Potatoes are an important and irreplaceable crop. This kind of crop is very important not only for it is use but also for a nutrition composition. There are also a prominent source of vitamins, minerals and antioxidants. Outside substances beneficial to health and potatoes contain harmful substances. These substances are foreign or naturally occurring, which include toxic glycoalkaloids. Glycoalkaloids are secondary metabolites of plants. Glycoalkaloids in potatoes have protective function it can increase the synthesis for example in case of pest infestations, mechanical damage or in case of to much light and heat. The potatoes were found several glycoalkaloides. Main, which constitutes 95 % of their content, are alpha-chaconine and alpha-solanine. Their toxicity is inhibition of acetylcholinesterase and breaking the cell membranes. The potato tuber is their content is distributed unevenly. The quantity of glycoalkaloids is affected by manny factors as for example place, year, kind, the way how the crops are grown and storage. In Czech Republic the maximum allowed limit of glycoalkaloides in potatoes were made by legislation on 200 mg/kg fresh potato matter. In the commonly grown varieties of the amount is far below the hygienic limit. The methods for isolation of glycoalkaloids in potatoes are mainly chromatographic. The most commonly used HPLC (high performance liquid chromatography). In performed experiment was determined the content of majority glycoalkaloid alpha- chaconinu and alpha-solaninu at four different kinds -Milva, Marabel, Laura and Valfi. Drought stress has been studied for their content, assuming their accumulation in comparison with the other two variants - irrigation watering and drip irrigation. The glycoalkaloides content were messured by the UHPLC/MS/MS. The obtained results concluded that the content of glycoalkaloids is the variety dependent. Drought stress can probably increase their content. In our experiment, it positively did not. Important is the choice of kind in case if expectation a hot and dry year of growing. Kinds Milva and Marabel are very good in these conditions. In the case of general principles for cultivation, storage and cooking, the glykoalkaloids does not vision a risk for the consumer.

Objetivo: Avaliar a farmacocinética da vildagliptina em animais sadios e diabéticos, através da análise dos níveis plasmáticos totais e livres teciduais, empregando-se a técnica de microdiálise. Metodologia: A doença foi induzida nos animais através da administração de 42mg/kg de aloxano através da via intravenosa (i.v.). A vildagliptina foi administrada nas doses de 50 mg/kg (n = 6) e 75 mg/kg (n = 6) via i.v. nos animais diabéticos e na dose de 50 mg/kg (n = 6) nos animais sadios. As concentrações plasmáticas foram quantificadas por CLAE-EM-EM em método desenvolvido e validado. A ligação às proteínas plasmáticas foi determinada por microdiálise, assim como a avaliação tecidual. As sondas de microdiálise foram calibradas in vitro através de diálise e retrodiálise e in vivo utilizando retrodiálise. Para determinação das concentrações teciduais, uma segunda metodologia foi desenvolvida e validada em CLAE-EM-EM. Avaliações compartimentais (software Scientist ®) e não compartimentais (software Excel ®) foram realizadas. Resultados e Discussão: A ligação as proteínas plasmáticas apresentou um valor médio de 9,44 % ± 3,23, condizente com valores encontrados na literatura. Os valores de Ke, clearance, tempo de meia vida, MRT e VDss não apresentaram diferença estatística significativa entre as diferentes doses administradas nos animais diabéticos e entre os animais sadios. As calibrações in vitro por diálise e retrodiálise apresentaram uma recuperação média de 30%, sem diferença estatística entre as duas metodologias empregadas (α = 0,05). A recuperação in vivo também apresentou o mesmo valor médio de recuperação. A penetração tecidual do fármaco em animais diabéticos para as diferentes doses estudadas apresentou mesmo valor nos tecidos estudados, uma média de 0,20. A penetração tecidual semelhante no animal diabético pode ser devido ao dano similar entre os órgãos sofrido durante a indução da doença. Já os animais sadios apresentaram penetração tecidual similar no músculo sem diferença estatística significativa em relação aos diabéticos, entretanto no fígado foi observada uma penetração quarenta e quatro vezes inferior a observada no músculo. Essa disparidade pode ser atribuída a diferença de expressão de proteínas transportadoras no fígado do animal diabetico quando comparado ao sadio. O perfil farmacocinético plasmático foi semelhante entre os dois grupos avaliados, sendo que os parâmetros não diferiram estatisticamente (α = 0,05). Foi empregado o modelo de dois compartimentos para prever as concentrações teciduais. A previsão supõe concentrações superiores as encontradas experimentalmente, contradizendo dados de literatura. Esses dados são inéditos na literatura e demostram a importância da determinação do fármaco em tecidos alvo, uma vez que nem sempre modelos matemáticos conseguem prever a realidade fisiológica. Conclusões: As metodologias analíticas para quantificação da vildagliptina em microdialisado e plasma foram desenvolvidas e validadas, seguindo os requisitos do FDA. O perfil farmacocinético plasmático foi adequadamente descrito pelo modelo de 2 compartimentos. Os perfis teciduais obtidos nesse trabalho podem contribuir para o melhor entendimento dos mecanismos farmacológicos envolvidos e contribuir para futura otimização de terapias. / Objective: To evaluate the pharmacokinetics of vildagliptin in healthy and diabetic animals using a microdialysis technique. Methodology: Diabetes was induced in animals by administration of 42 mg/kg of alloxan intravenously (iv). Vildagliptin was administered intravenously as 50 mg/kg (n = 6) and 75 mg/kg doses (n = 6) in the diabetic animals and as a 50 mg/kg dose (n = 6) in healthy animals. Plasma concentrations were quantified by a HPLC-MS-MS method developed and validated. The plasma protein binding was determined by microdialysis and tissue evaluation. Microdialysis probes were calibrated in vitro using dialysis and retrodialysis and in vivo using retrodialysis. A second method was developed and validated using HPLC-MS-MS to determine tissue concentrations. Results and Discussion: Mean plasma protein was 9.44% ± 3.23, consistent with values reported in the literature. The values of Ke, clearance, half-life, MRT and Vdss showed no statistical difference between the evaluated doses in diabetic animals and between healthy animals (α = 0.05). Calibrations in vitro by dialysis and retrodialysis showed mean recovery of 30%, with no statistical difference between the two methodologies. Mean recovery in vivo also showed the same value. The tissue penetration of the drug in diabetic animals for the different doses studied showed the same value in both tissues studied, an mean of 0.20. The tissue penetration similar in diabetic animals could be due to the similar damage suffered between organs during induction of the disease. The healthy animals showed similar muscle penetration, compared with diabetics animals, although the liver showed a penetration forty four times lower than muscle. This discrepancy can be attributed to differential expression of transporter proteins in the liver of diabetic animals, when compared to the healthy group. The plasma pharmacokinetic profile was similar between the investigated groups, and the parameters did not differ. The two-compartment model was employed to describe the data and used to predict the concentration in the tissues. This is the first study to present these tissue profiles, which presented concentrations below the estimated by the model. These data demonstrate the importance of determining the drug inside the target tissue, as the mathematical models sometimes cannot predict physiology. Conclusions: The analytical methods for the quantification of vildagliptin in microdialysate and plasma were developed and validated by following the requirements of the FDA. The plasma pharmacokinetic profile was correctly described by the model of two compartmental models. The novel tissue profiles obtained in this study may contribute to a better understanding of the pharmacological mechanisms involved and contribute to optimization of future therapies.

Objetivo: Avaliar a farmacocinética da vildagliptina em animais sadios e diabéticos, através da análise dos níveis plasmáticos totais e livres teciduais, empregando-se a técnica de microdiálise. Metodologia: A doença foi induzida nos animais através da administração de 42mg/kg de aloxano através da via intravenosa (i.v.). A vildagliptina foi administrada nas doses de 50 mg/kg (n = 6) e 75 mg/kg (n = 6) via i.v. nos animais diabéticos e na dose de 50 mg/kg (n = 6) nos animais sadios. As concentrações plasmáticas foram quantificadas por CLAE-EM-EM em método desenvolvido e validado. A ligação às proteínas plasmáticas foi determinada por microdiálise, assim como a avaliação tecidual. As sondas de microdiálise foram calibradas in vitro através de diálise e retrodiálise e in vivo utilizando retrodiálise. Para determinação das concentrações teciduais, uma segunda metodologia foi desenvolvida e validada em CLAE-EM-EM. Avaliações compartimentais (software Scientist ®) e não compartimentais (software Excel ®) foram realizadas. Resultados e Discussão: A ligação as proteínas plasmáticas apresentou um valor médio de 9,44 % ± 3,23, condizente com valores encontrados na literatura. Os valores de Ke, clearance, tempo de meia vida, MRT e VDss não apresentaram diferença estatística significativa entre as diferentes doses administradas nos animais diabéticos e entre os animais sadios. As calibrações in vitro por diálise e retrodiálise apresentaram uma recuperação média de 30%, sem diferença estatística entre as duas metodologias empregadas (α = 0,05). A recuperação in vivo também apresentou o mesmo valor médio de recuperação. A penetração tecidual do fármaco em animais diabéticos para as diferentes doses estudadas apresentou mesmo valor nos tecidos estudados, uma média de 0,20. A penetração tecidual semelhante no animal diabético pode ser devido ao dano similar entre os órgãos sofrido durante a indução da doença. Já os animais sadios apresentaram penetração tecidual similar no músculo sem diferença estatística significativa em relação aos diabéticos, entretanto no fígado foi observada uma penetração quarenta e quatro vezes inferior a observada no músculo. Essa disparidade pode ser atribuída a diferença de expressão de proteínas transportadoras no fígado do animal diabetico quando comparado ao sadio. O perfil farmacocinético plasmático foi semelhante entre os dois grupos avaliados, sendo que os parâmetros não diferiram estatisticamente (α = 0,05). Foi empregado o modelo de dois compartimentos para prever as concentrações teciduais. A previsão supõe concentrações superiores as encontradas experimentalmente, contradizendo dados de literatura. Esses dados são inéditos na literatura e demostram a importância da determinação do fármaco em tecidos alvo, uma vez que nem sempre modelos matemáticos conseguem prever a realidade fisiológica. Conclusões: As metodologias analíticas para quantificação da vildagliptina em microdialisado e plasma foram desenvolvidas e validadas, seguindo os requisitos do FDA. O perfil farmacocinético plasmático foi adequadamente descrito pelo modelo de 2 compartimentos. Os perfis teciduais obtidos nesse trabalho podem contribuir para o melhor entendimento dos mecanismos farmacológicos envolvidos e contribuir para futura otimização de terapias. / Objective: To evaluate the pharmacokinetics of vildagliptin in healthy and diabetic animals using a microdialysis technique. Methodology: Diabetes was induced in animals by administration of 42 mg/kg of alloxan intravenously (iv). Vildagliptin was administered intravenously as 50 mg/kg (n = 6) and 75 mg/kg doses (n = 6) in the diabetic animals and as a 50 mg/kg dose (n = 6) in healthy animals. Plasma concentrations were quantified by a HPLC-MS-MS method developed and validated. The plasma protein binding was determined by microdialysis and tissue evaluation. Microdialysis probes were calibrated in vitro using dialysis and retrodialysis and in vivo using retrodialysis. A second method was developed and validated using HPLC-MS-MS to determine tissue concentrations. Results and Discussion: Mean plasma protein was 9.44% ± 3.23, consistent with values reported in the literature. The values of Ke, clearance, half-life, MRT and Vdss showed no statistical difference between the evaluated doses in diabetic animals and between healthy animals (α = 0.05). Calibrations in vitro by dialysis and retrodialysis showed mean recovery of 30%, with no statistical difference between the two methodologies. Mean recovery in vivo also showed the same value. The tissue penetration of the drug in diabetic animals for the different doses studied showed the same value in both tissues studied, an mean of 0.20. The tissue penetration similar in diabetic animals could be due to the similar damage suffered between organs during induction of the disease. The healthy animals showed similar muscle penetration, compared with diabetics animals, although the liver showed a penetration forty four times lower than muscle. This discrepancy can be attributed to differential expression of transporter proteins in the liver of diabetic animals, when compared to the healthy group. The plasma pharmacokinetic profile was similar between the investigated groups, and the parameters did not differ. The two-compartment model was employed to describe the data and used to predict the concentration in the tissues. This is the first study to present these tissue profiles, which presented concentrations below the estimated by the model. These data demonstrate the importance of determining the drug inside the target tissue, as the mathematical models sometimes cannot predict physiology. Conclusions: The analytical methods for the quantification of vildagliptin in microdialysate and plasma were developed and validated by following the requirements of the FDA. The plasma pharmacokinetic profile was correctly described by the model of two compartmental models. The novel tissue profiles obtained in this study may contribute to a better understanding of the pharmacological mechanisms involved and contribute to optimization of future therapies.

Orientador: Susanne Rath / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-10T13:13:12Z (GMT). No. of bitstreams: 1 Paschoal_JonasAugustoRizzato_D.pdf: 2545620 bytes, checksum: 047e1d72fd2de11da7411d954b23d23f (MD5) Previous issue date: 2007 / Resumo: Os antimicrobianos são largamente empregados na medicina veterinária, e resíduos destes podem permanecer nos alimentos de origem animal, acima de valores considerados seguros, quando não são respeitadas as boas práticas veterinárias. Este trabalho teve por objetivos: (i) desenvolver e validar métodos analíticos para a determinação de multi-resíduos de quinolonas (enrofloxacina, ciprofloxacina, danofloxacina, sarafloxacina, ácido oxolínico e flumequina) em carne de peixe, usando a cromatografia líquida de alta eficiência associada a detecção por fluorescência (HPLC-FL) e cromatografia líquida associada a espectrometria de massas em tandem por interface de ionização por electrospray e (LC-ESI-MS/MS Q-ToF); (ii) desenvolver e validar métodos analíticos para a determinação de oxitetraciclina (OTC) em ração e carne de tilápias por HPLC-DAD e HPLC-FL, respectivamente e (iii) realizar um ensaio com tilápias (Oreochromis niloticus) para avaliar a depleção da OTC na carne desses peixes. De modo geral, a extração das quinolonas e da OTC da carne de peixes foi conduzida por extração sólido líquido seguida da limpeza do extrato em cartuchos de extração em fase sólida. A separação cromatográfica dos antimicrobianos foi realizada em coluna de fase reversa octadecil híbrida. Os métodos foram validados mediante avaliação dos seguintes parâmetros: faixa linear, linearidade, sensibilidade, seletividade, limites de detecção e quantificação, precisão intra e inter-ensaios e exatidão. Para o método LC-MS/MS foi também avaliado o efeito matriz. Todos os métodos foram considerados adequados aos objetivos propostos neste trabalho. Para avaliar a depleção de OTC na carne de tilápias, os peixes (peso médio de 93 a 115 g) receberam o fármaco via ração na dose de 80 mg OTC/kg peso vivo/dia, por cinco dias consecutivos. A temperatura da água durante o ensaio variou de 16,5 a 24,5 °C. A curva de depleção se ajustou a um modelo exponencial de primeira ordem. O tempo de meia vida de eliminação foi de 2,5 dias e o período de carência estimado foi de 5 dias / Abstract: Antimicrobials are widely employed in veterinary medicine, and their residues could remain in food of animal origin above values considered safe if good veterinary practices are not followed. The aim of this work is to address (i) the development and validation of analytical methods using high performance liquid chromatography with fluorescence detection (HPLC-FL) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS Q-Tof) for the determination of multi residues of quinolones (enrofloxacin, ciprofloxacin, danofloxacin, sarafloxacin, oxolinic acid and flumequine) in fish; (ii) the development and validation of analytical methods for the determination of oxytetracycline (OTC) in fish feed and tilapia fish fillets using HPLC-DAD and HPLC-FL, respectively, and (iii) the study of tilapias (Oreochromis niloticus) in order to evaluate the depletion of OTC from the fish fillet. In general, the extraction of the quinolones and OTC from the fish matrix was conducted by solid-liquid extraction followed by clean-up on solid phase extraction cartridges. The antimicrobials chromatographic separation was performed on a reverse phase octadecyl hybrid column. The methods were validated through the following parameters: linear range, linearity, sensitivity, selectivity, detection limit, quantitation limit, intra- and inter-assay precision and accuracy. For the LC-MS/MS method, the matrix effect was also evaluated. All methods were adequate to the proposed objectives. In order to evaluate the depletion of OTC in the tilapia fillets, the fish (weight range 93 ¿ 115 g) were given medicated feed in a concentration of 80 mg OTC/kg body weight/day for five consecutive days. The water temperature was between 16.5 to 24.5 °C during the treatment. The depletion curve was fitted to an exponential first order model. The elimination half-life was 2.5 days and the withdrawal period was estimated as five days / Doutorado / Quimica Analitica / Doutor em Ciências

La consommation excessive d’alcool présente des risques élevés pour l’individu et pour la société ; elle est fréquemment associée à une augmentation du risque d’accidents, d’actes de violence, et peut également conduire à court et/ou à long terme à de graves maladies et à des problèmes sociaux. Dès lors, l’utilisation de marqueurs fiables permettant de détecter une consommation excessive d’alcool, ponctuelle ou chronique, s’avère nécessaire pour prévenir des conséquences néfastes de l’abus d’alcool. L’éthylglucuronide (EtG) est un marqueur d’alcoolisation utilisé en toxicologie clinique (alcoologie) et médicolégale. Par rapport aux marqueurs indirects d’alcoolisation (CDT, &#947;-GT), ce métabolite mineur de l’éthanol est très spécifique et est quantifiable dans diverses matrices biologiques. La production d’EtG est catalysée par des enzymes de la famille des UDP-glucuronosyl-transférases (UGT). Cependant, les UGT impliquées dans la glucuronoconjugaison de l'éthanol, ainsi que les sources potentielles de variabilité interindividuelle de la production d'EtG, sont encore mal connues. Nos travaux ont ainsi consisté à (1) développer et valider une méthode de dosage de l’EtG dans différentes matrices biologiques par chromatographie en phase gazeuse couplée à la spectrométrie de masse en tandem, (2) identifier les UGT humaines impliquées dans la glucuronoconjugaison de l’éthanol et étudier la contribution relative de chaque isoforme active au niveau hépatique, (3) étudier l’impact de substances fréquemment utilisées par les consommateurs d’alcool sur la production d’EtG in vitro, (4) étudier l’impact de polymorphismes génétiques fonctionnels des UGT sur la production hépatique d’EtG, et enfin (5) évaluer l’impact de la consommation de cannabis et d’autres drogues sur la production d’EtG à l’aide de prélèvements post-mortem. Ces travaux ont notamment permis de montrer que (1) l'éthanol est glucuronoconjugué principalement par le foie, puis dans une moindre mesure par les reins et par l'intestin, (2) les UGT1A9 et 2B7 sont les deux enzymes majoritairement impliquées dans la glucuronoconjugaison de l’éthanol, quel que soit l’organe considéré, (3) la morphine, la codéine, la nicotine et la cotinine n’entraînent aucune modification des taux de production d’EtG in vitro ; le lorazépam et l'oxazépam augmentent légèrement cette production (p = 0,2 et 0,065, respectivement) ; le cannabidiol inhibe la glucuronoconjugaison de l’éthanol par un mécanisme non-compétitif (CI50 = 1,17 mg/L; Ki = 3,1 mg/L), alors que le cannabinol augmente cette glucuroconjugaison de manière concentration-dépendante (p <0,05), (4) les SNP c.-900G>A affectant l’UGT2B7 et IVS1+399T>C affectant l’UGT1A9 augmentent légèrement la production d’EtG in vitro. Enfin (5) le rapport des concentrations sanguines EtG/éthanol apparaît significativement plus élevé chez des co-consommateurs de cannabis et/ou d’autres drogues que chez des consommateurs d’alcool seul. L’ensemble de ces résultats démontre l’existence de plusieurs facteurs pouvant potentiellement influencer la production d’EtG et devraient donc être pris en considération lors de l’interprétation de sa concentration in vivo. / Alcohol abuse is frequently associated with an increased risk of road accidents and violence, and can also lead to serious social and health problems. Therefore, the use of reliable markers to detect excessive punctual and/or chronic consumption of alcohol is necessary to prevent the harmful consequences of alcohol abuse. Ethylglucuronide (EtG) has been proposed as a marker of alcohol consumption in a variety of clinical and forensic contexts. Compared with the indirect markers (e.g. CDT, &#947;-GT), this minor metabolite of ethanol is very sensitive and specific, and is quantifiable in various biological matrices. It is formed by conjugation of ethanol with uridine 5’-diphosphate glucuronic acid (UDP-GA) via the action of UDP-glucuronosyltransferase (UGT) enzymes. However, the knowledge of the UGTs involved in the glucuronidation of ethanol, and the potential sources of the interindividual variability of EtG production are still not clearly established. The aims of our work were (1) to develop and validate a method for the determination of EtG in different biological matrices by gas chromatography coupled with tandem mass spectrometry, (2) to identify the human UGT isoforms involved in the glucuronidation of ethanol, and then to evaluate qualitatively and quantitatively their specific contribution in the formation of EtG, (3) to study the impact of the co-administration of drugs frequently used by ethanol consumers on the in vitro production of EtG, (4) to study the impact of functional genetic polymorphisms of two UGTs on the hepatic production of EtG, and finally (5) to study the impact of the consumption of cannabis and other drugs on the production of EtG using post-mortem samples. The main results of our study showed that (1) ethanol is primarily glucuronidated by the liver and, to a lesser extent, by kidneys, (2) UGT1A9 and 2B7 were identified as the main human UGTs involved in ethanol glucuronidation, (3) morphine, codeine, nicotine, and cotinine did not modify EtG in vitro formation rate; lorazepam and oxazepam produced a minor, but not significant, increase of EtG formation. Only cannabinol and cannabidiol significantly affected ethanol glucuronidation; cannabinol significantly increased the glucuronidation of ethanol in a concentration-dependent manner, whereas cannabidiol inhibited the glucuronoconjugaison of ethanol by a non-competitive mechanism (CI50 = 1.17 mg / L; Ki = 3.1 mg / L), (4) the SNP c.-900G>A and IVS1+399T>C affecting UGT2B7 and UGT1A9, respectively, seem to increase the in vitro production of EtG, and (5) cannabis and/or drugs consumption (mainly opioids, benzodiazepines, and paracetamol) seem to be associated with ratios of blood concentrations of EtG/ethanol significantly higher than those observed among only alcohol consumers. Taken together, these results show the existence of several factors that could potentially influence the production of EtG, and that should be taken into account when interpreting its concentration in vivo.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22487号 / 農博第2391号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5267(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 梅澤 俊明, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

High-throughput liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methodology for the determination of methamphetamine (METH), amphetamine (AMP), 4-hydroxymethamphetamine (4-OH-METH), and 4-hydroxyamphetamine (4-OH-AMP) was developed and validated using simple trichloroacetic acid sample treatment. The method was validated in rat serum, brain, and testis. Lower limits-of-quantitation (LOQ) for METH and AMP were 1 ng·mL-1 using positive ion electrospray tandem mass spectrometry (MS/MS). The accuracy of the method was within 25% of the actual values over a wide range of analyte concentrations. The within-assay precision was better than 12% (coefficient of variation). The method was linear over a wide dynamic range (0.3-1000 ng·mL-1). Quantitation was possible in all 3 matrices using only serum standards because of minimal matrix-associated ion effects or the use of an internal standard. Finally, the LC-MS/MS method was used to determine serum, brain, and testis METH and AMP concentrations during a subcutaneous infusion (5.6 mg kg-1 day-1) of METH in rats. Concentrations of 4-OH-AMP and 4-OH-METH were below the LOQ in experimental samples. The bias introduced by using serum calibrators for the determination of METH and AMP concentrations in testis and brain was less than 8% and insignificant relative to the interanimal variability.

While ketamine is traditionally administered for anesthesia or pain management, illicit usage is often seen in forensic cases either as a recreational drug or as a tool in drug-facilitated sexual assault. Xylazine is an anesthetic agent used in veterinary medicine and does not have FDA approval for use in humans. However, it has recently been observed as a cutting agent in heroin. Post-mortem specimens present many challenges when it comes to toxicological analysis. Due to compound degradation and decomposition factors, analytes present at trace levels may be missed in blood and urine. Hair, bone, and insects have recently been investigated as alternative matrices for postmortem analysis due to their increased durability compared to more traditional matrices. However, this durability increases the difficulties in extracting and isolating compounds of interest from these matrices via traditional extraction and chromatography methods. These methods require lengthy extraction times and extensive cleanup steps in order to obtain samples suitable for analysis. Utilizing multiple instrumentation combinations, analysts are able to detect compounds at trace levels. Through the use of multidimensional chromatography, several time-consuming extraction steps can be eliminated while still retaining the ability of trace level detection and quantitation. Using Waters Oasis® HLB PRiME solid phase extraction cartridges using a methanol pH10 loading and an acetonitrile pH3 elution, a solvent extraction yielded linear dynamic ranges of 2pg/mL-1ng/mL and 5pg/mL-1ng/mL for xylazine and ketamine respectively. Rat specimens utilized in this project were treated as per an Institutional Animal Care and Use Committee (IACUC) protocol. The test rodents received an acute dosage of 2mg/mL of xylazine and 24mg/mL of ketamine approximately half an hour prior to death. The 14 test samples were placed outside directly on the ground at the Boston University Forensic Anthropology Outdoor Research Facility (Holliston, MA, U.S.A.) for a period of 6 months. A 15th rat was kept in -20°C until analysis to serve as a Time=0 sample. The outdoor samples were recovered and de-fleshed along with the Time=0 sample manually. Drug-free hair samples were donated anonymously as per Internal Review Board (IRB) protocols.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19517号 / 理博第4177号 / 新制||理||1600(附属図書館) / 32553 / 京都大学大学院理学研究科化学専攻 / (主査)教授 藤井 紀子, 教授 三木 邦夫, 教授 杉山 弘 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19752号 / 工博第4207号 / 新制||工||1649(附属図書館) / 32788 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 梅田 眞郷, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM