/MS/MS method for the determination of ampicillin antibiotic in fish muscle tissue was developed and accredited according to Commission Decision 2002/657/EC. The metabolism of ampicillin in tilapia was them studied in serum, liver and muscle. The homogenized fish tissue was first extracted with MeOH-H2O(4:1), C18 sorbent was added to remove lipids and impurities, the extract was then evaporated to dryness with a steam of nitrogen gas at 38 ¢XC. The residue was redissolved with H2O, filtered and analyzed by LC/MS/MS equipped with an Agilient HC-C18(5£gm, 150mm ¡Ñ4.6mm), the mobile phase A was 10mM ammonium acetate containing 0.1% formic acid, while the mobile phase B was methanol. The determination of ampicillin was performed with electrospray ionization-tandem mass spectrometry in positive mode using multiple reation monitoring(MRM) for detection. Average recoveries were 81¡V86%, the limit of detection was 6.00 £gg kg-1¡Adecision limit(CC£\) of ampicillin in fish muscle sample was 63.65 ¡Ó 7.99 £gg kg-1. In the metabolism study, the oral administered dose to talipia was 20 mg/kg¡DBW. The maximum concentration of ampicillin in each tissues was obserned at 0.5 hour after oral administration, the maximum concentration in serum, liver and muscle was 27.53 mg L-1, 66.75 mg kg-1 and 1.33 mg kg-1, respectively. The concentration of ampicillin in muscle was 0.04 mg kg-1 24 hours after oral administration, which is lower than the 0.05 mg kg-1 MRL value of European Union resolutions. No residual ampicillin was detected in tilapia 48 hours after oral treatment, which conformed to the drug regulations for aquaculture ainmals in Taiwan.

The purpose of this research is that investigate the effects of time and concentration of exposure for the accumulation and depletion of malachite green and leucomalachite green in tilapia by pharmacokinetics. LC-MS/MS was used as the analytical instrument in this research, and the detection limit of malachite green and leucomalachite green is 0.51 ppb and 0.48 ppb. The results show that malachite green is unstable at high temperature. Addition of TMPD into the standard can stabilize malachite green. The malachite green in exposure water is easy to adhere to the fiberglass tub and cause the decreases of concentration of malachite green in water. The concentrations of malachite green and leucomalachite green in tilapia are positive related to the time and concentration of exposure. In experiments of exposure of malachite green, the highest concentration of malachite green occurs in liver, accumulation rate constant is 21.62 h-1. Liver is also the major organ for transforming malachite green into leucomalachite green, the net leucomalachite green accumulation rate constant is 213.67 h-1. In the period of water bath, Gill is the fastest organ for eliminating malachite green and leucomalachite green, the elimination rate constant is 0.7799 h-1 and 0.4658 h-1; the leucomalachite green concentration in fat is still increase until 6h of water bath.

The study felld into three sections. The first section that a liquid chromatography method with fluorescence detection was developed for simultaneous determination of 11 quinolones (QNs; marbofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, nalidixic acid and flumequine) in chicken, pork, fish and shrimp. The analytes were extracted with 0.3% metaphosphoric acid: acetonitrile (1:1, v/v), followed by a HLB cartridge clean-up procedure. The HPLC separation was carried out on a symmetry column C18 (250 mm x 4.5 mm i.d., 5 £gm) with linear gradient elution of 0.1% formic acid: acetonitrile as mobile phase and programmable fluorescence detection. The method was validated by spiking blank animals tissues at three different levels (25, 50 and 250 ng/g; except 6.25, 12.5 and 62.5 ng/g for DAN) and linearity, detection limit, quantification limit, precision and accuracy were checked. Mean recoveries of 11 QNs from edible animal tissues were 71.7-105.3%. The limits of quantification in different muscle tissues ranged from 5.0 to 28.0 ng/g. The results showed it was simple, rapid, sensitive and suitable for routine test. The second section that a LC-ESI-MS/MS method was developed for determining 18 (fluoro)quinolone (QNs) residues in milk, chicken, pork, fish and shrimp. This method is capable of screening and confirming the presence of 12 amphoteric QNs (marbofloxacin, norfloxacin, enrofloxacin, ciprofloxacin, desethylene ciprofloxacin, lomefloxacin, danofloxacin, sarfloxacin, difloxacin, ofloxacin, orbifloxacin and enoxacin) and 6 acidic QNs (oxolinic acid, nalidixic acid, flumequine, cinoxacin, piromidic acid and pipemidic acid). The drugs were extracted from matrix using acetonitrile with 1% formic acid, diluted in 10% acetonitrile and defatted by extraction with hexane. The LC separation was conducted on a XDB C8 (150 x 4.6 mm, 5£gm) column with gradient elution of 20 mM ammonium formate with 0.1% formic acid¡Vacetonitrile as the mobile phase. Mass spectral acquisition was completed in the positive ion mode by applying multiple reaction mode (MRM). The decision limit (CC£\) and detection capability (CC£]) stated in the Decision No. 2002/657/EC and the ISO standard No.11843, has been calculated in the case of the nonauthorized substance. The values of CC£\ ranged from 0.18 to 0.68 ng/g and CC£] ranged from 0.24 to 0.96 ng/g under specified conditions. The third section that the pharmacokinetics of ENR and its active metabolite (CIP and des-CIP) were estimated in tilapia after intravenous (i.v.) and oral (p.o.) administration of a single dose of 2.5 and 10 mg/kg body weigh, respectively. At prefixed time points, from 0.25 h to 7 days after administration, whole blood and main tissue (liver, kidney, bile and muscle) from 4 individuals in each were collected. The concentration of ENR and its active metabolites in the main tissue were simultaneously detected by LC/MS/MS method. Limited of quantitation (LOQ) of this method were 0.01£gg/g. Pharmacokinetic parameters from both routes were described to have a two- compartment open model with first-order elimination. After i.v. administration, the area under the drug concentration-time (AUC), elimination half-life (t1/2£]), maximum plasma concentration (Cmax ), total body clearance (Cltot) and apparent volume of distribution at steady-state (Vss) of ENR were 109.6 ¡Ó 31.33 £gg.h/mL, 55.17 ¡Ó 22.84 h, 4.70 ¡Ó 0.36 £gg/mL, 14.82 ¡Ó 4.24 L/h/kg, 1105 ¡Ó 223.40 L/kg ,respectively. After oral administration, the AUC , t1/2£], Tmax , Cmax of ENR were 599.42 ¡Ó 76.19£gg.h/mL , 75.95 ¡Ó 12.94 h, 0.601¡Ó0.06h, 9.75 ¡Ó 0.46£gg/mL, respectively. After p.o. administration, CIP could be detected in liver, kidney and bile. Regarding des-CIP, the main active metabolite of CIP, could be detected in 120¡ã168 h bile among tissue. ENR and CIP had significance enterohepatic cycle in Tilapia and easily accumulated in bile. It seems reasonable to explain the phenomenon of ENR and CIP maintenance of high concentration in blood and muscle during the test time.