Tulathromycin is a macrolide antibiotic approved for use in cattle and swine respiratory disease. Extra-label use of tulathromycin occurs in bison and deer and significant interspecies differences in pharmacokinetics warrant specific investigation in these species. This study involved investigation of the pharmacokinetics of tulathromycin in bison and white-tailed deer following a single 2.5 mg/kg bw subcutaneous injection (n=10) of Draxxin (Pfizer Inc.) to provide important information regarding tulathromycin dosage regimens in these species. As well, tulathromycin distribution and depletion in deer muscle and lung tissues following a 2.5 mg/kg bw subcutaneous injection of Draxxin was investigated to obtain pilot information regarding withdrawal time of tulathromycin in deer. For the pharmacokinetic studies, serial blood samples were collected at baseline and up to 25 days post-injection. Pharmacokinetic parameters were estimated using non-compartmental methods. For the tissue pilot study, deer (n = 2 to 3) were slaughtered at 0, 1, 2, 6, 7, and 8 weeks post-injection. A quantitative analytical liquid chromatography-mass spectrometry method for measuring tulathromycin was developed and validated in bison and deer serum and deer lung and muscle according to international guidelines. Samples were processed by solid-phase extraction. Reverse-phase chromatography was performed by gradient elution. Positive electrospray ionization was used to detect the double charged ion [M+2H]+2 at m/z 403.9 and monitored in selected ion monitoring mode. Tulathromycin demonstrated early maximal serum concentrations, extensive distribution, and slow elimination characteristics in deer and bison. In bison, mean Cmax (195 ng/mL) was lower compared to cattle (300 to 500 ng/mL) and half-life (214 hours) longer (cattle, 90 to 110 hours). In deer, mean Cmax (359 ng/mL) is comparable to cattle, but half-life (281 hours) was much longer. Tissue distribution and clinical efficacy studies are needed in bison to confirm extensive distribution of tulathromycin into lung and the appropriate dosage regimen. Tulathromycin was extensively distributed to deer lung and muscle, with tissue levels peaking within 7 to 14 days after injection. Drug tissue concentrations were detected 56 days after treatment, longer than the established withdrawal time of 44 days in cattle. This prolonged drug concentration in the tissue is supportive for the administration of tulathromycin as a single injection therapy for treatment of respiratory disease of deer. While more study is needed to establish a recommended withdrawal time, the long serum and tissue drug half-life and extensive interindividual variability in tissue levels suggests a withdrawal period well beyond 56 days may be required in deer.

The initial research of this thesis involves the evaluation of different strategies for developing diverse chemistries of highly stable coatings for the automated 96-blade (thin-film) solid phase microextraction (SPME) system. Thin-film geometry increases the volume of extractive phase, and consequently improves the sensitivity of the analysis. Sol-gel technology was used for the preparation of octadecyl (C18)-silica gel thin-film coating. The evaluation of the C18-silica gel SPME extractive phase resulted in stable physical and chemical characteristics and long-term reusability with a high degree of reproducibility. Biocompatible polyacrylonitrile (PAN) polymer was used for the preparation of particle-based extractive phases in order to improve the biocompatible characteristics of SPME coatings for the extraction from biological samples. Three different immobilization strategies were evaluated for developing highly stable coatings for the automated 96-blade SPME system. The spraying was found to be the optimal method in terms of stability and reusability for long-term use. The optimized C18-PAN coating demonstrated improved biocompatibility, stability, and reusability for the extraction of benzodiazepines from human plasma in comparison with those of C18-silica gel coating. To improve the biocompatible properties of the C18-PAN SPME coating for long-term direct analysis from whole blood, different modification strategies were studied and evaluated. The modification of the coating with an extra layer of biocompatible polyacrylonitrile resulted in significant improvement in the blood compatibility in long-term use. ‘Extracted blood spot’ (EBS) sampling was introduced as a novel approach to overcome the limitations of dried blood spot sampling. EBS includes the application of a biocompatible SPME coating for spot sampling of blood or other biofluids. The compatibility of EBS sampling with different analytical methods was demonstrated. The utilization of EBS as a fast sampling and sample preparation method resulted in a significant reduction of matrix effects through efficient sample clean-up. Modified polystyrene-divinylbenzene (PS-DVB)-PAN and phenylboronic acid (PBA)-PAN 96-blade SPME coatings were developed and evaluated for the extraction of analytes in a wide range of polarity. These coatings demonstrated efficient extraction recovery for both polar and non-polar groups of compounds, and presented chemical and mechanical stabilities and reproducible extraction efficiencies for more than 100 usages in biological sample.

Thesis (Ph. D.)--University of Missouri--Rolla, 2007. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 23, 2008) Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 15, 2008) Vita. Includes bibliographical references.

Thesis (M.S.) -- Mississippi State University. Department of Chemistry. / Title from title screen. Includes bibliographical references.

Thesis (M.S.)--Baylor University, 2009. / Includes bibliographical references (p. 83-88).

Lisdexanfetamina (LDX) é um pró-fármaco estimulante de longa duração indicado para o tratamento dos sintomas do transtorno do déficit de atenção e hiperatividade e do transtorno da compulsão alimentar periódica. A hidrólise da ligação amida da LDX ocorre in vivo liberando a molécula terapeuticamente ativa d-anfetamina (d-ANF) e o aminoácido l-lisina. Visto que a LDX se biotransforma à d-ANF – um potente estimulante do sistema nervoso central com destaque tanto na clínica quanto na toxicologia – existe potencial para uso inadequado, abuso e desvio para fins não terapêuticos. Nos laboratórios de toxicologia, amostras biológicas com resultados positivos para anfetamina (ANF) são um desafio, uma vez que alguns testes toxicológicos podem detectar ANF devido à utilização de alguns medicamentos, dificultando a sua interpretação. Assim, são necessários métodos bioanalíticos eficientes aliados ao conhecimento farmacocinético, que permite a verificação da possibilidade de detecção, a estimativa da janela de detecção e as concentrações que podem ser alcançadas em diferentes matrizes biológicas. Dessa forma, neste trabalho, foram desenvolvidos métodos bioanalíticos para quantificação simultânea da LDX e de seu produto de biotransformação, a ANF, nas matrizes biológicas fluido oral, plasma e urina utilizando a cromatografia líquida acoplada a detector de massas sequencial (CL-EM/EM). A preparação de amostra é simples, utilizando a precipitação de proteínas para o plasma, com pouca quantidade de solvente orgânico, a diluição para o fluido oral e a filtração para urina, ambas com nenhuma quantidade de solvente orgânico. As curvas de calibração utilizando o padrão interno ANF deuterada apresentaram linearidade entre 1 e 128 ng/mL para o fluido oral e o plasma e entre 4 e 256 ng/mL para a urina. A menor concentração das curvas de calibração é igual ao limite inferior de quantificação. Precisão e exatidão intra e interdia ficaram dentro dos limites de ± 15% para os controles e ± 20% para o limite de quantificação. Os métodos foram seletivos e sem efeito residual, porém apresentaram um leve efeito matriz, frequentemente encontrado em métodos de CL-EM/EM. O método foi aplicado para análise das amostras do estudo farmacocinético da LDX e ANF nas matrizes biológicas fluido oral, plasma e urina após administração oral de LDX. Seis voluntários do sexo masculino coletaram amostras de fluido oral e plasma em tempos pré-determinados durante 72 horas e amostras de urina em intervalos pré-determinados durante 120 horas. Os dados foram avaliados de maneira não-compartimental e compartimental. Considerando a análise não-compartimental, a concentração máxima média da d-ANF foi quase seis vezes inferior no plasma em relação ao fluido oral e ocorreu em 3,8 e 4 horas, respectivamente, após a administração oral. A LDX atingiu a concentração máxima no plasma e no fluido oral em 1,2 e 1,8 horas após a administração oral, respectivamente, com um valor médio de pico de concentração quase duas vezes mais elevado no plasma em comparação com o fluido oral. A eliminação da d-ANF a partir do plasma e a partir do fluido oral foi semelhante, porém para LDX a eliminação a partir do fluido oral foi mais lenta, mesmo com concentrações mais baixas do que no plasma. A detecção da d-ANF ocorreu até 48-72 horas no plasma e fluido oral e até 120 horas em urina. Já para a LDX, a detecção ocorreu até 3, 5 e 12 horas no plasma, fluido oral e urina, respectivamente. LDX intacta e d-ANF foram detectadas nas três matrizes avaliadas. Na análise compartimental, o melhor ajuste de modelo foi observado para 1 compartimento para ambos os analitos tanto no plasma quanto no fluido oral. Houve uma correlação entre as concentrações do fluido oral e do plasma para d-ANF e entre as proporções de LDX intacta/d-ANF pelo tempo no plasma e no fluido oral. O método analítico desenvolvido pode ser aplicado em diferentes áreas do conhecimento a fim de certificar os resultados de uma análise de triagem positiva para ANF. Porém, para interpretação das situações tanto de triagem quanto de confirmação é necessário aliar o conhecimento farmacocinético gerado no trabalho, que demonstra se há a possibilidade de detecção na matriz analisada e por quanto tempo após a administração da LDX. Isto auxilia na diferenciação do uso de outros medicamentos derivados da ANF e do uso ilegal, para que as devidas providências legais e de manejos clínicos de tratamento e controle de dependência sejam tomadas quando necessário. / Lisdexamfetamine (LDX) is a long-acting prodrug stimulant indicated for the treatment of attention-deficit/hyperactivity disorder and binge-eating disorder symptoms. In vivo hydrolysis of lisdexamfetamine amide bond releases the therapeutically active d-amphetamine (d-AMPH) and the amino acid l-lysine. Since LDX biotransformation gives rise to d-AMPH - a potent stimulant of the central nervous system that stands out in clinical and toxicology - there is potential for misuse, abuse and diversion for non-therapeutic purposes. In laboratories of toxicology, biological samples with positive results for amphetamine (AMPH) are a challenge, since some toxicological tests can detect AMPH due to the use of some medications hindering the interpretation. Therefore, we need efficient bioanalytical methods combined with the pharmacokinetic knowledge, which allows to verify the possibility of detection, to assess the detection window and the concentrations that can be reached in different biological matrices. Hence, bioanalytical methods were developed for simultaneous quantification of LDX and its main biotransformation product AMPH in the biological matrices oral fluid, plasma and urine by liquid chromatography-mass spectrometry (LC-MS/MS). The sample preparation is simple, using protein precipitation for plasma, with a small amount of organic solvent, dilution for oral fluid and filtration to urine, both with no amount of organic solvent. Calibration curves using deuterated AMPH internal standard showed linearity between 1 and 128 ng/mL for oral fluid and plasma, and between 4 and 256 ng/mL for urine. The lowest concentration of the calibration curve is the lower limit of quantification. Intra and interday precision and accuracy were within the limits of ± 15% for controls and ± 20% for the limit of quantification. The methods were selective and no carry-over was observed, however with some matrix effect, often found in LC-MS/MS methods. The method was applied to analyze samples from LDX and AMPH pharmacokinetics study in the biological matrices oral fluid, plasma and urine following oral administration of LDX. Six male volunteers collected oral fluid and plasma samples at predetermined times during 72 hours and urine samples at pre-determined intervals during 120 hours. Data were evaluated through non-compartmental and compartmental analysis. Considering the noncompartmental analysis, the mean maximum concentration of d-AMPH was almost 6-fold lower in plasma than in oral fluid and occurred at 3.8 and 4 hours, respectively, after LDX administration. LDX maximum concentration was reached at 1.2 and 1.8 hours after LDX oral administration for oral fluid and plasma, respectively, with a mean peak concentration almost 2-fold higher in plasma when compared with oral fluid. Elimination of d-AMPH from oral fluid and from plasma were similar, albeit for LDX elimination from oral fluid was slower even with lower concentrations than plasma. Detection occurred until 48 to 72 hours in plasma and oral fluid and until 120 hours in urine for d-AMPH. Whereas for LDX, detection could be done for up to 3, 5 and 12 hours in plasma, oral fluid and urine, respectively. Intact LDX and d-AMPH were detected in the three evaluated matrices. In compartmental analysis, the best model fit was observed for 1-compartment model for both analytes in plasma and in oral fluid. There was a correlation between oral fluid and plasma d-AMPH concentrations and between intact LDX/d-AMPH ratios along time in plasma as well as in oral fluid. The bioanalytical methods developed can be applied in different fields of knowledge in order to ensure the results of a positive screening analysis for AMPH. Nevertheless, for interpretation of situations in both screening and confirmation tests is necessary to combine the pharmacokinetic knowledge produced in this study, which shows if there is the possibility of detection in the analyzed matrix and for how long after the administration of LDX. This results aid in the differentiation from other AMPH derived drugs use and from illegal use, so that appropriate legal action and clinical management strategies for treatments and control of dependence be taken when necessary.

Lisdexanfetamina (LDX) é um pró-fármaco estimulante de longa duração indicado para o tratamento dos sintomas do transtorno do déficit de atenção e hiperatividade e do transtorno da compulsão alimentar periódica. A hidrólise da ligação amida da LDX ocorre in vivo liberando a molécula terapeuticamente ativa d-anfetamina (d-ANF) e o aminoácido l-lisina. Visto que a LDX se biotransforma à d-ANF – um potente estimulante do sistema nervoso central com destaque tanto na clínica quanto na toxicologia – existe potencial para uso inadequado, abuso e desvio para fins não terapêuticos. Nos laboratórios de toxicologia, amostras biológicas com resultados positivos para anfetamina (ANF) são um desafio, uma vez que alguns testes toxicológicos podem detectar ANF devido à utilização de alguns medicamentos, dificultando a sua interpretação. Assim, são necessários métodos bioanalíticos eficientes aliados ao conhecimento farmacocinético, que permite a verificação da possibilidade de detecção, a estimativa da janela de detecção e as concentrações que podem ser alcançadas em diferentes matrizes biológicas. Dessa forma, neste trabalho, foram desenvolvidos métodos bioanalíticos para quantificação simultânea da LDX e de seu produto de biotransformação, a ANF, nas matrizes biológicas fluido oral, plasma e urina utilizando a cromatografia líquida acoplada a detector de massas sequencial (CL-EM/EM). A preparação de amostra é simples, utilizando a precipitação de proteínas para o plasma, com pouca quantidade de solvente orgânico, a diluição para o fluido oral e a filtração para urina, ambas com nenhuma quantidade de solvente orgânico. As curvas de calibração utilizando o padrão interno ANF deuterada apresentaram linearidade entre 1 e 128 ng/mL para o fluido oral e o plasma e entre 4 e 256 ng/mL para a urina. A menor concentração das curvas de calibração é igual ao limite inferior de quantificação. Precisão e exatidão intra e interdia ficaram dentro dos limites de ± 15% para os controles e ± 20% para o limite de quantificação. Os métodos foram seletivos e sem efeito residual, porém apresentaram um leve efeito matriz, frequentemente encontrado em métodos de CL-EM/EM. O método foi aplicado para análise das amostras do estudo farmacocinético da LDX e ANF nas matrizes biológicas fluido oral, plasma e urina após administração oral de LDX. Seis voluntários do sexo masculino coletaram amostras de fluido oral e plasma em tempos pré-determinados durante 72 horas e amostras de urina em intervalos pré-determinados durante 120 horas. Os dados foram avaliados de maneira não-compartimental e compartimental. Considerando a análise não-compartimental, a concentração máxima média da d-ANF foi quase seis vezes inferior no plasma em relação ao fluido oral e ocorreu em 3,8 e 4 horas, respectivamente, após a administração oral. A LDX atingiu a concentração máxima no plasma e no fluido oral em 1,2 e 1,8 horas após a administração oral, respectivamente, com um valor médio de pico de concentração quase duas vezes mais elevado no plasma em comparação com o fluido oral. A eliminação da d-ANF a partir do plasma e a partir do fluido oral foi semelhante, porém para LDX a eliminação a partir do fluido oral foi mais lenta, mesmo com concentrações mais baixas do que no plasma. A detecção da d-ANF ocorreu até 48-72 horas no plasma e fluido oral e até 120 horas em urina. Já para a LDX, a detecção ocorreu até 3, 5 e 12 horas no plasma, fluido oral e urina, respectivamente. LDX intacta e d-ANF foram detectadas nas três matrizes avaliadas. Na análise compartimental, o melhor ajuste de modelo foi observado para 1 compartimento para ambos os analitos tanto no plasma quanto no fluido oral. Houve uma correlação entre as concentrações do fluido oral e do plasma para d-ANF e entre as proporções de LDX intacta/d-ANF pelo tempo no plasma e no fluido oral. O método analítico desenvolvido pode ser aplicado em diferentes áreas do conhecimento a fim de certificar os resultados de uma análise de triagem positiva para ANF. Porém, para interpretação das situações tanto de triagem quanto de confirmação é necessário aliar o conhecimento farmacocinético gerado no trabalho, que demonstra se há a possibilidade de detecção na matriz analisada e por quanto tempo após a administração da LDX. Isto auxilia na diferenciação do uso de outros medicamentos derivados da ANF e do uso ilegal, para que as devidas providências legais e de manejos clínicos de tratamento e controle de dependência sejam tomadas quando necessário. / Lisdexamfetamine (LDX) is a long-acting prodrug stimulant indicated for the treatment of attention-deficit/hyperactivity disorder and binge-eating disorder symptoms. In vivo hydrolysis of lisdexamfetamine amide bond releases the therapeutically active d-amphetamine (d-AMPH) and the amino acid l-lysine. Since LDX biotransformation gives rise to d-AMPH - a potent stimulant of the central nervous system that stands out in clinical and toxicology - there is potential for misuse, abuse and diversion for non-therapeutic purposes. In laboratories of toxicology, biological samples with positive results for amphetamine (AMPH) are a challenge, since some toxicological tests can detect AMPH due to the use of some medications hindering the interpretation. Therefore, we need efficient bioanalytical methods combined with the pharmacokinetic knowledge, which allows to verify the possibility of detection, to assess the detection window and the concentrations that can be reached in different biological matrices. Hence, bioanalytical methods were developed for simultaneous quantification of LDX and its main biotransformation product AMPH in the biological matrices oral fluid, plasma and urine by liquid chromatography-mass spectrometry (LC-MS/MS). The sample preparation is simple, using protein precipitation for plasma, with a small amount of organic solvent, dilution for oral fluid and filtration to urine, both with no amount of organic solvent. Calibration curves using deuterated AMPH internal standard showed linearity between 1 and 128 ng/mL for oral fluid and plasma, and between 4 and 256 ng/mL for urine. The lowest concentration of the calibration curve is the lower limit of quantification. Intra and interday precision and accuracy were within the limits of ± 15% for controls and ± 20% for the limit of quantification. The methods were selective and no carry-over was observed, however with some matrix effect, often found in LC-MS/MS methods. The method was applied to analyze samples from LDX and AMPH pharmacokinetics study in the biological matrices oral fluid, plasma and urine following oral administration of LDX. Six male volunteers collected oral fluid and plasma samples at predetermined times during 72 hours and urine samples at pre-determined intervals during 120 hours. Data were evaluated through non-compartmental and compartmental analysis. Considering the noncompartmental analysis, the mean maximum concentration of d-AMPH was almost 6-fold lower in plasma than in oral fluid and occurred at 3.8 and 4 hours, respectively, after LDX administration. LDX maximum concentration was reached at 1.2 and 1.8 hours after LDX oral administration for oral fluid and plasma, respectively, with a mean peak concentration almost 2-fold higher in plasma when compared with oral fluid. Elimination of d-AMPH from oral fluid and from plasma were similar, albeit for LDX elimination from oral fluid was slower even with lower concentrations than plasma. Detection occurred until 48 to 72 hours in plasma and oral fluid and until 120 hours in urine for d-AMPH. Whereas for LDX, detection could be done for up to 3, 5 and 12 hours in plasma, oral fluid and urine, respectively. Intact LDX and d-AMPH were detected in the three evaluated matrices. In compartmental analysis, the best model fit was observed for 1-compartment model for both analytes in plasma and in oral fluid. There was a correlation between oral fluid and plasma d-AMPH concentrations and between intact LDX/d-AMPH ratios along time in plasma as well as in oral fluid. The bioanalytical methods developed can be applied in different fields of knowledge in order to ensure the results of a positive screening analysis for AMPH. Nevertheless, for interpretation of situations in both screening and confirmation tests is necessary to combine the pharmacokinetic knowledge produced in this study, which shows if there is the possibility of detection in the analyzed matrix and for how long after the administration of LDX. This results aid in the differentiation from other AMPH derived drugs use and from illegal use, so that appropriate legal action and clinical management strategies for treatments and control of dependence be taken when necessary.

Magister Pharmaceuticae - MPharm / Tuberculosis and HIV are arguably South Africa’s largest and most important health issues. With drug-resistant strains of tuberculosis on the increase and little research on new drugs, there is an urgent need for research around the drugs presently available to ensure their optimal use and to minimise their sometimes serious and significant side effects. Treatment of drug-resistant tuberculosis is expensive and lengthy, and is complicated by a limited choice of drugs with lower efficacies and higher toxicities. Treatment is further complicated in patients with HIV due to several factors including drug interactions. While some authors suggest that HIV and malabsorption might be associated with poor clinical outcomes, other researchers have found no link. Patients may benefit from Therapeutic Drug Monitoring in order to ensure that their doses of antituberculosis drugs are reaching the required minimum effective concentrations, without attaining toxic levels in the plasma which may cause unpleasant side effects. There is little research concerning drug levels in HIV patients with TB in South Africa, let alone in patients with drug-resistant forms of tuberculosis, and there are no studies in this country that use Liquid Chromatography-Mass Spectrometry to investigate the plasma levels of pyrazinamide in patients with MDR-TB. This study aimed to investigate whether or not there is a difference in the pharmacokinetics of PZA in MDR-TB patients with HIV, and those without HIV infection. It also aimed to establish whether LC-MS could be used to study the levels of pyrazinamide in the plasma of patients with multidrug-resistant tuberculosis with and without concurrent HIV infection. The plasma levels of pyrazinamide in 32 MDR-TB patients (23 HIV negative and 9 HIV positive), were successfully 2 analysed using LC-MS, and the pharmacokinetics of PZA in these 2 populations was described. It was established that the Tmax of pyrazinamide was significantly higher in HIV-negative patients than in HIV-positive patients. Although there was a difference between the Ka in the two populations, this difference did not quite reach statistical significance. There were no statistically significant differences between HIV-negative and HIV-positive patients with regards to the other pharmacokinetic parameters investigated. Our findings established that there was little evidence to suggest that there is a difference between the pharmacokinetics of the antimycobacterial drug pyrazinamide in HIV-positive patients and that in HIV-negative patients. We were also able to successfully develop and validate an assay for the analysis of PZA in plasma using LC-MS, and this finding could be very valuable for further studies. Although our study failed to prove this, the possibility still exists that HIV-positive patients could exhibit altered kinetics of antiTB drugs and this has not been fully investigated in South Africa. The clinical impact of low plasma levels of antimycobacterial drugs is still largely unexplored and further research with larger sample sizes should be done in order to establish which factors may contribute to low plasma levels of anti-tuberculosis drugs in MDR-TB patients, and whether or not these low levels are increasing the risk of treatment failure or other poor clinical outcomes.

Acid whey, a byproduct of Greek yogurt production, has little commercial value due to its low protein content and is also environmentally harmful when disposed of as waste. However, as a product of microbial fermentation, acid whey could be a rich source of beneficial metabolites associated with fermented foods. This study increases understanding of acid whey composition by providing a complete metabolomic profile of acid whey. Commercial and lab-made Greek yogurts, prepared with three different bacterial culture combinations, were evaluated. Samples of unfermented yogurt mix and cultured whey from each batch were analyzed. Ultra-high-performance liquid chromatography/tandem mass spectrometry metabolomics were employed to separate and identify 477 metabolites, including many with potential health benefits similar to those provided by yogurt, such as creatine and acetylcarnitine. Examples of other metabolites identified in the acid whey include beneficial phospholipids (1,2-dilinoleoyl-sn-glycero-3-phosphocholine) and sphingolipids; compounds with neuroprotective (glycerophosphorylcholine) or cardiovascular (betaine) benefits; antimicrobial compounds (benzoic acid), and anti-inflammatory compounds (citrulline). Compared to uncultured controls, acid whey showed decreases in some metabolites associated with microbial metabolism and increases in others. Metabolite production was significantly affected by combinations of culturing organisms, and production location. Differences between lab-made and commercial samples could be caused by different starting ingredients, or environmental factors or both.